Preface

Chloroplasts, vital plant organelles, are being studied for potential applications in biotechnology, agriculture, biopharming, and renewable energy, leveraging advancements in genome sequencing technologies.

The book, *Chloroplast Structure and Function*, consists of seven chapters. In Chapter 1, "Introductory Chapter: Biology and Biotechnological Applications of Chloroplasts", Dr. Khan highlights the potential of green plant pigments in agriculture, biopharming, renewable energy, and beyond. Further, he demonstrates the essential role of the pigments in plant adaptation and response. In Chapter 2, Dr. Liu et al. explain the characteristics of the chloroplast genome of several plants on the Qinghai–Tibet Plateau. Further, they use the chloroplast genome in molecular evolution and phylogenetics to study plant species and their genetic information. In Chapter 3, Dr. Soumaya et al. confirm the domestication of date palms in the Arabian Peninsula and demonstrate an important gene flow with North African palm populations, considering chloroplast DNA as the best molecule for determining the evolutionary history of plant species. In Chapter 4, Dr. Espinoza Sánchez et al. explain the importance of plastome engineering, which offers potential for food security, drug production, and sustainable energy; however, challenges remain in expanding to other crops. In Chapter 5, Dr. Khan et al. explain chloroplast recycling and stress tolerance in plants. Further, they briefly describe how plastids regulate plant responses to stress by producing pigments, metabolites, and phytohormones, communicating with the nucleus, releasing nutrients, and degrading proteins, and improving stress tolerance and adaptation. In Chapter 6, Dr. Kucheli explains that guard cells, containing chloroplasts and stomata, regulate plant productivity by controlling stomatal pore, CO2 uptake, and water loss. Further, overexpression of Rieske FeS protein improves quantum efficiency and growth rates. In Chapter 7, Drs. Saefudin and Basri highlight how chlorophyll and tannin production from forestry waste can boost selling-price capacity, reduce environmental impact, and provide economic opportunities for small businesses, enhancing fabric coloring, food safety, and health benefits.

> **Muhammad Sarwar Khan** Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan

#### **Chapter 1**

## Introductory Chapter: Biology and Biotechnological Applications of Chloroplasts

*Muhammad Sarwar Khan*

#### **1. Introduction**

Chloroplasts are semi-autonomous, photosynthetic organelles that play an essential role in photosynthesis. Beyond their basic role in photosynthesis, these extraordinary structures are critical to a variety of important cellular processes, including the response to stress and the synthesis of various biomolecules including vitamins, lipids, amino acids, phytohormones, and products of secondary metabolism [1]. Chloroplast transformation presents numerous benefits, such as the precise integration of a transgene at a predetermined location within the plastome through homologous recombination. It avoids issues such as gene silencing and position effects, ensuring elevated levels of transgene expression due to the high ploidy plastome. Furthermore, maternal inheritance prevents transgenic leakage through pollen, making transplastomic GMOs environmentally friendly. Numerous valuable traits have been purposefully incorporated to enhance the agronomic performance of crop plants. The chloroplast transformation system has proven instrumental in the expression of industrial enzymes and therapeutic proteins, indicating it as a promising solution to address persistent issues surrounding food security, drug manufacturing, and sustainable energy generation [2, 3]. Chloroplasts have firmly established their capabilities by proficiently expressing transgenes, and packaging recombinant proteins, thus protecting them from cellular degradations. This makes it easier to produce highly useful proteins on a large scale. Various segments of the chloroplast genome have served as stable markers for identification and evolutionary research. The ability of chloroplasts to produce tannin and chlorophyll also provides a sustainable pathway for the synthesis of a variety of natural pigments [4]. This boosts not only the market value of these plants but also endows them with biologically beneficial and environment-friendly attributes. Additionally, chloroplasts interact with the nucleus and other organelles of the cell to organize the acquisition of essential molecules needed for their resistance to stressful environmental conditions. They act as sensitive environmental sentinels that effectively produce substances to mitigate stress and, particularly, induce the activation of genes that are encoded by the nucleus, to develop stress resistance (**Figure 1**).

*Graphical representation of chloroplast biology and biotechnological interventions through plastome engineering.*

#### **2. Advances in chloroplast genomics**

Plastid genomes, the 'plastomes' contain 120–130 genes and the majority of them are responsible for encoding critical parts of the organelle's gene-expression machinery and substances necessary for photosynthesis. These genes are neatly arranged within nucleoids. Tobacco was the first higher plant whose plastid genome was sequenced, opening the door for the sequencing and characterization of hundreds of new higher plants. Researchers now have access to an extensive amount of plastid genome data that will help them better understand how the genes in these plastids are functionally characterized. Many of the genes discovered in chloroplasts have undergone in-depth analysis to see how they affect the stability and metabolic functions of these organelles. A collection of genes called hypothetical chloroplast open reading frames (ycfs) is still unknown to have all of its specialized roles fully understood. Some of these ycfs have been classified as non-essential, while others have been classified as essential elements [5].

Recent advancements in sequencing technologies, such as next-generation sequencing (NGS), have made it possible to quickly and affordably conduct complete chloroplast genome sequencing. This is because chloroplast genomes contain an enormous amount of valuable information that is useful for species identification, phylogenetic inferences, and population genetic studies. Chloroplast genome analysis is more effective in understanding the complexities of progeny evolution and their evolutionary relationships. This decision is based on the haploid structure, maternal inheritance, and unusually well-preserved genes [6]. Furthermore, in recent times, chloroplast genomics has emerged as a highly promising tool extensively employed in phylogenetic investigations, owing to its exclusive maternal inheritance and absence of recombination events [7]. The study of chloroplast genetics has played a pivotal role in unraveling the dynamics of gene distribution, cytoplasmic diversity, and population divergence. Recently, chloroplasts have been associated with a number of vital functions within numerous plant processes, including plant defense mechanisms, the synthesis of defensive compounds, as well as the regulation of plant physiology, development, and the alternative splicing of transcripts [8, 9].

*Introductory Chapter: Biology and Biotechnological Applications of Chloroplasts DOI: http://dx.doi.org/10.5772/intechopen.113272*

#### **3. Plastids as a source of natural and engineered products**

Innovative advances in the synthesis of chlorophyll and tannin have led to the development of their liquid, paste, and dry forms. This improvement in the production of natural pigments raises their market value while also promoting biologically favorable qualities that are in line with their sustainability and health advantages. Chlorophyll and tannin are abundantly available in wood waste, especially from forestry operations that use mangrove trees. These organic substances have the potential to replace synthetic pigments, in sustainable ways which are frequently connected to environmental issues. Utilizing wood waste in this way could promote and provide business opportunities in forest villages, mangrove industry hubs, and small- to medium-sized batik and weaving businesses. Batik artists who use coastal and inland motifs seem to prefer using natural chlorophyll/tannin hues. Therefore, research into different natural sources of tannin that are safe for food and drink as well as the human body is crucial for fabric and batik coloring.

Over the past few years, advances in chloroplast engineering have shown great promise in addressing critical global challenges such as ensuring enough food, producing medicine, and generating sustainable energy for our growing world population. Chloroplasts have proven to be effective at expressing transgenes and protecting the recombinant proteins from cellular processes, resulting in highly functional proteins. This characteristic has also been useful in the field of RNA interference technology. In addition to the practical advantages of chloroplast transformation, such as the elimination of positional effects, the capacity for polycistronic expression, and significant protein production, this method indicates a development in biosafety; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite rigorous efforts, a few important crops have shown to be resistant to chloroplast transformation, which limits their ability to be grown more widely. This chapter covers the most recent developments in this field as well as the challenges that still need to be overcome before this method can be applied to more crops and become an important tool in the field of plant biotechnology.

#### **4. Plastid engineering for crop improvement**

The incorporation of specific genes into the chloroplast genome has proven effective in enhancing important agricultural qualities in crop plants. Due to its numerous benefits when compared to nuclear transformation, plastid modification has emerged as a powerful method for the genetic engineering of significant crops. Potato [10] and sugarcane [11] are the most recent examples. Many initiatives to increase food yields concentrated on improving plants' capacity for photosynthetic activity. Rubisco, the key enzyme at the center of photosynthesis, is made up of two parts: a large subunit encoded in chloroplasts and a tiny subunit encoded by the nucleus, which is later imported into the chloroplast. Similar to this, efforts have been made to change either the RuBisCO big subunit, the small subunit, or both parts [12] attempted to express complete RuBisCO protein in tobacco from *Synechococcus elongatus* and found CO2 fixation rate and carboxylase activity of the RuBisCO to be increased. Another option for enhancing photosynthetic carbon fixation and agricultural yield is to increase the concentration of CO2 in plastids. The introduction of the cyanobacterial bicarbonate transporter into the tobacco plastid genome failed to yield a significant enhancement

in photosynthetic efficiency. However, when fructose-1, 6-sedoheptulose-1, and 7-bisphosphatase were expressed in both lettuce and tobacco chloroplasts, a notable boost in the productivity of these genetically modified plants became evident. The *chlB* gene from *Pinus thunbergii*, which is chloroplast-encoded, was also discovered to encourage tobacco root growth and early chlorophyll pigment development [13]. In order to increase biomass production, research is being done to convert C3 plants to C4 by modifying the RuBisCO large subunit and photo respirational pathway [14].

Since 1994, insect-resistant crops have flourished in fields, but Bt crops are raising concerns about the development of resistance. The development of insect-resistant transplastomic plants is a promising strategy, leveraging the benefits of transplastomic technology that have already been mentioned [10]. On the other hand, dsRNA expression, which targets an important insect gene in transplastomic plants, has been tested as a unique non-Bt-type insect resistance method. Within 5 days of feeding, RNA interference caused the target gene to be disrupted, killing all adult beetles and larvae [15]. When the agglutinin gene (pta) was expressed in leaf chloroplasts, aphids, lepidopteran insects, and bacterial and viral diseases were all successfully resisted [16]. The integration of the CeCPI gene (derived from sweet potatoes) and chitinase from *Paecilomyces javanicus* into tobacco plants conferred resistance not only to a wide range of pests and diseases but also rendered the plants resilient to salinity, osmotic stress, and oxidative challenges [17]. Proposing a potential defense against damage from excessive oxidation, increasing the activity of the mdar gene in tobacco plastids, and merging these chloroplasts with Petunia cells were suggested. This modification also seemed to make tobacco plants more resilient to oxidative stress. Additionally, when tobacco plants were genetically altered with the cyanobacterial flavodoxin (fld), they became better at handling oxidative stress. Furthermore, tobacco plants modified with the panD gene showed not only a 30–40% boost in their growth but also proved to be more resistant to high temperatures. Similarly, when arabitol dehydrogenase (ArDH) was introduced into tobacco chloroplasts, it allowed the plants to survive even in environments with extremely high salt concentrations, such as 400 mM NaCl [18]. This may open up novel opportunities for understanding how to cultivate resilient plants that can withstand stress and the value of transplastomic technologies.

#### **5. Conclusion**

In conclusion, chloroplasts are not only the most active cell organelle metabolically but also hold an immense potential for various biotechnological applications. Chloroplast being the main site of photosynthesis plays a pivotal role in a plant's responsiveness and mitigation to biotic as well as abiotic stresses. Studying its basic biological attributes helps to unfold new possibilities of innovations in agriculture, biopharming, renewable energy, and beyond.

*Introductory Chapter: Biology and Biotechnological Applications of Chloroplasts DOI: http://dx.doi.org/10.5772/intechopen.113272*

#### **Author details**

Muhammad Sarwar Khan Center of Agricultural Biochemistry and Biotechnology, Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan

\*Address all correspondence to: sarwarkhan\_40@hotmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[2] De-la-Pena C, Leon P, Sharkey TD. Editorial: Chloroplast biotechnology for crop improvement. Frontiers in Plant Science. 2020;**13**:848034. DOI: 10.3389/ fpls.2022.848034

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[4] Rascón-Cruz Q, González-Barriga CD, Iglesias-Figueroa BF, Trejo-Muñoz JS, Siqueiros-Cendón T, Sinagawa-García SR, et al. Plastid transformation: Advances and challenges for its implementation in agricultural crops. Electronic Journal of Biotechnology. 2021;**51**:95-109

[5] Khan MS, Riaz R, Majid M, Mehmood K, Mustafa G, Joyia FA. The tobacco chloroplast YCF4 gene is essential for transcriptional gene regulation and plants photoautotrophic growth. Frontiers in Plant Science. 2022;**13**:1014236. DOI: 10.3389/ fpls.2022.1014236

[6] Khan AL, Asaf S, Lee IJ, Al-Harrasi A, Al-Rawahi A. First chloroplast genomics study of *Phoenix dactylifera* (var. Naghal and Khanezi): A comparative analysis. Plos One. 2018;**13**. DOI: 10.1371/journal. pone.0200104

[7] Liu H, He J, Ding C, Lyu R, Pei L, Cheng J, et al. Comparative analysis

of complete chloroplast genomes of Anemoclema, Anemone, Pulsatilla, and Hepatica revealing structural variations among genera in tribe Anemoneae (Ranunculaceae). Frontiers in Plant Science. 2018;**9**:1097. DOI: 10.3389/ fpls.2018.01097

[8] Singh NV, Patil PG, Sowjanya RP, Parashuram S, Natarajan P, Babu KD, et al. Chloroplast genome sequencing, comparative analysis, and discovery of unique cytoplasmic variants in pomegranate (*Punica granatum* L.). Frontiers in Genetics. 2021;**12**:704075. DOI: 10.3389/fgene.2021.704075

[9] Toufexi A, Duggan C, Pandey P, Savage Z, Segretin ME, Yuen LH, et al. Chloroplasts navigate towards the pathogen interface to counteract infection by the Irish potato famine pathogen. BioRxiv. 2019;**2019**:516443

[10] Hossain MJ, Bakhsh A, Joyia FA, Aksoy E, Gökçe NZÖ, Khan MS. Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, *Leptinotarsa decemlineata* (Coleoptera: Chrysomelidae). Transgenic Research. 14 Sep 2023. DOI: 10.1007/ s11248-023-00366-6

[11] Mustafa G, Khan MS. Transmission of engineered plastids in sugarcane, a C4 monocotyledonous plant, reveals that sorting of preprogrammed progenitor cells produce heteroplasmy. Plants*.* 2021;**10**(1):26. DOI: 10.3390/ plants10010026

[12] Lin MT, Occhialini A, Andralojc PJ, Parry MA, Hanson MR. A faster RuBisCO with potential to increase photosynthesis in crops. Nature. 2014;**513**:547-550. DOI: 10.1038/ nature13776

*Introductory Chapter: Biology and Biotechnological Applications of Chloroplasts DOI: http://dx.doi.org/10.5772/intechopen.113272*

[13] Nazir S, Khan MS. Chloroplastencoded chlB gene from *Pinus thunbergii* promotes root and early chlorophyll pigment development in *Nicotiana tabaccum*. Molecular Biology Reports. 2012;**39**:10637-10646. DOI: 10.1007/ s11033-012-1953-9

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#### **Chapter 2**

## Chloroplast Genome Characteristics of Plants on the Tibetan Plateau

*Ying Liu, Jinping Qin, Zhengsheng Li, Lijun Zhang and Xinyou Wang*

#### **Abstract**

Located in the interior of Asia, the Qinghai-Tibet Plateau is the largest plateau in China and the highest plateau in the world. It is also known as the "Roof of the world" and the "third pole". The various nature reserves on the Qinghai-Tibet Plateau are a treasure house of natural resources with the strangest ecological environment and the richest biological resources on the roof of the world. They are of high scientific value. This chapter will describe the chloroplast genome characteristics of several plants on the Qinghai-Tibet Plateau, such as *Aster, Asterothamnus centraliasiaticus, Aster altaicus, Corethrodendron multijugum*, *Clematis nannophylla*, and so on.

**Keywords:** chloroplast genome, *Aster*, *Asterothamnus centraliasiaticus*, *Aster altaicus*, *Corethrodendron multijugum*, *clematis nannophylla*

#### **1. Introduction**

Chloroplasts are important organelles in plants that help in photosynthesis [1]. Chloroplasts of higher plants are metabolic centers that sustain life on Earth through photosynthesis, as a semi-autonomous organelle with own DNA that encodes and has an independent genetic system [2]. For several reasons, cp genomes are more commonly used to study plant molecular evolution and phylogeny than mitochondrial genomes. The researchers note that differences in chloroplast genome sequences and their highly differentiated regions between plant species and individual plants not only enable a more comprehensive study of the taxonomy and phylogeny of these species, but also facilitate the identification, breeding, and conservation of valuable biological genetic information among related species [3, 4]. Here, we collected representative plants of alpine meadow, alpine steppe, desert steppe grassland types on the Qinghai-Tibet Plateau and determined their chloroplast genome sequences to understand their genome characteristics and phylogenetic evolution.

#### **2. Species morphological characteristics and distribution information of five** *Aster* **species**

*Aster* is a perennial herb of the Compositae family. Most *Aster* plants have well-developed roots, rhizoid type, and rapid reproduction [5]. With large root surface area, a large number of nutrients in soil can be absorbed and utilized for ecological restoration, giving full play to their ornamental and ecological value. It is vigorously used in greening and beautifying the environment to give full play to its ecological functions [6].

*Aster* has a flower head, with ligulate of purple or bluish-purple flowers and tubular of yellow flowers, which is of high ornamental value. *Aster* plants is one of the traditional Chinese herbs, and most *Aster* species contain a large number of important medicinal components such as flavonoids and quercetin [7–9], and the root and stem of *Aster* plants are commonly used as medicines. *Aster* species has a long history of medicinal use, there are many varieties of *Aster* distributed on the Qinghai-Tibet Plateau, and it is used in traditional Tibetan medicine as a medolome to treat seasonal epidemic diseases and clear heat and detoxification [10–12].

China is the main origin and distribution center of Eurasian *Aster* [12, 13], with about 123 species, including 82 endemic species. *Aster* has many species and abundant resources in China, widely distributed in northeast, northwest, southwest, and south China [5]. The Qinghai-Tibet Plateau has a unique geographical location, rich grassland resources, and high species diversity; according to the Flora of China, the Qinghai-Tibet Plateau is also one of the distribution centers of *Aster*. Here, we selected Guoluo Prefecture, the source of the Yellow River in the hinterland of the Qinghai-Tibet Plateau, to conduct a comprehensive survey of *Aster* germplasm resources and investigate the morphological characteristics of *Aster* species and determine the chloroplast genome of *Aster* species. Combined molecular tools with classical morphological information could prove a valuable information for a more accurate classification of Aster species.

#### **2.1 Morphological characteristics of five species of** *Aster*

#### *2.1.1* Aster yunnanensis *var.* labrangensis *(Hand. -Mazz.) ling*

*A. yunnanensis* var. *labrangensis* is usually perennial herbs, and its height is about 20-70 cm (**Figure 1A**); Rhizome; stems erect and upper branched, stems 2–5 branched; both surfaces of leaves hairy and glandular, half clasped; Involucres hemispheric, 4-7 cm in diameter. The Ray florets are bluish-purple, the disk florets are yellow, the sparsely to moderately villous are 2 layers, short outside and long inside. Flowering period is July-August, and fruit period September-October. Generally found in hillside meadows, alpine meadow or thicket, elevation 3300–4300 m.

#### *2.1.2* Aster farreri *W. W. Sm. et J. F. Jeffr*

*A. farreri* is usually perennial herbs, its height is about 30–60 cm (**Figure 1B**); rhizome long; stems erect, simple, sparsely to moderately villous. The leaves are basal and cauline, and sparsely villous, eglandular, margin entire or sparsely serrulate, villous-ciliate;blade oblanceolate to narrowly oblanceolate, 1.5–22 × 0.7–2.3 cm, leaf base gradually narrow, apex acute; middle cauline leaves linear-lanceolate, 7–13 x 0.7–2 cm, base rounded, subvalvate to valvate, apex acuminate; upper blade linear,

*Chloroplast Genome Characteristics of Plants on the Tibetan Plateau DOI: http://dx.doi.org/10.5772/intechopen.112100*

#### **Figure 1.**

*Morphology and habitat map of five species of* Aster*. (A)* Aster yunnanensis *var.* labrangensis*; (B)* Aster farreri*; (C)* Aster souliei*; (D)* Aster asteroids*; (E)* Aster poliothamnus*.*

about 2 x 0.1 cm, apex sharp. The capitulum is terminal, solitary, 5–8 cm in diameter, and the involucre is hemispherical, 2–2.4 cm in diameter; ray florets are purplish blue or lilac, disk florets are yellow; Pappus 2 layers, white or smudge white, outer layer very short. Flowering period is in July-August, and fruit period is in August-September. Generally distributed in Alpine, subalpine slope grassland, sand and gravel, elevation 2600–4200 m.

#### *2.1.3* Aster souliei *Franch*

*A. Souliei* is usually perennial herbs, its height is about 2–45 cm (**Figure 1C**), sometimes caespitose; rhizomes robust, woody. Stems solitary, erect. The leaves are basal and cauline, the leaf surface is smooth to sparsely hairy, the margin is whole, and the basal leaves present at flowering stage are spatulate or obovate to oblanceolate; inflorescence terminal and solitary, 3–6 cm in diameter, involucral hemispherical, 6–8 mm; floret 25–55 radiate, bluish-purple to purple, tube glabrous, lamina 12–25 × 2–3 mm, glabrous, glandular; disk florets yellow,3–5 mm, pappus 1 layer, purplish brown. The flowering period is from July to August, and the fruit period is from August to September. It is generally distributed in alpine thickets, meadow, and hillside meadows, with an altitude of 2700–4500 m.

#### *2.1.4* Aster asteroids *(DC.) O. Kuntze*

*A. asteroids* is usually perennial herbs, its height is about 2–25 cm (**Figure 1D**); rhizomes short; roots tuberoid, near ground surface, with 2–6 tuberous roots, radish shape, stems erect, solitary, 2–15 cm tall to 30 cm. Basal leaves are dense and live at flowering stage, obovate, or oblong, 1–4 cm long, 0.4–0.8 thin, 1.7 cm wide, leaf base tapered into a short stalk, subentire, with few fine teeth; middle leaf oblong or oblong spatulate, apex obtuse or acuminate, sessile, upper leaf linear. All leaves are with long hairs that are sparse or dense above, glabrous below or hairy only along veins, with long marginal hairs, with three veins off base. The capitula terminal are solitary at the end of the stem and are 2–3.5 cm in diameter. Involucral hemispherical, 0.7–1.5 cm in diameter; involucral bracts 2–3 layers, subequal, linear-lanceolate, 5.5–7 mm long, 1–1.5 mm wide, apical acuminate, abaxially and margin with purplish-brown dense hairs. Ray florets1 layer, about 30–60, tongue bluish-purple, 10–20 mm long, 1–2 mm wide, apical point; disk florets orange-yellow, tube 1 mm long, split 1–2 mm long, with black or colorless glandular hairs; Achenes oblong, up to 3 mm long, white sparsely hairy or silky. The flowering period is from June to July, and the fruit period is from July to August. It is generally distributed in Alpine meadow, thickets, and hillside grassland, with an altitude of 2750–4800 m.

#### *2.1.5* Aster poliothamnus *diels*

*A. poliothamnus* is usually perennial subshrubs, and its height is about 15–100 cm (**Figure 1E**), sometimes shrublike, caespitose, caudex woody. Stems branched, branches erect, there are dense leaves. Lower leaf withered; middle leaf oblong or linear oblong, about 1–2 cm long and 0.2–0.5 cm wide, entire margin, blade base slightly narrow or sharply narrow, apex blunt or pointed, leaf margin flat or slightly reversed; upper leaf small, elliptic. All leaves are short strigose above, pilose below, glandular on both sides, midvein raised below, lateral vein not visible. Inflorescence densely corymbose or solitary at branch ends; inflorescence peels are thin, 1–2.5 cm long, with sparse bracteoles. The involucre is broad campanulate, 5–7 mm long, 5–7 mm in diameter; involucral bracts 4–5 layers, imbricate arranged, outer oval or oblong-lanceolate, 2–3 mm long, all or upper grassy, apically apical, outer or only along midvein densely puberulent and glandular; inner layer up to 7 mm long, 0.7 mm wide, subleathery, upper grassy and reddish purple, marginal hairs. 10–20 Ray florets, lilac, oblong, 7–10 mm long, 1.2–2 mm wide. The disk florets are yellow, 5–6 mm long and 1.6–2 mm long. The crest is dirty white and about 5 mm long. Achenes oblong, 2–2.5 mm long, often ribbed on one side, covered with white silky hair. The flowering period is from July to August, and the fruit period is from August to September. It is generally distributed in arid slopes, alpine timberline, glades, and so on, with an altitude of 1800–3800 m.

#### **2.2 Chloroplast genome characteristics of** *Aster* **species**

We sequenced the chloroplast genome of five *Aster* leaves collected from Maqin County, Guoluo Prefecture, Qinghai Province, China (36°010 N, 103°450 E, altitude 3970 m). The chloroplast genome was sequenced. And the assembled chloroplast genome and its detailed annotation were submitted to GenBank (*Aster yunnanensis* var. *labrangensis*: OQ569735.1; *Aster diplostephioides*: OQ603807.1; *Aster farreri*: OQ603808.1; *Aster souliei*: NC\_073537.1; *Aster asteroides*: NC\_073536.1; *Aster poliothamnus*: OQ658689.1).

The complete chloroplast genome sequence lengths varied from 152,549 to 153,087 bp of five species of Aster (**Figure 2**), which has the quadripartite structure typical for most higher plants and highly conserved, respectively, which was divided into a LSC (84218-84,742 bp) and an SSC (18165–18,307 bp) regions separated by a pair of inverted repeats (IR, 24960–25,031 bp). In addition, the overall GC content of the chloroplast genome sequence was 37.3%. Since all the rRNAs were located in the IR

#### **Figure 2.**

*Chloroplast genome map of five* Aster *species. Genes inside the circle are transcribed clockwise, and those outsides are transcribed counter-clockwise. Genes of different functions are color-coded. The darker gray in the inner circle shows the GC content, while the lighter gray shows the AT content. The red and blue lines indicate GC skew, the red means GC skew greater than zero, while the blue means GC skew smaller than zero. Same as the chloroplast genome characterization below.*

regions, the GC content of the IR regions (42.9%) was higher than that of the LSC (35.2%) and SSC (31.2%) regions, respectively. A total of 130 genes were annotated successfully, including 85 protein-coding genes, 37 tRNAs, 8 rRNAs, respectively.

The CDS and tRNA of the five *Aster* species were mainly distributed in the LSC region, rRNA was only distributed in the IR region, and only one tRNA was distributed in the SSC region. Nineteen genes had two copies, which comprised of seven CDS coding genes, seven tRNA genes, and all four rRNA species (rrn16, rrn23, rrn4.5, and rrn5). In the genome, eighteen genes contain introns, among them 17 contains 1 intron and pafI contains 2 introns. The start codon of all CDS is ATG except rps19, where the start codon of rps19 is GTG, respectively.

Similarity in codon usage and amino acid frequencies were observed in five *Aster* species. And the results showed that about twenty-five thousand amino acids were detected in the chloroplast genome of five *Aster* species, in which Leucine was the most abundant, with two thousand and seven hundred codons (10.8%), followed by Isoleucine with two thousand and one hundred codons (8.4%), Serine and Glycine, with about 1960 and 1750 codons (7.7% and 6.9%), and Cysteine was the least abundant. There are 29 codons with RSCU values greater than 1 in five Aster. Methionine and Tryptophan had RSCU values equal to 1, but the most preferred codon was TTA, encoding Leucine (Leu), with an RSCU value of about 1.85.

SSRs are highly polymorphic molecular genetic markers, widely used, especially for population, evolutionary, and conservation genetics studies and forensics [14]. SSRs are composed of one or a few consecutive repeated nucleotides. By analysis of SSR dynamics in chloroplast genomes of five *Aster* species show that the pentanucleotide SSRs was only found in *A. asteroids* and *A. poliothamnus*, while the types and numbers of SSRs varied across species of *Aster*, indicating genetic diversity among species. A6 contains less mononucleotides and trinucleotides than the other four species of *Aster*. In five *Aster* species chloroplast genome, mononucleotide repeats are the most abundant repeats, followed by tetranucleotides, dinucleotides, and trinucleotides repeat, while pentanucleotides and hexanucleotides repeats rarely occur, and all of the mononucleotide repeats consisted of either A or T bases. SSR repeats in LSC region were much higher than SSC region and IR region in five *Aster* species.

The chloroplast genomes are of great significance in the reconstruction of plants phylogenetic relationships and evolutionary history [15]. In our study, we constructed a phylogenetic tree using the sequences of the whole chloroplast genomes of 41 species in the family *Aster*, including 24 (including *Heteropappus* and *Symphyotrichum*) species and using 15 species in *Senecio, Diplostephium*, *Asterothamnus, Artemisia*, and *Erigeron* as outgroups. The alignment of plastomes was generated by MAFFT. The maximum-likelihood (ML) analysis was performed using MEGA11, of which the bootstrap values were calculated using 1000 replicates with the best selected GTR + G model [14]. The result showed that all clades were strongly supported, *A. ageratoides* var. *scaberulus* is sister to a clade formed by *A. yunnanensis* var. *labrangensis*, *A. farreri*, *A. asteroids*, *A. souliei*, and *A. batangensis* according to the current sampling extent, and *A. poliothamnus* was sister to *A. sampsonii.*

#### **2.3 Discussion and conclusion**

In this chapter, the architecture of the basic characteristics, codon usage bias, SSRs and phylogenetic relationships of chloroplast genomes of five *Aster* species are studied. Five species of *Aster* have a typical quadripartite structure and 130 functional *Chloroplast Genome Characteristics of Plants on the Tibetan Plateau DOI: http://dx.doi.org/10.5772/intechopen.112100*

genes have been annotated. Moreover, in the coding strand, the bias of T over A existed in all five Aster species, while the bias of G over C differed. These findings, in combination with identified introns, codon usage bias, and SSRs, enrich our knowledge on chloroplast biology and genetic diversity of five *Aster* species and lay a strong foundation for further studies on molecular marker development, phylogenetic analysis, population studies, and chloroplast genome engineering.

#### **3. Morphological characteristics and chloroplast genome analysis of** *Aster altaicus* **Willd**

#### **3.1 Species morphological characteristics and distribution information**

*Aster altaicus* Willd., depicted in **Figure 3** [16], is a perennial herbaceous plant. It was previously classified as *Heteropappus altaicus* when it was part of the *Heteropappus* genus. However, due to its unique feature of ray floret having very short crown hairs, it was often associated with *Aster*, although some other *Aster* species also exhibit this trait. Eventually, it was reclassified solely under the *Aster* category [17]. Typically, these plants grow to be 15–40 cm tall and possess transversely or vertically woody roots. The stems originate at the base and give rise to multiple branches, which can be either erect or obliquely branched. These branches are covered with upwardly curved or spreading hairs and contain glands situated distally. The leaf blade is linear, oblong, or oblanceolate, measuring 3–35 mm in length and 1–7 mm in width. The anterior ends of the leaves can be blunt or acute and are usually complete, featuring short coarse or fine hairs on both upper and lower surfaces, accompanied by glandular dots. At the uppermost parts of the branches, multiple heads grow either individually or in an umbellate fashion. The involucre, approximately 1–1.5 cm in diameter, takes the shape of a hemispherical

#### **Figure 3.**

*Morphology and habitat map of* Aster altaicus*. From Yueliang Bay park in Guide County, Hainan Tibetan autonomous prefecture, Qinghai Province, China (101°43*′*95" E, 36°04´61" N), photograph by Ying Liu.*

structure and consists of 2–3 layers of nearly equal-length involucral bracts. These bracts are oblong-lanceolate or linear, approximately 5 mm long and 1.5 mm wide, with tapered pointed tips. They exhibit narrow membranous margins and glandular hairs on the abaxial side. The flower head contains 15–20 ray florets with tubes measuring around 2.5 mm. The ligules of these florets are blue, linear-oblong, reaching a length of 15 mm and a width of approximately 2 mm. The ray florets themselves are yellow, about 5 mm long, featuring five lobes of varying lengths and small external hairs. The achenes are oblong-obovate and hairy, with reddish-brown crown hairs, approximately 4 mm long and thicker. Flowering and fruiting occur from July to October [18].

*A. altaicus* Willd. thrives in various environments such as grasslands, deserts, sands, and arid mountainous areas at altitudes ranging from 0 to 4000 meters. This plant is widely spread in northern China and is mainly produced in Asia [17]. It is an important medicinal plant, widely used in traditional Chinese medicine and Mongolian medicine [19], and holds some forage value [20].

#### **3.2 Characterization and phylogenetic appreciation of the chloroplast genome of**  *A. altaicus*

The chloroplast genome of *A. altaicus* leaves, collected from Yueliang Bay Park in Guide County, Hainan Tibetan Autonomous Prefecture, Qinghai Province, China (101°43′95" E, 36°04´61" N), was sequenced. Subsequently, the assembled chloroplast genome, along with detailed annotations, was submitted to GenBank under accession number NC072176. The analysis revealed that the *A. altaicus* chloroplast genome is 152,473 bp in size and possesses an average GC content of 37.3%. It exhibits a typical four-part structure, including a large single-copy region (LSC) spanning 84,235 bp, a small single-copy region (SSC) spanning 18,218 bp, and two inverted repeat regions (IRs) spanning 25,013 bp (**Figure 4**). A total of 129 genes were successfully annotated, encompassing 85 protein-coding genes, 8 ribosomal RNA genes, and 36 transfer RNA genes. Among these genes, 17 contain one intron, while 3 contain two introns (**Figures 3** and **4**).

In this study, a phylogenetic analysis was conducted using the complete chloroplast genome of *A. altaicus* and 25 other *Aster* species, and two *Medicago* species (*Medicago monspeliaca* and *Medicago monspeliaca*) of Fabaceae (Two *Medicago* species were used as outgroups). The results showed that *A. altaicus* and *Aster altaicus* var. *uchiyamae* have a strong sister relationship.

#### **3.3 Discussion and conclusion**

*A. altaicus* Willd. belongs to the *Aster* genus of the *Asteraceae* family. Previous studies have explored the complete chloroplast genomes of various *Aster* species [21–25]. In this study, we present the first successfully assembled and annotated complete chloroplast genome of *A. altaicus* Willd., collected from the Tibetan Plateau. With a length of 152,473 bp, its sequence exhibits a typical tetramerization structure observed in other *Aster* species. Additionally, for systematic evolutionary tree analysis, we incorporated chloroplast whole genome data from 25 other *Aster* species. The results indicate a strong relationship between *A. altaicus* Willd. and *A. altaicus* from Korea, as well as *A. altaicus var. uchiyamae* and the samples used in this study. This research contributes to valuable genetic resources for future investigations on *A. altaicus* Willd. and proves to be essential in studying the phylogenetic relationships of *A. altaicus* Willd. It is crucial to continue exploring the genetic

*Chloroplast Genome Characteristics of Plants on the Tibetan Plateau DOI: http://dx.doi.org/10.5772/intechopen.112100*

 **Figure 4.**  *Chloroplast genome map of* Aster altaicus *.* 

information of *Asteraceae* and advancing classification research within the *Asteraceae* family, including the *Aster* genus.

#### **4. Morphological characteristics and chloroplast genome analysis of**   *Asterothamnus centraliasiaticus*

#### **4.1 Species morphological characteristics and distribution information**

*Asterothamnus centraliasiaticus* Novopokr. [ 26 , 27 ] ( **Figure 1** ), also known as Aster centraliasiaticus and Aster alyssoides, is a perennial deciduous half-shrub of the family Asteraceae [ 28 ], native to arid and semi-arid areas of the Qinghai-Tibetan Plateau and northwestern Mongolia in China. It is a perennial deciduous half-shrub of the family Asteraceae [ 28 ], native to the arid and semi-arid regions of the Qinghai-Tibetan Plateau and northwestern Mongolia in China, and naturally distributed in the areas of 1300–3900 meters above sea level. *A. centraliasiaticus* is an ecologically important species in its range, especially for its role in soil stabilization and as a food resource for livestock and wildlife. In addition, *A. centraliasiaticus* has brightly

colored flowers and can also be used as an ornamental plant. *A. centraliasiaticus* is a much-branched half-shrub, with plants up to 120 cm tall. Its stems are cespitose, branched below, with inflorescence branches above. *A. centraliasiaticus* is a not very tall half-shrub with many branches below, and a root neck buried in the soil, from which branches emanate. Its axial roots are deeply embedded in the soil up to about 1 m. Most of the adventitious roots grow from the branches, forming a rather extended root system with increased space for water and nutrient uptake in dry and early environments, and the width of the root system is usually several times the width of the aboveground crown. Aboveground older branches are highly lignified, grayish-yellow, and stout, and newer branches are slender and grayish-green. Centraliasiaticus is a super-arid desert plant found in deserts and desert grassland zones, preferring to grow on loose, gravelly alluvial and floodplain soils. It often forms communities along dry riverbeds and flowlines, and is also found on stony hills and pre-hill floodplain slopes (**Figure 5**) [26].

#### **4.2 Characterization and phylogenetic appreciation of the chloroplast genome of**  *A. centraliasiaticus*

We sequenced the chloroplast genome of *A. centraliasiaticus* leaves collected from Yueliang Bay Park in Guide County, Hainan Tibetan Autonomous Prefecture, Qinghai Province, China (101°53′47" E, 36°09´23" N, 2050 m a.s.l.). The chloroplast genome was sequenced. And the assembled chloroplast genome and its detailed annotation were submitted to GenBank with the accession number OP909739. The

#### **Figure 5.**

*Morphology and habitat map of* Asterothamnus centraliasiaticus*. From Yueliang Bay park in Guide County, Hainan Tibetan autonomous prefecture, Qinghai Province, China, photograph by Zheng-sheng Li.*

#### *Chloroplast Genome Characteristics of Plants on the Tibetan Plateau DOI: http://dx.doi.org/10.5772/intechopen.112100*

results indicate that the complete cp genome of *A. centraliasiaticus* is 152,205 bp in length ( **Figure 6** ) and comprises a pair of inverted repeats (IR) regions of 25,031 bp each, a large single-copy (LSC) region of 83,956 bp and a small single-copy (SSC) region of 18,187 bp. The GC content of *A. centraliasiaticus* is 37.32%. A total of 130 genes were successfully annotated containing 85 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. Among these genes, 21 genes have one intron each, and two genes contain two introns.

 The complete chloroplast sequences of *A. centraliasiaticus,* and other seventeen species in six genera (eight *Aster* species, two *Artemisia* species, one *Pericallis* species, one *Guizotia* species, two *Ambrosia* species, and three *Cynara* species) within *Asteraceae* , were used in the phylogenetic analysis. Two *Oryza* species (Oryza punctata and Oryza minuta) of Poaceae were used as outgroups. The phylogenetic analysis indicated that *A. centraliasiaticus* was close to *Aster hypoleucus* and *Aster lavandulifolius.*

 The complete chloroplast sequences of *A. centraliasiaticus,* and other seventeen species in six genera (eight *Aster* species, two *Artemisia* species, one *Pericallis* species, one *Guizotia* species, two *Ambrosia* species, and three *Cynara* species) within *Asteraceae* were used in the phylogenetic analysis. Two *Oryza* species

 **Figure 6.**  *Chloroplast genome map of* Asterothamnus centraliasiaticus*.* 

(Oryza punctata and Oryza minuta) of Poaceae were used as outgroups. The phylogenetic analysis indicated that *A. centraliasiaticus* was close to *Aster hypoleucus* and *Aster lavandulifolius.*

#### **4.3 Discussion and conclusion**

Sequencing chloroplast genomes from various plants has provided valuable insights into chloroplast biology, biodiversity conservation, and genetic information that can be harnessed for improving agronomic traits or developing high-value agricultural and biomedical products. In this study, we assembled the chloroplast genome of *A. centraliasiaticus* using Illumina HiSeq2500 sequences. Our findings reveal that the complete cp genome of *A. centraliasiaticus* spans a length of 152,205 bp. Consistent with previous studies, it exhibits a standard quadripartite structure, consisting of a pair of inverted repeat (IR) regions of 25,031 bp each, a large singlecopy (LSC) region of 83,956 bp, and a small single-copy (SSC) region of 18,187 bp. We successfully annotated a total of 130 genes, including 85 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. Moreover, the chloroplast genome sequence has been widely utilized for determining evolutionary relationships among plants. In this study, the maximum-likelihood (ML) phylogenetic analysis based on the complete chloroplast genome data strongly supported the close relationship between *A. centraliasiaticus* and *A. hersileeoides*. These findings provide valuable genetic information for germplasm protection and informed development strategies. Furthermore, our analysis revealed that some species of the genus Aster are more distantly related at the chloroplast genome level compared to *A. centraliasiaticus*. This discovery challenges the current botanical species classification of *A. centraliasiaticus* and offers new insights and theoretical support for future plant taxonomy research.

#### **5. Ecology, structural characteristics, and chloroplast genomes of**  *Corethrodendron multijugum* **(maxim.) species**

#### **5.1 Morphological characteristics and distribution information**

*Corethrodendron multijugum (Maxim.)* [29] (**Figure 7**), formerly known as *Hedysarum multijugum* [30, 31], also known as *Hedysarum multijugum f. albiflorum*. This genus *Hedysarum* was recently revised to the genus *Corethrodendron* based on morphological and molecular evidence of several barcoding regions, including plastid and nuclear regions [29]. Now, this species belongs to the *Corethrodendron* of *Fabaceae* in *Fabales.*

*C. multijugum* is a semishrub or herb, woody only at the base, 0.3–1 m tall. Roots lignified. Stem erect, much branched, slenderly striate, densely white pilose, longitudinally furrowed. Leaves 6–18 cm long; stipules brown scaly, ovate-lanceolate, 2–5 mm long, base connate, apex free, abaxially pilose; leaf rachis grooved, densely gray-white pubescent; leaflets 15–35, elliptic, ovate, or obovate, 5–12 mm long, 3–6 mm wide, apex obtuse or slightly concave, base subrounded or rounded-cuneate, ventrally glabrous, abaxially densely appressed pubescent; The petiole is very short and hairy. Racemes growing in axils of branches, 20–35 cm long, 9–25 flowers growing sparsely; bracts caducous; pedicels 2–3 mm long, pilose; calyx obliquely campanulate, 5–6 mm long, calyx teeth subulate or acute, 3–4 times shorter than calyx tube, lower calyx teeth slightly longer than or twice as long as upper calyx teeth, usually

*Chloroplast Genome Characteristics of Plants on the Tibetan Plateau DOI: http://dx.doi.org/10.5772/intechopen.112100*

#### **Figure 7.**

*Morphology and habitat map of* Corethrodendron multijugum*. From Yueliang Bay park in Guide County, Hainan Tibetan autonomous prefecture, Qinghai Province, China (101°43*′*95" E, 36°04´61" N), photograph by Ying Liu.*

splitting between upper calyx teeth deeper below middle of calyx tube, sometimes splitting deeper between both calyx teeth and upper calyx, outside covered with Corolla 15–19 mm long, purple-red or rose-red, with yellow spots; flagellum obovate, slightly concave at first end, 14–18 mm long, claw short; wing petals narrow, 6–8 mm long, ca. 1 mm wide, claw half as long as limb, auricles nearly as long as petiole; keel petal slightly shorter than flagellum, anterior lower angle bow-shaped curved. Ovary linear, pubescent. Pods flattened, usually l-3-noded; ovate or semiorbicular nodding pods ca. 5 mm long and 4 mm wide, sparsely pubescent, with reticulate and small spines on sides. Fl. Jun-Aug, fr. Jul-Sep [12, 29–32].

The plant is widely distributed in northwestern, northern, and southwestern of China, including Sichuan, Tibet, Xinjiang, Qinghai, Gansu, Ningxia, Shaanxi, Shanxi, Inner Mongolia, Henan, and Hubei, and grows mainly in gravelly floodplains and riverbanks in desert areas around 1800–3800 m above sea level, on sunny slopes, gullies, embankments, gravelly lands, gravelly slopes in grassland areas and in certain deciduous broad-leaved forest areas on dry mountain phi and gravelly river banks in some deciduous broad-leaved forest areas [12, 29–34]. It is distributed in all states and counties of Qinghai Province [12, 29]. It is distributed abroad in Mongolia and Russia. The type specimens were collected from the western part of the Hexi Corridor in Gansu [29–32]. This plant has deep roots, strong cold, and drought tolerance [35–37]. It is not only a widely used in traditional Chinese medicine, but also an excellent feed, soil, and water conservation plant, which has important medicinal and economic values [38–42].

#### **5.2 Characterization and phylogenetic appreciation of the chloroplast genome of**  *Corethrodendron multijugum*

The complete chloroplast genome was sequenced using the Illumina Hiseq 2500 platform (Illumina, SanDiego, CA) with paired-end reads of 150 bp by Genesky

Biotechnologies Inc., Shanghai, China. And the complete chloroplast genome structure of *C. multijugum* was a circular DNA molecule ( **Figure 8** ), with a length of 122,994 bp (GenBank accession no. NC069301). Unlike the typical tetrad structure of most angiosperm chloroplast genomes, the *C. multijugum* chloroplast genome does not have the typical tetrad structure consisting of a large single copy (LSC), a small single copy (SSC), and a pair of inverted repeats (IRs), and similarly in *Hedysarum polybotrys* var. *alaschanicum* results [ 43 ]. The overall G + C content of the whole genome is 34.5%, and the genome presented a negative AT-skew (−0.002) on the J-strand. The genome contains 110 genes, including 76 protein-coding genes (PCGs), 30 transfer RNA genes (tRNAs), 4 ribosomal RNA unit genes (rRNAs). There are 17 genes containing one intron (s) and one trans-splicing genes rps12, and two genes (ycf2 and ycf3) contain two introns. A total of 65 simple sequence repeat (SSR) markers ranging from mononucleotide to tetranucleotide repeat motif were identified in *C. multijugum* chloroplast genome, and mononucleotide had the most repeats.

 *Chloroplast genome map of* Corethrodendron multijugum *.* 

*Chloroplast Genome Characteristics of Plants on the Tibetan Plateau DOI: http://dx.doi.org/10.5772/intechopen.112100*

 All of the 20 chloroplast genome sequences of Fabaceae were obtained from GenBank and used for phylogenetic analysis, and two chloroplast genome sequences of Rosaceae were used as outgroups. Phylogenetic analysis indicated a strong sister relationship with *C. multijugum* and *Hedysarum petrovii* ( **Figure 9** ). This study will contribute to a better understanding of the evolutionary pattern of chloroplast genomes in *C. multijugum* and provide more basis for the identification and application of *Corethrodendron* plants.

#### **5.3 Discussion and conclusion**

 In this study, the chloroplast genome of *C. multijugum* is reported for the first time. We described the sequence structures and annotated genes in the genome. Its sequence length was found to be 122, 994 bp, similar to that of other *Corethrodendron* ( *Hedysarum* ) species. What's different from other studies is that compared with other previously published chloroplast genomes of *Corethrodendron* ( *Hedysarum* ) species, the *C. multijugum* chloroplast genome does not have the typical quadripartite structure, and similar genetic structure results have been demonstrated in both genus *Hedysarum* [ 43 ] and genus *Astragalus* [ 44 ]. What is special about this study is that the phylogenetic tree was reconstructed to confirm the phylogenetic of *C. multijugum* for the first time. The chloroplast genome of *C. multijugum* will contribute to a better understanding of the evolutionary mode of the chloroplast genome and provide more evidence for the identification and application of *Corethrodendron* species.

#### **Figure 9.**

 *Chloroplast phylogeny of 16 Fabaceae species based on the complete chloroplast genome sequences. The asterisk represents the assembled plastome sequence in this picture. The clades of species are represented with black lines.* 

### **6. Morphological characteristics and chloroplast genome analysis of**  *Clematis nannophylla* **Maxim**

#### **6.1 Morphological characteristics and distribution informations**

*Clematis* is belong to widely Ranunculaceae family and distributed worldwide [ 21 , 45 ]. *Clematis nannophylla* is Small perennial shrub, and has high ornamental, ecological, and medicinal value [ 22 , 45 ] *. C. nannophylla* is normally found dry or gravelly slopes; 1200–3200 m. Gansu, SW Nei Mongol, Ningxia, Qinghai, Shaanxi ( **Figure 10** ).

*Clematis nannophylla* is usually small shrubs, erected 30–100 cm tall. The branches are reddish-brown and ribbed, and the branchlets are densely appressed puberulous and fall off later. Leaves are simple opposite or several fascicled, almost sessile or up to 4 mm long; leaf blade contour subovate, 0.5–1 cm long, 3–8 mm wide, leaf blade pinnately lobed, with lobes 2–3 or 4 in pairs, or lobes subdivided 2–3, lobes or lobules elliptic to broadly obtusely cuneate or lanceolate, 1–4 mm long, with varying 2–3 notched small teeth or entire margin, glabrous or pubescent. Flowers solitary or cyme with 3 flowers; sepals 4, yellow, oblong to obovate, 0.8–1.5 cm long, 5–7 mm wide, pubescent outside, margin densely villous, interior pubescent to nearly glabrous; stamens glabrous, filaments lanceolate, longer than anthers. Achenes oval, about 5 mm long, puberulent . The flowering period is from July to August, and the fruit period is from August to September [ 22 ].

#### **6.2 Characterization and phylogenetic appreciation of the chloroplast genome of**   *C. nannophylla*

 The chloroplast genome of *C. nannophylla* was 159,801 bp in length and divided into four distinct regions, such as large single copy region (LSC, 79,526 bp), small single copy region (SSC, 18,185 bp), and a pair of inverted repeat regions (31,045 bp). The genome annotation predicted a total of 133 genes, including 89 protein-coding

#### **Figure 10.**

 *Morphology and habitat map of* clematis nannophylla *. From Yueliang Bay park in Guide County, Hainan Tibetan autonomous prefecture, Qinghai Province, China (101°43*′*95" E, 36°04´61" N), photograph by Ying Liu.* 

#### *Chloroplast Genome Characteristics of Plants on the Tibetan Plateau DOI: http://dx.doi.org/10.5772/intechopen.112100*

genes, 36 tRNA genes, and 8 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that *C. nannophylla* is nested in Sect. *Fruticella* of family Ranunculaceae and has a close relationship to *C. fruticosa* and *C. songorica* (**Figure 11**)*.*

#### **6.3 Discussion and conclusion**

In summary, the complete cp genome sequence of *C. nannophylla* was sequenced and compared with other related species, providing an important reference for the phylogeny of *C. nannophylla*. Although the cp genomes of *C. nannophylla* was identical to each other *Clematis* species in genome structures, gene contents, and GC contents, phylogenetic analysis showed that *C. nannophylla* is closely related to *C. fruticose*, *C. tomentella*, and *C. songarica*, and the well-resolved phylogenetic tree showed monophyletic origin of genus *Clematis* and genus *Aconitum* as its sister genus. The cp genome information in this study provides reference data for molecular marker development, phylogenetic analysis, population study, and cp genome processes, as well as for better exploitation and utilization of *C. nannophylla*. The results can guide more efficient germplasm resource utilization, conservation, and breeding strategy development.

**Figure 11.** *Chloroplast genome map of* clematis nannophylla*.*

### **Funding**

This work was supported by the Qinghai Science and Technology Department of the senior scientist responsibility system project (2024-SF-101).

### **Conflict of interest**

The authors declare no conflict of interest.

### **Abbreviations**


### **Author details**

Ying Liu\*, Jinping Qin, Zhengsheng Li, Lijun Zhang and Xinyou Wang Qinghai University, Qinghai Academy of Animal and Veterinary Sciences, Qinghai Provincial Key Laboratory of Adaptive Management on Alpine Grassland, Key Laboratory of Superior Forage Germplasm in the Qinghai-Tibetan Plateau, Xining, China

\*Address all correspondence to: liuying\_yanhong@sina.com

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## Sequences Evolution and Population Structure of Tunisian Date Palm (*Phoenix dactylifera* L.) Revealed by Chloroplast DNA Markers

*Rhouma-Chatti Soumaya, Choulak Sarra and Chatti Khaled*

#### **Abstract**

Date palm is present among the vital crops of arid and semiarid countries of North Africa and the Middle East. Chloroplast DNA is the best molecule for finding the evolutionary history of plant species. In the present study, cpDNA variation in date palm was estimated using the *trn*L*-trn*F intergenic spacer and *psb*Z*-psb*C region. The high AT values in both molecular markers may clarify the high proportion of transversions observed in this species. The neutrality tests, expansion parameter estimation (mismatch distribution), and haplotype network patterns proposed that demographic expansion had occurred in recent times. Furthermore, the taxa distribution is not related to geographical origins; neighbor-joining trees are clustered independently either from their geographic origin or from the sex of trees, suggesting a common genetic basis between different cultivars. Statistical analysis of chloroplast germplasm provides a means of assessing cytoplasmic gene flow, which occurred in Tunisian *Phoenix dactylifera* L. In fact, *N*m was important between Tunisia and Eastern Arabic region (*N*m = 2.57), which reflects high levels of connectivity between these population pairs. In conclusion, genomic studies prove date palm domestication happened in the Arabian Peninsula and showed an important gene flow with North African palm populations.

**Keywords:** *Phoenix dactylifera* L*.*, *trn*L*-trn*F spacer, *psb*C*-psb*Z region, population expansion, molecular evolution, gene flow

#### **1. Introduction**

Date palms (*Phoenix dactylifera*, Arecaceae) are of major economic and ecological importance to the oasis agriculture of arid and semiarid zones. The past distribution area of this fruit crop covers Mauritania in the west to Pakistan in the east and northwestern India [1]. It is moreover existing in sub-Saharan Africa and has been hosted in California, Peru, Australia, and other countries [2]. The presence of this crop in these areas gives it an undeniable ecological role by limiting the progression of steppe areas and the silting of farmland.

*P. dactylifera* was brought to Tunisia by Phoenicians before the Roman occupation and has played a vital part in establishing oases. In Tunisia, date palm cultivation covers over 46 thousand ha, with a total number of palm trees of approximately 5.4 million [3]. One hectare of land host an average of 120 date palm trees [4]. Further, the Tunisian date palm germplasm is distinct by a remarkable richness, represented by the high number of cultivars (over 250) [5, 6]. Throughout the plant domestication procedure, breeding actions, selection, migration, and admixture have given growth to cultivated populations distinct from the inherited gene pools [7]. Humans have particularly selected qualities associated with production, fruit quality, and fertility [8].

Understanding the population genetics and domestication history of cultivated species is certainly important for the genetic improvement of crops relying on the conservation and usage of the germplasm [9]. In order to contribute to the varietal improvement of date palms and to offer novel perceptions on the influence of geographic origins and human action on the genetic structure of the date palm, this study investigated the diversity of the species using chloroplast DNA.

The small, sensibly constant size, and conservative evolution of chloroplast DNA (cpDNA) make it an ultimate molecule for finding the evolutionary history of plant species. In angiosperm, the use of chloroplast DNA sequences for intra-specific phylogenetic research is now routine [10]. These investigations have been simplified by the abundant number of whole chloroplast genome sequences that are accessible from an extensive variety of angiosperms and the development of universal PCR primers in conserved coding as well as noncoding regions.

Commonly, in terms of its size, organization, and sequence, cpDNA is the most recognized conservatively evolving genome. It has been used for genetic analysis in plants and provides an accessible and well-characterized source of comparative sequence data. Moreover, identifying the footmark of positive selection is an imperative assignment in evolutionary genetic studies [11]. Otherwise, different modes of selection may result in divergent patterns of the nature and extent of genetic variation. The neutral theory affirms that all observable mutations in populations have little or no effect on an organism's fitness, and their evolutionary dynamics are entirely measured by genetic drift [12, 13]. For such mutations, the evolution continues as equilibrium among the forces of mutation pressure, natural selection, and genetic drift. New molecular techniques based on single and combined sequences data sets have provided a vast understanding of the evolution of flowering plants [14]. Noncoding regions are usually less sensitive to natural selection than coding regions and then may be more beneficial for studying plant evolution.

The chloroplast genome can be classified into three functional categories: (1) protein-coding genes, (2) introns, and (3) intergenic spacers. It has greater phylogenetic potential than nuclear DNA because it is sufficient variable but conserves to be less variable within than between species [15]. The noncoding regions are leading systematic molecular, phylogeographic, and DNA barcoding studies for plants [16, 17]. Chloroplast DNA markers are used for systematic studies of plant species [18–23] and are particularly used to study phylogeny.

In *Paris* genus, Song et al. [24] described eight most variable barcodes, to discriminate between the different species, including psbC-trnS-psbZ region. Moreover, the psbC-trnS intergenic spacer was successfully used for phylogeographic study in *Lolium* species [25]. In 2013, Ballardini et al. confirmed that the psbZ-trnfM (CAU) region could be considered a good basis for the establishment of a DNA barcoding system in *Phoenix*, and is potentially useful for the identification of the female parent in *Phoenix* hybrids.

*Sequences Evolution and Population Structure of Tunisian Date Palm (*Phoenix dactylifera *L.)… DOI: http://dx.doi.org/10.5772/intechopen.111835*

The date palm chloroplast genome is a typical circular double-stranded DNA molecule, and it shares a common quadri-partite structure: a pair of IRs (27.276 bp) separated by the LSC region (86.198 bp) and the SSC regions (17.712 bp) [26]. An important set of primer pairs improved for PCR amplification and covering sequencing in monocotyledons were optimized, which are distributed throughout the whole chloroplast genome, including exons, introns, and intergenic spacers (IGS).

Thus, in the present study, we undertake to find out the level of cpDNA variation in date palm using the *trn*L*-trn*F intergenic spacer and *psb*Z*-psb*C region and to accurate evolution process, which controls the pattern of polymorphism among the species.

#### **2. Material and methods**

#### **2.1 Plant material and DNA extraction**

Twenty-four accessions (20 cultivars and four male trees) of Tunisian date palm, listed in **Table 1**, were used in this study. Each cultivar was represented by one tree. Five varieties accommodated in Tunisian plantations ("Ghars Mettig" and "Tantabecht" from Algeria, "Berhi" and "Khadhraoui" from Iraq, and "Abou Meaan" from the United Arabic Emirates) were used in this survey. Young leaves were frozen until their use for DNA purification. Extraction of the total DNA was determined as stated by Dellaporta et al. [27] protocol. DNA concentration and integrity were checked by 0.8% agarose gel electrophoresis according to Sambrook et al. [28].

#### **2.2 Amplification and DNA sequencing**

Chloroplastic DNA was sequenced for the *trn*L*-trn*F (intergenic spacer) and *psb*C*psb*Z (intergenic spacer + gene). Target regions were amplified using universal primers: (Fw1: 5′GGTTCAAGTCCCTCTATCCC3′; Rv1: 5′ATTTGAACTGGTGACACGAG3′) and (Fw2:5′CAACCTTGGCAAGAACG3′; Rv2: 5′TTGACCAACCATCAGRAGA3′) *trnL-trnF* spacer and for *psb*C*-psb*Z region, respectively. Amplification was carried out for 35 cycles, all comprising of a denaturation phase at 95°C for 1 min, annealing at 50°C for 1 min, and an extension step at 72°C for 2 min.

The total volume of PCR reaction was 25 μL, which contained 25 mM of MgCl2, 2 mM of dNTP mix, 1.6 mM of each primer and 1 unit of DNA Taq polymerase, and 20 ng of DNA. Agarose-gel electrophoresis (1.5%) was used to check the PCR products.

The purified PCR products for the *trnL-trnF* and *psb*C*-psb*Z regions were sequenced in both strands according to the automated Sanger method [29] using automated sequencer ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems). The process consists of the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerases during in vitro DNA replication. In Sanger sequencing, target DNA is copied multiple times, producing fragments of varying lengths. Fluorescent "chain-terminating" nucleotides mark the ends of the fragments so that the sequence can be determined. It is the most widely used method for detecting single nucleotide variations.

#### **2.3 Sequence analysis**

The identity of both sequenced regions was confirmed through a BLASTN search in NCBI database [30]. Nucleotide sequences were aligned by Mega 5.2.2 [31].


#### *Chloroplast Structure and Function*

#### **Table 1.**

*Date-palm accessions studied, their origin, accession numbers, and their variation in length, GC and AT contents of the psbC-psbZ region and the combined sequences.*

Several genetic parameters were determined with DnaSP program [32]. Haplotype diversity (Hd) [33] and genetic diversity (Pi) [34] were calculated to evaluate genetic diversity. The average of nucleotide differences (k), the minimum number of recombination events (Rm), and the average number of nucleotide differences among cultivars were also detected. By means of selective neutrality tests, we checked the hypothesis of the mutation/drift equilibrium for a supposedly neutral polymorphism. Selection neutrality for the detected mutations was tested by both Tajima's D [35] and Fu and Li's D\* and F\* methods [36], using the DNAsp program. Demographic parameters were assessed using the distribution of pairwise sequence differences (mismatch distribution) of Rogers and Harpending [37] and site-frequency spectra of Tajima [35].

*Sequences Evolution and Population Structure of Tunisian Date Palm (*Phoenix dactylifera *L.)… DOI: http://dx.doi.org/10.5772/intechopen.111835*

Moreover, Fu's Fs statistics [38] was used to confirm the assumption of population growth and range expansion as revealed by the mismatch distribution of Rogers and Harpending [37]. In addition, we calculated the Harpending's raggedness index (r) corresponding to an estimate of the fluctuation in the frequency of differences between haplotype pairs [39]. In a complementary way, the R2 statistic [40] was calculated based on the differences between the number of singleton-type mutations and the average of the nucleotide differences. These analyses were executed using coalescent simulations implemented in DnaSP software, with 1000 simulated resampling replicates.

To study the genetic relationships between the studied sequences (haplotypes), we used the reduced median network analysis available in the NETWORK software [41]. The phylogenetic relationships between the studied chloroplast haplotypes were reconstructed using the neighbor-joining (NJ) method [42]. NJ builds a tree from a matrix of pairwise evolutionary distances relating to the set of taxa being studied. Gene flow (*Nm*) was estimated with the mean number of migrants per generation among populations. *N*m values were calculated with 1000 data permutations using the software DnaSP v 5.10.01. With reference to the standard for gene flow, we described genetic flow as low for Nm < 1, high for 1 < Nm < 4, and very high for Nm > 4 [43]. In gene flow analysis, we consider three different geographic regions of *P. dactylifera* L*.* Tunisia, Algeria, and Eastern Arabic (Iraq and UAE).

#### **3. Results**

#### **3.1 Sequences variation in the psbC-psbZ region**

The DNA sequencing of the generated bands has been successfully performed and the blast search allowed confirming the identity of the sequences. DNA sequence varied from 951 bp for "*Besser Helou"* cultivar to 967 bp for "*Gasbi"* cultivar (**Table 1**) with an average of 964.5 pb length. The nucleotide composition frequencies are 0.274 (A), 0.325 (T), 0.204 (C), and 0.196 (G). In addition, the GC content of the amplified sequences varied from 39.7% to 40.7%, and the AT content from 59.3% to 60.3% (**Table 1**).

The polymorphism pattern of the *psb*C*-psb*Z sequence in *P. dactylifera* L. reveals a high level of mutation with 55 polymorphic sites (**Table 2**). We observed 33 singleton variable sites and 22 parsimony-informative ones. Moreover, a low transitional/ transversional ratio (ti/tv = 0.68) occurs in the *psbC-psbZ* sequence. In addition, 22 haplotypes were detected among 24 cultivars analyzed, yielding a haplotype diversity of 0.989 (**Table 2**). Sequence variation observed between cultivar groups and nucleotide diversity was estimated (Pi = 0.00862) (**Figure 1a**). The average number of nucleotide differences "k" was estimated to be 8.264. Furthermore, seven regions of conserved DNA were detected using DnaSP program yielding a value of sequence conservation C = 0.913. Mismatch distribution within the species was unimodal for the *psb*C-*psb*Z marker (**Figure 2a**), suggesting demographic expansion had occurred in recent times.

A phylogenetic tree was constructed using *NJ* methods (**Figure 3a**). The evolutionary distances were estimated by the maximum composite likelihood method [44]. Haplotypes of the *psb*C-*psb*Z regions made it possible to make moderately sustained phylogenetic trees (Bootstrap values ≤74%). This dendrogram supported the varieties' organization into two main clusters, which were subdivided into different subclusters. Clearly, the obtained clustering is made independently from the geographical origin of cultivars since the foreign varieties, newly introduced in the


#### **Table 2.**

*Sequences polymorphism and divergence within date palm cultivars.*

Tunisian growing areas, did not remarkably separate from the autochthonous ones. In addition, the male and female trees did not diverge from each other.

In a complementary way, a genetic network based on the *psb*C-*psb*Z sequences was reconstructed (**Figure 3b**). All haplotypes are connected to the most frequent haplotype H1. It represented the ancestor sequence of the other ecotypes, which mutate during evolution. Several putative haplotypes, corresponding to intermediate evolutionary steps, were detected in our network (mv in **Figure 3b**). The haplotype patterns (star-like pattern) reflect a signature of a recent expansion in Tunisian date palm.

#### **3.2 The combined region trnL-trnF and psbC-psbZ spacers**

The combined sequence varied from 1347 bp for "*Gasbi"* cultivar to 1382 bp for "*Kharroubi"* cultivar (**Table 1**). Current sequences do not contain insertions or deletions (indels). These sequences revealed 69 polymorphic sites and defined 24 haplotypes. Among the 69 variable sites, 31 were parsimoniously informative and 38 were singletons sites. The means of the haplotype (*Hd*) and nucleotide (*Pi*) diversity (**Figure 1b**) were higher than for *trn*L-*trn*F spacer [16] and the *psb*C*-psb*Z region taken separately. In fact, these values were 1 ± 0.014 and 0.00861 ± 0.001, respectively. The average of pairwise

*Sequences Evolution and Population Structure of Tunisian Date Palm (*Phoenix dactylifera *L.)… DOI: http://dx.doi.org/10.5772/intechopen.111835*

#### **Figure 1.**

*Variability of nucleotide diversity (Pi) and segregating sites (S) for the psbC-psbZ chloroplastic DNA (a) and the combined sequences (b).*

#### **Figure 2.**

*Mismatch distribution of chloroplastic DNA sequences of the date palm cultivars based on pairwise nucleotide differences in the psbC-psbZ region (a) and the combined trnL-trnF spacer and psbC-psbZ (b). In addition, graphical representation of the site frequency spectrum of chloroplast DNA sequences. Solid lines in the sitefrequency spectra indicate the expected distributions under neutrality and at equilibrium.*

#### **Figure 3.**

*Summary of phylogenetic analysis within Phoenix dactylifera based on psbC-psbZ sequences: (a) neighbor-joining tree of 24 Tunisian date-palm cultivars. (b) Median-joining network of the haplotypes inferred from the analyzed sequences. Nodes are proportional to haplotype frequencies and branch length is proportional to the number of mutations. The circles represent haplotypes; circle diameters are proportional to the frequencies. Black bands correspond to mutational steps.*

nucleotide differences (*k*) was equal to 11.471 (**Table 2**). In addition, the GC content of the combined sequences varied from 37.88% to 38.44% (**Table 1**). The polymorphism pattern in *P. dactylifera* L. revealed an elevated level of mutation points with a low transitional/transversional ratio (ti/tv = 0.63).

Mismatch distributions within the species were generally unimodal for markers, suggesting population expansions (or past selective sweeps) (**Figure 2b**). Numerous *Sequences Evolution and Population Structure of Tunisian Date Palm (*Phoenix dactylifera *L.)… DOI: http://dx.doi.org/10.5772/intechopen.111835*

**Figure 4.** *Neighbor-joining tree based on trnL-trnF spacer and psbC-psbZ gene sequences.*

significant negative values of *Fs* and Tajima's *D* in *trn*L-*trn*F [16], *psb*C-*psb*Z spacers, and the combined region (**Table 2**) rule out the hypothesis of a constant population and propose either selective sweeps or past demographic expansion (**Table 2**). In addition, low values of *R2* and Harpending's raggedness index (*r*) will be characteristic of a recent expansion.

For gene flow estimation, *N*m was high (*N*m > 1.0) between Tunisia and Algeria (*N*m = 1.8) and Tunisia/Eastern Arabic accessions (*N*m = 2.57) indicating that few differentiations could be established among them. On the other hand, *Nm* was weak between Algeria and Eastern Arabic (*N*m = 0.66), this reflects low levels of connectivity between these population pairs.

Genetic relationships among date palm cultivars are investigated, also, using the variation observed in the combined (*trn*L*-trn*F and *psb*C*-psb*Z) sequences. As illustrated by **Figure 4**, the phylogeographical patterns were very similar between the *psb*C*-psb*Z gene and the combined sequences. The *NJ* dendrogram supported the variety organization into three monophyletic branches and one cluster. The first branch is composed of "*Lagou"* (Lg), the second one *CRPh5* (C5) Tunisian male tree, while the third is made of Iraquian variety "*Berhi"* (Br).

The remaining cultivars are ranged in the unique cluster, which is divided into four subgroups. In fact, genotypes' clustering is independent of the sex of trees, and it is not structured according to geographical origin. Indeed, with the exception of the foreign cultivar "*Berhi"* (Br) divergence, the other foreign cultivars did not significantly diverge from the Tunisian plantations (**Figure 4**).

#### **4. Discussion**

In the present survey, we tested the reliability of the noncoding chloroplast markers to identify date palm cultivars. Therewith, we sought an indication of the level of genetic variation and genetic classification within the date palm cultivars grown in Tunisia.

In this object, we represented the evolution of the *psb*C-*psb*Z gene and the combined sequences (*trn*L-*trn*F spacer/*psb*C-*psb*Z) to reveal polymorphisms in *P. dactylifera* L. The high AT values in both molecular markers may clarify the high proportion of transversions (ti/tv = 0.67 and ti/tv = 0.63) observed in this species. This result corroborates the discovery in angiosperm chloroplast noncoding regions with a ratio ti/tv not exceeding one for any of the examined taxa [45–50]. Base content explains the relatively high proportion of transversions [45].

The use of coalescent theory [51, 52] for the *P. dactylifera* L. sequences has permitted inferences about past and present population size. It has been confirmed that demographic expansions conduct to star-shaped genealogies [53], an excess of rare mutations [54], and a unimodal mismatch distribution [37]. Haplotype network patterns, the neutrality tests, and the expansion parameter estimations (mismatch distribution) proposed that demographic expansion had occurred in recent times as indicated by the excess of mutations type singletons in the *P. dactylifera* sample.

Furthermore, there was no geographical structure in the relationships among the haplotypes; neighbor-joining trees are clustered independently either from their geographic origin or from the sex of trees, indicating a common genetic basis between different cultivars. A low genetic structure is usually related to natural habitat change or to human activities that increase gene flow between populations [55]. Human impact on these regions may be the reason for these findings. Actuality, in Tunisian localities, cultivars are manipulated by farmers after continuous selection, cloning, and exchange of varieties. Similar results have been observed using other molecular markers, where *P. dactylifera* ecotypes clustering made independently from either the tree's sex or their geographic origin in spite of their great distribution [16, 56–60]. This result is contrasted with Zehdi-Azzouzi et al. (2015), where *NJ* classification was clearly coherent with a geographical structuring into two clusters: Eastern pool (Djibouti, Oman, Iraq, the UAE) and the Western pool (North Africa accessions). Also, using three chloroplast regions of rbcL, matK, and trnH-psbA, Iranian date palm cultivars were separated into clades corresponding to their geographical distribution [61].

Statistic analysis of chloroplast germplasm provides a means of assessing cytoplasmic gene flow, which occurred in Tunisian *P. dactylifera* L. The pattern observed confirmed that gene flow is designed to be a significant evolutionary factor in date palm history, allowing a variability of the genetic diversity at different scales (local and foreign). In fact, *N*m was important between Tunisia and Eastern Arabic region (*N*m = 2.57), which reflects high levels of connectivity between these population pairs. Zehdi-Azzouzi et al. [9] proved the movement of gene flows between Eastern and Western origins, mostly from east to west, following a human-mediated diffusion of the species.

In addition, genomic studies prove date palm domestication has happened in the Arabian Peninsula and that the North African population has mixed ancestry with components from Middle Eastern *P. dactylifera* [62]. In this subject, Gros-Balthazard et al. [63] noted that a first domestication event was shown to be improbable, given the very similar form of seeds in cultivars from Africa and the

*Sequences Evolution and Population Structure of Tunisian Date Palm (*Phoenix dactylifera *L.)… DOI: http://dx.doi.org/10.5772/intechopen.111835*

Middle East. In fact, travelers and pilgrims brought many cultivars of date palms grown in Tunisian oases, particularly from the east [64]. The origins of domestication, the direction of germplasm flows, and the breeding history within the traditional cultivation are sincerely the reason for the observed exchange.

In addition, the mean number of migrants per generation between Tunisia and Algeria (*N*m) was important; this could be the result of the relative proximity of geographical sites. In fact, *Deglet Nour* cv. was introduced into Tunisia plantations four centuries ago from western Algeria [64, 65], and became the most valuable cultivar. After this period, the human migrations between Algeria and the south of Tunisia were perturbed by the French occupation of Algeria [64]. Despite that, other works prove that in Algeria, chloroplast diversity presents about 70% of the eastern Arabic chloroplast. In Tunisia and Morocco, this proportion only ranges from 11–42%, although Algerian nuclear diversity is similar to that obtained in Tunisia [66].

In conclusion, environmental change, over-exploitation of water reserves, and an increasing tendency toward *Deglet Nour* cv. Monoculture consists of some of the difficulties of date palm culture in Tunisia. Those are significant issues, which need in-depth research. Considering these, it is decisive to comprehend the genetic diversity and variation among and within accessions, and extensive programs must be prepared, to include collection, estimation, and preservation of plant genetic resources. Determining the gene flow and the demographic expansion of this population should be possible with our two molecular markers, nevertheless, the exploitation of additional nuclear markers clearly provides more accurate results. Actually, with the availability of the date palm nuclear and chloroplast genome sequences, more molecular markers are accessible to explore systematic, cultivar relatedness, and genetic map structure. Furthermore, a webpage regrouping all information about *P. dactylifera* germplasm conservation and utilization has an obligation to be planned in collaboration with all producer countries in order to engender bases for inhibiting its genetic erosion.

#### **Conflict of interest**

The authors declare that they have no conflict of interest.

#### **Abbreviations**


*Chloroplast Structure and Function*

### **Author details**

Rhouma-Chatti Soumaya\*, Choulak Sarra and Chatti Khaled Laboratory of Genetics Biodiversity and Valorisation of Bioresources (LR11ES41), Higher Institute of Biotechnology of Monastir, University of Monastir, Tunisia

\*Address all correspondence to: rhoumasoumaya@yahoo.fr

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Sequences Evolution and Population Structure of Tunisian Date Palm (*Phoenix dactylifera *L.)… DOI: http://dx.doi.org/10.5772/intechopen.111835*

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#### **Chapter 4**

## Chloroplasts: The Future of Large-Scale Protein Production

*Brenda Julian Chávez, Stephanie Solano Ornelas, Quintín Rascón Cruz, Carmen Daniela González Barriga, Sigifredo Arévalo Gallegos, Blanca Flor Iglesias Figueroa, Luis Ignacio Siañez Estrada, Tania Siqueiros Cendón, Sugey Ramona Sinagawa García and Edward Alexander Espinoza Sánchez*

#### **Abstract**

Chloroplast engineering has matured considerably in recent years. It is emerging as a promising tool to address the challenges related to food security, drug production, and sustainable energy posed by an ever-growing world population. Chloroplasts have proven their potential by efficiently expressing transgenes, encapsulating recombinant proteins, and protecting them from cellular machinery, making it possible to obtain highly functional proteins. This quality has also been exploited by interfering RNA technology. In addition to the practical attributes offered by chloroplast transformation, such as the elimination of position effects, polycistronic expression, and massive protein production, the technique represents an advance in biosafety terms; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite efforts, other essential crops have remained recalcitrant to chloroplast transformation, which has limited their expansion. In this chapter, we address the most recent advances in this area and the challenges that must be solved to extend the transformation to other crops and become the de facto tool in plant biotechnology.

**Keywords:** chloroplast, engineering, agronomical traits, biofuels, biopharmaceuticals, mass protein production

#### **1. Introduction**

It has recently been projected that the world population will reach 9.8 billion people in the next three decades, considering that plants provide about 80% of the food that is consumed and that traditional agriculture is inefficient [1–3], meeting their food and public health needs constitutes a challenge. Therefore, seeking to meet the requirements of this growth, biotechnology has used plants as platforms for the

production of proteins, and this has been exploited by different industries such as energetic, pharmaceutical, and agricultural, which have already obtained fortified foods, vaccines, antibodies, plants that exhibit resistance to pests and diseases as well as plants with low content of cell wall components [4].

Modified plants have usually been developed by inserting genes directly into the nuclear genome because of the facilities this offers, such as the possibility of using different transformation methods, high transformation frequencies, and the fact that different plant species can be transformed using a single vector type. However, the use of chloroplast transformation seems to be increasing because, although it has lower levels of transgene integration and there are difficulties in obtaining homoplasmic plants [5], it allows the containment of transgenes, which can even be expressed in a polycistronic way, it avoids position effects, and above all, increase the production levels of recombinant proteins. In addition, an advantage that can be exploited is that chloroplasts can function as biocapsules, accumulating proteins of therapeutic interest, and these can pass through the digestive tract without disturbance [6, 7].

Although chloroplast transformation has been implemented in a broader set of plant species, it has not yet been satisfactorily implemented in important crops, such as cereals, considering that these provide about 50% of the proteins in the human diet [8], limits the expansion of this technology. However, although chloroplast transformation still has challenges, it offers an opportunity to lower the production costs of recombinant proteins without biosafety concerns. In this chapter, we explore the advances in the genetic engineering of chloroplasts to produce proteins of industrial interest and the challenges that must be overcome to establish this technology as the preferred tool in the expression of recombinant proteins.

#### **2. Genetics of the chloroplast**

Chloroplasts are small organelles primarily associated with their role in the photosynthetic process, which is possible because they can obtain electrons from water [9, 10]. However, other major metabolic processes also occurred within them, such as the biosynthesis of phytohormones, vitamins, pigments, starch, or phenols [11, 12]. Also, the biochemical processes that occur in the chloroplast largely determine the plant's adaptation to its environment [13, 14].

Even though the chloroplasts are semi-autonomous, the control of chloroplast biogenesis and the chloroplast metabolic processes is mainly under nuclear control because, from the ~3000 proteins that there are in the chloroplast, 97% of them are encoded by the nuclear genome and imported via the Toc/Tic machinery [15–17]. Nevertheless, some critical proteins are produced in the chloroplast. There are involved in the transcription, replication, and translation, as well as the proteins that make up the complexes of NADPH plastoquinone oxidoreductase, cytochrome *b*6*f*, and the ATP-synthase subunit, as well as the photosystems I and II, envelope membrane proteins, ß-subunit of acetyl-CoA-carboxylase, and cytochrome C biogenesis [16, 18].

The proteins produced in the chloroplast are encoded by ~120 genes, which are arranged in a circular double-strand genome of quadripartite structure [16, 19, 20] of which, depending on the species and the tissue age, there are multiple copies [21], *e.g.,* 22 copies in potato leaves and 900 copies in wheat leaves. However, the reason for this is not yet known [22, 23].

It has been considered that most chloroplast genomes are highly conserved in terms of their organization and content. However, the length genome varies from ~150 to 220 kb and always appears to be associated with the contraction or expansion of the Inverted Repeat regions [11, 16, 24], which are essential to gene conservation, replication initiation, stabilization, and the evolution of the chloroplast genome [21, 25, 26]. Nevertheless, this is still under discussion because there are species of angiosperms (pea and alfalfa) and gymnosperms (pine) that lack these regions and species such as *Euglena gracilis* that have three sequences but, like repeated sequences clustered in tandem [27–29].

Chloroplast genomes are organized into nucleoids whose number, size, composition, and structural organization are varied, even between adjacent nucleoids [16]. These nucleoids are attached to the thylakoid membrane through different proteins such as MFP1, TCP34, and pTAC16, and seem to be involved in the regulation of gene expression through their association with different SWIB-domain proteins, CND41, sulfite reductase, and WHIRLY proteins [30–33]. In addition, there is strong evidence that nucleoids serve as platforms for forming ribosomes [34, 35]. Therefore, it is currently accepted that nucleoids participate in replication, transcription, translation, post-translational regulation of gene expression, and repair of chloroplast genomes.

Although the number of nucleoids and genomes per nucleoid is variable, it is considered that a nucleoid comprises 10 to 20 copies of plastid DNA. The large number of chloroplast genomes with the possibility of polycistronic expression has made chloroplast genetic engineering rapidly growing [36, 37].

#### **3. Incorporation of new traits in plants through chloroplast transformation**

The growing demand for agricultural products has influenced the search for alternatives that maximize their production, including developing modified plants [38]. These crops have allowed an increase in agricultural production, which has also translated into higher profits for farmers [39, 40], to such a degree that by 2018, the commercialization/planting of modified plants had already been adopted by 71 countries [41], which is a rapid growth rate given that it was introduced to the market in 1995, just 23 years prior. The incorporation of new characteristics to plants has maintained an interest in modified crops, so much so that to date, 88% of cotton and 82% of corn grown in the United States is genetically modified [42], and this trend continues, in March 2023 Brazil approved the wheat-HB4 (*Triticum aestivum*) crop that exhibits the *Hahb-4* transcription factor from *Helianthus annuus* that confers drought stress tolerance, as reported by the International Service for the Acquisition of Agri-biotech Applications (event: IND-ØØ412–7) [43].

The development of the first modified plants was achieved by nuclear transformation with the intention of conferring resistance to biotic and abiotic stresses [44–46], and although this method of improvement has been maintained the leadership of plant genetic engineering, to date, we are still grappling with problems such as gene silencing, random transgene integration, inappropriate gene expression regulation, genomic instability, interference with other genes, and selection issues. These problems can affect the expression and function of the transgene, as well as its stability and heritability. Also, there is an ecological risk of the crop-to-crop gene flow [47–49].

To reduce the problems associated with nuclear transformation, biotechnology has gone deeper into the genetic engineering of chloroplasts, which considerably improves the expression levels of recombinant proteins and decreases the unwanted effects associated with modified crops.

The genetic engineering of chloroplasts seems to be growing rapidly, which is possible thanks to the standardization of DNA delivery protocols, the understanding of the mechanisms that govern transformation efficiency, as well as the increase in sequenced chloroplast genomes, which have gone from 6768 in 2021 to 10,712 reported in the RefSeq database from the National Center for Biotechnology Information (NCBI) today, and of which 1072 of them have been reported only so far in 2023.

Although establishing chloroplast transformation in different crops has been challenging, it has already been successfully established in different species (**Table 1**), and currently, been developed plants that express enzymes of industrial value, antigenic



#### **Table 1.**

*Species transformed by stable chloroplast transformation.*

proteins for vaccine production, proteins of pharmaceutical interest, and proteins for the production of biofuels and biomaterials [36, 94–97], as well as proteins that confer resistance to pests and diseases [77, 98, 99].

#### **3.1 Chloroplast transformation to confer resistance to pests**

Annually, about 40% of agricultural products are lost before harvest due to attacks by insects, weeds, and plant pathogens, increasing by 20% after harvest [100]. Therefore, using pesticides has been one of the alternatives that have allowed controlling these losses, increasing their use in cropland from 1.55 kg Ha−1 in 1990 to 2.69 kg Ha−1 in 2019 [101].

The use of pesticides has contributed significantly to agricultural production. However, the emergence of pests resistant to these chemicals (>17,000 cases of resistance amongst 612 species globally by 2020) [102] has resulted in a greater reliance on higher doses and the introduction of new pesticides, which is a worrying trend since it can lead to environmental contamination, and more significant risks for human health; by the way, each year, millions of people around the world experience unintentional acute poisoning by pesticides (~385 million reported cases), of which 11,000 cases end in death [103]. Although this figure is already worrying in itself, it must be considered that 50% of pesticides are organophosphates. These are of particular concern since they pass from the roots to the leaves and can be consumed by the public, which increases the risk of poisoning [104–106].

Pursuing sustainable and effective alternatives to protect crops without the negative consequences of excessive pesticide use resulted in the introduction of the first insecticidal gene, Cry1Ab, to plants through nuclear transformation [44]. Since then, numerous studies have demonstrated the efficacy of Cry proteins against various pests, including *Plutella xylostella Sesamia inferens*, *Chilo suppressalis*, *Herpitogramma licarisalis*, *Mycalesis gotama*, *Cnaphalocrocis medinalis*, *Scirpophaga incertulas*, *Naranga anescens*, *Parnara guttata,* and *Elasmopalpus lignosellus* [107–111]. Cry proteins have also been tested to control nematodes, Phthiraptera, Orthoptera, mites, Coleoptera, Lepidoptera, Diptera, Hymenoptera, Hemiptera, and protozoa [112, 113]. The usefulness of these proteins has led to their continued study and exploration, with over 700

insecticidal proteins already reported (http://www.lifesci.sussex.ac.uk/home/Neil\_ Crickmore/Bt/). However, insects' resistance to Bt crops has been reported (from 3 cases in 2005 to 16 in 2016), affecting crops containing Cry1Ab, Cry1Ac, Cry1A.105, Cry1Fa, Cry2Ab, Cry3Bb, mCry3A, eCry3.1Ab, and Cry34/35Ab [114, 115]. On the other hand, there is a direct observation of resistance by *Diabrotica virgifera virgifera* to the eCry3.1Ab protein in the field before the plants were commercialized [116, 117]; such resistance may increase through cross-resistance.

Although to reduce the risks of insect resistance to Bt crops have been used strategies such as multiple toxins, protein engineering, ultra-high doses, and refugees [118], the existence of other risks of the nuclear transformation, such as ecological risks and collateral effects in the expression of the genes, have motivated the use of chloroplast engineering for the expression of proteins with insecticidal potential, which began with the expression of the Cry1Ac protein in tobacco chloroplast obtaining protection against lepidopteran *Heliothis virescens, Helicoverpa zea* and *Spodoptera exigua* [45].

Other Cry proteins such as Cry9Aa2, Cry1Ia5, Cry2Aa2, Cry1C*,* and Cry1Ab have also been expressed in chloroplast from soybean, cabbage, and poplar, including protecting the plants against *Phthorimaea operculella, Hyphantria cunea, P. xylostella, Lymantria dispar, Anticarsia gemmatalis* and *Helicoverpa armigera* [77, 119–123], and until today, the insects did not show resistance to Cry proteins compartmentalized in the chloroplast; therefore, the accumulation proteins in the chloroplast, either through their direct expression within it or by redirecting nuclear-expressed proteins to the chloroplast for storage, seems to be a viable option to avoid the development of resistance. Although this last option it as already been analyzed with the expression of Tvip3A\*, Cry1Ac, Cry1Ah, and Cry2A proteins [124–127], and is very attractive because it benefits from the facilities offered by nuclear transformation, the compartmentalization of proteins does not eliminate the risks of environmental contamination, nor any other undesired effect caused by gene insertion into the nuclear genome. Therefore it requires a more detailed analysis.

Other proteins with insecticidal activity have been expressed using chloroplasts, such as a chloroperoxidase from *Pseudomonas pyrrocinia* and the *pta* gene from *Pinellia ternata* agglutinin being effective in the control of *Alternaria alternata, Aspergillus flavus, Escherichia coli, Fusarium moniliforme, Pseudomonas syringae*, *Verticillium dahliae, Colletotrichum destructivum*, and *Fusarium verticillioides* [128–130].

Although the Cry proteins with which Bt crops have been nuclearly armed have shown excellent performance in controlling different pests, the expression of lectins and other proteins with insecticidal capacity from *Bacillus toyonensis* and *Lysinibacillus sphaericus* has also been shown to be effective in controlling pests such as *Alphitobius diaperinus, Spodoptera exigua, Cydia pomonella, Anthonomus grandis*, *Aedes aegypti,* and *Myzus persicae* [131, 132]. Furthermore, while the expression of these proteins in chloroplasts has not been thoroughly investigated, it is important to note them because despite the efficacy of Bt crops in controlling Lepidoptera and Coleoptera, control of Hemiptera has not been entirely successful, as many Hemipteran species have now become significant pests of Bt crops [133, 134].

#### *3.1.1 Expression of RNAi for pest control*

In recent years, it has been proposed that the expression of interference RNA (RNAi) for pest control in the chloroplast is a promising alternative to protein expression, and although the RNAi has been achieved previously by nuclear transformation using both double-stranded RNA (dsRNA) and long hairpin RNA (hpRNA)

#### *Chloroplasts: The Future of Large-Scale Protein Production DOI: http://dx.doi.org/10.5772/intechopen.111829*

[135–137], the obtained level protection in plants has been insufficient for practical application [138].

One problem in achieving high levels of protection has been the impossibility of accumulating large amounts of unprocessed RNAi in the host because the nuclearexpressed RNAi are processed by the cellular machinery leaving little raw RNAi available for ingestion by the host [139, 140]. Furthermore, when RNAi is ingested, the host's digestive system degrades another part of the ingested RNAi, resulting in a reduced amount of RNAi available for effective interference [141]. Therefore, the chloroplast has been visualized as a viable solution because it offers three crucial advantages 1) there is no RNAi processing machinery in these organelles, 2) they can produce and accumulate large amounts of RNAi [142–144], and 3) the chloroplast acts as a natural bioencapsulation method to protect the RNAi when consumed by insects [145, 146].

There is limited information on RNAi expression in the chloroplast, but the published reports show an area of high potential. For example, recently [138] expressed dsRNA targeting the *MpDhc64C* gene, a newly identified target gene whose silencing causes the lethality of the green peach aphid *Myzus persicae*. Results revealed that transplastomic plants exhibited significant resistance to aphids, which in turn showed reduced survival, decreased fecundity, and decreased weight of survivors, a similar effect to the obtained by Ren, Cao [147] with the use of dsRNA β*-Actin to control of the beetle Henosepilachna vigintioctopunctata in transplastomic potato plants.*

Other RNAi studies in chloroplasts have shown results that go the same way. In 2015, Jin et al. [141] silenced the V-ATPase, chitin synthase (*Chi* gene), and cytochrome P450 monooxygenase (*P450* gene) from *H. armigera* using dsRNA, reducing the weight and growth of larvae. Also, a hpRNA targeting acetylcholinesterase (*ACE* gene) of *H. armigera* and a dsRNA targeting β-actin and Vps32 (*ACT* and *SHR* genes, respectively) from Colorado potato beetle provided substantial protection against *H. armigera* and reduced growth of larvae of Colorado potato beetle in transplastomic tobacco and potato plants [140, 143].

Although the RNAi expression in chloroplast has been successfully established with promising results of accumulation and stability, future studies should be focused on elucidating the ideal length of the RNAi since there are reports of dsRNA with a length of 200 nt more protective than a dsRNA of 60 nt or > 200 nt; also, it has been reported that the cellular machinery can degrade the long RNAi producing siRNA that have a less insecticidal effect [136, 140]. Therefore, the adequate length of the RNAi that must be expressed is still not entirely clear, and this is important because the length and type of RNAi, whether it is hpRNA or dsRNA, dramatically influences the accumulation [138, 142, 144].

Other aspects that should also be considered are the suitable target genes and the implementation of efficient RNAi delivery methods, even though topical RNAi has recently been reported and seems a cost-effective alternative [148]. Currently, microinjection and overall ingestion are the most used. Moreover, this last, in practice, should be carefully considered because there are pests that do not have direct contact with chloroplasts and considerably impair RNAi efficacy [149].

In 2017, was approved the first variety of transgenic maize Smartstax PRO®, which nuclearly express a dsRNA of the *Snf7* (Sucrose non-fermenting 7) gene from *D. virgifera virgifera* [150–152], whose commercial launch was in 2022, which supports the idea that RNAi technology has great potential, and although there is no product developed from chloroplast transformation on the market yet, RNAi technology may help chloroplast engineering in this process.

#### **3.2 Chloroplast transformation to confer abiotic stress tolerance**

Reactive oxygen species (ROS) are forms of oxygen partially reduced and produced during biotic and abiotic stress. Although ROS are commonly associated with stress, they are also produced under cellular respiration and photosynthetic processes [153]. Nevertheless, although ROS are produced in normal metabolic processes, the uncontrolled production of ROS causes an alteration of the correct function of the cells. Therefore, decreasing ROS in plants is still an essential target in biotechnology.

Nuclearly have been expressed genes to promote the tolerance to different abiotic stresses such as cold stress (*CsWRKY46* gene) [154], oxidative stress (*ScVTC2* gene) [155], drought and salt stress tolerance (*ZmSNAC13* and *ZmWRKY86* gene, respectively) [156, 157], metal tolerance (*OsMYB-R1* and *SbMT*-*2* gene) [158–160], ROS decrease (*CfAPX* gene) [160] and tolerance to waterlogging (*HaOXR2* gene) [161] and have been obtained satisfactory results; nevertheless, trying to improve the results obtained by nuclear expression, genes have been expressed in chloroplasts aimed at increasing the antioxidant pathway, intervening in the glycine betaine (GB) pathway such as *codA* gene from *Arthrobacter globiformis* [98, 162, 163].

Other proteins have also been expressed in the chloroplast, such as flavodoxin (*fld* gene) [164], arabitol dehydrogenase (*ArDH*) [165], *otsB-A* operon (trehalose phosphate synthase/phosphatase) [166], γ-tocopherol methyltransferase (*TMT* gene) [167], betaine aldehyde dehydrogenase (*badh* gene) [68], dehydroascorbate reductase (*DHAR* gene), superoxide dismutase (*MnSOD* gene), glutathione reductase (*gor* gene) [168], glutathione-S-transferase (*GST* gene) [162, 169], homogentisate phytyltransferase (*HPT* gene), conferring tolerance to salt, cold, UV-B radiation, heavy metal, and osmotolerance.

Despite the potential and benefits of plastid transformation in protecting plants, it is challenging to confer abiotic stress resistance due to the involvement of various metabolic pathways. Therefore, providing a robust resistance would require multiple gene expressions. These must be carefully selected since the expression of certain types of genes can cause pleiotropic effects because the encoded proteins could interfere with the structure and function of the thylakoids or decrease the levels of ATP production [170, 171]. Nevertheless, despite the challenges of achieving stress resistance, the population's constant growth requires finding strategies to address this need.

#### **3.3 Expression of hydrolytic enzymes in chloroplasts**

Lignocellulose residues are the most abundant raw material and a highly renewable carbon source on earth [172], and due to particularities as its abundance, availability, and sustainability is believed to be a solution to solve fossil fuel shortage [173–175], to such a degree that global ethanol and biodiesel production is projected to rise to 132 billion liters and 50 billion liters, respectively, by 2030 [176]. However, although lignocellulosic compounds are abundant [177, 178], a large part of this renewable energy is beyond our reach; that is, despite being produced each year more than 40 million tons of non-edible plant material, lignocellulose is not the most important feedstock for biofuel production because it is not efficiently processed [176, 179].

The processing problem is caused by the presence of lignin that imbibes both the cellulose as well as hemicellulose and limits its degradation [180–182]. Therefore, physical and chemical methods have been used to increase biomass degradation.

However, apart from the fact that these methods can represent a potential ecological risk, they increase production costs.

In the search for alternatives to improve biomass degradation, the use of multiple enzymes has been recurring, and in this sense, different organisms have been the source of them [183]; nevertheless, the hydrophobic nature of the substrate, the enzymes cost, the concentration of required enzymes, as well as the release of phenolic compounds during the enzymatic reaction such as xylan that can inhibit enzyme activity, have limited their use [184–186], forcing the search for new and more efficient enzymes as well as an efficient method for their accumulation; therefore, the protein expression in the chloroplast it has presented as a promissory strategy to aboard the challenges of the degradation of lignocellulosic biomass.

The expression of enzymes capable of degrading cell wall polymers into chloroplast has already been tested successfully. However, although different genes of pectinases, manganese peroxidase, cutinase, and laccase enzymes from *Fusarium solani*, *Streptomyces thermocarboxydus, Phanerochaete chrysosporium*, *Trichoderma reesei*, and *Pleurotus ostreatus* has been tested with promissory results [187–190], cellulases have been the subject of the most intensive search because they represent 40% of plant biomass [177, 178].

Different cellulase enzymes have already been expressed in the chloroplastic compartment with satisfactory results, *e.g.,* xylanases (*xynA, xyl, xyn2*, *Xyl10B, xynA, xyn10A,* and *xyn11B* genes) from *Bacillus subtilis, Trichoderma reesei, Clostridium cellulovorans, Thermotoga maritima, Clostridium cellulovorans, and Alicyclobacillus acidocaldarius* [191–194], as well as endo-, exo-glucanases, and β-glucosidases (*cel6A, cel9A, cel6B, Cel6, Cel7, EndoV, CelK1, Cel3, TF6A, Bgl-1, bgl1C, EGPh, celA,* and *celB* genes) from *Trichoderma reesei, Thermomonospora fusca, Pyrococcus horikoshii, Chaetomium globosum, Paenibacillus* sp., *Phanerochaete chrysosporium*, *Aspergillus niger, and Thermotoga neapolitana* [36, 195–200]. Despite the expressed proteins being functional, it should be considered that the microorganisms that possess efficient mechanisms for cellulose degradation have redundant genes often [201]. Therefore, high degradation depends not on a particular protein but on its enzymatic cocktail, which should be considered to improve enzymatic processes.

Regardless of the intense search for cell wall hydrolytic enzymes that have been carried out, there is still a need to identify efficient cellulose enzymes [184] because they are critical factors in paper recycling, cotton processing, juice extraction, detergent production, food industry, animal feed additives and largely determine the price of biofuels [202–204]. For this reason, it is necessary to use strategies that reduce cellulase enzyme production costs as much as possible to make them commercially viable on a larger scale. Further studies will continue to be carried out to express cellulases in chloroplasts.

#### **3.4 Chloroplast engineering for the biopharmaceutical industry**

Population growth implies a constant demand for medicines, representing a lucrative opportunity for the pharmaceutical industry, which only in 2022 billed ~1.48 trillion U.S. dollars, an increase of 4.23% concerning 2021. However, most of the world's population cannot access medicines due to unaffordable prices [205–207]. Therefore, products with nutritional and pharmaceutical value are gaining importance to such an extent that there are currently 1775 products with different formulations, biosimilars, and biobetters approved by the Foods and Drugs Administration (FDA) and European Medicines Agency (EMA) for use in humans [208], which can reduce the costs of medical treatments.

Chloroplasts have already begun to contribute to the production of plasma proteins, vaccines, antibodies, and enzymes [97, 209, 210], which is especially relevant since two of the current challenges in treatments with proteins for oral consumption are obtaining high protein concentrations and the possible degradation that they may experience when passing through the digestive system [206]. These challenges could be overcome by the accumulation and bioencapsulation of proteins in chloroplasts [211].

Two decades have passed since the first candidate antigen against a human disease was expressed [212], and since then, other proteins have been expressed, *e.g.,* A27L immunogenic protein (*A27L* gene) [213], ESAT6, Mtb72F [214, 215]*, Angiotensinconverting enzyme 2 (ACE2* gene), Angiotensin (1–7) (*Ang-*[*1–7*] gene) [7, 211], Coagulation factor VIII (*F8* gene) [216], E7 Human papillomavirus antigen (*E7* gene) [217], Coagulation Factor IX (*F9* gene) [96], SAG1 Surface antigen (*SAG1* gene) [95], EDIII-1, EDIII-3, EDIII-4 (*ediii-1, ediii-3*, and *ediii-4* genes) [218], KETc1, KETc7, KETc12, GK1, TSOL18/HP6-Tsol [219], Griffithsin protein (*grft* gene) [220], S1D [221], and Human epidermal growth factor (*hEGF* gene) [94] in plants; and recently, was cloned the gene *IL29* in alga to produce human interleukin 29. The proteins expressed in the chloroplastic compartment have been obtained in high concentrations retaining their functionality, showing the potential value of expressing biopharmaceutical proteins in chloroplasts [222].

One of the challenges currently faced by therapeutic proteins is to stimulate their passage through the epithelial mucosa [223]; however, this challenge has been efficiently addressed by expressing the proteins in the chloroplast fused with the cholera toxin B subunit (CTB), and this has already been tested with the expression of exendin-4 (CTB-EX4) and acid alpha-glucosidase (CTB-GAA) [6, 224]. Fusing the proteins to CTB is an important approach as it has been reported that it could help promote oral tolerance; however, other non-toxic fusion proteins have been reported, such as human transferrin, although there are no reports with this fused protein in the chloroplast.

There is a need for biobetters/biosimilars on the market, and expressing therapeutic proteins in the chloroplast is not only feasible but could solve delivery and efficacy issues. To date, no one plant/chloroplast-based vaccine against human diseases is available to the public. On the other hand, although transplastomic plants have been cultivated for just over a decade with the consent of the United States Department of Agriculture Animal and Plant Health Inspection Service USDA-APHIS, they have not been scaled to the commercial level even though these plants do not fit USDA-APHIS regulation 7 CFR part 340 [206].

#### **4. Conclusions**

Given that the world population has been projected to increase by just over 20% in 30 years, it becomes evident that it will be necessary to adopt new technologies to face this situation in areas such as agriculture, energy, and pharmaceuticals, which are key sectors to guarantee well-being and sustainability in the future. For almost three decades, chloroplast transformation has shown important qualities that can help us address the challenges, offering greater efficiency and performance in the expression of modified genes and more precision and control in genetic modification accompanied by greater environmental safety and biosafety. While some challenges need to be addressed, such as the optimization of regulatory regions, limited expression of

proteins in non-photosynthetic tissues, the understanding of the mechanisms that govern pleiotropic effects, and the current inability to transform cereals efficiently, it is a fact that it can play a fundamental role in the production of products.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Brenda Julian Chávez1 , Stephanie Solano Ornelas1 , Quintín Rascón Cruz1 , Carmen Daniela González Barriga2 , Sigifredo Arévalo Gallegos1 , Blanca Flor Iglesias Figueroa1 , Luis Ignacio Siañez Estrada1 , Tania Siqueiros Cendón1 , Sugey Ramona Sinagawa García and Edward Alexander Espinoza Sánchez1 \*

1 Biotechnology Laboratory I, Faculty of Chemical Sciences, Autonomous University of Chihuahua, Chihuahua, Mexico

2 Tissue Culture Laboratory, Division of Engineering and Sciences, Monterrey Institute of Technology and Higher Education, Chihuahua, Mexico

\*Address all correspondence to: eaespinoza@uach.mx

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Chloroplast Recycling and Plant Stress Tolerance

*Faiz Ahmad Joyia, Ghulam Mustafa and Muhammad Sarwar Khan*

#### **Abstract**

Plastids have emerged as pivotal regulators of plant's response to biotic and abiotic stresses. Chloroplasts have the ability to synthesize a variety of pigments, secondary metabolites, and phytohormones which help plant cells to withstand adverse conditions. Further, plastids communicate with the nucleus and other cellular organelles for the acquisition of essential molecules to survive under unfavorable conditions. They act as environmental sensors which not only synthesize molecules for stress tolerance but also induce nucleus-encoded genes for stress resilience. Senescence is a key developmental process in this context and plays an important role in the release of essential nutrients. Chloroplast proteolytic machinery plays a crucial role in the degradation or remodeling of plastid proteins resulting in the generation of numerous endogenous peptides which are present in the plant secretome. Plastid chaperone system is also activated for the repair/refold of damaged proteins resulting in improved tolerance to stresses. Autophagy is a conserved process that involves large-scale breakdown of chloroplast through piecemeal degradation and chlorophagy. The piecemeal degradation occurs through Rubisco-containing bodies (RCBs) and senescenceassociated vacuoles (SAVs), whereas chlorophagy targets chloroplasts as a whole. Though information about chloroplast recycling is limited, the present work provides a comprehensive review on chloroplast recycling and its role in stress mitigation and adaptation in climate change scenarios.

**Keywords:** chloroplast proteolytic machinery, leaf senescence, chlorophagy, autophagy, stress tolerance

#### **1. Introduction**

Leaf senescence is a complex biological process characterized by the active breakdown of cellular macromolecules and the subsequent remobilization of their components. During this process, proteins like Rubisco and other chloroplast proteins undergo gradual degradation, serving as a vital source of nitrogen for recycling. This degradation is closely linked with a decline in photosynthetic activity [1]. Notably, under conditions of darkness-induced starvation, chloroplast proteins, including Rubisco, can be degraded with their carbon skeletons being utilized for respiration [2]. As the levels of these essential components diminish, chloroplasts undergo

structural changes, eventually transitioning into gerontoplasts. In gerontoplasts, thylakoid membranes break down, plastoglobules accumulate, and the overall population of chloroplasts in the cell declines.

The vacuole within leaf mesophyll cells, constituting a substantial portion of the total cell volume, is rich in a diverse array of lytic hydrolases. A significant proportion of the proteolytic activity responsible for degrading Rubisco is localized within the vacuolar fraction. Notably, senescence-associated induction of various vacuolar cysteine proteases has been observed [3]. Previous studies have proposed that sequential degradation of chloroplasts within the vacuole serves as the primary pathway for chloroplast protein degradation during leaf senescence. In electron microscopy observations, chloroplasts were found to either reside within the vacuole or within tonoplast invaginations in mesophyll protoplasts from senescing wheat leaves, suggesting a mechanism resembling phagocytosis [1]. This process of delivering cytoplasmic components to the vacuole for degradation is now recognized as autophagy.

Interestingly, the decline in Rubisco protein levels occurs more rapidly than the decrease in the chloroplast population size during senescence. Furthermore, the reduction in major chloroplast proteins does not proceed uniformly; for instance, Rubisco declines more quickly than light-harvesting complex II (LHCII) [4]. Both of these proteins are primarily synthesized during leaf expansion and exhibit lower turnover rates thereafter. These observations suggest the existence of alternative pathways or mechanisms beyond whole chloroplast autophagy that contribute to Rubisco degradation. Consequently, the study of Rubisco degradation has largely focused on chloroplast proteases rather than autophagy [5]. Notably, several proteases within chloroplasts have crucial roles in chloroplast development and maintenance. Genome-wide studies have uncovered prokaryotic-origin proteases within chloroplasts, with ATP-dependent proteases such as Clp, FtsH, and Lon being prominent enzymes involved in gradual protein degradation into amino acids and oligopeptides [6]. While some of these proteases are likely to play pivotal roles in senescence-associated bulk degradation of chloroplast proteins, the exact mechanisms remain a topic of debate [7].

#### **2. Chloroplast recycling**

Chloroplasts serve as vital organelles of the plant cell performing photosynthesis as well as production and storage of an array of biomolecules and waste materials. Pigments and light-harvesting proteins make up a significant portion of their nitrogen and Rubisco content. In the advanced stages of aging, chloroplast numbers dwindle. Studies employing electron microscopy have hinted at an intriguing process – chloroplast recycling, the complete self-consumption of chloroplasts, in leaves undergoing senescence induced by aging as well as biotic or abiotic stresses [1].

#### **2.1 Chloroplast recycling in senescence**

Senescence, the final stage of leaf development before cell death, includes the disintegration of chloroplast as well as the loss of protein, nucleic acid, pigments, lipids, and polysaccharides (such as starch). Because of this seer degradation, chloroplast ultimately loses either all or a majority of their ability to photosynthesis. The senescence of leaves and other green plant organs results in the annual breakdown of millions of tons of chlorophyll and photosynthetic protein [8]. This results in a loss of photosynthetic capacity, releasing nutrients like nitrogen for re-use in other parts of the plant. During

#### *Chloroplast Recycling and Plant Stress Tolerance DOI: http://dx.doi.org/10.5772/intechopen.114852*

senescence, chloroplast loses their photosynthetic mechanism, and nutrients are redirected to young tissues and storage organs. For instance, nitrogen extracted from mature grains in field-grown rice may represent 60% of the nitrogen released by sensing leaves [9]. In some species, abscission marks the end of senescence, requiring precisely timed petiole senses. As leaves degrade, mesophyll tissue starts to lose its green color and turn yellow, or tissue starts to change color. This is caused by the preferential degradation of chlorophyll over carotenoids, as well as other factors and the synthesis of new compounds such as anthocyanins and phenolic [10]. While chloroplast changes early in senescence, they are last to collapse as the part of this developmental program [11]. Key characterization of chloroplast degeneration includes altered volume and morphology, a transition from ellipsoid to circular shape, and internal membrane reorganization. The three steps that Tamari et al. [12] determined to be the most significant ultrastructural alterations connected with the transformation from mature chloroplast to gerontoplast are thylakoid membrane breakdown, increased plastoglobulus size and number, and modification and disorderness of the plastid envelope. Senescence in chloroplasts in terms of pigment composition resembles with the chromoplasts, yet differs in development, division capacity, retention of the genome, and biosynthetic ability. Chromoplasts have their own DNA, produce carotenoid, are capable of division, and create iron or young chloroplast, but on the other hand, senescent chloroplasts only form from mature chloroplast, are unable to divide, have eliminated all metabolic activity, and do not have their own DNA [13]. The term "gerontoplasts" was introduced to highlight these distinctions. Detecting the exact transition from chloroplast to gerontoplasts is challenging due to difficulties in identifying the senescence onset. During senescence, the natural process of aging and degradation in plant chloroplast undergoes dynamic changes to adapt protein import and export mechanisms to cellular demands. During their light-induced differentiation, chloroplasts import pre-proteins and amino acids from the cytosol. Nuclear-encoded proteins are imported into chloroplast through membrane transport complexes, and protein import is crucial for the regulation of plant adaptation processes. Under adverse stress and environmental conditions including senescence, to ensure regular cell function, the damaged chloroplast or one of its specialized components needs to be dismantled quickly. Similar to this way, during leaf senescence, chloroplast also exports organic materials including proteins and amino acids [14].

#### **2.2 Chloroplast recycling in stress tolerance**

Plastids have emerged as pivotal regulators of plant's response to biotic and abiotic stresses. Chloroplasts have the ability to synthesize a variety of secondary metabolites and phytohormones which help out plant cells to withstand adverse conditions. Further, plastids communicate with the nucleus and other cellular organelles for the acquisition of essential molecules for survival under unfavorable conditions.

Chloroplasts act as environmental sensors which not only synthesize molecules for stress tolerance but also induce nucleus-encoded genes to be activated and produce stress-resilient proteins. The MEcPP (methylerythritol cyclodiphosphate) is an important constituent of isoprenoid biosynthesis in plastids and also acts as a retrograde signal to activate the expression of stress-responsive nucleus-encoded genes. MEcPP was first identified by screening for mutants with an elicited expression of HPL (hydroxyperoxide lyase). HPL is a nucleus-encoded stress responsive gene but encodes for a plastid enzyme, which plays a fundamental role in the synthesis of jasmonic acid and other stress-related proteins.

Constitutive expression of HPL led to hyperaccumulation of methylerythritol cyclodiphosphate and regulation of a series of nuclear genes [15]. Likewise, PAP (phosphonucleotide 3′-phosphoadenosine 5′-phosphate) is another important plastid metabolite that has explored retrograde signals for stress tolerance in plants [16]. Plastidic SAL1 (chloroplast 3′ (2′), 5′ -bisphosphate nucleotidase) can dephosphorylate PAP to AMP (adenosine monophosphate). The activity of SAL1 is inhibited, and as a result, PAP accumulation is increased as PAP has been explored to play a crucial role in drought stress tolerance and in the regulation of a number of nucleus-encoded stress-responsive genes [17]. Chloroplasts are sensitive to high-temperature stress, and more than 200 genes have been found to be upregulated in response to heat stress. Plants also induce leaf chlorosis wherein the activity of chlorophyll degrading enzymes is increased during photosynthesis. This rapid degradation of chlorophyll, in response to heat stress, is linked with the activation of genes encoding for pheophytinase and chlorophyllase [18].

Senescence is a key developmental process that has effectively been utilized by the plants to cope with biotic and abiotic stresses. The first organelle to be degraded during senescence is chloroplast which contains more than 75% of the leaf nitrogen. Meanwhile, nucleus and mitochondria remain active and provide energy for the progression of senescence. Most of the nitrogen is in the form of proteins, so proteolysis is the core step for its remobilization particularly during the reproductive stage. The degradation of chloroplast components involves different vesicular pathways which can be dependent and/or independent of the ATG (autophagy-related genes). ATG (autophagy-related gene)-dependent degradation pathways involve RCB (RubisCOcontaining bodies) and ATI-PS (ATG8-interacting protein 1-plastid associated bodies). Compared with RubisCO-containing bodies, ATG8-interacting protein does not require functional autophagic machinery. ATI1 interacts with thylakoid localized proteins (PsbS, LHCA4, APE1, and PrxA), whereas RCB transports stroma proteins [19]. Under stress conditions, ATG-dependent pathways help in the removal of damaged chloroplast contents resulting in accelerated senescence and increased sensitivity to abiotic stresses. Hence, these proteins play a crucial role in the adaptation to adverse climatic conditions [20].

Under stress conditions, intensive degradation or remodeling of plastid proteins results in the generation of numerous endogenous peptides which are present in the plant secretome. The stress may lead to an increase in misfolded plastidic proteins which activates the chaperone system for the repair/refolding of damaged proteins as well as induces proteases for the degradation of proteins unable to be refolded. Hence, activity of chloroplast proteolytic machinery plays a crucial role in stress tolerance. Nature has blessed plastids with highly specialized proteases, that is, MAPs (methionine aminopeptidases) for the repair and maturation of not only chloroplast-encoded proteins but also of the nucleus encoded proteins [21]. Hence, plants manage to withstand stresses (biotic and abiotic) through recycling of their chloroplasts which involves degradation of the misfolded proteins as well as the repair/refolding of the damaged proteins [22].

#### **3. Recycling and repair pathways**

There are two pathways of plastid recycling and repair, that is, piecemeal degradation where chloroplasts are degraded gradually, while the other pathway is known as chlorophagy in which the whole chloroplast is degraded for recycling.

#### **3.1 Piecemeal degradation of chloroplasts**

In the process of piecemeal degradation and chloroplast recycling, a specific mechanism for releasing Rubisco from chloroplasts and subsequently breaking it down in other cell compartments has been proposed as an explanation for the early decline in Rubisco levels compared to the chloroplast population. As previously summarized [23], ATG-dependent autophagy via Rubisco-containing bodies (RCBs), which are sent to the central vacuole for degradation, and an ATG-independent alternative pathway involving senescence-associated vacuoles (SAVs) are currently evident to be at least two distinct transport pathways responsible for breaking down stromal proteins outside the chloroplast.

#### *3.1.1 Rubisco-containing bodies (RCBs)*

The RCBs were initially discovered in naturally aging wheat leaves (*T. aestivum*) [4]. According to thorough immunoelectron-microscopic investigations, Rubisco is occasionally contained within tiny spherical structures (RCBs), which are mainly found in the cytoplasm and occasionally in the vacuole. The stromal protein Gln synthetase is found in RCBs, which also have an electron density like that of chloroplast stroma. Notably, RCBs lack major membrane proteins including cytochrome f, LHCII, and the -subunits of ATPase coupling factor 1 as well as thylakoid structures. The isolation membranes (phagophores) that surround the double membranes of RCBs in the cytoplasm are thought to be derived from the chloroplast envelope. In the early phases of leaf senescence, when Rubisco levels begin to decline but chlorophyll content stays largely constant, RCBs are frequently observed.

The phenomenon of gradual chloroplast breakdown through RCBs (Rubiscocontaining bodies) seems to be prevalent across various plant species, serving as a crucial mechanism for recycling proteins during both growth and in response to environmental challenges. The RCBs have also been identified in young tobacco leaves (*Nicotiana tabacum*), where they are known as Rubisco-vesicular bodies (RVBs), and in rice leaves (*Oryza sativa*) exposed to salt stress conditions. Interestingly, the RCBs found in rice leaves under salt stress exhibit distinctive structural features compared to previously documented RCBs. These rice RCBs possess inner membrane structures, potentially formed from vesicles originating from invaginations of the chloroplast's inner envelope. Furthermore, they occasionally contain crystalline inclusions that develop within chloroplasts under osmotic stress and disappear during the recovery phase. It is plausible that RCBs play a role in breaking down these crystalline inclusions, and the possible mechanisms involved in RCB formation during salt stress have been extensively detailed [24].

The connection between RCBs (Rubisco-containing bodies) and autophagy has been unveiled in Arabidopsis through the use of reverse genetics and live-cell imaging techniques. In this process, RCBs can be visualized by employing stroma-targeted green fluorescent protein (GFP) (or RFP) or a fusion involving a small subunit of Rubisco (RBCS) with GFP (or RFP). This fusion interacts with the plant's endogenous large Rubisco subunits (RbcL) and RBCS molecules, resulting in the creation of Rubisco-GFP (or RFP) [25]. When Arabidopsis leaves expressing these fluorescent markers are treated with concanamycin A, which suppresses vacuolar lytic activity, spherical structures displaying GFP or RFP fluorescence, devoid of chlorophyll fluorescence, appear within the vacuolar lumen. These structures are referred to as RCBs. Additionally, the accumulation of RCBs is disrupted in autophagy-defective mutants

known as atg5 and atg4a4b [25, 26]. In normal, wild-type cells, stroma-targeted RFP and the GFP-ATG8a fusion, serving as a marker for autophagosomes and autophagic bodies, are observed together within autophagic bodies located in the vacuole [27]. These findings provide evidence supporting the notion that RCBs constitute a distinct type of autophagic body containing Rubisco and potentially other proteins localized within the stroma.

#### *3.1.2 Senescence-associated vacuoles (SAVs)*

A novel type of diminutive lytic vacuole, known as the senescence-associated vacuole (SAV), has come to light within the cytoplasm of aging leaves in plants like soybean (*Glycine max*), Arabidopsis, and tobacco. This discovery has unveiled an alternative route for the breakdown of chloroplast proteins, distinct from the conventional chloroplast-based degradation [28]. Remarkably, SAVs exclusively emerge in photosynthetic tissues undergoing senescence, harboring a unique senescencespecific cysteine-protease, SAG12 (senescence-associated gene 12).

Comparable to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) containing bodies (RCBs), SAVs encompass stromal proteins like Rubisco and glutamine synthetase, and stromal-targeted GFP. However, they lack thylakoid proteins such as the LHCII. Additionally, unlike RCBs, SAVs are laden with chlorophyll a. Intriguingly, there is no substantiated evidence thus far to suggest that SAVs engage autophagic machinery. Notably, it was postulated that SAVs still manifest even in the absence of autophagy, as demonstrated in the autophagy-defective atg7 mutant, albeit supporting data were conspicuously absent in the original study [29]. This underscores a semblance in the content of RCBs and SAVs, while their formation mechanisms remain unequivocally distinct. A recent investigation unveiled that SAV buildup coincides with the induction of autophagy in the Arabidopsis des1 mutant, which lacks L-cysteine desulfhydrase 1, an enzyme crucial for cysteine degradation [30]. However, further research is imperative to elucidate the intricate interplay between ATG-dependent autophagy and SAVs.

The visual contrast between SAVs (stroma-associated vacuoles) and RCBs (Rubisco-containing bodies) is quite remarkable and can be easily appreciated under the microscope. The RCBs are normally found enclosed by a double membrane and display internal electron density similar to that of the chloroplast stroma. In sharp contrast, SAVs are enclosed by a single membrane. The electron density within SAVs resembles that of the central vacuole and is notably lower than that found in the chloroplast stroma. Additionally, SAVs often contain dense aggregates within their interior, potentially composed of partially degraded cellular components.

The exact mechanism by which stromal proteins are directed to SAVs remains a mystery. As previously discussed in a comprehensive review [31], one possible pathway involves stromal proteins initially traversing the chloroplast envelope before being transferred directly to SAVs through a yet-to-be-revealed mechanism. Alternatively, similar to the process of piecemeal microautophagy of the nucleus in yeast, it is plausible that SAVs themselves encapsulate a portion of the chloroplast or stromule, forming a structure similar to RCBs. The SAVs exhibit a higher level of acidity compared to the central vacuole and possess potent proteolytic capabilities. Thus, chloroplast particles enclosed within SAVs would undergo rapid degradation, resembling the fate of RCBs within the central vacuole. It is important to note that this phenomenon can only be observed in the presence of concanamycin A.

#### *3.1.3 Protein import/export and chloroplast recycling*

The majority of chloroplast proteins originate from nuclear genes and are guided into chloroplast by TOC (translocon at the outer chloroplast membrane) and TIC (translocon at the inner chloroplast membrane) complexes under normal conditions. The process of transporting these nuclear-encoded proteins into the chloroplast is a highly orchestrated series of steps that rely on specific molecules and energy sources. These proteins contain a distinct molecular tag called a transit peptide, which acts as the navigation signal, directing them to the appropriate location within the chloroplast. Several key stages are involved in the import of protein: firstly, they are identified by transit peptide specialized proteins called cytosolic chaperones and targeting factors. These molecules create a protective complex around the protein, preventing it from folding prematurely and maintaining it in a state ready for import. After identification and binding, the protein-chaperon complex intratas with molecular machinery situated at the outer membrane of the chloroplast, referred to as the TOC complex [32]. This nitration triggers the movement of the protein across the outer chloroplast membrane. Upon successfully crossing the outer membrane, the protein chaperon complex encounters another translocon, the TIC complex, embedded in the chloroplast inner membrane [33]. This complex assists in transporting the protein across the inner membrane, ensuring its access to the inner space of the chloroplast. Once the protein has reached the inner space of the chloroplast, the transit peptide is cleaved off. This step is vital for the protein to adopt its final functional shape. Furthermore, molecular chaperones within the chloroplast environment aid in correctly folding the protein, ensuring its effective participation in the intricate biochemical process of organelles. In essence, the process of chloroplast protein import involves a sequence of precisely coordinated events, safeguarding the integrity and functionality of the transported proteins within the dynamic environment. While most proteins are imported, there are situations when specific proteins need to be exported. Some of these exported proteins play roles in photosynthesis, plastid division, or inter-organelle signaling. The export mechanism typically involves recognizing sorting signals and their integration with the export machinery [34].

A controlled physiological process is involved in the gradual breakdown of cellular components leading to significant alterations in chloroplasts' structure and function, thereby influencing the intricate protein import and export process. Prominent observations highlight the changes that take place in such process during senescence. Firstly, studies indicate that the rates of protein import into chloroplast vary as senescence advances. These changes often connect to modifications in the expression of essential components in the TOC and TIC complexes, which play vital roles in facilitating efficient protein translocation across the chloroplast membrane [35]. As chloroplast age during senescence, the importance of quality control mechanisms becomes more prominent. These mechanisms identify and transport mis-folded protein out of the chloroplast to prevent their buildup and potential damage. This involves the retrograde transport of malfunctioning proteins to the cytoplasm for degradation. Senescence introduces distinct sorting signals known as senescenceassociated sorting signals. These signals guide proteins to specific locations within the chloroplast, highlighting the unique nature of protein transport during senescence. Moreover, senescence stimulates substantial shifts in gene expression and signal transduction pathways [36]. Regulatory proteins managing this process might need to be exported from chloroplast toward organelles like vacuoles and nucleus. This export plays a vital role in coordinating the different events associated with senescence.

In the process of chloroplast protein degradation, two vesicles' categories play vital roles: Rubisco-containing bodies (RCBs) and senescence-associated vacuoles (SAVs) (**Figure 1**). The RCBs are smaller, sphere-shaped vesicles, and enclosed by dual membranes that consist of Rubisco, excluding thylakoid proteins. They are considered as the autophagosomic bodies responsible for transporting stomata proteins toward the vacuoles [37]. Notably, RCBs operate during darkness, senesce, and leaf carbon homeostasis, participating in Rubisco reduction under stall stress in soybean [38]. In contrast, SAVs are characterized by elevated proteolytic and acidic activity compared to central vacuole. These vessels are prominent during senescence with chloroplastcontaining cells, notably accumulating in senescence leaves. They facilitate the breakdown of soluble photosynthesis proteins within the chloroplast stroma, including glutamine synthetase and a large unit of Rubisco. Unlikely autophagy, SAVs can specifically degrade chloroplast components, yet the mechanism of their formation and translocation of chloroplast components to SAVs remains unknown [39].

Recent research has uncovered that SAVs are implicated in the chlorophyll, and PSI degradation throughout senescence, supported by their strong cysteine protease activity, and the presence of the senescence-specific protease perhaps contribute to the breakdown of the Rubisco during leaf senescence [40]. These vesicles are crucial for the proteolysis of chloroplast proteins. Chloroplast proteins need to be moved outside of the plastid for the previously mentioned pathways to function [41]. Lately, a significant gene chloroplast vesiculation (CV) has been recognized, involving translocation of chloroplast proteins pathway external to plastid. This gene's expression has been shown to elevate during ordinary and abiotic stress induced senescence.

#### **Figure 1.**

*Cellular pathways involved in chloroplast protein import showing protein trafficking. SAVs represent senescenceassociated vacuoles, RCBs are for rubisco-containing bodies, and chloroplast-containing vesicles (CCVs) are for CV-containing vesicles.*

#### *Chloroplast Recycling and Plant Stress Tolerance DOI: http://dx.doi.org/10.5772/intechopen.114852*

Once chloroplast proteins enter the plastids, CV destabilizes the chloroplast, prompting the formation of CCVs during senescence, which subsequently deliver chloroplast proteins to the vacuoles for proteolysis [42]. The CV protein interrelates with several chloroplast proteins, primarily thylakoid components, in plastids, where it localizes. Intra-plastidic vesicles, or CCVs, are produced as a result of the CV gene. These CCVs eventually die from the plastid and travel to the central vacuole, carrying proteins from stromal and thylakoid membranes. Importantly, this degradation process through CCVs operates independently of autophagy and SAVs. The CV protein interacts mainly with thylakoid components, triggering the development of vesicles. The RD26 transcription factor has recently been linked to the induction of the CV-related gene expression. The RD26 regulates the direct degradation of chloroplast proteins during senescence [43]. As chlorophyll breakdown initiates within chloroplasts in senescent leaves, non-fluorescent chlorophyll proteins are released and ultimately broken down in vacuoles. This suggests that senesces or stress can stimulate the degradation of chloroplast protein, even the entire chloroplast, leading to peptide accumulation within the chloroplast, the cytoplasm, and various vesicles. Notably, these peptides could potentially be transported into the extra-cellular space through vesicles and vacuole fusion, aided by outer and inner peptidases, as well as peptide and amino acid transporters.

#### **3.2 Role of autophagy in chloroplast recycling**

Autophagy is a highly conserved biological mechanism in eukaryotic organisms. This process involves the engulfing of cellular components within membrane-bound vesicles, followed by their subsequent degradation through lytic processes. Whole organelles can also be enclosed within these membrane structures as part of autophagy [44]. These cellular components wrapped in membrane-bound vesicles are taken to special compartments called vacuoles or lysosomes. Inside these compartments, enzymes called hydrolases break down both the vesicles and their contents. There are two different forms of autophagy, known as micro-autophagy and macro-autophagy, which have been seen in various organisms, including plants [45]. In micro-autophagy, parts of the cell's fluid are directly taken in by the folding of the vacuolar membrane. In the more common form called macro-autophagy, often just referred to as autophagy, the cell's content is enclosed within a double-layered structure called an autophagosome. The fusion event between the outer membrane of the autophagosome and the vacuolar membrane facilitates the transfer of the enclosed inner membrane-defined structure, known as the autophagic body, into the interior space of the vacuole.

In more recent times, a breakthrough occurred with genome-wide studies, which paved the way for a molecular examination of plant autophagy. These studies identified numerous genes in plants that are similar to yeast ATGs and also explored the effects of disrupting these genes in Arabidopsis (*Arabidopsis thaliana*). Researchers have explored the roles of Arabidopsis ATGs by utilizing T-DNA insertional knock-out mutants and RNA interference knockdown mutants. Additionally, they have established a live-cell system for monitoring autophagy in plants. This system involves the use of a GFP fused with ATG8, which acts as a marker to track the presence of autophagosomes. These molecular methods have demonstrated the crucial function of plant autophagy in response to food deficiency, abiotic stressors, and pathogen infection. They have also confirmed that the central autophagy apparatus functions in plants but not in yeast [46]. Additionally, it has been demonstrated that target of rapamycin (TOR) kinase functions as a negative regulator in yeast and Arabidopsis [47].

Plant autophagy was initially studied for its ability to respond to nutrient deprivation, a critical role similar to that of yeast. Autophagy is triggered when cultured plant cells lack externally supplied sucrose. During this period, key ATGs linked to Ub-like conjugation systems experience temporary increases in activity [48]. Scientists have examined the role of autophagy in nutrient recycling by studying autophagy-deficient (atg) mutant plants. Essentially, Arabidopsis atg mutants can complete their life cycles [1].

However, they struggle to survive extended periods of nitrogen and/or carbon starvation. Additionally, they exhibit accelerated leaf aging and cell death, along with reductions in chlorophyll and photosynthetic proteins, even when nutrient and growth conditions are favorable. This premature aging affects seed production, leading to lower yields in atg mutants. Consequently, it was previously concluded that while autophagy plays a vital role in nutrient recycling during starvation and senescence in plants, it does not primarily target chloroplasts, despite their abundance in leaf degradation [45].

#### **3.3 Chlorophagy: autophagy of whole chloroplasts**

In the advanced stages of aging, chloroplast numbers dwindle. Studies employing electron microscopy have hinted at an intriguing process – the complete self-consumption of chloroplasts, an occurrence termed "chlorophagy," in leaves undergoing senescence induced by darkness [1]. In the world of Arabidopsis, a plant's individual leaf darkened for experimentation experiences a rapid onset of senescence. Remarkably, within a few short days, both the quantity and size of chloroplasts within these leaves diminish significantly. However, in the case of individually darkened leaves (IDLS) from the atg4s mutant, senescence, evident as chlorosis, unfolds much like in the wildtype, yet the drop in chloroplast count and to some extent, their size, is hindered [49].

What's more, beyond the realm of conventional chloroplast behavior, small chloroplasts retaining their chlorophyll fluorescence come into view inside the vacuole after 3 days of the IDL treatment in wild-type specimens, but this phenomenon remains absent in the atg4 mutant. Given that the formation of Rubisco-containing bodies (RCBs) consumes elements from both the stroma and the chloroplast envelope, it is a logical assumption that this process contributes to the shrinking of chloroplasts. These shrunken chloroplasts, reminiscent of gerontoplasts, are then ferried into the vacuole through the machinery of autophagy.

Notably, mutations that disrupt the normal functioning of chloroplasts can trigger the wholesale degradation of these organelles, a process likely attributable to chlorophagy. This degradation appears to occur independently of senescence or starvation. For instance, partially degraded chloroplasts make their home within the vacuoles of cotyledon cells belonging to the Arabidopsis ppi40 mutant, a genetic variant deficient in a protein homologous to the 40 kDa protein found in the inner envelope membrane of chloroplasts' translocon (Tic complex). In the advanced stages of aging, chloroplast numbers dwindle. Studies employing electron microscopy have hinted at an intriguing process – the complete self-consumption of chloroplasts, an occurrence termed "chlorophagy," in leaves undergoing senescence induced by darkness [1]. As chloroplast transfer to the vacuole is observed even under well-fed conditions, it is posited that plants utilize autophagy to eliminate defective plastids, ensuring quality control of their organelles.

The Arabidopsis mex1 mutant, which lacks the maltose transporter in the chloroplast envelope, presents a fascinating scenario. This mutant accumulates

#### *Chloroplast Recycling and Plant Stress Tolerance DOI: http://dx.doi.org/10.5772/intechopen.114852*

higher-than-normal levels of maltose and starch within its chloroplasts and displays a chlorotic phenotype even when it's not undergoing senescence. What's particularly intriguing is that various components of chloroplasts, such as thylakoid membranes, starch granules, and plastoglobules, have been found inside the vacuole of the mex1 mutant [50]. These discoveries have given rise to a hypothesis suggesting that the increased maltose concentration in the mex1 mutant disrupts chloroplast function, possibly setting off a form of retrograde signaling that initiates the degradation of chloroplasts.

#### **4. Conclusions**

Green photosynthetic organisms including plants, algae, and cyanobacteria are the source of energy and carbon fixation through photosynthesis. Green pigments containing plastid "chloroplasts" are the hub of photosynthesis. It supplies energy to all living organisms. Photosynthesis products, through various biochemical pathways, produce numerous macromolecules. Moreover, chloroplasts become an abundant source of necessary organic nitrogen and other nutrients through the process of chloroplast recycling in senescing leaves. Such nutrients are utilized in growth, reproduction, storage organs, and new plant organs. The protein translocation mechanism is essential for chloroplast function and plays a critical role in cellular adaptations during senescence. The dynamic changes observed in these processes during senescence highlight their significance in plant adaptation to changing environmental conditions and developmental stages. Though limited literature is available, a deep understanding of the protein import/export during senescence and/or chloroplast recycling can potentially lead to the progression of strategies to enhance crop productivity and stress tolerance, contributing to agriculture sustainability and food security.

### **Author details**

Faiz Ahmad Joyia, Ghulam Mustafa and Muhammad Sarwar Khan\* Faculty of Agriculture, Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan

\*Address all correspondence to: sarwarkahn\_40@hotmil.com

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## The Role of Guard Cells Chloroplasts toward the Enhancement of Plant Growth

*Batta Kucheli*

#### **Abstract**

Guard cells contain chloroplasts, and the stomata through which exchange of gas takes place. They control the stomatal pore, which serves as a channel for exchange of gas by balancing between CO2 uptake for photosynthesis and water loss through transpiration. As a result, chloroplasts in the guard cells have become potential tool for manipulation toward improvement of plant productivity through photosynthesis. The role of the guard cells chloroplasts can, therefore, be elucidated through manipulations of enzymes for photosynthesis by using molecular means. The cytochrome *b*6*f* complex catalyzes the transfer of electrons between the two photosynthetic reaction centers, Photosystems II and Photosystem I, while at the same time, transferring protons across the thylakoid used to synthesize ATP for the Calvin cycle. In this study, the overexpression of the Rieske FeS protein in Arabidopsis exhibited phenotypes, which resulted in substantial improvements of quantum efficiency of PSII. Transgenic lines were significantly higher in early development of the plants. Phenotypes observed in the transformed plants also showed faster initial growth rates evidenced by larger leaf area and faster rosette increases, which may suggest that Rieske might be of importance for enhanced plant growth. The result obtained proves more opportunities await the exploitation of guard cells chloroplasts metabolism toward the improvement of plants.

**Keywords:** Rieske FeS, guard cells, photosynthesis, chloroplasts, plant growth

#### **1. Introduction**

Stomatal conductance determines the flux of gases between the inside of the leaf and the external atmosphere, which influences photosynthetic carbon assimilation and water use. Understanding the structure, function, and signaling mechanism in stomata in response to changing environmental conditions is, therefore, critically important if we are to manipulate the processes for optimal plant use (**Figure 1**) [1–3].

Stomata has, therefore, attracted the attention of scientists for almost three centuries [4], and a great deal of knowledge related to the structure, development, and physiology of stomata has been acquired. In order to balance between carbon supply and the ability to optimize or sustain plant growth in an ever-fluctuating environment, an understanding of the external and internal responses is required, as

#### **Figure 1.**

*Guard cells, stomata, and chloroplasts as potential tools for manipulation. (A) Guard cells from an epidermal peel showing chloroplasts, which are the site of photosynthesis and the stomata through which exchange of gas takes place. The guard cells control the stomatal pore, that is. The opening and closing of the stomata. (B) Schematic diagram of a single chloroplast illustrating (a) the thylakoid membrane where electron transport chain takes place. The cytochrome b6f between the PSII and PSI shuttles electrons, which are used for the synthesis of NADPH and ATP as energy for fueling the Calvin cycle. (b) Calvin cycle found in the stroma of the chloroplast where sedoheptulose-1, 7-bisphosphatase (SBPase) a key component in the regeneration of RuBP in the Calvin cycle functions. (source: Picture taken from PhD research of Batta kucheli 2018).*

well as new approaches that integrate both molecular and physiological approaches [5, 6]. Genetic potentials for maximum yield still lie unrealized, and the need for better adaptation of plants to climatic factors in which the plants are grown are still enormous [7].

In view of this, this chapter highlights or provides a way to elucidate the role of guard cell chloroplasts by using molecular tools and techniques to manipulate chloroplast metabolism specifically in the guard cells.

#### **1.1 Guard cell chloroplasts**

Photosynthesis takes place primarily in the mesophyll tissue as epidermal cells generally lack chloroplasts. However, guard cells, which developed from protodermal cells, also contain photosynthetically active chloroplasts in most but not all species [8–12]. Guard cell chloroplasts are smaller and have less granum, hence could be said to be less developed than the mesophyll cells. Guard cell chloroplasts have a reduced thylakoid network and chlorophyll contents compared to the mesophyll [13] and have functional photosystems I and II, electron transport, oxygen evolution, and photophosphorylation [8, 14]. Elucidating the role of the guard cell chloroplasts and guard cell photosynthesis presents a serious challenge for researchers in the field but the following roles have been proposed for guard cell chloroplasts.

*The Role of Guard Cells Chloroplasts toward the Enhancement of Plant Growth DOI: http://dx.doi.org/10.5772/intechopen.114204*


#### **1.2 Electron transport in guard cells**

Linear electron transport and photophosphorylation in the guard cells have been documented. The quantum efficiency for PSII photochemistry in guard cells has been shown to the rate of 70 to 80% that of mesophyll cells when subjected to a range of light levels thereby suggesting a similarity of operation in mechanisms in both guard and mesophyll cell [14, 21]. The pigment composition of guard cells is also similar to that of the mesophyll cells [22], which leads to enhance ATP production. As a result, such electron transport rate could provide sufficient energy to drive ions for stomatal opening in the absence of CO2 fixation. Using the high-resolution chlorophyll fluorescence imaging, [14, 23] found that Rubisco was a major sink for the products of electron transport suggesting that guard cell electron transport can be mediated by [CO2] meaning Calvin cycle activity does take place in the guard cell [24].

#### **1.3 Advances, tools, and techniques for chloroplasts and guard cells manipulation toward plants improvements**

Recent developments in technology have opened opportunities to explore guard cell mechanisms, and therefore potentials for regulating WUE and enhancing plant productivity. Efficient, simple, and fast cloning techniques are available for the design of desired single or multiple genes to be expressed in plants for genetic manipulation of metabolites involved in photosynthetic metabolism, which could lead to changes in stomatal behavior and potentially improve photosynthesis and water use efficiency in plants [25].

Specific cell metabolisms in guard and mesophyll cells can now be targeted and the possible coordination between mesophyll metabolites in relation to stomatal functions determined. The development of guard cell-specific promoters has made these manipulations of expression of specific gene transcripts possible, which provides opportunities to manipulate guard cell-specific metabolisms or specific stomatal traits in order to elucidate their functions or interactions. For instance, KST1 and MYB60 promoters, which are guard cell-specific promoters developed by Müller-Röber et al. [26], have been used to drive the expression of target genes specifically in guard cells. The use of a specific guard cell promoter has demonstrated by Wang et al. [27], Kucheli [25] through an enhanced light-induced stomatal opening, greater photosynthesis, and improved growth rate in Arabidopsis overexpressing H+ -ATPase among others.

Prior studies have targeted both the Calvin cycle and electron transport chain enzymes to expressions in order to identify their control on carbon assimilation [28–30]. It is now possible to demonstrate single or more enzyme transformations, which have shown to enhance photosynthetic rates in varieties of crops. For instance, increased sedoheptulose-1,7-bisphosphatase activity resulted in tobacco plants with

improvements in carbon assimilation by 6–12% [28, 31]. It is in line with this that we elucidated the role of the guard cells chloroplasts using guard cell-specific promoters (Myb60 and KST1) to drive expression specifically in the guard cells in order to produce transgenic plants with manipulation in electron transport chain.

#### **1.4 Design and development of golden gate construct to manipulate expression of cytochrome B6F (Rieske) in** *Arabidopsis thaliana*

An electron transport chain is a series of complex reactions that transfer electrons from electron donors to electron acceptors alongside the transfer of protons H+ ions across a membrane. The cytochrome *b*6*f* complex also known as the plastoquinolplastocyanin reductase is an enzyme found in the electron transport chain on the thylakoid membrane of chloroplasts. In photosynthesis, the cytochrome b6f complex catalyzes the transfer of electrons between the two photosynthetic reaction centers, Photosystems II and Photosystem I, while at the same time, transferring protons across the thylakoid used to synthesize ATP from ADP for the Calvin cycle [32, 33].

The overexpression of the Rieske FeS protein in Arabidopsis resulted in substantial improvements of quantum efficiency of PSI and PSII and electron transport, which lead to significant impacts on plant yield [30].

Transgenic approaches have demonstrated striking results of the manipulation of the Calvin cycle where energy conversion led to increasing yield potential [29, 34–37] suggesting that these studies may imply improvements in photosynthesis through overexpressing the activity of individual enzymes. Already, evidence supporting this hypothesis from single manipulations has been demonstrated from transgenic tobacco plants over-expressing SBPase [28] and also the combined multigene approach of over-expressing SBPase and FBPA [38]. These photosynthetic manipulations resulted in increased carbon assimilation, enhanced growth, and increased cumulative biomass, hence the genetic potentials that lie there. It is, therefore, obvious that number of sense and antisense plants with increased and reduced levels of SBPase and Rieske have varying photosynthetic capacity and have altered carbohydrate status at the whole leaf level, thus leading to modifications in growth and development.

Transgenic plants with guard cells specific manipulation are, therefore, important for identification and characterization of the signal transduction mechanisms [25]. Therefore, guard cell photosynthesis demands genetic analyses by guard cell-specific manipulation of photosynthesis. Multiple targets have been identified and could be manipulated to aid more understanding to maximize crop production. Some of these targets are the SBPase and Rieske enzymes, which have demonstrated of their significance in controlling photosynthetic processes.

#### **2. Materials and methods**

#### **2.1 Design and construction of constructs for use in** *Arabidopsis thaliana* **in the chloroplasts of guard cells**

The sequences of the genes of interest Rieske in *Arabidopsis thaliana* were retrieved from the TAIR database and primers designed. The plasmid vectors used for the plant transformation were constructed using the golden gate cloning and the Moclo system [39, 40]. Constructs were designed to alter expression of the Rieske genes in

a cell-specific manner driven by the KST1 and MYB60 promoters. YFP tags were also included in the construct to demonstrate cell specificity.

#### **2.2 Screening of mutants**

Arabidopsis mutants were identified by PCR screening. Arabidopsis stable transformants carrying the transgenes were also screened using antibiotic and/or herbicide. Plants were germinated on agar plates containing antibiotics. For seeds germinated in soil, the soil was treated with 0.82 mM of glufosinate-ammonium and were watered with this until selection was obvious and seedling selected with plants transplanted into individual pots. The presence of the transgene was reconfirmed by genomic DNA PCR screening.

#### **3. Results**

#### **3.1 Selection of** *Arabidopsis* **transformants**

Transformed floral plants in the Arabidopsis plants were allowed to mature to seed. Seeds were collected and planted for the screening of the T1 generation and selection of positive transformants achieved. Selection of positive transformants was identified by the application of BASTA watered on the soil in which the T1 germinated seedlings as transformants were resistant to the herbicide (**Figure 2**).

The resulting successful transgenic plants were selected on the herbicide glofusinate ammonium (BASTA) and subsequent screening for homozygous lines began by PCR followed by confirmation by iDNA technology.

#### **4. DNA analysis of transformed plants**

The result selected from transformed individual plants was checked for the presence of the transgene by PCR analysis. The result of the DNA analysis produced PCR fragment sizes exactly as the gene of interest in all the lines screened. Constructs have shown all ten lines selected positive for the presence of the transgene (**Figure 3**).

#### **Figure 2.**

*Hebicide (BASTA) selection of transformed Arabidopsis plants. Resistant transformants were selected by growing on soil and spraying with BASTA (presence of bar gene confers resistance to the glofusinate ammonium herbicide BASTA). (a) WT control showing complete death of cotyledons grown on BASTA, (b) WT control grown without BASTA, and (c) selection of resistant transformants grown on soil watered with BASTA. Plants growth conditions maintained under controlled-environment growth room with (22°C, 8 h light, 16 h dark cycle). White scale bar represents 1 cm.*

#### **Figure 3.**

*Genomic DNA PCR screening of transformants for presence of the transgene. The presence of transgenes was checked by PCR analysis of genomic DNA of plants). Ten lines were screened per construct. WT DNA (WT + red) and plasmid DNA containing the gene of interest (P+) were used as negative and positive controls, respectively. PCR products were run alongside molecular weight marker (DNA generuler ladder mix from thermoscientific) in base pairs.*

#### **5. Fluorescence microscopy to detect YFP expression in chloroplasts of guard cells**

Rieske YFP mutants were rapidly screened for the presence and localization of the yellow fluorescence protein (YFP) specifically in guard cells using high-resolution chlorophyll fluorescence microscope. Constructs fused to the yellow fluorescence protein confirmed that expressions driven by the cells specific promoters were confined to the chloroplasts in the guard cells. Constructs tagged with either the MYB60 promoter or KST1 promoter revealed the chloroplasts with the YFP in them while wild-type control had no signal (**Figure 4**).

### **6. Photosystem II operating efficiency and growth analysis**

The first obvious thing observed in the transgenic lines was the clear evidence of phenotypes in all the constructs selected for further analysis.

Observations revealed developmental phenotypes in the early stage of plants growth between the WT and transgenic lines. The total rosettes or leaf area of the

#### **Figure 4.**

*Specific YFP expression in the guard cell of transformed plants. Localization of the YFP in the chloroplasts of guard cells of Arabidopsis transformants. (a) Wild-type (Col-0) tissue showing no signal while (b) expression of the constructs tagged with YFP and driven by the MYB60 promoter in leaf tissue was checked using the high-resolution microscope. Images acquired by exiting with 515 nm LEDs and emission collected with a band pass filter 530 ± 20).*

#### *The Role of Guard Cells Chloroplasts toward the Enhancement of Plant Growth DOI: http://dx.doi.org/10.5772/intechopen.114204*

transgenic plant of the lines within the construct 4-pL2B-BAR-(pKST1)-AtRiesketHSP evidently showed larger leaf area (**Figure 5**).

Chlorophyll fluorescence imaging was also performed on the plants and the operating efficiecny of PSII photochemistry (*Fq*′*/Fm*′) determined. Chlorophyll fluorescence imaging of WT and mutant lines of construct 4-(pL2B-BAR-(pKST1)- AtRieske-tHSP to changes in light intensity subjected to 150 and 600 μmol m −2 s−1 showed significant differences between the wild type and mutants in the first 3 and 4 weeks of recording confirming also the phenotype found in the images observed above. However, as the plants advanced in the next 2–3 weeks, the reductions in *Fq'/ Fm*′ of the wild type seemed to catch up with the transgenic lines in all constructs suggesting that the expression of these genes might be most critical in their early stages of development. There were no significant differences found in the later weeks of the experiment between the wild type and the transgenics (**Figure 6**).

Chlorophyll fluorescence imaging used to determine the maximum PSII operating efficiency of photosynthesis (*Fq*′*/Fm*′) values of the whole plant was also used to capture images at the time of analysis. These images further tell more of the PSII efficiency differences indicated by colors from the scale, which ranges from green (lowest value) to orange (highest value). The more the value, the more efficient is the PSII efficiency. Images likewise show the growth differences, which were evident between the wild type and transgenic lines. The PSll photosynthetic differences of all the four constructs at week 3 are shown in the **Figure 7** below, which clearly

#### **Figure 5.**

*Growth phenotype of WT and homozygous mutant lines of construct 4- (pL2B-BAR-(pKST1)-AtRieske-tHSP plants grown on soil. 4–weeks old plants were germinated and grown for 14 days on soil before picked out and transferred individually to pots. Plants were grown under identical conditions in a controlled-environment growth room with (22°C, 8 h light, 16 h dark cycle).* Arabidopsis thaliana *(Col-0) WT and mutant lines are shown. White scale bar represents 5 cm.*

#### **Figure 6.**

*Chlorophyll fluorescence imaging comparison of WT and mutant lines of construct 4-(pL2B-BAR-(pKST1)- AtRieske-tHSP to changes in light intensity. The maximum PSII operating efficiency (Fq*′*/Fm*′*) values of the whole plant subjected to (a)150 μmol m -2 s-1 and (b) 600 μmol m -2 s-1 were measured. Plants were germinated and grown for 14 days on soil before picked out and transferred to individual pot on soil. These were grown for additional 5 weeks under identical conditions in a controlled environment growth room (22°C, 8 h light, 16 h dark cycle).* Arabidopsis thaliana *(Col-0) WT and mutants individual lines of the construct 4-(pL2B-BAR-(pKST1)- AtRieske-tHSP measured. Data were obtained using 10–15 individual plants from three independent transgenic lines and are derived from weeks 3, 4, 5, and 7. Columns represent mean values, and standard errors are displayed, respectively. Significant differences between WT and lines (P < 0.05) at weeks 3 and 4 at 150 μmol m -2 s-. Each line was significantly different from wild-type WT. At higher light level of 600 μmol m -2 s-.1 no difference between the wild type and transgenic found in all the lines.*

showed a faster growth phenotype with larger rosettes in the transgenic lines than the wild type.

Growth was slower in the wild type in the early stages as seen above. However, as the plants advanced (week 7), the reductions in rosette area of the wild type seemed to catch up with the transgenic lines, which might again be implying that the expression of these two genes might be more active in early developmental stages.

*The Role of Guard Cells Chloroplasts toward the Enhancement of Plant Growth DOI: http://dx.doi.org/10.5772/intechopen.114204*

#### **7. Discussion**

Transgenic studies have provided numerous evidence that manipulation of certain enzymes is potential route for the improvement of plant productivity [14, 28, 31, 36, 38, 41, 42].

In this work, we generated transgenic Arabidopsis (*Arabidopsis thaliana*) plants overexpressing the Rieske enzyme mutant lines with altered manipulation identified in *Arabidopsis thaliana*. The analysis carried out such as the DNA and herbicides screening plus the localization of the YFP have all revealed that these genes are present in the chloroplasts of the guard cells and had impact on the transgenic lines.

Phenotypes observed suggested that the transformed plants exhibited significant faster initial growth rates evidenced by larger leaf area and faster rosette increases which may suggest that Rieske might be of importance for enhanced plant growth. Studies have reported of increased yield in plant productivity by overexpressing these genes in the whole plants [28, 30, 36, 41]; however, it is interesting that we have observed similar enhancement of growth despite expression being limited to guard cells. The quantum efficiency of PSII photochemistry in the transgenic lines was also significantly higher in early development. These data imply that the photosynthetic efficiency of young plants may have a greater impact on plant development. These

#### **Figure 7.**

*Photosynthetic efficiency of PSll operating systems of images captured of WT and homozygous mutant Arabidopsis plants. 3–weeks old plants were germinated and grown for 14 days on soil before picked out and transferred to individual pots on soil. Plants were grown under identical conditions in a controlled-environment growth room with (22°C, 8 h light, 16 h dark cycle).* Arabidopsis thaliana *(Col-0) WT and mutants are shown. Scale bars represent 5 cm and* Fq′/Fm′ *values represented by colors as indicated.*

findings are consistent with earlier studies, which reported that the stimulatory effects of increased levels of SBPase occurred earlier in development [28], which may also demonstrate the different limitations witnessed on photosynthesis between developing and fully expanded leaves.

It is important also to keep in mind that little changes in photosynthetic capacity counts and can have a great impact on plant development [28]. Therefore, these results suggest that altered expression (assumed due to the expression of the construct) Reiske in guard cells alone in plants seem to improve the overall plant photosynthetic efficiency and growth in these plants compared with the wild type suggesting that genes manipulation in the guard cells may be playing roles in plant development.

In conclusion, numerous studies have shown that photosynthetic enzymes in carbon metabolism have yielded increased photosynthetic rates in plant at the whole leaf level [34, 36, 41–45].

This research was based on elucidating the role of guard cells chloroplasts in stomatal regulation, and the role these cells could play in enhancing plant productivity. A comprehensive understanding of the signals or metabolites synchronizing stomatal conductance and carbon assimilation is, therefore, paramount toward successful manipulations of stomatal behavior for enhancing water use efficiency and sustainable inputs in agriculture.

The molecular approach efforts to comprehend the involvement of guard cell photosynthesis in stomatal function required manipulating photosynthesis specifically in guard cells, which demonstrated the potential of transgenic plants with altered guard cell metabolism. We particularly have demonstrated specificity of the KST promotor and shown that expression was only in the guard cells. This has also shown the potential of this promotor for manipulating guard cell-specific metabolism.

#### **8. Additional information**

Parts of this chapter were previously published in a doctoral thesis by the same author titled "The role of guard cell chloroplasts in stomatal function and coordinating stomatal and mesophyll responses" at the University of Essex, UK 2018.

#### **Author details**

Batta Kucheli Adamawa State University, Mubi, Adamawa, Nigeria

\*Address all correspondence to: battakucheli@gmail.com

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Role of Guard Cells Chloroplasts toward the Enhancement of Plant Growth DOI: http://dx.doi.org/10.5772/intechopen.114204*

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#### **Chapter 7**

## Extraction of Renewable Natural Pigments in Indonesian Cultures for Coloring Batik Fabrics

*Saefudin and Efrida Basri*

#### **Abstract**

Nowadays, trends of Indonesian batik have increased to replace synthetic pigments with renewable natural pigments (chlorophyll, tannin, and others) due to strong consumer demands for more natural products. Production of renewable natural pigments from plant biomass (e.g., wood wastes) has been developed and produced in liquid, paste, and dry forms. Natural pigment production not only increases their selling-price capacity but should also exhibit biological activities that are environmentally friendly and beneficial for health. Abundant sources of chlorophyll/ tannin in wood wastes (including mangrove origins) from forestry industries could potentially substitute for synthetic pigments, which sound environmentally harmful. Wood waste utilization could encourage and create economic opportunities in forest villages, centers of mangrove wood industries, and small/medium-scale batik/weaving enterprises. Natural colors of chlorophyll/tannin seem preferred by coastal and inland motif batik crafters. Accordingly, exploring various natural sources of tannin is essential for fabric/batik coloring, safe for food/beverages, and not endangering health.

**Keywords:** utilization, natural pigments, batik fabrics, environmentally friendly, wood wastes

#### **1. Introduction**

Renewable natural pigments obtained from living creatures/biomass (e.g., plants), such as chlorophyll, tannin, and others, have attracted remarkable attention from the related industry and trade world for coloring purposes (especially textile/batik fabrics, food/beverage packaging, paper, etc). The demand for chlorophyll/tannin pigments tends to increase continuously, as they are renewable, beneficial for human health; seem more environmentally friendly; and also due to the people's growing awareness of allegedly the possible dangerous compounds in synthetic pigments. Chlorophyll is a complex organic compound containing C, N, H, O, and Mg elements, present in almost any plants (particularly leaves) that functions as a biocatalyst in photosynthesis, whereby CO2 and water (H2O) are combined together to produce very simple food (sugar/hexoses). Further, the chlorophyll could exhibit bluish-green colors. Such color appearances could be due to the presence of double bonds (e.g., –C=C– in conjugation or not, –C=O) and unsaturated aromatic rings with N elements [1].

Meanwhile, tannin belongs to polyphenols (with C, H, and O elements), regarded as one of the indirect products of plant photosynthesis, which is formed afterward through assimilation and other complex metabolism processes. Further, tannin is commonly found in particular plant tissues, for example, twigs, leaves, barks, woods, flowers, fruits, and roots. Further, the physiological functions of the tannin presence in the plants are still unknown, possibly protecting the plant tissues against microorganism attacks [2]. The tannin could reveal brownish-yellow to reddish-brown colors, which could be attributed to phenolic, benzene, C=O, and conjugated –C=C– bond groups. Further, such presences in tannin as well as in chlorophyll enable both pigments to absorb light at a particular visible (colored) wavelength range (4000–8000 A), thereby exhibiting various and specific colors [1, 3]. Accordingly, chlorophyll and tannin could play essential roles in the coloring culture of weaving and crafting textile materials (especially the batik fabrics) and others (paper, food/beverage packaging, etc.) in Indonesia.

Batik makers, weavers, and eco-printers can look for chlorophyll and tannins around their homes. Color variations are different for each type and part of the plant, but the performance can be adjusted according to the customer's order. The presence of natural pigments is often together with other pigments such as flavonoids, anthocyanins, and carotenoids [4].

Chemically chlorophyll contains polar groups (e.g., CHO, Mg+2 ions, and COO); however, its large nonpolar portions are insoluble in water. Further, plants commonly have two types of chlorophyll (i.e., a and b). Chlorophyll b is more soluble (in water and other polar solvents) than a because the former contains aldehyde (CHO) groups, while the latter is not so. Meanwhile, the tannin is only partially water-soluble, owing to the presence of hydrolyzable (more hydrophilic) structures and condensed/ nonhydrolyzable (less hydrophilic) structures in particular proportions. However, chlorophyll and tannin are soluble in alcohols, ethers, and hot water (moreover, with the addition of detergent or alkali).

Regarding other plant pigments (e.g., flavonoid, anthocyanin, and carotenoid), they mostly could be, among others, polyphenols (with highly condensed structures), and the compounds with the presence of double bonds (–C=C– and –C=O) and phenolic groups, as well as macromolecules (with sizable nonpolar portions, despite containing polar groups). Consequently, most of them are insoluble or only partially soluble either in water. However, as occurred to chlorophyll/tannin, those other pigments could be expectedly solubilized in hot water. This is because the hot water could induce more kinetic molecular movement and hence cause more intensive melting of pigments, thereby enhancing their water solubility.

The solubility properties and other characteristics of chlorophyll/tannin as well as other pigments are essential for coloring purposes (e.g., textile/batik fabrics, paper products, and food/beverage packaging). This is because the coloring works, beginning from the pigment extraction (from the plants), the coloring processes, until the finalization, usually employ aqueous liquid media. Further, the presence of double bonds, carbonyl (–C=O), and phenolic groups in those other pigments, as also the case in chlorophyll/tannin, renders them to also absorb visible light at a particular wavelength and hence to appear colored [1, 4]. Further, similar to tannin, those other pigments (e.g., flavonoids, saponine, and anthocyanin) could possibly be formed as indirect products of photosynthesis through further assimilation/other complex metabolisms. The function of those pigments for the plant themselves might be,

#### *Extraction of Renewable Natural Pigments in Indonesian Cultures for Coloring Batik Fabrics DOI: http://dx.doi.org/10.5772/intechopen.112448*

among others enhancing the plant-tissue durability, releasing specific smells (for the arrival of wanted animals/insects), and attracting the insects to come (especially those in the flowers with their attractive colors) to enhance the plant/flower pollinations. Pollination occurs mostly in the flowers, which refers to the transfer of pollen grains to the surface of the stigma, aiming mainly to develop future seeds. Those seeds would further develop into young plants and ultimately mature plants. As the plants grow mature/older, the reserve food (resulting from photosynthesis) stored mostly in the plant parenchyma tissues might exceed the needs for plant assimilation and other regular metabolism activities. This situation renders the excess food in the parenchymas to be transformed (under no or little oxygen) into the so-called plant extractives, including also the synthesized natural pigments [2].

Communities in forest villages can produce chlorophyll and tannins from plants such as betel nuts (*Areca catechu*), jackfruits (*Artocarpus heterophylla*), bananas (*Musa paradiciaca*), mangroves (*Rhizophora mucronata*), pine (*Pinus mercusii*), and *Uncaria gambir*. Those in the highlands can get it from the tea plant (*Camelia sinensis*), while those in the lowlands can get it from *Rhizophora mucronata, Xylocarpus granatum, Avicenia marina, and Terminalia catappa*. Especially in the coastal area are the species of *Rhizopora apiculata, R. macrophylla, Pterospermum ferroginum, Ceriops candolleana*, etc.

The source of tannin and chlorophyll comes from tree waste which was produced as much as logging forests, amounting to around 928,934.81 m3 /year [5]. Another source of log production from plantation forests reaches 40,945,378.90 m3 per year (Ministry of Environment and Forestry of the Republic of Indonesia/Keputusan Menteri Lingkungan Hidup dan Kehutanan, 2018). The source of tannin, chlorophyll, and other pigments mainly comes from the bark of the mangium wood, which can reach 216.49 tons per day or around 9% of logs by weight [6].

Further, wood wastes in the forms of wood slabs, sawdust, barks, etc., are supposedly rich in various natural pigments, such as tannin, flavonoid, anthocyanin, chlorophyll, and carotenoid. So far, those alleged pigment-rich wood wastes are still not utilized optimally, instead just abandoned or discarded. Consequently, the waste utilization into renewable natural pigments could be a better solution/recommendation and hence deserve consideration.

In obtaining/producing renewable natural pigments, after their extraction from plant biomass portions (e.g., barks, woods, roots, leaves, and flowers/fruits) or wood wastes, they could be produced in three forms/shapes (i.e., dry-solid extracts, concentrated pastes, or liquid shapes). Whatever their shapes, attempts should be thoroughly performed, thereby enabling the pigment to meet the standards of quality, security, and uses or benefit principles. Further, the pigment's raw materials should be consistently available and easily assessed. The pigment products should bear their specification, especially the color standard, color consistency, information about the ingredients as well as other matters added in the pigment formulation, and essentially their shapes. Accordingly, pigment testing deserves to be carried out.

Regarding the shapes/forms of pigment products, however, for the sake of efficiency in long-termed use, especially the trade, it would be better to process/produce the chlorophyll/tannin as dry-solid pigments. Dry natural pigments could last longer or be more durable during storage, rapidly packaged, and easy in case of coloring work/activities (just dissolve them in the water). The essential coloring activities (chiefly for the fabrics/batik) with the pigments, for example, chlorophyll/tannin, consist mainly of mordanting, coloring, and fixating. Mordanting aims to eliminate fat, oil, and other foreign matters, which could adhere or stick to the fabric fibers, thereby otherwise interfering with the fiber-pigment bonds. Also, such elimination

could greatly facilitate pigment infiltration/penetration into the pores, voids, or other microscopic fiber structures (micropores/microvoids) during the fabric coloring. Mordanting is usually performed before the fabric coloring, using typol, tawas/alum, tannic acid, and acetic acid/vinegar (all in aqueous solution). Fabric coloring is carried out by immersing the mordanted fabrics in an aqueous coloring-pigment solution (e.g., chlorophyll/tannin). Afterward, the fixation could be conducted, whereby the pigment-colored fabrics are submerged (immersed) in the fixative solution. The fixation intends to strengthen the color fixing at the pigmented fabrics [7]. Further, to examine the pigment performance (e.g., chlorophyll/tannin) at the pigment-colored fabrics, then their color-leaching/fading resistance should be tested against washing, rubbing, and exposure to sunlight, which refers to the recognized standards (e.g., ISO/international standard organization and SNI/Indonesia's national standards).

Accordingly, research into diverse natural sources of pigments (e.g., tannin and chlorophyll) that are safe for food/drink as well as the human body is crucial for fabric and batik coloring. Relevantly, this chapter presents, elaborates, and assesses the results of exploration, extraction, and application of natural pigments (e.g., chlorophyll/tannin and others) extracted from various plant species/portions/wood wastes and motivation of batik, weaving, and eco-print crafters.

#### **2. Current main situations and elaborated approaches**

Natural renewable pigments now become one of the prominent complements in substituting for synthetic pigments for coloring purposes, such that the coloring results become the community's partial lifestyle. Natural pigments from wherever they come are preferred a lot by consumers (users) because of their superiority, which is inherently exclusive; renewable; environmentally friendly; and appear very typical, distinct, and ethnic, thereby affording high selling values. Natural pigments could become potential superior products regionally and globally. Consequently to achieve such, it is necessary to conduct research in order to get even better results from pigment extracts as well as pigment-colored products (e.g., especially, fabrics/batik, paper, and food/beverage).

The research which have been performed partially revealed that many ethnic groups throughout the world have utilized particular plant species/portions (and vegetation/wood wastes) in natural renewable pigments, including in Indonesia. For such, Indonesia's ethnic groups have utilized specific plant biomass, among others: ketapang roots (*Terminalia catappa* L), teak leaves (*Tectona grandis* Lf.), barks of *Rhizophora* sp., annato seeds (*Bixa orellana* L.), tea leaves (*Camelia sinensis* L), and betels nuts (*Areca catechu* L), as they feel concerned with securing the environments. Meanwhile, several countries, such as India, Singapore, Malaysia, and China, have conducted intensive research and begun performing the trade transaction dealing with renewable natural pigments because they mostly get attracted to environmentally friendly and renewable products (e.g., plant pigments). Relevant to such favorable achievements, this still needs to be enhanced toward substituting natural pigments for synthetic pigments, as elaborated in the following related essential topics, respectively.

#### **3. Production of renewable natural pigment**

In the production aspects, it is essentially related to how renewable natural pigments are obtained. Renewable natural pigments (e.g., chlorophyll, tannin, flavonoid,

#### *Extraction of Renewable Natural Pigments in Indonesian Cultures for Coloring Batik Fabrics DOI: http://dx.doi.org/10.5772/intechopen.112448*

saponine, carotenoid, and anthocyanin) could be obtained commonly through the extraction process from almost any parts of the plant tissues/biomass allegedly rich in such pigments, for example, tree barks, roots, leaves, fruits, seeds, exudates/saps, and fruit skins [8, 9]. The terms extraction and production are often used interchangeably. In the extraction/production of natural pigments (e.g., chlorophyll/tannin), currently, the particular plant portions considered worth for such are the wood wastes generated abundantly from the tree cutting/felling in the forests and from the wood sawing in the sawmilling industries. So far, those wastes are still underutilized, instead as described before, just abandoned in the forests, merely left accumulated on wood-industry sites, or just burnt uncontrollably, thereby potentially polluting the environment and hence arousing the idea for waste utilization for the production of renewable natural pigments.

Further, those would-be natural pigments (e.g., tannin, flavonoids, saponine, anthocyanin, chlorophyll, and carotenoid), besides already naturally present in the plant biomass or wood wastes, could also be accidentally formed during their heating or storage. This is because during such, chemical and biological changes could occur, such as degradation, depolymerization, respiration, fermentation, and other physiological processes, thereby chemically converting the more complex and higher molecular weight (MW) plant components to simpler and lower MW pigments [10]. Accordingly, those phenomena could also affect or contribute to the pigment productions.

The production of chlorophyll/tannin and other pigments should be preceded by the exploration of the allegedly pigment-rich plants (or wood wastes) which grow or exist in the surrounding community. The simplest way of performing the exploration is through the identification process using specific chemicals. For example, the extracts from the fruits, leaves, and stem barks of mangrove (*S. alba*) trees, after identification using phytochemical tests with FeCl3, turned their colors to blackish green or strong blue, which indicated the tannin presence. Still relevant, the barks of *S. alba* stems, after being dried, maintained their colors similar to those before being dried but then exhibited dry and hard textures [11, 12]. Unfortunately, still, not many of the extracted pigments (e.g., tannin) are ready for use (usable), continuous, and standardized, and nor is their availability in the markets.

To cope with such pigment drawbacks (e.g., its ready use, incontinuity, standardization, and market availability), among the ways, it is necessary to enhance the pigment yields that result from the extraction. This is because the pigment yields are affected by the species origins of the plant biomass (e.g., wood and wood wastes) and the kind of cooking/extracting chemicals. For example, this occurred, when woods of various mangrove species were extracted in hot water (near 100°C), other polar solvents (alcohol/acetone), or aqueous alkali solution (0.5–1.0% Na2CO3). It turned out that the extraction using 0.5–1.0% Na2CO3 (alkali) afforded higher chlorophyll and tannin yield, viscosity, and solid content than using water. As such, the highest solid content (7.866%) in the tannin was obtained from the woods of *A. officinalis*, using a 1.0% Na2CO3 solution. Meanwhile, correspondingly the highest tannin yield (31.003%) was found at *R. apiculata* woods [13]. These results indicated again chlorophyll and tannin pigments contained less water-soluble portions, more sizable less-polar structures, and greater molecular weight compounds, which could only be effectively solubilized in hot water or aqueous alkali solution. The data/information about the pigment yields (e.g., tannin) is essential because it is closely related to the pigment potency.

The potency of natural pigments (e.g., chlorophyll/tannin) in particular plants growing at particular area could be predicted from the density of plant stands there (e.g., number of stands per ha; or average biomass weight of the overall stands per ha). As such, as an example, research has been conducted on the tannin pigment in the plant stands that grew on rehabilitated lands at Rembang (Central Java). First of all, the tannin content/yield in the plant biomass was determined (w/w) through a laboratory experiment. Further, by measuring the stand density (i.e., number of stands per ha or stand-biomass weight per ha), then the tannin potency could be approximated/predicted, that is, roughly 105.93 kg/ha/year. Still related, after the third year of stand growth, the tannin potency reached 146.36 kg/ha/year [14]. Consequently, knowing the potency of natural pigments (e.g., tannin) is essential because, further, the tannin contains specific compounds that could potentially exhibit exclusive tastes/smells.

Among such exclusive tastes/smells is that the tannin could render its compounds to taste bitter and astringent, which could be toxic, thereby besides protecting the plant tissues against organisms (as presumed before), also decreasing the plant digestibility and hence deterring the scavenging herbivores and other pests to consume them. Such bitter/astringent tastes are often found in young fruits. Changes in tannin compounds could exert a significant role in fruit ripening. Accordingly, it is strongly alleged physiologically, perhaps that tannin serves as plant-growth regulator. Further, tannin content in the plant biomass (e.g., slabs, twigs, and woods), inadvertently dissolved and leached by the rainwater (with various kinds of humus), renders the colors of the flooded/stagnant water in the swamps or peat marshes to appear blackish brown like those of tea water, popularly known as black water. The tannin also causes bitter/astringent tastes and imparts dark brown colors to the tea water.

Relevantly, imparting the colors by natural pigments (e.g., tannin/chlorophyll and others) at the fabrics (e.g., batik) should proceed in stages. This is because most of the pigments are, as described before, insoluble or only partially so in the water. Therefore with stages, it could render the pigments more water-soluble. Moreover, in the coloring works, as has been described, it employs mostly aqueous liquid media. The first stage in the fabric coloring (after the fabric cleaning and mordanting) starts with immersing the cleaned/mordanted fabrics in the aqueous pigment solution, followed by the fixation stage. In this stage, the pigmented fabrics are fixated with specific inorganic salts such as kapur/lime (CaCO3), tawas/alum (Al2[SO4]3), and tunjung/ferrous sulfate (FeSO4). Fixing agents direct chlorophyll and tannins into light or dark colors and are more resistant to washing, sun exposure, and ironing. The common procedures in coloring the fabrics are as follows: 50 grams of any chemical fixatives (lime, alum, or tunjung) are dissolved in 1 liter of water. The resulting fixative solution is then allowed to settle/precipitate for 24 hours until two layers are formed (upper and lower). The upper/supernatant layer is further used as the fixative solution. The fixation is performed by immersing the pigmented fabrics in the fixative solution for approximately 15 minutes. Afterward, the fabrics are removed, allowed to dry, and ready for color detection. As such, the dried fabrics are immersed again in the specific color-detecting aqueous liquid. The color detection/identification test procedures refer to the RHS (royal horticultural society) color charts. Afterward, the color-identified fabrics are properly documented by inscribing specific labels, such as kinds of pigments, kinds of fixatives, appearances of the imparted colors, 2022.

As of this occasion, despite strenuous attempts in imparting various pigment colors/motifs at the materials (e.g., fabrics) with the fixative aids, unfortunately, those available varying sources of renewable natural pigments which are abundant in Indonesia and more environmentally friendly have not yet provided maximal solution, especially as a substitute for synthetic pigments. Further, in spite of strong

#### *Extraction of Renewable Natural Pigments in Indonesian Cultures for Coloring Batik Fabrics DOI: http://dx.doi.org/10.5772/intechopen.112448*

suggestions and appeals for Indonesia's batik crafters/weavers to use natural pigments, based on such positive allegations, pitifully, in fact, the majority of domestic batik crafters/weavers still use synthetic pigments. Accordingly, systematic and thorough attempts deserve carried out to reduce the dependence on imported synthetic pigments, among others, by establishing renewable natural pigment industries that utilize chlorophyll (green), anthocyanin (red-violet), xanthophyll (yellow), tannin (brown, reddish brown), and carotene (orange), which are abundantly available or existed in the plants.

Further, in order that Indonesia's abundant natural pigments could be obtained and produced into usable or ready-for-use pigments in feasible operation (technically as well as economically), then it is necessary to explore the genetic and biochemical aspects of the sources of natural pigments (especially the plants), which can bring essential economy values. Accordingly, a breakthrough is urgently performed with respect to the effectiveness, efficiency, and rules to regulate the utilization of those natural biopigment sources. In addition, the involvement of the batik-crafting community and traditional fabric weavers should get a priority. The aims are that the pigment-industry continuation and environment sustainability could be realized.

#### **4. Variations in colors and motifs at batik fabrics**

Batik fabrics are one of the pigment-colored and motif-decorated products. As such, the traditions performed by the batik crafters and fabric weavers to impart the fabric colors and motifs using natural pigments are by cooking (boiling in the water) the plant biomass (e.g., barks, leaves, wood slabs, sawdust, and flowers), until the colors appear in the boiled water (as pigment solution). When the colors become thickened or viscous enough, the fabrics are immersed in the pigment solution to undergo the coloring, as systematically shown in **Figure 1**. These local coloring procedures are conducted a lot by the forest village's community, which further become a guidance/reference, adopted by consecutively the regional authority, Indonesia's state-owned forest enterprise (Perhutani) in Central and East Java; and wood enterprises in Kalimantan, Sumatra, Nusa Tenggara, and Bali.

The colors of natural pigments (e.g., chlorophyll, tannin, and others) become essential to seek and arrange the motifs at the pigmented fabrics, which should be compatible with the desires of the batik/fabric crafters; and also with the tastes of the ordering users/consumers. If we want motifs other than chlorophyll and tanning

#### **Figure 1.**

*Illustration of coloring the fabrics, using the pigments extracted from consecutively leaves dan barks of avocado (Persea americana Mill), jambu seeds (Psidium guajava L.), mahoni (Swietenia mahagoni; Jacq), manggous (Mangifera indica L.), and coffee (Coffea arabica L), (using the fixatives: Alum, lime, and ferrous sulphate).*

pigments, we can use waste from plant species or other sources of wood waste. Plant biomass and wood wastes produce a variety of colors. The leaves were dominated by chlorophyll can create green. The presence of flavonoids can create yellow. If tannins dominate the leaves and bark, they give brown, brownish-yellow, or reddish-brown [15, 16].

The varying colors imparted by those several kinds of natural pigments should be confirmed more convincingly with regard to the pigment source origins and their positive presence. As such, source origins that cover tree roots, barks, stems, and leaves from mangrove (*Avicennia* sp.) plants are prevalently very rich in chlorophyll, tannin, phenolic, and flavonoid compounds, where their presence is indicated, after the detection tests, by a strongly positive category [++] to a very strongly positive category [+++] [17, 18]. This implies that if a part of those mangrove plants is dissolved in hot water, then the resulting colors could appear very strong, that is, blackish brown, resembling the tea-water colors. The presence of particular pigments (e.g., chlorophyll, tannin, and flavonoid) with high content in mangrove plant parts allows the extracts to be used as a natural pigment for fabric coloring.

Besides mangroves, other plant species (e.g., old teak trees) could also exhibit specific colors, which were due to the presence of extractives, predominantly 2-methyl anthraquinone (as polyphenols), where its content was quite great (reaching 13.54%). In teak wood, the quinones together with wood lignin allegedly enhance wood durability and also imparts attractive appearance/color to wood surface as well as seemingly renders easier the wood working (e.g., sanding, planing, and polishing) into favorable teakwood products [19]. However, the teak extractives contained tannin only a little [20, 21]. Further, the tannin content in the extracts from *Acacia mangium* barks could reach 15–20% [22]. The batik fabrics which were colored with *Acacia amngium*'s tannin, were resistant against the high-intensity sunlight and also against the pH changes [15].

Likewise, coloring the cotton fabrics with the extracts from teak (*Tectona grandis*) leaves, previously mordanted with alum and acetic acid (vinegar) separately, brought the fabrics with different color motifs, expressed in brightness values, that is, 55.56% (less bright) and 77.22% (brighter), respectively [23]. On other occasions, chlorophyll and tannin pigments were extracted from the barks of *Acacia mangium*, three mangrove species (*Terminalia catappa*, *Rhizophora apiculate*, and *R. mucronata*), and *Tectona grandis*, and then used concurrently for fabric coloring, followed with the fixation (using alum, lime, and ferrous sulfate, separately). It turned out that these procedures that incorporated chlorophyll and tannin could be used either as basic color motifs or as supplementing the colors of the resulting batik fabrics [24–26]

In imparting the fabric colors, it turned out that tannin is the most dominantly used in batik fabrics. Also, tannin is used in the coloring aspects and the color variations, where the two latter cases are the most important in designing and developing the batik colors and motifs, especially the inland motifs [27]. On other occasions, research results revealed that different colors in the colored fabrics could occur using particular pigments due to the use of different fixatives. This case was commensurate with the previous statement that the appearance of different colors in the pigmented fabrics could be due to different fixatives. The fixative use actually aimed to strengthen the bonds of fabric-fibers-to-pigments.

Further, research results in using a fixative solution, after the batik-fabric coloring, revealed that the fabric colors would not easily fade or leach out and become more resistant to rubbing/ironing [25]. The fixatives used in this research consisted of tawas/alum, kapur/lime, and tunjung/ferrous sulfate. Those three fixatives were used

*Extraction of Renewable Natural Pigments in Indonesian Cultures for Coloring Batik Fabrics DOI: http://dx.doi.org/10.5772/intechopen.112448*

because they had been utilized a lot; moreover, the fixative prices were affordable, inexpensive, and easily obtained in the markets. Meanwhile, the natural pigments used for such were the chlorophyll and tannin extracts from *Acacia mangium* and two mangrove species (*Terminalia catappa* and *Rhizophora apiculate*).

Relevantly, tests have been conducted on 73 plant species, which supposedly could serve as natural pigment sources. It turned out 26 species were identified containing natural pigments (allegedly tannins) that imparted yellowish brown, reddish brown, or strong brown colors for basic colors of batik fabrics [1, 13, 14, 28]. Those tannins originated from the particular parts of those 26 species, such as leaves, barks, flowers, and twigs (**Table 1**).

The natural pigments (i.e., tannin) extracted from the root parts of those particular plant species (**Table 1**) for fabric coloring, then treated with three different fixatives (tunjung, alum, and lime) brought the fabrics with three different colors. As such, after being fixated on tunjung, the fabric colors appeared dark, in average black to gray colors. Meanwhile, correspondingly after being fixated with alum, the fabric colors became brighter than the original pigmented-fabric colors (without fixative treatment). Likewise after lime fixation, the pigmented-fabrics generated blackish colors [27].

The dark/blackish colors (**Table 1**) appearing at the pigmented fabrics (after tunjung fixation)) indicated that a reaction occurred between the fixative and tannin pigments (plant extractives) that produced complex salts. The complex



*Sources: Jamal [29]; Basri et al. [30]; Saefudin et al. [1]; Susanto (1980); Prayitno et al. [28], Poedjirahajoe et al. [14]; Suhendry et al. [13]*

#### **Table 1.**

*Parts of the particular plant species, regarded as sources of tannin's natural pigments for coloring the batik fabrics and other woven fabrics.*

salts, being large-sized compounds, could additionally provide a bridging (intermediary) between the pigment and the fabric fibers, thereby further creating strong fiber-pigment bonds (fixing) and, accordingly, imparting strong color adherence at the pigmented fabrics. Meanwhile, the different colors (not dark/ blackish colors, but brighter colors) of the pigmented fabrics after the lime as well as alum fixations, supposedly did not produce complex salts, thereby rendering the fibers-pigment bonds not too strong. However, the lime as well as alum fixatives, could afford ionic bonding with either fabric fibers or pigments, thereby still imparting the fabric-pigment bonds, although not so strong as the bonds exerted by the complex salts [31].

Further, it appeared that the use of different fixatives (i.e., alum, lime, and tunjung/ferrous sulfate) separately at particular fabrics brought the pigmented fabrics with the colors toward the brightest, then less brightness and ultimately the darkest, respectively (**Table 1**). In another case, different kinds of fabrics consecutively with cellulose-based fibers (i.e., mori, cotton, and cotton yarns) and with protein-based fibers (wool), but colored with the same natural pigment and then treated with the same fixatives, brought the pigmented-fabrics with different colors. This difference occurred because mori/cotton fabrics and cotton yarns comprise mostly cellulose polymers, thereby rendering the fabric/yarn fibers to easily adsorb the pigments and fixatives. However, the wool (as the animal/ sheep hair fibers) comprises mostly protein polymers, which could have different characteristics in interacting/adsorption with the pigments/fixatives. Accordingly, cellulose-based fabrics/yarns are widely used as pigment-colored media by batik crafters/weavers [32].

*Extraction of Renewable Natural Pigments in Indonesian Cultures for Coloring Batik Fabrics DOI: http://dx.doi.org/10.5772/intechopen.112448*

#### **5. Opportunities and challenges**

Several batik and eco-print artisans in Central Java and Yogyakarta have started cultivating, producing and selling natural chlorophyll and tannin pigments commercially. Available dry extracts include: Indigofera tinctoria, Camelia chinensis, Intsia bijuga. Other plants that are also rich in natural pigments (tannin and chlorophyll) are soga tingi (*Ceriops candolleana*), tegeran (*Cudraina javanensis*) and noni (*Morinda citrifolia*), jambal (*Pelthophorum ferruginum*), kesumba (*Bixa orelana*), and guava (*Psidium guajava*) (Susanto, 1980; [1]). Although those extracted natural pigments have been known a lot by fabric crafters/weavers; unfortunately, only a few have utilized them. The main obstacles for such few users are the limitation of pigment varieties that are ready for use (usable), lack of the crafter/weavers' knowledge about pigments' raw materials, and insufficiency in ready-for-use technology. Besides, the availability of natural pigments is affected much by the seasons, environments, and collecting workers, thereby causing a not-continuous pigment supply to the crafters/ weavers. Despite their drawbacks, the natural pigments could prospectively afford high market potencies as Indonesia's superior commodity to enter into the global markets due to their attractiveness with unique, ethical, and exclusive characteristics. Those pigment drawbacks and prospects could become notable challenges for the batik crafters and related researchers.

The challenge for the researchers is that it is necessary to explore the natural pigment sources ready for use. The creativity of fabric crafters/weavers to seek fabric motifs that should conform to the user's tastes and desires could become their own problems. These classical problems also occur to the producers of batik and other woven fabrics scattered in many Indonesia's provinces. The batik crafters should obtain pigment-rich plants, which are fast growth, shortages, enormous available, and easily obtained in their home vicinities. To obtain the plant species rich in natural pigments with such favorable plant-growth characteristics necessitates thorough development for the present or future.

The development of natural pigments in the future should focus on research in the design motifs to anticipate local and global markets. Kinds of laboratory researches that use experiment methods should try hard to invent efficacious/unique formulae to develop such pigments in the crafter-community vicinity. Commonly, natural pigments are obtained/extracted by cooking the plant biomass/wood wastes in hot water (near 100°C). Further, to make the colors of the pigmented stuffs (e.g., fabrics) last long or as color protection against fading/leaching, it should use fixatives (alum, lime, tunjung, etc.). Plant biomass and wood wastes are allegedly rich in natural pigments (e.g., tannin/chlorophyll, anthocyanin, xanthophyll, and quinone). Evaluation results alleged that tannin's natural pigment was the most prospective for coloring purposes (e.g., fabrics).

Despite the reported success/prospects of using natural pigments (especially tannin), the data and information about the pigment potency (e.g., from forests/wood wastes) are still lacking or inadequate and have not yet been assessed. Furthermore, using tannin as a natural pigment from forests/wood wastes could become an added value for the tannin's plant sources and essential information for the community who love Indonesia's batik fabrics and other woven materials colored with the tannin.

The plant tannin, besides already being intended as fabric coloring, has long been widely used as a tanning agent to convert animal bones and skins into leathers (under high temperatures), which are stable, strong, and elastic/flexible. The tannin, as high molecular weight polyphenols, contains numerous OH and other polar groups (e.g., CO and COO) and is, therefore, able to impart multiple strong hydrogen bonds not only with fabric fibers but also with hydroxyl/other polar groups at the bone/skin proteins. Tannin presence in the plant leaves is concurrent with the chlorophyll presence, thereby allegedly causing the changes in leaf colors. The leaves of whatever plant species could also undergo the color changes sooner or later because the leaves perform a double function. As such, the leaf chlorophyll absorbs the red and blue colored light from the sunlight while concurrently conducting photosynthesis. Then, gradually reducing the capacity of chlorophyll to absorb sunlight, reducing the intensity of the green color of the leaves, until they become old, dry out and finally fall. Meanwhile, the carotene that imparts red colors to the leaves is more stable than the chlorophyll, thereby enabling the leaf 's reddish-orange colors to stay longer, although the leaf 's green colors disappear [33]. Other factors that could affect the pigment stability (e.g., their colors) of cations, oxygen, pH, sulfur dioxide (SO2), proteins, and enzymes. All factors should then be considered.

Considering such, it is essential to know that the attractive varying colors imparted by plant's natural pigments (e.g., chlorophyll, anthocyanin, flavonoid, carotenoid, etc.) have attracted enthusiastic attention among many genetic experts. This is because such pigments might have meaningful relations with the various morphology aspects in plant species (but in the same genus), which are still close relatives. That information is essential for the taxonomist to determine and combine the plant's evolution lines into one group. Regarding flavonoid pigments, their form in the plants could be induced by the blue-colored light radiation (e.g., from the sunlight) to increase the plant resistance against ultraviolet radiation.

#### **6. Conclusion and suggestions**

Indonesia is endowed with abundant availability of renewable and environmentally friendly natural pigments, with respect to their huge potencies, numerous pigment kinds (e.g., tannin, chlorophyll, flavonoids, saponine, anthocyanin, and carotenoid), and their varying colors (e.g., green, red-violet, yellow, brown, reddish brown, and orange). Expectedly those could serve as substitutes for synthetic pigments, which are mostly environmentally unfriendly.

Those natural pigments could be obtained from almost any part of the plant biomass (e.g., tree barks, roots, leaves, fruits, seeds, exudates, and fruit skins) and wood wastes through commonly the extraction process, which is further beneficial for imparting colors and motifs to, for example, specially woven fabrics/batik, paper products, and food/beverage packaging, commonly performed by the synthetic pigments.

Unfortunately, the majority of Indonesia's users/consumers still use synthetic (inorganic) pigments, which are mostly imported. One way to overcome such is by establishing industries to produce natural pigments, which could last longer and be ready for use. Accordingly, the natural pigments (as the pigment-colored products, e.g., batik fabrics) should incorporate the fixation process (to strengthen the fabric fiber-pigment bonds), using, for example, alum, lime, and tunjung/ferrous sulfate fixatives); and undergo the quality tests (among others the color resistance against the leaching/fading, due to mainly the detergent washing, rubbing/ironing, and sunlight exposure).

#### *Extraction of Renewable Natural Pigments in Indonesian Cultures for Coloring Batik Fabrics DOI: http://dx.doi.org/10.5772/intechopen.112448*

The success in utilizing the plant biomass and wood wastes into pigments, which are not only confined to chlorophyll and tannin (currently the dominant natural pigment for especially batik coloring and tanning agents), but extended to others (flavonoids, carotenoid, anthocyanin, etc.), could be beneficial and safe for health, thereby expectedly encouraging and creating economic opportunities as well as welfare, for example, pigment industries in small/medium and large scale levels, users/ consumers, and enthusiastic community.

#### **Author details**

Saefudin1,2\* and Efrida Basri1,2

1 National Research and Innovation Agency, Research Center for Biomass and Bioproducts, Bogor, Republic of Indonesia

2 Cibinong Science Centre, Bogor (West Java), Indonesia

\*Address all correspondence to: saefudinkahfi@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Muhammad Sarwar Khan*

Plants contain green organelles called chloroplasts, which are inherited from the mother plant in most cultivated species. These organelles have a double-stranded, circular genome with inverted repeats that duplicate genes, increasing their expression levels. Chloroplasts are ideal manufacturing units for pharmaceuticals and vaccines due to their polyploidy at both the organelle and genome levels. A mature leaf mesophyll cell contains approximately 100 chloroplasts, each carrying 100 genome copy numbers, making 10,000 genome copies per cell. The availability of chaperon proteins in chloroplasts allows expressed proteins to accumulate in biologically active form with chemical structures. The chloroplast genome has been manipulated to investigate gene functions, biology, and transgene expression for diverse applications. This book, *Chloroplast Structure and Function*, provides a comprehensive overview of chloroplast biology, chloroplast genome and biotechnological applications, and chloroplast applications in plant growth and stress tolerance.

### *Tomasz Brzozowski, Physiology Series Editor*

Published in London, UK © 2024 IntechOpen © 123dartist / iStock

Chloroplast Structure and Function

IntechOpen Series

Physiology, Volume 26

Chloroplast Structure

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*Edited by Muhammad Sarwar Khan*