The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase Plant Drought Tolerance

*Ilya Zlobin, Marina Efimova, Natalia Permykova, Irina Sokolova, Vladimir Kuznetsov and Elena Deineko*

## **Abstract**

Due to climate aridization, the need to increase the resilience of plant productivity lo water stress becomes urgent. Abscisic acid and cytokinins have opposing biological roles during water deficit and post-drought recovery, but both these regulators can be utilized to maintain plant productivity under water stress. Downregulation of abscisic acid biosynthesis and signaling can aid in the maintenance of photosynthesis, growth, and productivity in plants, although increasing the susceptibility to severe stress. Cytokinin upregulation can maintain photosynthesis and productivity during water stress and aid recovery processes, whereas downregulation can lead to increased root growth, thus improving plant water balance, nutrient absorption, and hence productivity in water-limited conditions. The use of modern genome editing methods makes it possible to specifically modify genes involved in the implementation of complex traits in plants, such as resistance to stress factors. This review will examine the main areas of work on genome editing of gene families involved in plant responses to water deficiency using CRISPR/Cas technologies. Our current work on editing the ABF gene family, encoding transcription factors for ABA (AREB1/ABF2, AREB2/ABF4, and ABF3), as well as the CKX gene family (CKX1 and CKX4), encoding cytokinin oxidase/dehydrogenases, will be presented.

**Keywords:** stress factors, plant drought resistance, molecular methods, plant genome modification, genome editing, abscisic acid, cytokinin signaling

## **1. Introduction**

Drought is the most important abiotic factor challenging plant survival, performance, and productivity on the planet. The rapidly increasing risk of coupled negative effects of water deficit and heat stress implies that we need to adapt the physiology of major crop plants to a hotter and drier future. Generally, the adaptation of annual crop plant to water stress can be confined to two capabilities:


It is clear that these capabilities depend on different or even conflicting plant traits. Survival during water deficiency depends mainly on the ability to prevent the irreversible desiccation of plant tissues and maintain the hydraulic integrity of a plant [1]. Plants need to minimize water losses, whereas photosynthesis and growth can be drastically diminished during this period without the major threat for plant survival during drought. In contrast, recovery occurs when water is plenty again and depends on the ability to recover photosynthesis, growth, and resource allocation to productive organs. Therefore, it is likely that opposing regulatory mechanisms would be required to make plants more tolerant to water stress per se and to make them abler to recover form water stress [2]. A clear example of such opposing pair of regulatory mechanisms are abscisic acid (ABA) and cytokinins (CKs). This chapter is devoted to the effects of these regulatory molecules on plant performance during water stress and recovery, on their regulatory modes, and on the usage of genome editing technologies to change plant ABA and CK balance to increase drought tolerance and post-drought recovery.

## **2. Abscisic acid**

The response to water deficit is the major biological function of abscisic acid, and ABA can be considered as a versatile hormone that regulates plant water status in an integrated fashion. Abscisic acid increases water acquisition by affecting root growth and plant osmotic balance, affects water transport from the root surface to leaf tissues through regulation of aquaporin genes, and regulates water spending through the regulation of stomatal conductance, possibly influencing cuticular conductance. In case of stress severe enough to exert a substantial degree of dehydration in plant cells, ABA regulates the biosynthesis of stress-protective compounds such as dehydrins [3], but such intensive stress is likely of minor importance for agricultural plants [4]. The major biological functions of ABA during water stress are considered below.

#### **2.1 Abscisic acid and plant water spending**

The most-studied biological effect of ABA is stomatal closure, which enables plants to greatly diminish water losses, thus making a major contribution to the maintenance of plant water status during drought [5]. The paramount importance of ABA for stomatal regulation is clearly illustrated by ABA-deficient mutants, which are extremely sensitive to increasing vapor pressure deficit even under well-watered conditions [6]. In more ancient plant lineages, ABA biosynthesis is rather slow, likely due to a reliance on non-specific enzymes during ABA biosynthesis, and therefore, ABA accumulation can occur only if water stress is rather prolonged [7–9]. For example, in [10], ABA increment in drying leaf tissues in several coniferous species occurred only after 2-h of dehydration. In contrast, angiosperms can induce ABA biosynthesis rapidly within few tens of minutes through the activation of NCED gene expression in ABA-producing tissues [7]. ABA catabolism genes are often upregulated simultaneously with ABA biosynthesis genes, but the expression of the former is lower than

## *The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

the latter, resulting in net ABA accumulation under inductive conditions [11]. ABA accumulation in dehydrating cells occurs due to the decreasing of cell volume, rather than of turgor or water potential [12]. After water stress relief, the activity of ABA biosynthesis genes stayed elevated in guard cells, thus achieving drought memory effects favorable in case of subsequent droughts [13]. ABA controls memory processes through signaling pathway SnRK2/ABF/ABRE [14]. ABA biosynthesis in roots also occurs under water stress, but leaf is likely a major site of ABA biosynthesis [15, 16]. There are conflicting evidences whether leaf ABA biosynthesis occurs mainly in vascular buds and guard cells or in mesophyll tissues, with solid evidences in favor of mesophyll as the main site of ABA biosynthesis [7, 10, 15]. Additionally, in angiosperms, rapid ABA-induced stomatal closure occurs within seconds to minutes, thus indicating the presence of ABA-dependent non-transcriptional mechanisms in stomatal closure [17]. Abscisic acid is among the key mechanisms underlying the difference between isohydric (R-type) and anisohydric (P-type) strategies under drought stress, as isohydric plants achieve high leaf ABA levels during stress, whereas anisohydric plants respond to stress with an initial peak and subsequent decline of ABA content [5, 18, 19]. In ferns or lycophytes, the stomatal closure is independent of ABA [5], thus indicating that stomatal regulation by ABA is a relatively late evolutionary achievement.

Besides regulation of stomatal conductance, ABA probably plays a role in the regulation of residual cuticular conductance. This type of conductance, although quantitatively minor in well-watered plants, becomes the major determinant of plant survival during prolonged water deficiency, when water absorption by the plant root system reaches zero, and the ability to preserve the water already present in tissues becomes crucial [1]. Cuticle is often viewed as a rather stable structure hard to be modified, but in fact, recent assimilates can be incorporated rapidly in the cuticle [20], indicating that cuticle can be probably abler to modifications than it is thought currently. Plant minimum leaf conductance can decrease under drought stress from −4 to −70%, with a decrease of 30–40% being typical [21]. It is known that ABA can change the chemical composition of cuticle, but whether it aids in decreasing minimal conductance is unknown and requires further clarification [21].

### **2.2 Abscisic acid and regulation of water acquisition and transport**

ABA effect on root growth is biphasic, with mild ABA increase stimulating root growth through ethylene-dependent mechanisms, whereas higher ABA concentrations inhibit growth through auxin signaling pathway [22, 23]. The ABA-dependent increase in main root elongation concomitantly with the inhibition of lateral root formation aids plants in reaching deep water-containing soil horizons with minimal carbon expenditure on root growth, and the biological effects of ABA and drought on root growth are similar [24]. However, in [25], ABA increased lateral root number and length at mild water deficit, likely also suppressing primary root growth to a certain extent. Not only the biological effects of ABA but also the source of ABA in root remain somewhat controversial. Mild drought leads to local ABA accumulation in roots [26]. Earlier, root tip was thought to be the main source of ABA biosynthesis during water stress, but now, it is clear that leaf-derived ABA plays a major role in shaping root growth [27], whereas ABA biosynthesis in roots can be limited by carotenoid substrate limitation under water stress [10]. ABA effects on root growth take place via interacting network with cytokinins, ethylene, and auxin [23]. Synthesis of ABA in roots of transgenic poplar increased root growth and drought tolerance [28].

ABA is involved in the stimulation of reversible suberization of root endodermis, which is required to regulate the apoplastic movement of water [17]. The decrease in root hydraulic conductance, in turn, leads to stomatal closure and water economy, aiding in adaptation to water deficiency [29]. The decrease in ABA accumulation in root tissues during water stress can be also observed, probably due to increased ABA translocation to the above-ground plant part [30]. In addition to root growth, ABA can also positively affect osmotic adjustment, thus increasing plant water absorbing capacity [31].

Abscisic acid participates in regulation of the plant aquaporin system [32]. ABA positively affects root hydraulics [25] and is involved in jasmonate-mediated increase of root hydraulic conductivity [33]. However, not only promoting but also inhibiting effects of ABA on root hydraulic conductivity are observed [26]. Also, ABA can play a role in increasing the water-transporting ability of mycorrhizal fungi [26]. ABA is among the key regulators of expression of aquaporin genes [26]. In Zea mays, ABA increases both gene expression and protein content of different PIP aquaporins, although the results can vary between studies [34]. Also, ABA participates in the regulation of aquaporin activity through phosphorylation [32]. Although ABA is generally viewed as hormone inhibiting the above-ground growth, the positive ABA influence on plant hydraulic conductance through the regulation of aquaporin system can translate into positive effect on leaf extension growth, thus making the total ABA effect on growth less straightforward [34]. ABA-induced decrease of leaf hydraulic conductivity can participate in the regulation of stomatal closure [35].

The role of ABA in the regulation of axial water transport through xylem is less well studied, compared to cell-to-cell transport through aquaporins. ABA is wellknown to regulate the blockage of plasmodesmata in dormant cambium, making it unresponsive to activating environmental signals, and is involved in the termination of wood differentiation [36]. Exogenous ABA treatment often leads to reduced stem growth through inhibition of cambial activity, whereas occasional reports of secondary growth stimulation by ABA treatment likely stem from specific experimental approach rather than from ABA effects per se [37]. In cambial and xylem tissues of *Eucommia ulmoides* trees, the seasonal dynamics of ABA and IAA was the opposite, and ABA negatively influenced cambium reactivation by IAA [38]. ABA treatment decreases the hydraulic diameter of vessels, which negatively affects xylem hydraulic conductance [39]. Therefore, ABA likely plays a negative role in the formation of water-transporting tissues during plant secondary growth.

### **2.3 Trade-offs of ABA effects on plant performance**

The above-described integrative positive effects of ABA on plant drought tolerance are linked with several important trade-offs. Although ABA biosynthesis is down-regulated quite rapidly during post-stress period, the major increment of ABA in leaves can sustain for prolonged period after drought release [5]. This limits a plant's ability to rapidly restore gas exchange and photosynthesis and underlies, at least partially, the hysteresis between stomatal conductance and other leaf hydraulic characteristics post-drought [40], although these limitations can be also unrelated to ABA accumulation. However, it should be noted that sustained ABA accumulation may aid the recovery processes by facilitation of embolism repair by decreasing stomatal conductance and water loss, which favors embolism refilling processes [41, 42]. Also, memory effects due to ABA increase during the first stress encounter can increase the tolerance to subsequent stresses and yield [43]. The negative

### *The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

influence of ABA on leaf growth can be mainly due to the inhibition of assimilation resulting in source limitation of growth [10]. However, direct negative ABA effects on growth processes through ABF transcription factors is also well-known [44]. The ABA-induced increase of biosynthesis of osmolytes and protective compounds would distract these resources from growth and reproduction. Also, allocation of belowground growth to the deeper root system would probably lead to deterioration of mineral nutrition, since the deeper soil layers are deprived with mineral nutrients compared to upper layers [45].

Given these trade-offs, it is not surprising that constitutively increased ABA biosynthesis and ABA signaling results in depressed growth and productivity in non-stressed conditions, whereas the suppression of ABA signaling increases growth in the absence of abiotic stressors [46]. Crop plants are usually grown in more favorable conditions compared to native plants, and severe water stress is less prevalent for agricultural ecosystems compared to native ones [4]. Also, the maintenance of productivity during mild water stress is obviously more important from the economical point of view compared to the ability to survive severe water deficiency, since in the latter case, the productivity would be anyway lost. Therefore, for annual crops, the downregulation rather than upregulation of ABA biosynthesis and/or signaling can be a more promising strategy to maintain productivity during mild stress, although making plants more susceptible to severe stresses, which are devastating for plant productivity no matter whether plants survive the stress period or are desiccated. However, it is known that the logarithmic character of dependence of carbon fixation on stomatal conductance means that plants can decrease stomatal conductance to a certain extent without trade-off with CO2 uptake and assimilation activity [5]. It can be therefore proposed that mild increase in ABA biosynthesis/signaling with the associated moderate decrease of stomatal conductance can result in substantially improved water use efficiency without compromising plant productivity, making such plants more effective from the economical point of view.

## **3. Cytokinins and their effects on plant performance during drought and recovery**

Generally, biosynthesis and signaling of cytokinins are negatively affected by drought, consistent with the view on CKs as negative regulators of drought tolerance [11]. However, the regulation of CK metabolism under water-stress conditions can be rather specific, with different IPT genes demonstrating differently directed regulation under drought, whereas for CK OXIDASES/DEHYDROGENASES (CKX), more uniform upregulation is observed [47]. The directional changes in CK biosynthesis and signaling can have rather contrasting effects on plant ability to tolerate drought and to recover from its impact. Both CK signaling mutants and transgenic plants with enhanced CK signaling often demonstrate increased drought tolerance (Hai 2020). CKs generally exacerbate water loss by plants, thus making them more prominent to severe drought, whereas decreased CK levels contribute to more parsimonious water spending and better maintenance of plant water status during stress [48]. Also, CKs are positive regulators of shoot meristem activity and hence shoot growth [11, 49], and the increased above-ground growth can be maladaptive under severe water deficiency. The decreased CK accumulation can be associated with higher tolerance of photosynthetic processes during drought [48]. CKs and ABA reciprocally downregulate the biosynthesis and signaling of each other, thus exerting contrasting

effects in plants under non-stressed conditions and under drought stress [50]. ABA decreases CK contents, which increase plant sensitivity to ABA, thus making plants abler to respond to water deficiency [50]. Cytokinins repress SnRKs as major components of ABA signaling, thus inhibiting ABA effects on plant under non-stressed conditions [50].

On the other hand, when water stress is not severe and plants are not at risk of desiccation, CKs can have numerous positive effects on plant drought and postdrought performance. Both exogenous CK treatment and modulation of endogenous CK levels were reported to positively affect plant drought tolerance [48]. Increased CK biosynthesis delayed drought-induced leaf senescence in tobacco and maintained photosynthesis, thus decreasing yield loss [4]. Under water deficiency, CKs promote stomatal conductance and chlorophyll biosynthesis [47], which can be detrimental under severe stress but is advantageous for productivity under relatively mild water stress conditions. CK-mediated inhibition of stomatal closure is the conserved response in diverse plant species [5]. CKs promote plant antioxidant defense by increasing the activity of antioxidant enzymes and decreasing the activity of ROSgenerating systems such as xanthine oxidase [47]. CKs may positively affect plant osmotic adjustment under water deficiency [51]; in contrary, in [4], much lower proline accumulation in tobacco plants with increased CK biosynthesis was observed, likely due to their higher drought tolerance and lower degree of stress compared to wild-type plants. Also, CKs positively influence cambial activity and radial growth, thus increasing stem hydraulic conductance [37]. Generally, many of the positive CK effects during mild stress can be due to a delay in activation of drought response, thus decreasing stress impact [48]; CKs are known to suppress SnRK2 functioning and thus stress response [50]. It can be very promising for agricultural plants, since plant productivity and not plant survival is of the most interest for agriculture, thus making CK-induced desensitization of plants to environmental stress a promising strategy to maintain crop productivity under relatively mild stresses, typical for agricultural conditions [4].

CKs have numerous positive effects on plant post-drought recovery processes, making plants with upregulated CK content superior in recovery compared to wild-type plants. CKs in plants decreases under drought [11] while increasing prominently during the recovery period, together with compensatory growth acceleration compared to non-stressed plants [48]. Higher CK content during the post-drought recovery period can elevate auxin content in leaves [48] and also in cambium [52], which is necessary for active post-drought growth. CKs positively affect stomatal opening in post-drought period [53], helping to restore photosynthesis and to minimize cumulative negative drought impact on assimilation. Therefore, despite negative effects of CKs on tolerance to severe stress, their upregulation can be a promising way to increase the performance and productivity of crop plants.

However, downregulation of CKs can also have positive effects on crop performance under water shortage. Cytokinins are negative regulators of root meristem activity [11, 54], suppressing both primary root elongation and root branching [50, 55]. Negative CK effect on primary root growth is exerted through increase in ethylene biosynthesis [54]. As a result, CKs decrease both drought tolerance and absorption of mineral nutrients [56], whereas reduction of endogenous CKs can have prominent positive effects on root growth, increasing the number and length of lateral roots and root biomass accumulation [47]. For example, root-specific expression of CKX gene in Zea mays improved both root growth and mineral nutrition of plants, which was surprisingly achieved without trade-offs with above-ground growth [57].

*The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

The fact that such prominent changes in whole-plant architecture were made possible by expressing a single gene is quite promising for plant improvement. Therefore, both decrease and increase in CK biosynthesis and signaling can be viewed as a potential way to increase the resilience of crop productivity to water shortage.

## **4. Methods for modifying plant genomes**

Plant genetic traits are inherited from parents from generation to generation and are encoded by genetic information contained in DNA. At the same time, genetic information is subject to constant changes due to the presence of spontaneous or induced mutations, errors arising during transcription, the activity of transposable elements, the processes of meiotic crossing over, and cross-fertilization. Some pathogenic and symbiotic bacteria, such as Agrobacterium spp. [58], can transfer part of their DNA into the genome of the host cell, thereby changing the functioning of the host cell to suit their needs. Thus, genome modification occurs constantly in a plant cell.

Plant breeding is the process of obtaining new varieties of plants that contain in their genome a set of genes that make it possible to grow plants that are suitable for agricultural production, processing, and consumption and at the same time have properties beneficial to humans and animals. Thus, plant breeding involves systematic selection among the entire population of plants of samples bearing target properties. It is estimated that humans have been successfully breeding plants for over ten thousand years [59] when seeds of plants with favorable features were saved for the next plantation, a practice known as domestication. The most significant advances in plant breeding techniques have been achieved as knowledge and understanding of plants and their genetic structures have accumulated. In the second half of the twentieth century, with an increase in the quantity and quality of food consumption, a revolution in plant breeding occurred, the key achievements of which were achieved in the creation of hybrids and transgenesis. The most important stage in plant breeding was the Green Revolution, which made it possible to dramatically increase the productivity of agricultural crops through the development of high-yielding varieties of cereals, particularly dwarf wheat and rice. Norman Borlaug, Nobel Prize laureate and father of the Green Revolution, emphasized that the key to the success of these semi-dwarf varieties was their wide adaptability, short plant height, high sensitivity to fertilizers, and resistance to disease, which ultimately made it possible to obtain more yield at a lower cost [59]. Later, these requests were addressed to the emerging technology of transgenesis, which led to its rapid development. Transgenic crops are now widespread globally and are increasingly accepted as food and feed. Transgenesis changes the genetic information of a plant cell, resulting in a so-called genetically modified organism (GMO) that carries in its genome a fragment of foreign DNA that gives the plant new useful traits that cannot be obtained by conventional breeding methods. However, GMO organisms were perceived ambiguously by society, which led to the fact that obtaining state registration for a GMO variety in some countries is significantly difficult or completely impossible.

#### **4.1 Development of the genome editing tools**

With the development of genetic engineering methods and the accumulation of data on plant genomes, gene editing technologies began to develop—making

it possible to make site-specific changes in the target site of the genome. The first methods that appeared were zinc-finger nuclease (ZFN) and later transcription activator-like effector nucleases (TALEN). Both TALEN and ZFN are composed of repeated tandem sequences of DNA-binding domains and an attached Fok1 nuclease protein, such that the recombinant protein can be targeted to recognize a target DNA sequence and therefore create double-strand breaks (DSBs) at the target site. For each target site, a new TALEN or ZFN protein must be prepared to recognize the target DNA sequence, which required labor-intensive genetic engineering and significantly limited the widespread use of these gene editing technologies [60, 61]. However, there are examples of successful use of ZFN to manipulate genes in tobacco, Arabidopsis, and maize [62–64]. TALENs, which are easier to target to a specific DNA region because each TALEN domain recognizes one target nucleotide, as opposed to ZFN, where each domain recognizes a triplet of nucleotides, have been successfully used in horticultural crops such as soybeans, wheat, rice, tomatoes, and potatoes [65, 66]. However, the major drawback related to ZFNs and TALENs are their off-targeting effects, prolonged screening process, toxicity to the host cell, and complex genetic engineering procedures, limiting their applicability. The most modern method of genome editing is CRISPR technology; the first article on the successful application of this technology on plant cells was published in 2013, and the first edited plants were *Arabidopsis thaliana* and *Nicotiana benthamiana* [67].

Typically, CRISPR/Cas9 is a complex consisting of two components: the Cas9 endonuclease protein and a single guide RNA (sgRNA) with 20-nucleotide homology to the target DNA region [68–70]. The Cas9 endonuclease binds to the protospacer adjacent motif (PAM) DNA sequence (for Cas9 the PAM site is NGG), the sgRNA complementarily binds to the DNA sequence adjacent to the PAM site, and if the binding is successful, Cas9 carries out a DSB in the target site [68, 71]. DSBs caused by the Cas9 endonuclease lead to the activation of DNA repair systems, which can take two pathways, the errorprone non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Errors of DNA repair system result in deletions, insertions, or substitutions of DNA at DSB sites, which in turn disrupt gene function or cause a reading frameshift, known as a gene mutation or knockout [68–70]. As a result of DSB repair via the NHEJ pathway, insertions/deletions (indels) of several bases are usually observed during plant genome editing based on CRISPR/Cas9. The use of the mechanism of HDR, in turn, makes it possible, using editing systems, to replace individual nucleotides in the DNA sequence and even obtain a site-specific insertion of a gene or group of genes.

At the moment, editing technologies have become so widely developed that they make it possible to influence any stage of the implementation of genetic information in a cell—at the level of transcription, translation, post-translation, epigenetic and so on [72]. Over the past 10 years, a number of different CRISPR-based tools have been developed, allowing editing at almost any desired location in the genome. Some examples include DNA base editors [73], epigenetic modifiers [74, 75], prime editors [76, 77], and transcription regulators [78, 79]. Fusion of various additional molecules with partially disrupted (nickase Cas9, nCas9) or nuclease-deficient (dead Cas9, dCas9) Cas9 has been used as a vehicle to deliver the CRISPR fusion protein to the target genomic site. RNA-targeting Cas proteins also enable a variety of RNA manipulations beyond simple RNA editing, such as RNA degradation, detection of ribonucleic acids and pathogens, single RNA base editing, and live imaging of RNA, which can be read in more detail in recently published reviews [72, 75]. Plants cope with stress through a range of finely tuned mechanisms, which involve both protein-coding genes and non-coding regions of the plant genome, along with various epigenetic

mechanisms realized through the control of DNA packaging. The CRISPR-based tools described in this section can exploit the full range of molecular mechanisms mediated by these genomic elements.

## **4.2 Editing genes associated with transport and signaling of ABA and CKs**

Major thriving areas of research include gene discovery (allele mining, investigation of cryptic genes) and introgression of new traits to achieve the desired goal—biotic/ abiotic stress-resilient crops. Today, there is already a fairly large pool of works devoted to editing genes associated with the transport and signaling of ABA and CKs [75, 80]. Editing and transgenesis have helped to establish the functions of a number of genes associated with the ABA signaling pathway and their participation in the response to stress [81, 82]. For example, the enzymes SAPK1 and SAPK2 belonging to the SnRK2 family are members of the ABA signaling pathway in rice. Loss-of-function mutants of SAPK2 generated by CRISPR/Cas9 were insensitive to ABA [81]. The SAPK2 mutants displayed high sensitivity to dehydration and ROS, highlighting the role of SAPK2 in drought stress, the same as how CRISPR-edited OsERA1 mutant lines displayed enhanced tolerance to drought stress [83]. Another example is the work with histone acetyltransferase (HAT) enzyme that relaxes chromatin folding and promotes enhanced gene expression fused with dCas9 protein. Tools for gene activation and epigenetic modification combined with the CRISPR system made it possible to create the dCas9-HAT system, which increased the expression of AREB1 and as a result increased the resistance of Arabidopsis plants to drought [84]. CRISPR/Cas9 was successfully used to create new alleles of the OST2 gene in Arabidopsis, and as a result, edited plant lines carrying the new alleles exhibited an enhanced response to stress due to changes in stomatal closure under drought stress [85]. A number of genes have been shown to be involved in the negative regulation of plant responses to salinity and other abiotic stresses. Reducing the expression level of the RR22 gene, which encodes a type B response regulator (ARR B) involved in CK signaling, using the CRISPR/ Cas9 system, made it possible to increase the tolerance of rice plants to soil salinity [86]. Additional examples of negative regulators research using editing tools include work in Arabidopsis and rice. Editing of the C/VIF1 gene encoding the fructosidase inhibitor protein 1 showed that it is a regulator of the response to ABA and is involved in the development of salt tolerance [87]. Editing of the RR9 and RR10 genes in rice, encoding proteins involved in the CK signaling pathway and associated with response regulators type A (ARR A), allowed to establish their function as negative regulators in response to salinity [88]. As recent work on AITR family genes has shown, targeting mutations in genes with redundant or unclear functions using CRISPR editing systems can help elucidate their role in plant stress biology [89, 90].

As it can be seen, various genome editing tools have been successfully used to study genes associated with plant stress resistance and to create stress-tolerant plants belonging not only to model plant species but also to plant species important for agriculture. The ever-expanding set of CRISPR tools allows you to make changes to any process occurring in a plant cell and thereby regulate the growth, development, and all life processes of plants, through precise and effective genetic engineering. Consistent changes and grouping of genes responsible for resistance to various types of stress, both biotic and abiotic, can help in the development of new lines for plant breeding. Accelerated identification of new genes, as well as the creation of geneedited crops that do not fall under the regulatory requirements developed for transgenic plants, could be a step toward the next Green Transformation.

#### **4.3 AREB/ABF and CKX gene families as potential targets for editing**

CKX genes, which are key regulators of the level of CKs in plant cells and, accordingly, can influence the homeostasis of CKs in the cell, have long attracted the attention of researchers as providing ample opportunities for improving crops. Most studies investigating the function of CKX genes have been carried out using RNAibased silencing or overexpression of CKX genes. Overexpression of AtCKX7 in the model plant results in shorter primary roots [91]. Overexpressing the AtCKS2 gene in oilseed *Brassica napus* increased the root-to-shoot ratio [92]. A number of studies have shown that reducing the expression level of CKX genes in some cases can lead to increased crop yields. For example, in barley, cotton, rice, and Arabidopsis, downregulation of CKX family genes through RNAi-based silencing or various genome editing systems, or with the help of mutations, has resulted in increased seed number and/ or seed weight [93–96]. Also, in a number of works on editing genes of the OsCKX family in rice, it was shown that OsCKX genes serve as a link between CK and other plant hormones, in particular ABA [97, 98]. The perspectives of utilization of genome editing technologies to improve crop performance were discussed recently [80, 99]. The findings support the critical role of CKs in a variety of model plants.

There are significantly fewer studies on the AREB/ABF family. There is work to increase ABF2 expression using dCas9-HAT [84], but most of the research has been done on T-DNA-induced mutations in Arabidopsis obtained in the early 2000s [100– 102]. In Arabidopsis, three members of the AREB/ABF family that respond to water stress and participate in the ABA signaling pathway, ABF2, ABF4 and ABF3, are the master transcription factors that co-regulate ABF-dependent ABA signaling and require ABA for full activation [100]. At the same time, the incomplete functional redundancy of ABF transcription factors gives reason to expect that differential manipulations of ABF can be used to create plants with the desired mode of ABA signaling, for example, to reduce trade-offs between ABA-induced stress tolerance and productivity.

Over the past few years, experimental evidence has been obtained on changes in DNA regions located at some distance from the site of T-DNA integration [103, 104]. This prompted a reconsideration of the relevance of using such mutations to identify the functions of genes of interest, since the manifestation of a mutation caused by the insertion of foreign DNA into the region of the gene under study and causing the loss of its function (knockout) can be masked by other insertions in regions remote from the region of the target gene. The development of new genome editing tools using CRISPR/Cas9 makes it possible to specifically make changes only in the target gene and obtain new series of knockouts for genes of interest. This work firstly examines the possibility of editing genes of the ABF family encoding the AREB1/ABF2, AREB2/ ABF4, and ABF3 transcription factors using *Arabidopsis thaliana* as an example, taking into account the possible participation of other genes included in the network of regulation of abscisic acid biosynthesis. Secondly, the possibility of multiplex editing of CKX1 and CKX4 genes of *Arabidopsis thaliana* to establish their role in the response of plants to abiotic stress. Crossing the resulting mutants will make it possible to establish the details of the interaction between ABA and CKs.

## **5. Conclusion**

The development and improvement of molecular biology methods by the beginning of the twenty first century stimulated the creation of modern tools that make it *The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

possible to modify plant genomes by targeted changes in the functioning of genes of interest. This opens up great opportunities for researchers to modify genes involved in the control of complex traits in plants, such as resistance to water deficiency. The use of genome editing to knockout individual genes that control plant response to various stress conditions, including water deficiency, will reveal the role of both regulatory genes encoding transcription factors for ABA biosynthesis and genes that provide interconnections between the signaling pathways of various phytohormones, in particular, the relationship between ABA and CKs.

## **Acknowledgements**

This work was supported by the development program of National Research Tomsk State University (Priority-2030, project no. 2.1.2.22 "Genome editing as an innovative technology for studying the mechanisms of stress tolerance and increasing plant productivity under unfavorable environmental and climate changes").

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Ilya Zlobin1,2, Marina Efimova1 , Natalia Permykova1,3, Irina Sokolova3 , Vladimir Kuznetsov1,2 and Elena Deineko1,3\*

1 National Research Tomsk State University, Tomsk, Russia

2 Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia

3 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia

\*Address all correspondence to: deineko@bionet.nsc.ru

© 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.

## **References**

[1] Hammond WM, Adams HD. Dying on time: Traits influencing the dynamics of tree mortality risk from drought. Tree Physiology. 2019;**39**(6):906-909. DOI: 10.1093/treephys/tpz050

[2] Verslues PE, Bailey-Serres J, Brodersen C, Buckley TN, Conti L, Christmann A, et al. Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress. The Plant Cell. 2023;**351**:67-108. DOI: 10.1093/plcell/koac263

[3] Agarwal PK, Gupta K, Lopato S, Agarwal P. Dehydration responsive element binding transcription factors and their applications for the engineering of stress tolerance. Journal of Experimental Botany. 2017;**68**(9):2135- 2148. DOI: 10.1093/jxb/erx118

[4] Avni A, Golan Y, Shirron N, Shamai Y, Danin-Poleg Y, Gepstein S, et al. From survival to productivity mode: Cytokinins allow avoiding the avoidance strategy under stress conditions. Frontiers in Plant Science. 2020;**11**:879. DOI: 10.3389/fpls.2020.00879

[5] Hasan MM, Gong L, Nie Z-F, Li F-P, Ahammed GJ, Fang X-W. ABA-induced stomatal movements in vascular plants during dehydration and rehydration. Environmental and Experimental Botany. 2021;**186**:104436. DOI: 10.1016/j. envexpbot.2021.104436

[6] Brodribb T, Brodersen CR, Carriqui M, Tonet V, Dominguez CR, McAdam S. Linking xylem network failure with leaf tissue death. New Phytologist. 2021;**232**(1):68-79. DOI: 10.1111/nph.17577

[7] Brodribb TJ, McAdam SAM, Carins Murphy MR. Xylem and stomata,

coordinated through time and space. Plant, Cell & Environment. 2017;**40**(6):872-880. DOI: 10.1111/ pce.12817

[8] Martins SCV, McAdam SAM, Deans RM, DaMatta FM, Brodribb TJ. Stomatal dynamics are limited by leaf hydraulics in ferns and conifers: Results from simultaneous measurements of liquid and vapour fluxes in leaves. Plant, Cell & Environment. 2016;**39**(3):694- 705. DOI: 10.1111/pce.12668

[9] Sussmilch FC, Schultz J, Hedrich R, Roelfsema MRG. Acquiring control: The evolution of stomatal signalling pathways. Trends in Plant Science. 2019;**24**(4):342-351. DOI: 10.1016/j. tplants.2019.01.002

[10] McAdam SAM, Brodribb TJ. Mesophyll cells are the main site of abscisic acid biosynthesis in waterstressed leaves. Plant Physiology. 2018;**177**(3):911-917. DOI: 10.1104/ pp.17.01829

[11] Salvi P, Manna M, Kaur H, Thakur T, Gandass N, Bhatt D, et al. Phytohormone signaling and crosstalk in regulating drought stress response in plants. Plant Cell Reports. 2021;**40**:1305-1329. DOI: 10.1007/s00299-021-02683-8

[12] Sack L, John GP, Buckley TN. ABA accumulation in dehydrating leaves is associated with decline in cell volume, not turgor pressure. Plant Physiology. 2018;**176**(1):489-495. DOI: 10.1104/ pp.17.01097

[13] Virlouvet L, Fromm M. Physiological and transcriptional memory in guard cells during repetitive dehydration stress. New Phytologist. 2015;**205**(2):596-607. DOI: 10.1111/nph.13080

*The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

[14] Sadhukhan A, Prasad SS, Mitra J, Siddiqui N, Sahoo L, Kobayashi Y, et al. How do plants remember drought? Planta. 2022;**256**(1):7. DOI: 10.1007/ s00425-022-03924-0

[15] Buckley TN. How do stomata respond to water status? New Phytologist. 2019;**224**(1):21-36. DOI: 10.1111/ nph.15899

[16] Flexas J, Carriquí M, Nadal M. Gas exchange and hydraulics during drought in crops: Who drives whom? Journal of Experimental Botany. 2018;**69**(16):3791- 3795. DOI: 10.1093/jxb/ery235

[17] Polle A, Chen SL, Eckert C, Harfouche A. Engineering drought resistance in forest trees. Frontiers in Plant Science. 2019;**9**:1875. DOI: 10.3389/ fpls.2018.01875

[18] Brodribb TJ, McAdam SAM. Abscisic acid mediates a divergence in the drought response of two conifers. Plant Physiology. 2013;**162**(3):1370-1377. DOI: 10.1104/pp.113.217877

[19] Brodribb TJ, McAdam SAM, Jordan GJ, Martins SCV. Conifer species adapt to low-rainfall climates by following one of two divergent pathways. National Academy of Sciences of the United States of America. 2014;**111**(40):14489-14493. DOI: 10.1073/ pnas.140793011

[20] Heinrich S, Dippold MA, Werner C, Wiesenberg GLB, Kuzyakov Y, Glaser B. Allocation of freshly assimilated carbon into primary and secondary metabolites after in situ 13C pulse labelling of Norway spruce (*Picea abies*). Tree Physiology. 2015;**35**(11):1176-1191. DOI: 10.1093/treephys/tpv083

[21] Duursma RA, Blackman CJ, Lopéz R, Martin-StPaul K, Cochard H, Medlyn BE. On the minimum leaf conductance: Its

role in models of plant water use, and ecological and environmental controls. New Phytologist. 2019;**221**(2):693-705. DOI: 10.1111/nph.15395

[22] Li X, Chen L, Forde BG, Davies WJ. The biphasic root growth response to abscisic acid in arabidopsis involves interaction with ethylene and auxin signalling pathways. Frontiers in Plant Science. 2017;**8**:1493. DOI: 10.3389/ fpls.2017.01493

[23] Rowe JH, Topping JF, Liu J, Lindsey K. Abscisic acid regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin. New Phytologist. 2016;**211**(1):225-239. DOI: 10.1111/nph.13882

[24] Ranjan A, Sinha R, Singla-Pareek SL, Pareek A, Kumar SA. Shaping the root system architecture in plants for adaptation to drought stress. Physiologia Plantarum. 2022;**174**(2):e13651. DOI: 10.1111/ppl.13651

[25] Rosales MA, Maurel C, Nacry P. Abscisic acid coordinates dosedependent developmental and hydraulic responses of roots to water deficit. Plant Physiology. 2019;**180**(4):2198-2211. DOI: 10.1104/pp.18.01546

[26] Calvo-Polanco M, Armada E, Zamarreño AM, García-Mina JM, Aroca R. Local root ABA/cytokinin status and aquaporins regulate poplar responses to mild drought stress independently of the ectomycorrhizal fungus Laccaria bicolor. Journal of Experimental Botany. 2019;**70**(21):6437-6446. DOI: 10.1093/ jxb/erz389

[27] McAdam SAM, Brodribb TJ, Ross JJ. Shoot-derived abscisic acid promotes root growth. Plant, Cell & Environment. 2016;**39**(3):652-659. DOI: 10.1111/ pce.12669

[28] Rosso L, Cantamessa S, Bergante S, Biselli C, Fricano A, Chiarabaglio PM. Responses to drought stress in poplar: What do we know and what can we learn? Life. 2023;**13**(2):533. DOI: 10.3390/ life13020533

[29] Domec J-C, King JS, Carmichael MJ, Overby AT, Wortemann RR, Smith WK, et al. Root water gates and not changes in root structure provide new insights into plant physiological responses and adaptations to drought, flooding and salinity. bioRxiv. 27 Oct 2020. DOI: 10.1101/2020.10.27.357251

[30] De Diego N, Rodríguez JL, Dodd IC, Pérez-Alfocea F, Moncaleán P, Lacuesta M. Immunolocalization of IAA and ABA in roots and needles of radiata pine (*Pinus radiata*) during drought and rewatering. Tree Physiology. 2013;**33**(5):537-549. DOI: 10.1093/ treephys/tpt033

[31] Brito C, Dinis L-T, Ferreira H, Moutinho-Pereira J, Correia CM. Foliar pre-treatment with abscisic acid enhances olive tree drought adaptability. Plants. 2020;**9**(3):341. DOI: 10.3390/ plants9030341

[32] Gambetta GA, Knipfer T, Fricke W, McElrone AJ. Aquaporins and root water uptake. In: Chaumont F, Tyerman S, editors. Plant Aquaporins: From Transport to Signaling. Cham: Springer; 2017. pp. 133-153. DOI: 10.1007/978-3-319-49395-4\_6

[33] Sánchez-Romera B, Ruiz-Lozano JM, Li G, Luu D-T, Martínez-Ballesta MC, Carvajal M. Enhancement of root hydraulic conductivity by methyl jasmonate and the role of calcium and abscisic acid in this process. Plant, Cell & Environment. 2014;**37**(4):995-1008. DOI: 10.1111/pce.12214

[34] Parent B, Hachez C, Redondo E, Simonneau T, Chaumont F, Tardieu F. Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rate: A trans-scale approach. Plant Physiology. 2009;**149**(4):2000-2012. DOI: 10.1104/pp.108.130682

[35] Prado K, Maurel C. Regulation of leaf hydraulics: From molecular to whole plant levels. Frontiers in Plant Science. 2013;**4**:255. DOI: 10.3389/fpls.2013.00255

[36] Aloni R. The role of hormones in controlling vascular differentiation. In: Fromm J, editor. Cellular Aspects of Wood Formation. Berlin, Heidelberg: Springer; 2013. pp. 99-139. DOI: 10.1007/978-3-642-36491-4\_4

[37] Buttò V, Deslauriers A, Rossi S, Rozenberg P, Shishov V, Morin H. The role of plant hormones in tree-ring formation. Trees. 2020;**34**:315-335. DOI: 10.1007/s00468-019-01940-4

[38] Mwange KNK, Hou H-W, Wang Y-Q, He X-Q, Cui K-M. Opposite patterns in the annual distribution and time-course of endogenous abscisic acid and indole-3-acetic acid in relation to the periodicity of cambial activity in Eucommia ulmoides Oliv. Journal of Experimental Botany. 2005;**56**(413):1017-1028. DOI: 10.1093/jxb/eri095

[39] Rodriguez-Zaccaro FD, Groover A. Wood and water: How trees modify wood development to cope with drought. Plants, People, Planet. 2019;**1**(4):346-355. DOI: 10.1002/ppp3.29

[40] Blackman CJ, Brodribb TJ, Jordan GJ. Leaf hydraulics and drought stress: Response, recovery and survivorship in four woody temperate plant species. Plant, Cell & Environment. 2009;**32**(11):1584-1595. DOI: 10.1111/j.1365-3040.2009.02023.x

[41] Lovisolo C, Perrone I, Hartung W, Schubert A. An abscisic acid-related

*The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

reduced transpiration promotes gradual embolism repair when grapevines are rehydrated after drought. New Phytologist. 2008;**180**(3):642-651. DOI: 10.1111/j.1469-8137.2008.02592.x

[42] Tombesi S, Nardini A, Frioni T, Soccolini M, Zadra C, Farinelli D. Stomatal closure is induced by hydraulic signals and maintained by ABA in drought-stressed grapevine. Scientific Reports. 2015;**5**(1):12449. DOI: 10.1038/srep12449

[43] Kambona CM, Koua PA, Léon J, Ballvora A. Stress memory and its regulation in plants experiencing recurrent drought conditions. Theoretical and Applied Genetics. 2023;**136**(2):26. DOI: 10.1007/ s00122-023-04313-1

[44] Zhang H, Zhao Y, Zhu JK. Thriving under stress: How plants balance growth and the stress response. Developmental Cell. 2020;**55**(5):529-543. DOI: 10.1016/j. devcel.2020.10.012

[45] Querejeta JI, Ren W, Prieto I. Vertical decoupling of soil nutrients and water under climate warming reduces plant cumulative nutrient uptake, wateruse efficiency and productivity. New Phytologist. 2021;**230**(4):1378-1393. DOI: 10.1111/nph.17258

[46] Waadt R, Seller CA, Hsu P-K, Takahashi Y, Munemasa S, Schroeder JI. Plant hormone regulation of abiotic stress responses. Nature Reviews Molecular Cell Biology. 2022;**23**(10):680- 694. DOI: 10.1038/s41580-022-00479-6

[47] Hai NN, Chuong NN, Tu NHC, Kisiala A, Hoang XLT, Thao NP. Role and regulation of cytokinins in plant response to drought stress. Plants. 2020;**9**(4):422. DOI: 10.3390/plants9040422

[48] Prerostova S, Dobrev PI, Gaudinova A, Knirsch V, Körber N, Pieruschka R. Cytokinins: Their impact on molecular and growth responses to drought stress and recovery in arabidopsis. Frontiers in Plant Science. 2018;**9**:655. DOI: 10.3389/ fpls.2018.00655

[49] Romanov GA. Perception, transduction and crosstalk of auxin and cytokinin signals. International Journal of Molecular Sciences. 2022;**23**(21):13150. DOI: 10.3390/ ijms232113150

[50] Cortleven A, Leuendorf JE, Frank M, Pezzetta D, Bolt S, Schmülling T. Cytokinin action in response to abiotic and biotic stresses in plants. Plant, Cell & Environment. 2019;**42**(3):998-1018. DOI: 10.1111/ pce.13494

[51] De Diego N, Pérez-Alfocea F, Cantero E, Lacuesta M, Moncaleán P. Physiological response to drought in radiata pine: Phytohormone implication at leaf level. Tree Physiology. 2012;**32**(4):435-449. DOI: 10.1093/ treephys/tps029

[52] Immanen J, Nieminen K, Smolander O-P, Kojima M, Serra JA, Koskinen P. Cytokinin and auxin display distinct but interconnected distribution and signaling profiles to stimulate cambial activity. Current Biology. 2016;**26**(15):1990-1997

[53] Hu L, Wang Z, Huang B. Effects of cytokinin and potassium on stomatal and photosynthetic recovery of Kentucky bluegrass from drought stress. Crop Science. 2013;**53**(1):221-231. DOI: 10.2135/cropsci2012.05.0284

[54] Qin H, He L, Huang R. The coordination of ethylene and other hormones in primary root development. Frontiers in Plant Science. 2019;**10**:874. DOI: 10.3389/fpls.2019.00874

[55] Waidmann S, Sarkel E, Kleine-Vehn J. Same, but different: Growth responses of primary and lateral roots. Journal of Experimental Botany. 2020;**71**(8):2397- 2411. DOI: 10.1093/jxb/eraa027

[56] Kurepa J, Smalle JA. Auxin/cytokinin antagonistic control of the shoot/ root growth ratio and its relevance for adaptation to drought and nutrient deficiency stresses. International Journal of Molecular Sciences. 2022;**23**(4):1933. DOI: 10.3390/ijms23041933

[57] Ramireddy E, Nelissen H, Leuendorf JE, Lijsebettens MV, Inzé D, Schmülling T. Root engineering in maize by increasing cytokinin degradation causes enhanced root growth and leaf mineral enrichment. Plant Molecular Biology. 2021;**106**:555-567. DOI: 10.1007/ s11103-021-01173-5

[58] Gelvin SB. Integration of agrobacterium T-DNA into the plant genome. Annual Review of Genetics. 2017;**51**:195-217. DOI: 10.1146/ annurev-genet-120215-035320

[59] Lee J, Chin JH, Ahn SN, Koh H. Brief history and perspectives on plant breeding. In: Current Technologies in Plant Molecular Breeding. Dordrecht: Springer; 2015. 343 p. DOI: 10.1007/978-94-017-9996-6

[60] Mahfouz MM, Li L. TALE nucleases and next generation GM crops. GM Crops. 2011;**2**(2):99-103. DOI: 10.4161/ gmcr.2.2.17254

[61] Wood AJ, Lo T-W, Zeitler B, Pickle CS, Ralston EJ, Lee AH, et al. Targeted genome editing across species using ZFNs and TALENs. Science. 2011;**333**:307. DOI: 10.1126/ science.1207773

[62] Shukla VK, Doyon Y, Miller JC, Dekelver RC, Moehle EA, Worden SE, et al. Precise genome modification in the crop species *Zea mays* using zinc-finger nucleases. Nature. 2009;**459**:437-441. DOI: 10.1038/nature07992

[63] Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature. 2009;**459**:442-445. DOI: 10.1038/nature07845

[64] Osakabe K, Osakabe Y, Toki S. Site-directed mutagenesis in arabidopsis using custom-designed zinc finger nucleases. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**:12034-12039. DOI: 10.1073/pnas.1000234107

[65] Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, et al. Efficient TALEN-mediated gene knockout in livestock. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**:17382-17387. DOI: 10.1073/ pnas.1211446109

[66] Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, et al. Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiology. 2013;**161**:20-27. DOI: 10.1104/ pp.112.205179

[67] Li J-F, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination–mediated genome editing in arabidopsis and *Nicotiana benthamiana* using guide RNA and Cas9. Nature Biotechnology. 2013;**31**:688-691. DOI: 10.1038/nbt.2654

[68] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A. A programmable dual-RNA—Guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816-822. DOI: 10.1126/science.1225829

*The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

[69] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/ Cas systems. Science. 2013;**339**:819-823. DOI: 10.1126/science.1231143

[70] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;**339**:823-826. DOI: 10.1126/ science.1232033

[71] Li W, Teng F, Li T, Zhou Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nature Biotechnology. 2013;**31**:684-686. DOI: 10.1038/nbt.2652

[72] Pramanik D, Shelake RM, Kim MJ, Kim JY. CRISPR-mediated engineering across the central dogma in plant biology for basic research and crop improvement. Molecular Plant. 2021;**14**:127-150. DOI: 10.1016/j.molp.2020.11.002

[73] Jeong YK, Song B, Bae S. Current status and challenges of DNA base editing tools. Molecular Therapy. 2020;**28**:1938-1952. DOI: 10.1016/j. ymthe.2020.07.021

[74] Shelake RM, Pramanik D, Kim JY. Evolution of plant mutagenesis tools: A shifting paradigm from random to targeted genome editing. Plant Biotechnology Reports. 2019;**13**:423-445. DOI: 10.1007/s11816-019-00562-z

[75] Shelake RM, Kadam US, Kumar R, Pramanik D, Singh AK, Kim JY. Engineering drought and salinity tolerance traits in crops through CRISPRmediated genome editing: Targets, tools, challenges, and perspectives. Plant Communications. 2022;**3**:100417. DOI: 10.1016/j.xplc.2022.100417

[76] Huang TK, Puchta H. Novel CRISPR/ Cas applications in plants: From prime

editing to chromosome engineering. Transgenic Research. 2021;**30**:529-549. DOI: 10.1007/s11248-021-00238-x

[77] Lin Q, Jin S, Zong Y, Yu H, Zhu Z, Liu G, et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nature Biotechnology. 2021;**39**(8):923-927. DOI: 10.1038/s41587-021-00868-w

[78] Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A, Al-Shareef S, et al. RNAguided transcriptional regulation in planta via synthetic dCas9 based transcription factors. Plant Biotechnology Journal. 2015;**13**:578-589. DOI: 10.1111/pbi.12284

[79] Rehman RS, Zafar SA, Ali M, Pasha AN, Naveed MS, Waseem M, et al. CRISPR-Cas mediated genome editing: A paradigm shift towards sustainable agriculture and biotechnology. Asian Plant Research Journal. 2022;**9**:27-49. DOI: 10.9734/aprj/2022/v9i130197

[80] Mandal S, Ghorai M, Anand U, Roy D, Kant N, Mishra T, et al. Cytokinins: A genetic target for increasing yield potential in the CRISPR era. Frontiers in Genetics. 2022;**13**:1-12. DOI: 10.3389/fgene.2022.883930

[81] Lou D, Wang H, Liang G, Yu D. OsSAPK2 confers abscisic acid sensitivity and tolerance to drought stress in rice. Frontiers in Plant Science. 2017;**8**:1-15. DOI: 10.3389/fpls.2017.00993

[82] He QY, Jin JF, Lou HQ, Dang FF, Xu JM, Zheng SJ, et al. Abscisic aciddependent PMT1 expression regulates salt tolerance by alleviating abscisic acid-mediated reactive oxygen species production in arabidopsis. Journal of Integrative Plant Biology. 2022;**64**:1803- 1820. DOI: 10.1111/jipb.13326

[83] Ogata T, Ishizaki T, Fujita M, Fujita Y. CRISPR/Cas9-targeted mutagenesis of

OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice. PLoS One. 2020;**15**:1-12. DOI: 10.1371/journal. pone.0243376

[84] Roca Paixão JF, Gillet FX, Ribeiro TP, Bournaud C, Lourenço-Tessutti IT, Noriega DD, et al. Improved drought stress tolerance in arabidopsis by CRISPR/dCas9 fusion with a histone acetyltransferase. Scientific Reports. 2019;**9**:1-9. DOI: 10.1038/ s41598-019-44571-y

[85] Osakabe Y, Watanabe T, Sugano SS, Ueta R, Ishihara R, Shinozaki K, et al. Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants. Scientific Reports. 2016;**6**:1-10. DOI: 10.1038/srep26685

[86] Zhang A, Liu Y, Wang F, Li T, Chen Z, Kong D, et al. Enhanced rice salinity tolerance via CRISPR/Cas9 targeted mutagenesis of the OsRR22 gene. Molecular Breeding. 2019;**39**:47. DOI: 10.1007/s11032-019-0954-y

[87] Yang W, Chen S, Cheng Y, Zhang N, Ma Y, Wang W, et al. Cell wall/vacuolar inhibitor of fructosidase 1 regulates ABA response and salt tolerance in arabidopsis. Plant Signaling & Behavior. 2020;**15**:4. DOI: 10.1080/15592324.2020.1744293

[88] Wang WC, Lin TC, Kieber J, Tsai YC. Response regulators 9 and 10 negatively regulate salinity tolerance in Rice. Plant & Cell Physiology. 2019;**60**:2549-2563. DOI: 10.1093/pcp/pcz149

[89] Chen S, Zhang N, Zhou G, Hussain S, Ahmed S, Tian H, et al. Knockout of the entire family of AITR genes in Arabidopsis leads to enhanced drought and salinity tolerance without fitness costs. BMC Plant Biology. 2021;**21**:1-15. DOI: 10.1186/s12870-021-02907-9

[90] Wang T, Xun H, Wang W, Ding X, Tian H, Hussain S, et al. Mutation of GmAITR genes by CRISPR/Cas9 genome editing results in enhanced salinity stress tolerance in soybean. Frontiers in Plant Science. 2021;**12**:1-15. DOI: 10.3389/ fpls.2021.779598

[91] Köllmer I, Novák O, Strnad M, Schmülling T, Werner T. Overexpression of the cytosolic cytokinin oxidase/ dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation. The Plant Journal. 2014;**78**:359-371. DOI: 10.1111/ tpj.12477

[92] Nehnevajova E, Ramireddy E, Stolz A, Gerdemann-Knörck M, Novák O, Strnad M, et al. Root enhancement in cytokinin-deficient oilseed rape causes leaf mineral enrichment, increases the chlorophyll concentration under nutrient limitation and enhances the phytoremediation capacity. BMC Plant Biology. 2019;**19**:1-15. DOI: 10.1186/ s12870-019-1657-6

[93] Zalewski W, Galuszka P, Gasparis S, Orczyk W, Nadolska-Orczyk A. Silencing of the HvCKX1 gene decreases the cytokinin oxidase/dehydrogenase level in barley and leads to higher plant productivity. Journal of Experimental Botany. 2010;**61**:1839-1851. DOI: 10.1093/ jxb/erq052

[94] Bartrina I, Otto E, Strnad M, Werner T, Schmülling T. Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in *Arabidopsis thaliana*. The Plant Cell. 2011;**23**:69-80. DOI: 10.1105/ tpc.110.079079

[95] Li S, Zhao B, Yuan D, Duan M, Qian Q, Tang L, et al. Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression.

*The Modification of Abscisic Acid and Cytokinin Signaling with Genome Editing to Increase… DOI: http://dx.doi.org/10.5772/intechopen.113928*

Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:3167-3172. DOI: 10.1073/ pnas.1300359110

[96] Zhao J, Bai W, Zeng Q, Song S, Zhang M, Li X, et al. The abscisic acid– responsive element binding factors MAPKKK18 module regulates abscisic acid–induced leaf senescence in arabidopsis. The Journal of Biological Chemistry. 2023;**299**:103060. DOI: 10.1016/j.jbc.2023.103060

[97] Tao Y, Miao J, Wang J, Li W, Xu Y, Wang F, et al. RGG1, involved in the cytokinin regulatory pathway, controls grain size in rice. Rice. 2020;**13**:76. DOI: 10.1186/s12284-020-00436-x

[98] Zhang W, Peng K, Cui F, Wang D, Zhao J, Zhang Y, et al. Cytokinin oxidase/ dehydrogenase OsCKX11 coordinates source and sink relationship in rice by simultaneous regulation of leaf senescence and grain number. Plant Biotechnology Journal. 2021;**19**:335-350. DOI: 10.1111/pbi.13467

[99] Mahto RK, Singh C, Chandana BS, Singh RK, Verma S, Gahlaut V. Chickpea biofortification for cytokinin dehydrogenase via genome editing to enhance abiotic-biotic stress tolerance and food security. Frontiers in Genetics. 2022;**13**:900324. DOI: 10.3389/ fgene.2022.900324

[100] Yoshida T, Fujita Y, Sayama H, Kidokoro S, Maruyama K, Mizoi J, et al. AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. The Plant Journal. 2010;**61**:672-685. DOI: 10.1111/j.1365-313X.2009.04092.x

[101] Du J, Zhu X, He K, Kui M, Zhang J, Han X, et al. CONSTANS interacts with

and antagonizes ABF transcription factors during salt stress under longday conditions. Plant Physiology. 2023;**193**:1675-1694. DOI: 10.1093/ plphys/kiad370

[102] Zhao J, Bai W, Zeng Q, Song S, Zhang M, Li X, et al. Moderately enhancing cytokinin level by downregulation of GhCKX expression in cotton concurrently increases fiber and seed yield. Molecular Breeding. 2015;**35**:60. DOI: 10.1007/ s11032-015-0232-6

[103] Pucker B, Kleinbölting N, Weisshaar B. Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis. BMC Genomics. 2021;**22**:1-21. DOI: 10.1186/ s12864-021-07877-8

[104] Edwards B, Hornstein ED, Wilson NJ, Sederoff H. High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes. BMC Genomics. 2022;**23**:1-20. DOI: 10.1186/s12864-022-08918-6

## **Chapter 2**

## Organic Farming to Mitigate Abiotic Stresses under Climate Change Scenario

*Saikat Biswas, Rupa Das and Lay Lay Nwe*

## **Abstract**

Climate change is resultant from modern-day chemical agriculture, which is creating negative impacts on crop production. Global agriculture is now facing various problems arising due to abiotic stresses such as flood, drought, temperature extremes, light extremes, salinity, heavy metal stress, nutrient toxicity/deficiency. These stresses not only hamper the growth and production but also reduce the quality of crops through morphological, physiological, biochemical changes and synthesis of ROS. Further, they negatively impact on entire environment specially soil health. Deterioration of yield and quality often occurs due to lack of essential inputs to plants under abiotic stresses. Although plants adopt defensive mechanisms, such abiotic stresses need to be addressed properly with various eco-friendly organic farming approaches. Different organic inputs like organic manures, biofertilizers, bio-priming with microorganisms, bio-stimulants (seaweed extracts, humic acid, micro-organisms, etc.), mulches, biochar are known to alleviate abiotic stresses under climate change scenario. Further, various organic agronomic practices viz. crop rotation, intercropping, tillage, sowing methods and time, nutrient, water and intercultural operations, use of PGPB, organic formulations, grafting, selection of resistant/tolerant varieties and other scientific/wise uses of organic inputs can mitigate/escape the negative impacts of abiotic stresses resulting in upliftment in crop production as well as the quality of produce.

**Keywords:** abiotic stresses, agronomic management, climate change, crop growth, organic farming practices, production

## **1. Introduction**

Food scarcity is a major challenge in today's agriculture. In order to meet the food demand of ever-increasing population, worldwide, farmers are aiming to improve agricultural productivity at the expanse of environment through application of chemical fertilizers and pesticides. Unscientific and over use of chemicals and other management practices degrades soil, water and other valuable natural resources leading to climate change scenario which is a great concern for sustainable agriculture. Further, shrinkage of agricultural land due to population growth, aim/migration for alternative job, urbanization, deforestation, anti-environmental anthropogenic activities, etc. are creating major issues of agriculture and urging for improvement of agricultural productivity and fulfillment of this urge is questionable under climate change scenario as plant has sessile growth habit. Crop growth mostly depends on interaction between genetic trait of variety with growing environment. Climate change, therefore, exerts various stresses on crops and affects crop negatively. The stresses can be biotic (living) and abiotic (non-living) stresses which often put both sole and combined impact on crop.

Abiotic stresses such as drought, flood, salinity, temperature extremes (hot/cold), heavy metals, light, wind, nutrients/chemicals, etc. due to climate change decide distribution of plants in various environmental conditions [1] and thereafter, affect crop growth especially at reproductive stage resulting in poor crop productivity throughout the world [2]. Abiotic stresses also trigger various biotic stresses leading to poor crop productivity through disruption of seed germination, vegetative growth, dry matter production and its translocation to reproductive parts [3]. World experiences around 70% yield loss due to abiotic stresses [4]. Severity in abiotic stresses causes imbalance between demand and supply of nutrients, inactivation of enzymatic activities, suppression of various genes responsible for the quality expression, etc. [5] resulting loss of yield and quality of crop through hampering crop from morphological to molecular levels [6].

These abiotic stresses indeed are serious barriers in front of food security of global population and therefore, suitable strategies are highly needed to cope with these and to achieve good crop growth, yield and quality under climate change scenario. Although few mechanisms like escaping stress, stress avoidance and stress tolerance are done by plants through making various molecular, cellular and physiological changes, there is need to explore and adopt various strategies like traditional and modern breeding approaches, agronomic management practices, exogenous applications of stress tolerating compounds, etc. to mitigate harmful impacts of abiotic stresses on crop to a high extent. Agronomic management strategies cover various technologies including organic farming approaches to alleviate abiotic stresses. Organic farming consists of chemical excluded farming practices which mostly rely on natural and organic inputs/products to improve crop growth and yield as well as other allied sectors. Various organic farming inputs (manures, biofertilizers, crop residues, bio-stimulants, etc.) and practices (selection of varieties, tillage, sowing, nutrient, weed, water management practices, etc.) play a key role in addressing various abiotic stresses and allows the crop to grow well by coping up the climate change situation. Although the published information is less in this regard, an insight knowledge on organic farming activates against abiotic stresses is highly needed. Therefore, an attempt was made in this chapter to highlight negative impacts of abiotic stresses on crop and their mitigation strategies through various organic farming approaches.

## **2. Various abiotic stresses**

Abiotic stress is resulted due to the negative influence of physical or chemical environment on biological organisms either alone or in various forms of interaction. In agriculture, crop production is highly hampered due to abiotic stresses. They individually or as combination impair the normal metabolisms and other physiological functions in plants and thereby, affect crop growth and development. Combined influence

of various stresses is more pronounced on crop production than their individual adverse effect. There are different types of stresses viz. drought, flood, heat and cold stresses, heavy metals, toxicity due to nutrients and pesticides, high light, low light, UV exposure, photo-inhibition, shade, wind velocity, air pollution, etc. which negatively impact on crop plants (**Figure 1**). In the following section, these stresses are briefly highlighted.

## **2.1 Drought stress**

Among the different natural resources, water is now highly precious and scarce for living organisms including crop. Water regulates various physiological and biochemical activities of plant like photosynthesis, transpiration, nutrient uptakes, translocation of assimilates, etc. Plant growth, internal activities are seriously hampered if water availability deviates from normal which is now a common phenomenon throughout the globe as an effect of climate change due to unscientific, non-ecofriendly anthropogenic activities. Water stress mostly occurs in the form drought and flood. Drought or water scarcity may arise due to various reasons such as long period of no occurrence or less intensity of rainfall from usual, low river and stream flows, reduced ground water table, etc. in a region. In agriculture, during crop growth stages, drought may arise due to late onset and early cessation of rainfall, break of monsoon for long period, less availability of irrigation water, faulty or no water conservation practices/structures resulting in serious damage to crop growth and yield. Moreover, Under the situation of soil moisture availability, if salt concentration is high in soil, plant can't uptake water from soil properly and even, exosmosis occurs. This situation, thus creates apparent drought. High temperature triggers evapotranspiration as a part of internal cooling process, resulting in drought or water deficiency. Further, drought can also be resulted from low temperature, under which water freezes in the intercellular spaces creating protoplasmic dehydration and death of cell and eventually, the plant. Altogether, drought affects plant's germination and normal functioning.

## **2.2 Flood stress**

When water availability becomes unnecessarily high as compared to normal for a particular period in an area, flood occurs. It may be resulted from sudden outburst of cloud coupled with excessive rainfall for a short time period (flash flood) or due to

**Figure 1.** *Classification of various biotic and abiotic stresses [7].* continuous rainfall for few days or high water-table or overflow of river, ponds and dams associated with less drainage facility. Flash flood lasts for a very less time period from a day to only few weeks. However, deep water flood lasts for a longer period of time.

#### **2.3 Salinity stress**

Throughout the globe specially during arid and semi-arid areas, salinity is a major issue. It arises in areas where potential evapotranspiration is greater than the rainfall as well as insufficient leaching of salts beyond rhizospheric zone owing from poor rainfall. Presence of excess salts in soil drastically hampers the crop growth [8]. Soil salinity can be developed by both natural phenomena (Weathering of rocks, flooding and intrusion of sea water to agricultural land, seepage of saline water, wind blow, etc.) and human induced activities (poor water quality of irrigation, deforestation, overgrazing, intensive cropping, etc.). Salinity is indicated by electrical conductivity (EC). Usually, soil having EC > 4 dS/m, exchangeable sodium percentage (ESP) < 15.0 and pH <8.5 is called as saline soil [9]. Saline soil contains chloride, sulfate salts of sodium, magnesium and calcium ions. Presence of these salts in excessive quantities deteriorates soil health through changing cation exchange capacity, negatively impacting soil micro-organisms'survival, multiplication and activities, disrupting soil physical properties through deflocculation and reduction of hydraulic conductivity, etc.

#### **2.4 Temperature stress**

Temperature stress indicates both rise and fall of temperature from normal. Sudden change in temperature occurs due to climate change. Specifically, heat stress or high temperature situation arise due to due to global warming and anthropogenic activities resulting in change biodiversity, crop ecosystem, impairment of crop growth and production especially in areas of tropics and sub-tropics. Heat stress results in respiration greater than photosynthesis causing starvation injury through deficit of food reserves in plants. According to different degree of high temperature tolerance, plants are categorized as psychrophiles (up to 15–20°C), mesophiles (up to 35–45°C) and thermophiles (up to 45–100°C) [10].

In contrast to heat stress, an opposite phenomenon known as cold stress or low temperature occurs mostly in temperate areas. There are two types of cold stress viz. chilling stress and freezing stress both affecting the crop's physiological, biochemical activities and eventually, hampering crop's growth, yield and quality. Similar to heat stress, plants are also grouped into three based on cold stress tolerance: Chilling sensitive (Plants are extremely sensitive above 0°C and below 15°C), chilling resistant (plants can tolerate low temperature but highly suffer under formation of ice crystals in intra and inter cellular spaces) and frost resistant (plants are tolerant to extremely low temperature).

#### **2.5 Heavy metal toxicity**

Heavy metals impart mutagenic effects on plants by contaminating irrigation water, food chain and environment [11]. These are inorganic, non-biodegradable compounds with atomic mass >20 and density >5 g/cm<sup>3</sup> . The source of heavy metals in the soil is use of irrigation water from contaminated area, excessive application of

chemical fertilizer and pesticides. Plants absorb heavy metals from soil through roots. Ag, Cr, Cd, As, Sb, Pb, Se, and Hg are some major heavy metals which at high concentrations are non-essential and thereby, hamper soil quality and plant's normal functioning. Other than these, there are some essential elements viz. Zn, Cu, Ni, Fe, Co, etc. which at high concentrations create heavy metal toxicity in soil and plant.

## **2.6 Light stress**

Light is essential resource not only for plant growth but also for all life. In fact, harnessing of high amount of solar energy is the prime aim of crop production. However, excessive or low light can cause negative impact on crop such as poor crop growth, wilting, dwarfing, less photosynthesis, cell damage, low productivity and quality and even death of the plant.

#### **2.7 Wind velocity**

Wind plays a major role in maintenance of aeration, pollination, etc. in crop's microclimate. However, high wind velocity over the cropped area can exert stress on crop. Wind velocity occurs due to movement of wind from one direction to other at a particular speed. It can create high evapo-transpiration, sand injury, crop lodging, pollen shedding, loss of pollen through desiccation, etc.

## **2.8 Chemical toxicity**

Continuous dependence on chemical based inorganic fertilizers and pesticides specially after green revolution is a great concern now in present day intensive agriculture condition. Further, rapid industrialization and excessive use of untreated sewage water hampers crop's growth and productivity through exerting detrimental impacts of the chemical toxicity on the soil- plant-atmospheric continuum.

#### **2.9 Nutrient toxicity/deficiency**

Nutrient toxicity or deficiency resulted from excessive or scarce application of fertilizers and manures as well as soil own nutritional status impairs plant growth, productivity and quality of the crop. This situation is very common in today's intensive agriculture due to non-judicious, unscientific nutrient management practices by the farmers.

## **3. Negative impacts of abiotic stresses on crop**

Plants are negatively impacted by abiotic stress. In most cases, abiotic stresses exert combined impact on crop plants and it causes more harm over individual impact of stress. Hydrogen peroxide, hydroxyl radicals, superoxide radicals, singlet oxygen and other reactive oxygen species (ROS) are synthesized under various abiotic stresses, specially under drought stress. In combinations, these cause lipid peroxidation, protein oxidation etc. and affect nucleic acids and enzyme activity resulting in death of cell. Accordingly, plants adopt defensive mechanisms against stresses. For instance, under drought stress, partial or complete closure of stomata is the one such adoptive approach by plants, which further restricts entry of sunlight, CO2 and

impairs electron flow through electron transport chain, resulting in decline in photosynthesis. Various negative impacts of abiotic stress on plants are shown in **Figure 2** and **Table 1** and highlighted hereunder.

## **3.1 Drought stress**

Drought stress arises under water scarcity. It hampers seed germination as well as early stand establishment of a crop arising through depletion of seed reserves and mechanical obstruction by the hard soil under drought, resulting in poor vegetative growth and yield of crop. The various impacts of drought on physiological and biochemical activities of plants are shown in **Figure 3**. When drought arises, cell solutes concentrations increase due to less water uptake and it not only causes high intra- and inter-competitions for water among crop plants and between crop and weeds, but also exerts toxicity on plants. Further, under drought condition, nutrients show variations in their availability for plant's uptake. Few nutrients become more available (viz. nitrogen) and few become unavailable or less available (viz. phosphorus), while no distinct impact of drought occurs on some nutrients (viz. potassium). This creates alterations in nutrient uptake by plants resulting in impairment of nutrient metabolisms in cell [36]. Under drought stress, as the activities of enzymes such as nitrate reductase, glutamine synthetase, etc. decrease, ammonia assimilation to organic form is restricted. Among the different categories of plants, C4 ones suffer more than C3 plants due to closure of stomata resulting in less photosynthesis [37].

## **3.2 Flood stress**

When flood occurs, anaerobic situation arises due to water logging or submergence, which further causes depletion in oxygen as well as restriction of movement of oxygen and other gases in root zone of plant. As a consequence, chlorosis of plant leaves and decay/death of cell occur. Less root respiration, poor root proliferation and

**Figure 2.** *Influence of abiotic stress on plant [7].*


#### **Table 1.**

*Negative impacts of abiotic stresses on production of various crops.*

other physiological disorders are some common phenomena visible under flood condition. Various negative impacts of flood are shown in **Figure 3**.

#### **3.3 Salinity stress**

Salinity creates two prime impacts on plants viz. osmotic stress and ion toxicity. Under the situation of salinity, drought stress is aggravated due to limited water uptake by plants from the soil resulting from greater osmotic pressure to root cell (osmotic pressure of soil solution > osmotic pressure in plant's cell sap). Oxidative damage due to soil salinity include detrimental impact on protein, nucleic acid and certain enzymes of plant as there is synthesis of ROS [38]. Under soil salinity, even if

#### **Figure 3.**

*Negative impacts of water stress (drought and flood) on plants.*

uptake of water takes place, there is also intrusion of various salts (Na<sup>+</sup> , Cl, etc.) inside the plant along with water, which exert negative impact on plant's cell through by impairing activities of various essential enzymes. Plants show burnt like visual symptoms on leaves under excessive salt uptake. Salt stress not only increases the Na<sup>+</sup> , Cl, etc., but also causes deficiency of various essential elements like calcium (Ca2+), potassium (K<sup>+</sup> ), magnesium (Mg2+), nitrate (NO3 ), etc. in rhizospheric zone of soil. Calcium (Ca2+), potassium (K+ ), magnesium (Mg2+), nitrate (NO3 ) are known to influence photosynthesis and therefore, their limited uptake by plants under soil salinity results in less photosynthesis and translocation of assimilates from source to sink. Some major impacts of soil salinity include less leaf expansion, stunted growth, less dry weight of plant, sterility of florets, loss of pollen viability, high epidermal thickness, mesophyll thickness, palisade cell length and diameter, spongy cell diameter, reduction of intercellular space in leaves of plant [7]. Partial or complete of stomata under high salt situation causes less transpiration and cell division resulting in reduction in plant's growth, defoliation and senescence of aerial parts and eventually, plant dies [39]. Under salinity stress, Na<sup>+</sup> /K<sup>+</sup> ratio of the cell is excessively increased, resulting in reduction in cell turgidity, enzyme activity and membrane potential of plant. Further, due to abundance of Na<sup>+</sup> in cell, various essential enzymatic activities get downregulated resulting in impairments of cell expansion as well as division, membrane stability and cytosolic metabolism.

## **3.4 Temperature stress**

High temperature or heat stress increases evapotranspiration loss of water resulting in drought like situation. This is further triggered under increase of soil temperature coupled with drought. Due to high temperature, respiration exceeds photosynthesis resulting in depletion of food reserve or loss of carbon (respiration rate doubles with each 10°C rise in tissue temperature). It is also observed that sudden temperature rise causes relatively more harm than gradual increase in temperature due to higher reductions of biochemical, physiological and molecular activities of the plant by sudden temperature rise. Among the categories of plant, C3 plants suffer comparatively more than C4 plants due to fluctuations in energy supply and carbon metabolisms under high temperature (**Figure 4**) [40].

#### **Figure 4.**

*Negative impacts of temperature stress (heat and cold) on plants.*

On the other hand, cold stress or low temperature causes chilling and freezing injuries to the plant. Chilling injury results in disfunctioning of physiological properties, while freezing injury results in cell dehydration. Some major impacts of cold stress on plant include Wilting, bleaching through pigment photo-oxidation, leaf necrosis, browning, cell death, etc.

#### **3.5 Heavy metal toxicity**

When there is abundance of heavy metals in soil, plant's physiological, morphological, biochemical, molecular activities are highly affected. After being taken up by the plant's roots, these metals (Pb, Cu, Hg, etc.) move inside the plant through xylem due to transpiration pool and negatively impact nutrient distribution, photosynthesis, enzyme activities, Cu/Zn-SOD, ethylene receptors, etc. resulting in reduction of molecular oxygen content and increment of ROS [41]. Synthesis of ROS thereafter, damages the plant at cellular level.

#### **3.6 Other abiotic stresses**

Chemical toxicity/persistence in environment is a great concern today, which results from excessive and unscientific application of chemicals. Environmental hazard or pollution under chemical toxicity leads to poor ecosystem health and diversity. These chemicals not only include pesticides but also cover inorganic fertilizer. Unnecessary use of pesticides and fertilizers are creating climate change resulting in reductions of crop growth, yield and quality. Specifically, complete dependence on chemicals for crop production results in damage of soil health and eventually, soil productivity declines. Contamination of underground fresh water as well as surface water, air pollution, land pollution, etc. is commonly associated with chemical toxicity. Plants known to be grown in an area earlier, are facing trouble in adaptation to

changing climate in the same area. Changing climate is linked with various biotic and abiotic stresses which exert detrimental impacts on crop's germination, photosynthesis, translocation of assimilates, etc. and thereby, reduces crop yield. On a contrary, nutrient deficiency arises due to scarcity of nutrients in soil, resulting in their less uptake by plant roots and translocation inside the plants. As a consequence, plants show various deficiency symptoms and its growth diminishes, leading to poor yield and quality of crop.

When wind blows at high velocity over the crop field, plants specially the tall growing or weaker one lodges down, resulting in poor growth, shedding of flower, pollen, grains and thereby, reduction in yield. High wind velocity also causes soil erosion and washes the essential nutrients away from plants. Further, there is an increase in evapotranspiration loss of water under high wind velocity, which demands for frequent water application leading to high cost of cultivation and failure of supply of water leads to poor growth and yield of crop.

Light is one of the prime requisites for photosynthesis and therefore excessive light can disrupts photosynthetic apparatus (photoinactivation and photodamage), while scarcity of light reduces photosynthesis and dry matter production. Plant's growth reduces when light is less or shading by taller plants/trees or other structures occurs. Due to hot sunlight intensity, heat stress or drought stress occurs which alone or together, affects the crop growth. UV ray impairs DNA and causes leaf bleaching, oxidative stress through synthesizing ROS. Under excessive light, breakdown of D1 protein of PS II and decrement of PS I polypeptides like PsaA, PsaB, and PsaC occurs in plants [42].

## **4. Organic farming and its components**

Sustainable agriculture greatly relies on non-chemical, eco-friendly organic farming approaches. Organic farming is defined as holistic production management system which promotes and improves agro-ecosystem health covering bio-diversity, biological cycle and soil biological properties. It completely or largely excludes the use of synthetic off-farm inputs like fertilizers, pesticides, growth regulators, livestock feed additives, etc. and mostly relies on on-farm agronomic, biological and mechanical inputs such as crop rotations, crop residues, organic manures, biofertilizers, green manuring, organic wastes, mineral grade rock additives, biological means of nutrient mobilization and plant protection (botanical pesticides), etc. leading to improvement of soil health, crop growth and yield as well as safety of environment. The major components of organic farming are briefly highlighted below.

#### **4.1 Organic manures and biochar**

Organic manures are the sources of nutrients produced by decomposition of organic waste materials (crop residues, plant-based wastes from house/farm/market, etc., animal-based wastes like urine, dung, litter, excreta, etc.) through microbial actions. These are known as bulky organic manures (FYM, vermicompost, poultry manures, common compost, night soil, sewage and sludge, kitchen compost, etc.) as their requirements are high. Besides, there are concentrated organic manures like oilcakes, bone meal, blood meal, horn and hoof meals, fish meal, meat meal, etc. in which more nutrients are present and they, therefore, supply different nutrients relatively in large quantities from unit quantity applied than bulky ones. Apart from

solid organic manures, there are various organic liquid manures/ITK formulations such as *Jiwamrit*, *Beejamrit*, *Amrit pani*, *Kunapajala*, *Panchagavya*, *Sanjeevani*, etc. are also used to improve soil health and thereby, crop growth.

Biochar is an excellent soil ameliorant produced under high temperature through controlled pyrolysis of organic substances. Quality of biochar depends on feedstock, temperature and pyrolysis conditions and time. Application of biochar improves plant growth and yield by reviving soil health.

## **4.2 Crop rotation and other agronomic practices**

Crop rotation involves diversification of crops, that is, growing of different crops in succession on same field to avoid pest, disease and weed infestation, improve soil fertility, recycle nutrient reserves, utilize different resources properly, enhance crop productivity, profitability, etc. Besides, there are various other agronomic practices such as variety selection, land preparation, mulching, crop residue retention on soil surface, manure application, time and method sowing, seed rate, spacing and depth, physical, cultural or biological methods of weed, pest and disease control, timely and adequate irrigation, timely harvest and post-harvest operations, followed in organic farming to enhance crop productivity, quality and profitability in production.

## **4.3 Crop residue**

Crop residue is the remaining left after harvesting and separating the economic part from the entire plants. These residues are often burnt leading to environmental pollution. There can be multiple uses of these crop residues like mulching materials, livestock feed, raw materials for manure preparation, substrates for mushroom cultivation, roof thatching, etc. Crop residues can conserve soil moisture, reduce weed infestation and promote crop protection.

### **4.4 Bio-fertilizers**

These are the substances containing living organisms, that is, micro-organisms which are helpful for crop growth and productivity by improving soil health and fertility as well as uptake of nutrients and water by the plants. Seed inoculation or soil application of biofertilizer containing various bacteria (rhizobium, azotobacter, azospirillum, etc.), fungi (VAM, AMF, *Penicillium* sp., *Aspergillus awamori*, etc.), azolla, blue green algae, etc. can help the crop growth either by drawing nutrients and water near rhizospheric zone from distant area or by fixing atmospheric nitrogen in soil leading to enhancement of soil fertility.

#### **4.5 Bio-pesticides and other protection measures**

Bio-pesticides such as nicotine, pyrethrum, rotenone, subabilla, ryanin, margosa, neem, etc. are natural plant-based products containing secondary metabolites like alkaloids, terpenoids, phenolics and minor secondary chemicals. Besides, resistant variety selection, myco-pesticides, release of natural enemies or growing trap crop or plants which act as host for biocontrol agents can protect crops from disease and pest damages. Further, various agronomic approaches like mulching, soil solarization, stale seed bed technique, timely and line sowing, crop rotation, intercropping, smother crops, use of botanical extracts, etc. can suppress weed problem.

## **5. Different organic farming approaches to mitigate abiotic stresses**

Over the years, organic farming has served as an eco-friendly approach to improve agricultural productivity in a sustainable manner. Further, it acts as buffer against various biotic and abiotic stresses which are often less highlighted. In the following section, different organic farming practices having the potential to mitigate various abiotic stresses are mentioned.

*Mulching:* In organic farming, mulching with straw, compost and other crop residues plays a key role in mitigating drought stress along with associated salt accumulation on soil surface. Further, it reduces the chances of loss of surface soil nutrients by restricting the soil erosion arising due to direct impact of rainfall or high runoff velocity. Mulch materials also act as insulators which keep the soil cool during warm weather and hot during winter months and thereby, solving the issues of heat and cold injuries to a high extent. Decrease of soil temperature by 1–2°C has been reported by Král et al. [43]. Apart from their role in soil and moisture conservation and checking different abiotic stresses, mulch materials like crop residues, compost, etc. can improve soil microbial activities and add essential nutrients through decomposition over the time [43]. In reality, abiotic stresses are most often associated with biotic stresses. Mulching, beside alleviation of various abiotic stresses, also suppresses various weed infestations in crop field. All these benefits are directly reflected to high crop growth, yield and quality under climate change scenario.

*Sea weed extracts:* Sea weed extracts are now emerging as one of potential sources of nutrients in organic farming for crop production. They contain nutrients, plant growth promoting substances, enzymes as well as antioxidants which help the crop cope up with salinity, heat and drought stresses. Besides, the use of sea weed extracts for cold tolerance as well as associated nutrient deficiency is now emerging. Algal extracts trigger a number of pathways to enhance stress tolerance through scavenging ROS. These extracts improve soil properties to conserve water well and thus, allow crop to survive under drought. Earlier, the use of these extracts was successful on Kentucky bluegrass (*Poa pratensis* L. cv. Plush) to mitigate salinity stress also [44].

*Organic manures and biochar:* Organic manures as well as green manure plants contain various nutrients, growth regulators, micro-organisms, etc. which not only improve soil fertility by solving nutrient scarcity stress but also improve overall soil health to a high extent. Increasing soil porosity, aggregate stability, reduction of compactness, etc. increase water holding capacity of the soil and thereby, address the issues of drought stress. Further, moderation of soil temperature, greater aeration in root zone, beneficial micro-organisms' activities in soil improves crop growth and yield. It has been widely noted that organic matter through decomposition releases humic and fulvic acids which alleviate abiotic stresses.

Apart from the organic manures, biochar application can alleviate various abiotic stresses specially drought stress. Shashi et al. [45] observed positive result on maize from rice husk @ 20 t/ha biochar under drought condition by enhancing bacterial and fungal communities in soil. Biochar specially from poultry manure shows excellent properties to mitigate salinity stress by reducing Na and increasing CEC and SOC contents in soil. Further, biochar can protect plants from high and low temperatures as well as alleviates metal toxicity by immobilizing heavy metals, followed by reducing their mobility. Positive impact of biochar in mitigating different abiotic stresses in rice is summarized in **Table 2**.

*Biofertilizers and bio-stimulants:* Biofertilizer is known to improve soil fertility and overall soil health through accelerating beneficial micro-organisms' activities. Besides,


#### **Table 2.**

*Positive impact of biochar in mitigating different abiotic stresses in rice.*

biofertilizer is one of the key components of organic farming to alleviate various abiotic stresses. Various types of biofertilizer helps the crop to tolerate or overcome stresses resulting in good growth and productivity under stress situation. It has been also found that seed bio priming with micro-organisms alleviates various abiotic stresses through improving germination and early plant stand establishment [60]. Bio priming increases the osmolyte concentrations leading to high cell wall elasticity and turgid weight to dry weight ratio. Further, endophytic synthesis of alkaloids protects macromolecules through ROS scavenging activities. Plant growth promoting rhizobacteria (PGPR) improves drought responsive genes' expression through high ROS scavenging activities. It also synthesizes phytohormones like IAA, GA3, etc. resulting in high plant growth under stress. PGPR also synthesizes exopolysaccharides resulting in good soil structure and uptakes of nutrients and water. Various endophytic micro-organisms also confer abiotic stress tolerance in plants through activating host stress response as well as through synthesizing biochemicals against stresses. A specific category of microorganisms known as arbuscular mycorrhizal fungi (AMF) is well known to mitigate negative impacts of various abiotic stresses on crop by improving soil health and plant's defense mechanism. It makes symbiotic relationship with roots of around 90% of the plant types. Use of AMF as biofertilizer/bio-inoculant is an emerging strategy specially under climate change scenario. The fungal network extends as secondary root system and helps the crop to draw nutrients and water from distant areas. Further, it plays a key role in regulating anti-oxidant activities (CAT, POX, SOD, GST, etc.) of plants under specific or combined stress situation resulting in scavenging of ROS and improvement crop growth, yield and quality. Various stress alleviating properties of micro-organisms in the form of biofertilizers/priming are shown in **Table 3**.

Bio-stimulants are organic or inorganic substances rich in bioactive compounds and/or micro-organisms, which improve crop growth through developing root for



#### **Table 3.**

*Micro-organisms in the form of biofertilizers/priming against abiotic stresses.*

high absorption and assimilation efficiency of nutrients, regulating proper water balance in plants as well as tolerating various abiotic stresses by synthesizing proline, simple sugars, alcohols, abscisic acid, osmotic compounds and antioxidants (to scavenge ROS) [101]. Role of bio-stimulants in plants is shown in **Figure 5**. It increases the contents of carotenoids, phenolic compounds and other secondary metabolites in plants as defense against stresses. It is applied as soil drench (directly/through irrigation) or foliar spray or treatment of seeds. Mitigation of various abiotic stresses by biostimulants is listed in **Table 4**.

*Crop rotation and various agronomic interventions:* Crop rotation is one of the key principles of conservation agriculture. It is always suggested to add leguminous crop in rotation to revive soil fertility after cultivation of a soil exhaustive crop through fixing atmospheric nitrogen. Further, biomass incorporation in soil results in addition of SOC content and thereby, causes improvement of soil porosity, water holding capacity, soil fertility, etc. leading to protection of plants against drought, salinity, high temperature stress as well as nutrient deficiency. Growing a shallow rooted crop after deep rooted crop helps in utilization of nutrients and water from various depths of soil profile so that plant can't experience nutrient and water scarcity.

Various other agronomic practices also can protect the crop from being affected by abiotic stresses under climate change scenario (**Figure 6**, **Table 5**). For instance, proper selection of resistant/tolerant crop and varieties under a prevalent abiotic stress is one useful strategy. To achieve this, breeding activities should include identification of responsive genes. Grafting is another one, which is widely used in horticulture to counter various abiotic stresses specially, salinity, nutrient or water deficiency, heavy metal toxicity, etc. Here, scion susceptible to stress is grafted to stress tolerant root stock. Exogenous application of plant components such as amino acid, sugars, etc. and phytohormones such as ABA, GA3, jasmonic acid, salicylic acid, brassinosteriods, etc. protects crop from abiotic stresses. Application of citric acid and vitamin C exhibit antioxidant properties which inactivates heavy metals such as Cu, Pb, Al, etc. as well as helps crop to overcome salinity and drought stresses through ROS scavenging activities. Soil and foliar applications of humic substances, beneficial fungi, bacteria, chitosan, sea weed extracts, etc. can combat abiotic stresses. Tillage also plays key role in conserving moisture and nutrients as well as breaking hard pan and high percolation of water and thereby, mitigates drought, flood and salinity. Keeping the land fallow for a season or year can rejuvenate the soil fertility and

**Figure 5.** *Role of bio-stimulants in plants.*






**Table 4.**

*Mitigation of abiotic stresses through various bio-stimulants.*

**Figure 6.**

*Abiotic stress mitigation through various organic farming practices.*

moisture content for next crop. Timely and properly sowing, adequate seed rate, spacing and depth, seed treatment also allows the crop to grow and utilize resources properly resulting in surviving and withstanding of climate change scenario. For instance, wheat, if sown on time, can escape terminal heat stress. Further, adequate and timely water, nutrient and interculture (weeding) managements accelerate crop growth by conservating water, nutrients, light, etc. which otherwise could be utilized by weeds and thereby, mitigate drought, salinity, nutrient deficiency, etc. Tall variety is susceptible to lodge by high wind velocity, while dwarf, robust variety can withstand the wind stress. Shelterbelt also protects the crop from high wind. Sometimes, crop suffers from hot sunlight and requires shading from tall growing crop and thus, intercropping or agroforestry is beneficial. On a contrary, shading of tall weeds on crop affects crop growth and therefore, timely weed management is needed. Under saline condition, frequent flooding with irrigation water or irrigation to root by drip method, scraping of surface salts, application of plant growth promoting bacteria, etc. are the key mitigation practices. PGPB alleviates salinity through hydraulic conduct, osmotic accumulation, toxic sodium removal, higher osmotic activity. Further, use of organic product such as brewer's spent grain as soil amendment not only improves soil


**Table 5.**

*Agronomic management practices in organic farming to mitigate specific abiotic stress.*

fertility but also alleviates heavy metal, nutrient deficiency, salinity, drought stresses, etc. Intercropping/Mixed cropping also conserves soil and water, suppresses weeds, reduces salt accumulation on surface through evaporation and thereby, alleviates various stresses. Sometimes, allelopathic potential of many crops on weeds are utilized to suppress weeds resulting in conservation of resources and good crop growth. Under the scarcity of water, precise and wise use of water, clipping of leaves (to reduce transpiration water loss), organic anti-transpirants (like wax, panchagavya)

application, broadcasting of seeds, closer spacing, more plant population/hill, double transplanting, etc. are useful. Apart from drainage, double transplanting is also beneficial for flood condition where main field is too flooded to transplant seedlings on time. It is well known fact that various biotic stresses like pest, disease and weeds trigger abiotic stresses. Addressing these biotic stresses by botanical extracts, biopesticides, release of natural enemies or living organisms, trap cropping, etc. can help the crop to avoid various abiotic stresses.

## **6. Conclusion**

Abiotic stress is creating detrimental effect on living organisms specially on plants since long. Its negative impact on crop is becoming prominent in recent days in the context of climate change scenario. In most of the cases, an abiotic stress combines with other abiotic or biotic stresses to exert combined impact on crop growth, yield and quality and the extent of impact on crop varies from mild to severe resulting in hampering crop growth accordingly. Although plants adopt some internal defensive mechanisms to counter these stresses, in most of the times, they require external stimuli/practices/inputs to mitigate abiotic stresses. Due to population rise, crop yield loss through abiotic stresses cannot be accepted at this moment or future and therefore, suitable agronomic and breeding interventions are highly needed. Since chemical-based farming is a barrier against sustainable agricultural production as it deteriorates soil health and is hazardous to the environment due to toxic chemical footprint, organic farming is emerging as its potential alternative. Various organic farming inputs such as organic manures, biofertilizers, bio-priming with microorganisms, bio-stimulants (seaweed extracts, humic acid, micro-organisms etc.), mulches, biochar etc. have the potential to mitigate abiotic stresses under climate change scenario. Further, organic farming practices like crop rotation, inter cropping, tillage, time and method of sowing, nutrient, water and intercultural operations, use of PGPB, organic formulations, grafting, selection of resistant/tolerant varieties and other scientific/wise uses of organic inputs can help the crop to mitigate/escape the detrimental effects of various abiotic stresses to a great extent. Still, there is need on proper research or study on the abiotic stress potential of organic farming further. Available organic farming technologies as well as information/awareness about them are very also scanty at this moment. Therefore, proper multi-locational research experiments, transfusion of modern practices/awareness through strong extension services, policy interventions and advanced breeding approaches are highly required to address harmful abiotic stresses as well as to get high crop growth, yield and quality. Various strategies should be jointly implemented rather than using individually to get the best result from organic farming in making crop to cope up successfully with climate change scenario.

*Plant Physiology Annual Volume 2023*

## **Author details**

Saikat Biswas<sup>1</sup> , Rupa Das<sup>2</sup> \* and Lay Lay Nwe<sup>1</sup>

1 Department of Agronomy, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, India

2 Department of Seed Science and Technology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, India

\*Address all correspondence to: rupadasbiswas18@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.

## **References**

[1] Chaves MM, Maroco JP, Pereira JS. Understanding plant responses to drought- from genes to the whole plant. Functional Plant Biology. 2003;**30**: 239-264

[2] Rao IM, Beebe SE, Polania J, Ricaurte J, Cajiao C, Garc'ıa R, Rivera M. Can tepary bean be a model for improvement of drought resistance in common bean? African Crop Science Journal. 2013;**21**:265-281

[3] Prasad PVV, Pisipati SR, Momcilovic I, Ristic Z. Independent and combined effects of high temperature and drought stress during grain filling on plant yield and chloroplast protein synthesis elongation factor (EFTu) expression in spring wheat. Journal of Agronomy and Crop Science. 2011;**197**: 430-441

[4] Acquadh. Principle of Plant Genetics and Breeding. Oxford: Willey Blackwell; 2007

[5] Kumar S. Abiotic stresses and their effects on plant growth, yield and nutritional quality of agricultural produce. International Journal of Food Science and Agriculture. 2020;**4**(4): 367-378

[6] Fahad S, Bajwa AA, Nazir U, Anjum SA, Farooq A, Zohaib A, et al. Crop production under drought and heat stress: Plant responses and management options. Frontiers in Plant Science. 2017;**29**:8

[7] Das R, Biswas S. Influence of abiotic stresses on seed production and quality. In: Seed Biology Updates. IntechOpen; 2022. pp. 1-24

[8] Bockheim JG, Gennadiyev AN. The role of soil-forming processes in the definition of taxa in soil taxonomy and

the world soil reference base. Geoderma. 2000;**95**(1–2):53-72

[9] Abrol IP. Salt-affected soils: An overview. In: Chopra VL, Paroda SL, editors. Approaches for Incorporating Drought and Salinity Resistance in Crop Plants. New Delhi: Oxford and IBH Publishing Company; 1986. pp. 1-23

[10] Levitt J. Responses of plants to environmental stresses. Academic Press. 1980;**1**:496

[11] Wuana RA, Okieimen FE. Heavy metals in contaminated soils: A review of sources, chemistry, risks and best available strategies for remediation. ISRN Ecology. 2011;**2011**:1-20. DOI: 10.5402/2011/402647

[12] Farooq M, Nadeem F, Gogoi N, Ullah A, Alghamdi SS, Nayyar H, et al. Heat stress in grain legumes during reproductive and grain-filling phases. Crop & Pasture Science. 2017;**68**: 985-1005

[13] Bethke PC et al. Tuber water and pressure potentials decrease and sucrose contents increase in response to moderate drought and heat stress. American Journal of Potato Research. 2009;**86**:519

[14] Kang JS, Singh H, Singh G, Kang H, Kalra VP, Kaur J. Abiotic stress and its amelioration in cereals and pulses: A review. International Journal of Current Microbiology and Applied Sciences. 2017;**6**(3):1019-1045

[15] Singh S et al. Effect of water stress at different stages of grain development on the characteristics of starch and protein of different wheat varieties. Food Chemistry. 2008;**108**:130-139

[16] Di Caterina R et al. Influence of salt stress on seed yield and oil quality of two sunflower hybrids. Annual Applied Biology. 2007;**151**:145-154

[17] Liu Y, Li J, Zhu Y, Jones A, Rose RJ, Song Y. Heat stress in legume seed setting: Effects, causes, and future prospects. Frontiers in Plant Science. 2019;**10**:938

[18] Vaz Patto MC, Amarowicz R, Aryee ANA, Boye JI, Chung HJ, Martín-Cabrejas MA, et al. Achievements and challenges in improving the nutritional quality of food legumes. Critical Reviews in Plant Sciences. 2015;**34**:105-143

[19] Dong B, Zheng X, Liu H, Able JA, Yang H, Zhao H, et al. Effects of drought stress on pollen sterility, grain yield, abscisic acid and protective enzymes in two winter wheat cultivars. Frontiers in Plant Science. 2017;**8**(June):1-14

[20] Sekhon HS et al. Water use efficiency under stress environments. In: Climate Change and Management of Cool Season Grain Legume Crops. The Netherlands: Springer; 2010. pp. 207-227

[21] Ni Z, Li H, Zhao Y, Peng H, Hu Z, Xin M, et al. Genetic improvement of heat tolerance in wheat: Recent progress in understanding the underlying molecular mechanisms. Crop Journal. 2018;**6**(1):32-41

[22] Asseng S, Foster I, Turner NC. The impact of temperature variability on wheat yields. Global Change Biology. 2011;**17**(2):997-1012

[23] Kazai P, Noulas C, Khah E, Vlachostergios D. Yield and seed quality parameters of common bean cultivars grown under water and heat stress field conditions. AIMS Agriculture and Food. 2019;**4**:285-302

[24] Wilhelm I. Crop physiology and metabolism. Crop Science. 1999;**39**: 1733-1741

[25] Jagadish S, Craufurd P, Wheeler T. High temperature stress and spikelet fertility in rice (*Oryza sativa* L.). Journal of Experimental Botany. 2007;**58**(7): 1627-1635

[26] Redden RJ, Vara HJL, Prasad PV, Ebert AW, Yadav SS, O'Leary GJ. Temperature, climate change, and global food security. Temperature and Plant Development. 2014;**8**:181-202

[27] Vafa P et al. The effect of drought stress on grain yield, yield components and protein content of durum wheat cultivars in Ilam Province, Iran. International Journal of Agricultural and Biosystems Engineering. 2014;**8**:631-636

[28] Wardlaw IF et al. Contrasting effects of chronic heat stress and heat shock on kernel weight and flour quality in wheat. Functional Plant Biology. 2002;**29**:25-34

[29] Triboï E et al. Environmentally induced changes in protein composition in developing grains of wheat are related to changes in total protein content. Journal of Experimental Botany. 2003; **54**:1731-1742

[30] Flagella Z et al. Changes in seed yield and oil fatty acid composition of high oleic sunflower (*Helianthus annuus* L.) hybrids in relation to the sowing date and the water regime. European Journal of Agronomy. 2002;**17**:221-230

[31] Lin CJ et al. Influence of high temperature during grain filling on the accumulation of storage proteins and grain quality in rice (*Oryza sativa* L.). Food Chemistry. 2010;**58**:10545-10552

[32] Sadeghipour O. The influence of water stress on biomass and harvest

index in three mung bean (*Vigna radiata* L. (Wilczek)) cultivars. Asian Journal of Plant Sciences. 2009;**8**:245-249

[33] Silva JAB et al. Microtuberization of Andean potato species (*Solanum* spp.) as affected by salinity. Scientia Horticulturae. 2001;**89**:91-101

[34] Dornbos DL, Mullen RE. Soybean seed protein and oil contents and fatty acid composition adjustments by drought and temperature. Journal of the American Oil Chemists' Society. 1992;**69**: 228-231

[35] Hoegy P et al. Impacts of temperature increase and change in precipitation pattern on crop yield and yield quality of barley. Food Chemistry. 2013;**136**:1470-1477

[36] Samarah NH. Effects of drought stress on growth and yield of barley. Agronomy for Sustainable Development. 2005;**25**(1):145-149

[37] Alfonso SU, Brüggemann W. Photosynthetic responses of a C3 and three C4 species of the genus Panicum with different metabolic subtypes to drought stress. Photosynthesis Research. 2012;**112**:175-191

[38] Gomez JM, Jimenz A, Olmas E, Sevilla F. Location and effects of long term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea (*Pisum sativum* cv. Puget) chloroplasts. Journal of Experimental Botany. 2004;**55**:119-130

[39] Shannon MC, Grieve CM. Tolerance of vegetable crops to salinity. Scientia Horticulturae. 1999;**78**:5-38

[40] Salvucci ME, Crafts-Brandner SJ. Relationship between the heat tolerance of photosynthesis and the thermal stability of rubisco activase in plants

from contrasting thermal environments. Plant Physiology. 2004;**134**:1460-1470

[41] Polle A, Schützendübel A. Heavy metal signalling in plants: Linking cellular and organismic responses. In: Hirt H, Shinozaki K, editors. Plant Responses to Abiotic Stress. Topics in Current Genetics. Vol. 4. Switzerland: Springer Nature; 2003. pp. 187-215

[42] Jiao S, Hilaire E, Guikema JA. Identification and differential accumulation of two isoforms of the CF1-b subunit under high light stress in *Brassica rapa*. Plant Physiology and Biochemistry. 2004;**42**:883-890

[43] Král M, Dvořák M, Capouchová I. The straw as mulch and compost as a tool for mitigation of drought impacts in the potatoes cultivation. Plant, Soil and Environment. 2019;**65**(11):530-535

[44] Nabati DA, Schmidt RE, Parrish DJ. Alleviation of salinity stress in Kentucky bluegrass by plant growth regulators and iron. Crop Science. 1994;**34**(1):198-202

[45] Shashi M, Mannan M, Islam M, Rahman M. Impact of rice husk biochar on growth, water relations and yield of maize (*Zea mays* L.) under drought condition. The Agriculturists. 2018;**16**: 93-101

[46] Khan S, Wang N, Reid BJ, Freddo A, Cai C. Reduced bioaccumulation of PAHs by *Lactuca sativa* L. grown in contaminated soil amended with sewage sludge and sewage sludge derived biochar. Environmental Pollution. 2013; **175**:64-68

[47] Dong D, Feng Q, Mcgrouther K, Yang M, Wang H, Wu W. Effects of biochar amendment on rice growth and nitrogen retention in a waterlogged paddy field. Journal of Soils and Sediments. 2014;**15**(1):153-162

[48] Jinyang W, Pan X, Liu Y, Zhang X, Xiong Z. Effects of biochar amendment in two soils on greenhouse gas emissions and crop production. Plant and Soil. 2012;**360**(1–2):287-298

[49] Kamara A, Kamara HS, Kamara MS. Effect of rice straw biochar on soil quality and the early growth and biomass yield of two rice varieties. Agricultural Sciences. 2015;**6**:798

[50] Feng J, Cheng R, Qul AA, Yan QG, Li YG, Jian BL, et al. Effects of biochar on sodium ion accumulation, yield and quality of rice in saline-sodic soil of the west of Songnen plain, northeast China. Plant, Soil and Environment. 2018;**64**: 612-618

[51] Ran C, Gulaqa A, Zhu J, Wang X, Zhang S, Geng Y, et al. Benefits of biochar for improving ion contents, cell membrane permeability, leaf water status and yield of rice under saline-sodic paddy field condition. Journal of Plant Growth Regulation. 2020;**39**:370-377

[52] Liu Y, Lu H, Yang S, Wang Y. Impacts of biochar addition on rice yield and soil properties in a cold waterlogged paddy for two crop seasons. Field Crops Research. 2016;**191**:161-167

[53] Haefele S, Konboon Y, Wongboon W, Amarante S, Maarifat A, Pfeiffer E, et al. Effects and fate of biochar from rice residues in rice-based systems. Field Crops Research. 2011;**121**: 430-440

[54] Liu Y, Yang S, Lu H, Wang Y. Effects of biochar on spatial and temporal changes in soil temperature in cold waterlogged rice paddies. Soil and Tillage Research. 2018;**181**:102-109

[55] Huang M, Long F, Jiang LG, Yang SY, Zou YB, Uphoff N. Continuous applications of biochar to rice: Effects on grain yield and yield attributes. Journal of Integrative Agriculture. 2019;**18**: 563-570

[56] Kartika K, Lakitan B, Wijaya A, Kadir S, Widur LI, Siaga E, et al. Effects of particle size and application rate of rice-husk biochar on chemical properties of tropical wetland soil, rice growth and yield. Australian Journal of Crop Science. 2018;**12**:817-826

[57] Fahad S, Hussain S, Saud S, Hassan S, Tanveer M, Ihsan MZ, et al. A combined application of biochar and phosphorus alleviates heat-induced adversities on physiological, agronomical and quality attributes of rice. Plant Physiology and Biochemistry. 2016;**103**: 191-198

[58] He T, Meng J, Chen W, Liu Z, Cao T, Cheng X, et al. Effects of biochar on cadmium accumulation in rice and cadmium fractions of soil: A three-year pot experiment. BioResources. 2017;**12**: 622-642

[59] Bian R, Joseph S, Cui L, Pan G, Li L, Liu X, et al. A three-year experiment confirms continuous immobilization of cadmium and lead in contaminated paddy field with biochar amendment. Journal of Hazardous Materials. 2014; **272**:121-128

[60] Glick BR, Cheng Z, Czarny J, Duan J. Promotion of plant growth by ACC deaminase-producing soil bacteria. European Journal of Plant Pathology. 2007;**119**:329-339

[61] Chi F, Yang P, Han F, Jing Y, Shen S. Proteomic analysis of rice seedlings infected by *Sinorhizobium meliloti* 1021. Proteomics. 2010;**10**:1861-1874

[62] Hussain N, Mujeeb F, Tahir M, Khan GD, Hassan NM, Bari A. Effectiveness of Rhizobium under

salinity stress. Asian Journal of Plant Sciences. 2002;**1**:12-14

[63] Antoun H, Prevost D. Ecology of plant growth promoting rhizobacteria. In: Siddiqui ZA, editor. PGPR: Biocontrol and Biofertilization. Dordrecht: Springer; 2005. pp. 1-38

[64] Yao L, Wu Z, Zheng Y, Kaleem I, Li C. Growth promotion and protection against salt stress by *Pseudomonas putida* Rs-198 on cotton. European Journal of Soil Biology. 2010;**46**:49-54

[65] Egamberdiyeva D. The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different soils. Applied Soil Ecology. 2007;**36**:184-189

[66] Alavi P, Starcher MR, Zachow C, Müller H, Berg G. Root-microbe systems: The effect and mode of interaction of stress protecting agent (SPA) *Stenotrophomonas rhizophila* DSM14405T. Frontiers in Plant Science. 2013;**4**:141

[67] Kohler J, Caravaca F. An AM fungus and a PGPR intensify the adverse effects of salinity on the stability of rhizosphere soil aggregates of *Lactuca sativa* Roldan. Soil Biology and Biochemistry. 2010;**42**: 429-434

[68] Gill SS, Khan NA, Tuteja N. Cadmium at high dose perturbs growth, photosynthesis and nitrogen metabolism while at low dose it up regulates sulfur assimilation and antioxidant machinery in garden cress (*Lepidium sativum* L.). Plant Science. 2012;**182**:112-120

[69] Samuel S, Muthukkaruppan SM. Characterization of plant growth promoting rhizobacteria and fungi associated with rice, mangrove and effluent contaminated soil. Current Botany. 2011;**2**:22-25

[70] Baharlouei K, Pazira E, Solhi M. Evaluation of inoculation of plant growth-promoting rhizobacteria on cadmium. Singapore: International Conference on Environmental Science and Technology, IPCBEE. IACSIT Press; 2011; Vol. 6

[71] Bhardwaj D, Ansari MW, Sahoo RK, Tuteja N. Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microbial Cell Factories. 2014;**13**:66

[72] Ruiz-Sanchez M, Aroca R, Munoz Y, Polon R, Ruiz-Lozano JM. The arbuscular mycorrhizal symbiosis enhances the photosynthetic efficiency and the antioxidative response of rice plants subjected to drought stress. Journal of Plant Physiology. 2010;**167**: 862-869

[73] Hrkousse O, Simani A, Jadrane I, Aitboulahsen M, Mazri MA, Zouahri A, et al. Role of local biofertilizer in enhancing the oxidative stress defence systems of date palm seedling (*Phoenix dactylifera*) against abiotic stress. Applied and Environmental Soil Science. 2021;**6628544**:1-13

[74] Kohler J, Hernandez JA, Caravaca F, Rold´an A. Plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungi modify alleviation biochemical mechanisms in waterstressed plants. Functional Plant Biology. 2008;**35**(2):141

[75] Burd GI, Dixon DG, Glick BR. Plant growth-promoting bacteria that decrease heavy metal toxicity in plants. Canadian Journal of Microbiology. 2000;**46**: 237-245

[76] Madhaiyan M, Poonguzhali S, Sa T. Metal tolerating methylotrophic bacteria reduces nickel and cadmium toxicity and promotes plant growth of tomato (*Lycopersicon esculentum* L.). Chemosphere. 2007;**69**:220-228

[77] Safronova VI, Stepanok VV, Engqvist GL, Alekseyev YV, Belimov AA. Root-associated bacteria containing 1-aminocyclopropane-1 carboxylate deaminase improve growth and nutrient uptake by pea genotypes cultivated in cadmium supplemented soil. Biology and Fertility of Soils. 2006; **42**:267-272

[78] Wani PA, Khan MS, Zaidi A. Effect of metal tolerant plant growthpromoting Rhizobium on the performance of pea grown in metalamended soil. Archives of Environmental Contamination and Toxicology. 2008;**55**:33-42

[79] Ouledali S, Ennajeh M, Zrig A, Gianinazzi S, Khemira H. Estimating the contribution of arbuscular mycorrhizal fungi to drought tolerance of potted olive trees (Olea europaea). Acta Physiologiae Plantarium. 2018;**40**:1-81

[80] Pavithra D, Yapa N. Arbuscular mycorrhizal fungi inoculation enhances drought stress tolerance of plants. Groundwater for Sustainable Development. 2018;**7**:490-494

[81] Pedranzani H, Rodríguez-Rivera M, Gutiérrez M, Porcel R, Hause B, Ruiz-Lozano JM. Arbuscular mycorrhizal symbiosis regulates physiology and performance of *Digitaria eriantha* plants subjected to abiotic stresses by modulating antioxidant and jasmonate levels. Mycorrhiza. 2016;**26**: 141-152

[82] Rani B. Effect of arbuscular mycorrhiza fungi on biochemical parameters in wheat *Triticum aestivum* L. under drought conditions [Doctoral Dissertation]. Hisar: CCSHAU; 2016

[83] Nelsen CE, Safir GR. Increased drought tolerance of mycorrhizal onion plants caused by improved phosphorus nutrition. Planta. 1982;**154**:407-413

[84] Hajiboland R, Aliasgharzadeh N, Laiegh SF, Poschenrieder C. Colonization with arbuscular mycorrhizal fungi improves salinity tolerance of tomato *Solanum lycopersicum* L. plants. Plant and Soil. 2010;**331**:313-327

[85] Khalloufi M, Martínez-Andújar C, Lachaâl M, Karray-Bouraoui N, Pérez-Alfocea F, Albacete A. The interaction between foliar GA3 application and arbuscular mycorrhizal fungi inoculation improves growth in salinized tomato *Solanum lycopersicum* L. plants by modifying the hormonal balance. Journal of Plant Physiology. 2017;**214**:134-144

[86] Hajiboland R, Dashtebani F, Aliasgharzad N. Physiological responses of halophytic C4 grass, *Aeluropus littoralis* to salinity and arbuscular mycorrhizal fungi colonization. Photosynthetica. 2015;**53**:572-584

[87] Giri B, Kapoor R, Mukerji KG. Improved tolerance of *acacia nilotica*, to salt stress by arbuscular mycorrhiza, *Glomus fasciculatum*, may be partly related to elevated K/Na ratios in root and shoot tissues. Microbial Ecology. 2007;**54**:753-760

[88] Hashem A, Alqarawi AA, Radhakrishnan R, Al-Arjani AF, Aldehaish HA, Egamberdieva D, et al. Arbuscular mycorrhizal fungi regulate the oxidative system, hormones and ionic equilibrium to trigger salt stress tolerance in *Cucumis sativus* L. Saudi Journal of Biological Sciences. 2018;**25**: 1102-1114

[89] Hajiboland R, Joudmand A, Aliasgharzad N, Tolrá R,

Poschenrieder C. Arbuscular mycorrhizal fungi alleviate lowtemperature stress and increase freezing resistance as a substitute for acclimation treatment in barley. Crop & Pasture Science. 2019;**70**:218-233

[90] Mathur S, Jajoo A. Arbuscular mycorrhizal fungi protects maize plants from high temperature stress by regulating photosystem II heterogeneity. Industrial Crops and Products. 2020;**143**: 111934

[91] Ma J, Janoušková M, Ye L, Bai LQ, Dong RR, Yan Y, et al. Role of arbuscular mycorrhiza in alleviating the effect of cold on the photosynthesis of cucumber seedlings. Photosynthetica. 2019;**57**: 86-95

[92] Chu XT, Fu JJ, Sun YF, Xu YM, Miao YJ, Xu YF, et al. Effect of arbuscular mycorrhizal fungi inoculation on cold stress-induced oxidative damage in leaves of *Elymus nutans* Griseb. South African Journal of Botany. 2016;**104**: 21-29

[93] Kaldorf M, Kuhn AJ, Schröder WH, Hildebrandt U, Bothe H. Selective element deposits in maize colonized by a heavy metal tolerance conferring arbuscular mycorrhizal fungus. Journal of Plant Physiology. 1999;**154**: 718-728

[94] Kelkar TS, Bhalerao SA. Beneficiary effect of arbuscular mycorrhiza to *Trigonella foenum-graceum* in contaminated soil by heavy metal. Research Journal of Recent Sciences. 2013;**2**:29-32

[95] Jiang QY, Zhuo F, Long SH, Zhao HD, Yang DJ, Ye ZH, et al. Can arbuscular mycorrhizal fungi reduce Cd uptake and alleviate Cd toxicity of Lonicera japonica grown in Cd-added soils? Scientific Reports. 2016;**6**:21805

[96] Lingua G, Franchin C, Todeschini V, Castiglione S, Biondi S, Burlando B, et al. Arbuscular mycorrhizal fungi differentially affect the response to high zinc concentrations of two registered poplar clones. Environmental Pollution. 2008;**153**:137-147

[97] Li XL, Christie P. Changes in soil solution Zn and pH and uptake of Zn by arbuscular mycorrhizal red clover in Zncontaminated soil. Chemosphere. 2001; **42**:201-207

[98] Miller SP, Sharitz RR. Manipulation of flooding and arbuscular mycorrhiza formation influences growth and nutrition of two semiaquatic grass species. Functional Ecology. 2000;**14**:738-748

[99] Fougnies L, Renciot S, Müller F, Plenchette C, Prin Y, De Faria SM, et al. Arbuscular mycorrhizal colonization and nodulation improve flooding tolerance in Pterocarpus officinalis Jacq. seedlings. Mycorrhiza. 2007;**17**:159-166

[100] Solís-Rodríguez UR, Ramos-Zapata JA, Hernández-Cuevas L, Salinas-Peba L, Guadarrama P. Arbuscular mycorrhizal fungi diversity and distribution in tropical low flooding forest in Mexico. Mycological Progress. 2020;**19**:195-204

[101] Bulgari R, Franzoni G, Ferrante A. Biostimulants application in horticultural crops under abiotic stress conditions. Agronomy. 2019;**9**:306

[102] Pokluda R, Sękara A, Jezdinský A, Kalisz A, Neugebauerová J, Grabowska A. The physiological status and stress biomarker concentration of *Coriandrum sativum* L. plants subjected to chilling are modified by biostimulant application. Biological Agriculture and Horticulture. 2016;**32**:258-268

[103] Marfà O, Cáceres R, Polo J, Ródenas J. Animal protein hydrolysate as a biostimulant for transplanted strawberry plants subjected to cold stress. Acta Horticulturae. 2009;**842**: 315-318

[104] Polo J, Barroso R, Ródenas J, Azcón-Bieto J, Cáceres R, Marfà O. Porcine hemoglobin hydrolysate as a biostimulant for lettuce plants subjected to conditions of thermal stress. HortTechnology. 2006;**16**:483-487

[105] Botta A. Enhancing plant tolerance to temperature stress with amino acids: An approach to their mode of action. Acta Horticulturae. 2012;**1009**:29-36

[106] Korkmaz A, Korkmaz Y, Demirkiran AR. Enhancing chilling stress tolerance of pepper seedlings by exogenous application of 5 aminolevulinic acid. Environmental and Experimental Botany. 2010;**67**:495-501

[107] Kang SM, Khan AL, Waqas M, You Y-H, Hamayun M, Joo GJ, et al. Gibberellin-producing *Serratia nematodiphila* PEJ1011 ameliorates low temperature stress in *Capsicum annuum* L. European Journal of Soil Biology. 2015;**68**:85-93

[108] Petrozza A, Santaniello A, Summerer S, Di Tommaso G, Di Tommaso D, Paparelli E, et al. Physiological responses to Megafol® treatments in tomato plants under drought stress: A phenomic and molecular approach. Scientia Horticulturae (Amsterdam). 2014;**174**:185-192

[109] Xu C, Leskovar DI. Effects of *A. nodosum* seaweed extracts on spinach growth, physiology and nutrition value under drought stress. Scientia Horticulturae (Amsterdam). 2015;**183**: 39-47

[110] Mayak S, Tirosh T, Glick BR. Plant growth-promoting bacteria that confer

resistance to water stress in tomatoes and peppers. Plant Science. 2004;**166**: 525-530

[111] Goñi O, Quille P, O'Connell S. *Ascophyllum nodosum* extract biostimulants and their role in enhancing tolerance to drought stress in tomato plants. Plant Physiology and Biochemistry. 2018;**126**:63-73

[112] Kałuzewicz A, Krzesiński W, Spizewski T, Zaworska A. Effect of biostimulants on several physiological characteristics and chlorophyll content in broccoli under drought stress and rewatering. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2017;**45**: 197-202

[113] Petrozza A, Summerer S, Di Tommaso G, Di Tommaso D, Piaggesi A. An evaluation of tomato plant root development and morpho- physiological response treated with VIVA® by image analysis. Acta Horticulturae. 2013;**1009**: 155-160

[114] Heidari M, Golpayegani A. Effects of water stress and inoculation with plant growth promoting rhizobacteria (PGPR) on antioxidant status and photosynthetic pigments in basil (*Ocimum basilicum* L.). Journal of Saudi Society of Agricultural Sciences. 2012;**11**: 57-61

[115] Abd El-Mageed TA, Semida WM, Rady MM. Moringa leaf extract as biostimulant improves water use efficiency, physio-biochemical attributes of squash plants under deficit irrigation. Agricultural Water Management. 2017; **193**:46-54

[116] Asaf S, Hamayun M, Khan AL, Waqas M, Khan MA, Jan R, et al. Salt tolerance of *Glycine max*. L induced by endophytic fungus *Aspergillus flavus* CSH1, via regulating its endogenous

hormones and antioxidative system. Plant Physiology and Biochemistry. 2018;**128**:13-23

[117] Barnawal D, Bharti N, Pandey SS, Pandey A, Chanotiya CS, Kalra A. Plant growth-promoting rhizobacteria enhance wheat salt and drought stress tolerance by altering endogenous phytohormone levels and TaCTR1/ TaDREB2 expression. Physiologia Plantarum. 2017;**161**:502-514

[118] Kang SM, Khan MA, Hamyun M, Kim LR, Kwon EH, Kang YS, et al. Phosphate-solubilizing *Enterobacter ludwigii* AFFR02 and *Bacillus megaterium* Mj1212 rescues alfalfa's growth under post-drought stress. Agriculture. 2021;**11**: 485

[119] Niu X, Song L, Xiao Y, Ge W. Drought-tolerant plant growthpromoting rhizobacteria associated with foxtail millet in a semi-arid agroecosystem and their potential in alleviating drought stress. Frontiers in Microbiology. 2018;**8**:2580

[120] Batool T, Ali S, Seleiman MF, Naveed NH, Ali A, Ahmed K, et al. Plant growth promoting rhizobacteria alleviates drought stress in potato in response to suppressive oxidative stress and antioxidant enzymes activities. Scientific Reports. 2020;**2020**(10):1-19

[121] Begum N, Wang L, Ahmad H, Akhtar K, Roy R, Khan MI, et al. Coinoculation of arbuscular mycorrhizal fungi and the plant growth-promoting rhizobacteria improve growth and photosynthesis in tobacco under drought stress by up-regulating antioxidant and mineral nutrition metabolism. Microbial Ecology. 2022;**83**:971-988

[122] Chandra D, Srivastava R, Glick BR, Sharma AK. Drought-tolerant Pseudomonas spp. improve the growth

performance of finger millet (*Eleusine coracana* (L.) Gaertn.) under nonstressed and drought-stressed conditions. Pedosphere. 2018;**28**(2): 227-240

[123] Yasmin H, Nosheen A, Naz R, Bano A, Keyani R. L-tryptophan-assisted PGPR-mediated induction of drought tolerance in maize (*Zea mays* L). Journal of Plant Interactions. 2017;**12**(1):567-578

[124] Abdel Megeed TM, Gharib HS, Hafez EM, El-Sayed A. Effect of some plant growth regulators and biostimulants on the productivity of Sakha108 rice plant (*Oryza sativa* L.) under different water stress conditions. Applied Ecology and Environmental Research. 2021;**19**(4):2859-2878

[125] Francesca S, Cirillo V, Raimondi G, Maggio A, Barone A, Rigano MM. A novel protein hydrolysate-based biostimulant improves tomato performances under drought stress. Plants. 2021;**10**:1-13

[126] Ali S, Khan MA, Kim W-CJABC. *Pseudomonas veronii* KJ mitigates flood stress-associated damage in *Sesamum indicum* L. Applied Biological Chemistry. 2018;**2018**(61):575-585

[127] Rauf M, Awais M, Ud-Din A, Ali K, Gul H, Rahman MM, et al. Molecular mechanisms of the 1 aminocyclopropane-1-carboxylic acid (ACC) deaminase producing *Trichoderma asperellum* MAP1 in enhancing wheat tolerance to waterlogging stress. Frontiers in Plant Science. 2021;**11**:2213

[128] El-Bassiony AM, Ghoname AA, El-Awadi ME, Fawzy ZF, Gruda N. Ameliorative Effects of brassinosteroids on growth and productivity of snap beans grown under high temperature. Gesunde Pflanzen. 2012;**64**:175-182

[129] Nahar K, Hasanuzzaman M, Alam MM, Fujita M. Exogenous glutathione confers high temperature stress tolerance in mung bean (*Vigna radiata* L.) by modulating antioxidant defense and methylglyoxal detoxification system. Environmental and Experimental Botany. 2015;**112**:44-54

[130] Kaushal N, Gupta K, Bhandhari K, Kumar S, Thakur P, Nayyar H. Proline induces heat tolerance in chickpea (Cicer arietinum L.) plants by protecting vital enzymes of carbon and antioxidative metabolism. Physiology and Molecular Biology of Plants. 2011;**17**:203-213

[131] Kumar S, Kaushal N, Nayyar H, Gaur P. Abscisic acid induces heat tolerance in chickpea (*Cicer arietinum* L.) seedlings by facilitated accumulation of osmoprotectants. Acta Physiologiae Plantarum. 2012;**34**:1651-1658

[132] Khan MA, Asaf S, Khan AL, Jan R, Kang SM, Kim KM, et al. Extending thermotolerance to tomato seedlings by inoculation with SA1 isolate of *Bacillus cereus* and comparison with exogenous humic acid application. PLoS One. 2020; **15**(4):e0232228

[133] Kang SM, Khan AL, Waqas M, Asaf S, Lee KE, Park YG, et al. Integrated phytohormone production by the plant growth-promoting rhizobacterium *Bacillus tequilensis* SSB07 induced thermotolerance in soybean. Journal of Plant Interactions. 2019;**14**:416-423

[134] Quintero-Calderón EH, Sánchez-Reinoso AD, Chávez-Arias CC, Garces-Varon G, Restrepo-Díaz H. Rice seedlings showed a higher heat tolerance through the foliar application of biostimulants. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2021;**49**:1

[135] Niu C, Wang G, Sui J, Liu G, Ma F, Bao Z. Biostimulants alleviate

temperature stress in tomato seedlings. Scientia Horticulturae. 2021;**2022**(293): 110712

[136] Spinelli F, Fiori G, Noferini M, Sprocatti M, Costa G. A novel type of seaweed extract as a natural alternative to the use of iron chelates in strawberry production. Scientia Horticulturae (Amsterdam). 2010;**125**:263-269

[137] Cerdán M, Sánchez-Sánchez A, Jordá JD, Juárez M, Sánchez-Andreu J. Effect of commercial amino acids on iron nutrition of tomato plants grown under lime-induced iron deficiency. Journal of Plant Nutrition and Soil Science. 2013; **176**:859-866

[138] Papenfus HB, Kulkarni MG, Stirk WA, Finnie JF, Van Staden J. Effect of a commercial seaweed extract (Kelpak®) and polyamines on nutrientdeprived (N, P and K) okra seedlings. Scientia Horticulturae (Amsterdam). 2013;**151**:142-146

[139] Anjum K, Ahmed M, Baber JK, Alizai MA, Ahmed N, Tareen MH. Response of garlic bulb yield to biostimulant (Bio-cozyme) under calcareous soil. Life Sciences: An International Journal. 2014;**8**:3058-3062

[140] Barassi CA, Ayrault G, Creus CM, Sueldo RJ, Sobrero MT. Seed inoculation with *Azospirillum* mitigates NaCl effects on lettuce. Scientific Horticulturae (Amsterdam). 2006;**109**:8-14

[141] Del Amor FM, Cuadra-Crespo P. Plant growth-promoting bacteria as a tool to improve salinity tolerance in sweet pepper. Functional Plant Biology. 2012;**39**:82-90

[142] Del Pilar CM, Berrido SI, Ligero F, Lluch C. Rhizobium strain effects on the growth and nitrogen assimilation in Pisum sativum and *Vicia faba* plant

growth under salt stress. Journal of Plant Physiology. 1999;**154**:127-131

[143] Yildirim E, Taylor AG, Spittler TD. Ameliorative effects of biological treatments on growth of squash plants under salt stress. Scientific Horticulturae (Amsterdam). 2006;**111**:1-6

[144] Aydin A, Kant C, Turan M. Humic acid application alleviate salinity stress of bean (*Phaseolus vulgaris* L.) plants decreasing membrane leakage. African Journal of Agricultural Research. 2012;**7**: 1073-1086

[145] Ross R, Holden D. Commercial extracts of the brown seaweed *Ascophyllum nodosum* enhance growth and yield of strawberries. HortScience. 2010;**45**:S141-S141

[146] Guinan KJ, Sujeeth N, Copeland RB, Jones PW, O'Brien NM, Sharma HSS, et al. Discrete roles for extracts of *Ascophyllum nodosum* in enhancing plant growth and tolerance to abiotic and biotic stresses. Acta Horticulturae. 2013;**1009**:127-135

[147] Lucini L, Rouphael Y, Cardarelli M, Canaguier R, Kumar P, Colla G. The effect of a plant-derived biostimulant on metabolic profiling and crop performance of lettuce grown under saline conditions. Scientific Horticulturae (Amsterdam). 2015;**182**:124-133

[148] Mayak S, Tirosh T, Glick BR. Plant growth-promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiology and Biochemistry. 2004;**42**:565-572

[149] Demir K, Günes A, Inal A, Alpaslan M. Effects of humic acids on the yield and mineral nutrition of cucumber (*Cucumis Sativus*, L.) grown with different salinity levels. Acta Horticulturae. 1999;**492**:95-104

[150] Rady MM, Desoky ESM, Elrys AS, Boghdady MS. Can licorice root extract be used as an effective natural biostimulant for salt-stressed common bean plants? South African Journal of Botany. 2019;**121**:294-305

[151] Semida WM, Rady MM. Presoaking application of propolis and maize grain extracts alleviates salinity stress in common bean (*Phaseolus vulgaris* L.). Scientific Horticulturae (Amsterdam). 2014;**168**:210-217

[152] Rady MM, Varma B, Howladar SM. Common bean (*Phaseolus vulgaris* L.) seedlings overcome NaCl stress as a result of presoaking in *Moringa oleifera* leaf extract. Scientific Horticulturae (Amsterdam). 2013;**162**:63-70

[153] Abdel Latef AAH, Srivastava AK, Saber H, Alwaleed EA, Tran LSP. *Sargassum muticum* and *Jania rubens* regulate amino acid metabolism to improve growth and alleviate salinity in chickpea. Scientific Reports. 2017;**7**:1-12

[154] Arroussi HE, Benhima R, Elbaouchi A, Sijilmassi B, Mernissi NE, Aafsar A, et al. *Dunaliella salina* exopolysaccharides: A promising biostimulant for salt stress tolerance in tomato (*Solanum lycopersicum*). Journal of Applied Phycology. 2018;**30**: 2929-2941

[155] Semida WM, Abd El-Mageed TA, Hemida K, Rady MM. Natural bee-honey based biostimulants confer salt tolerance in onion via modulation of the antioxidant defence system. The Journal of Horticultural Science and Biotechnology. 2019;**94**:1-11

[156] Mesut Çimrin K, Türkmen Ö, Turan M, Tuncer B. Phosphorus and humic acid application alleviate salinity stress of pepper seedling. African Journal of Biotechnology. 2010;**9**:5845-5851

[157] Sapre S, Gontia-Mishra I, Tiwari S. Plant growth-promoting rhizobacteria ameliorates salinity stress in pea (*Pisum sativum*). Journal of Plant Growth Regulation. 2022;**41**:647-656

[158] Mellidou I, Ainalidou A, Papadopoulou A, Leontidou K, Genitsaris S, Karagiannis E, et al. Comparative transcriptomics and metabolomics reveal an intricate priming mechanism involved in PGPR-mediated salt tolerance in tomato. Frontiers in Plant Science. 2021;**12**:713984

[159] Alexander A, Singh VK, Mishra A. Halotolerant PGPR *Stenotrophomonas maltophilia* BJ01 induces salt tolerance by modulating physiology and biochemical activities of *Arachis hypogaea*. Frontiers in Microbiology. 2020;**11**:1-12

[160] Gupta S, Pandey S. ACC deaminase producing bacteria with multifarious plant growth promoting traits alleviates salinity stress in french bean (*Phaseolus vulgaris*) plants. Frontiers in Microbiology. 2019;**10**:1506

[161] Khan MA, Asaf S, Khan AL, Adhikari A, Jan R, Ali S, et al. Halotolerant rhizobacterial strains mitigate the adverse effects of Nacl stress in soybean seedlings. BioMed Research International. 2019; **2019**:9530963

[162] Ikram M, Ali N, Jan G, Iqbal A, Hamayun M, Jan FG, et al. *Trichoderma reesei* improved the nutrition status of wheat crop under salt stress. Journal of Plant Interactions. 2019;**14**(1):590-602

[163] Zhang S, Gan Y, Xu B. Application of plant-growth-promoting fungi *Trichoderma longibrachiatum* T6 enhances tolerance of wheat to salt stress through improvement of antioxidative defense system and gene expression. Frontiers in Plant Science. 2016;**7**:1405

[164] Hamayun M, Hussain A, Khan SA, Kim HY, Khan AL, Waqas M, et al. Gibberellins producing endophytic fungus *Porostereum spadiceum* AGH786 rescues growth of salt affected soybean. Frontiers in Microbiology. 2017;**8**:686

[165] Souza AC, Zandonadi DB, Santos MP, Canellas NOA, de Paula SC, da Silva Irineu LES, et al. Acclimation with humic acids enhances maize and tomato tolerance to salinity. Chemical and Biological Technologies in Agriculture. 2021;**8**(1):1-13

[166] Khan MS, Pandey MK, Hemalatha S. Comparative studies on the role of organic biostimulant in resistant and susceptible cultivars of rice grown under saline stress—organic biostimulant alleviate saline stress in tolerant and susceptible cultivars of rice. Journal of Crop Science and Biotechnology. 2018;**21**(5):459-467

[167] El Boukhari MELM, Barakate M, Bouhia Y, Lyamlouli K. Trends in seaweed extract based biostimulants: manufacturing process and beneficial effect on soil-plant systems. Plants. 2020;**9**(3):359

[168] Nawaz A, Shahbaz M, Asadullah M, Imran A, Marghoob MU, Imtiaz M, et al. Potential of salt tolerant PGPR in growth and yield augmentation of wheat (*Triticum aestivum* L) under saline conditions. Frontiers in Microbiology. 2020;**11**:2019

[169] Jan R, Khan MA, Asaf S, Lubna LIJ, Kim KM. Metal resistant endophytic bacteria reduces cadmium, nickel toxicity, and enhances expression of metal stress related genes with improved growth of oryza sativa, via regulating its antioxidant machinery and endogenous hormones. Plants (Basel). 2019;**8**(10):363

[170] Kang SM, Asaf S, Khan AL, Lubna KA, Mun BG, Khan MA, et al.

Complete genome sequence of *Pseudomonas psychrotolerans* CS51, a plant growth-promoting bacterium, under heavy metal stress conditions. Microorganisms. 2020;**8**(3):382

[171] Sahile AA, Khan MA, Hamayun M, Imran M, Kang SM, Lee IJ. Novel *Bacillus cereus* Strain, ALT1, enhance growth and strengthens the antioxidant system of soybean under cadmium stress. Agronomy. 2021;**11**:404

[172] Abdallah EF, Abeer H, Alqarawi AA, Hend AA. Alleviation of adverse impact of cadmium stress in sunflower (*Helianthus annuus* L.) by arbuscular mycorrhizal fungi. Pakistan Journal of Botany. 2015;**47**(2):785-795

## **Chapter 3**

## Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential and Limitations

*Renu Kathpalia and Anita Kamra Verma*

## **Abstract**

Artificial photosynthesis system (APS) uses biomimetic systems to duplicate the process of natural photosynthesis that utilizes copious resources of water, carbon dioxide and sunlight to produce oxygen and energy-rich compounds and has potential to be an alternative source of renewable energy. APS like natural photosynthesis includes the splitting of water into oxygen and hydrogen, and the reduction of carbon dioxide into various hydrocarbons such as formic acid (HCOOH), methane (CH4) and carbon monoxide (CO), or even pure hydrogen fuel. These processes are accomplished by a handful of device designs, including photoelectrochemical cells or photovoltaic-coupled electrolyzers which are driven by energy extracted from sunlight photons as well as suitable catalysts. Researchers are trying to combine advantageous components from both natural photosynthesis and artificial photosynthesis to create a semi-artificial photosynthesis system, involving the incorporation of enzymes or even whole-cell into synthetic devices. However, there are several limitations to the advancement of this field which are mainly centered on the inability to establish a system that is cost-effective, long-term durable and has the highest efficiency. Artificial photosynthesis devices can also function as atmospheric cleansers by extracting the excess amount of carbon dioxide and releasing back oxygen into the environment. Although there is still a long way to go to empower society with energy supplied through artificial photosynthesis, at the same time it is both desirable and necessary. To date, the efforts to commercialize APS have been fruitful, and it will soon be a viable alternative fuel source.

**Keywords:** artificial photosynthesis system (APS), biomimicry, photocatalyst, photosynthesis, photons, water oxidation

## **1. Introduction**

In this technological era, it has become mandatory to safeguard our natural resources and search for renewable sources of energy that can reduce the use of conventional fossil fuels. The usage of fossil fuel emits large quantities of carbon dioxide, sulfur dioxide and oxide particles as well as depletes natural resources. The International Panel on climate change recommended the urgent need to decrease

carbon dioxide emissions to zero on a global scale. To combat this grieving situation the development of sustainable and carbon-neutral energy technologies is the most compelling challenge faced by the entire humanity. Over the years, scientists have explored numerous alternatives that could possibly reduce our dependence on fossil fuels. Recently, efforts are centered toward the development of high-tech energygenerating systems inspired by nature itself. Nearly all natural resources are either depleting or getting contaminated except the solar energy which on conversion or utilization is a promising solution for energy-related problems.

One such phenomenon or reaction that takes place in plants, algae and photosynthetic bacteria to produce energy for themselves and provide energy to other organisms is photosynthesis. To mimic photosynthesis, the concept of artificial photosynthesis was introduced by Giacomo Ciamician way back in the year 1912 in a science paper entitled "The photochemistry of the future" [1]. He visualized and insisted on the use of technologies that can eliminate complete dependence on fossil fuels. Solar power stations could meet the challenges of sustainable energy more over the cost is lower than that of nuclear and thermal plants but the main bottleneck is the lack of efficient storage solution [2]. Different studies established systems to carry out photosynthesis similar to that of solar panels and convert it into electricity for direct application, as it was not possible to store energy for later use. So far, researchers have not been able to devise solar-driven catalysts for water oxidation and fuel production that are vigorous and use this abundant earth element.

Artificial photosynthesis system (APS) imitates the fundamental process of photosynthesis taking place in organisms for our societal needs. APS captures as well as stores solar energy in the form of fuel rather than glucose and is able to meet both the challenges of being carbon-negative and a source of solar fuel (**Figure 1**). Artificial photosynthesis devices involving semiconductors can absorb solar energy and store it by converting in the form of chemical energy which can be used later. Many

**Figure 1.** *An outline diagram to compare natural and artificial photosynthesis.*

## *Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

advancements have been achieved where artificial reaction center involving the movement of electrons (injected from a dye) into the conduction band of nanoparticles (such as titanium dioxide) on to electrode coupled with catalyst (such as platinum) or hydrogenase enzymes producing hydrogen gas. This system involves solar energy to split the water molecule into oxygen and hydrogen fuel; however, the efficiency is quite low and requires external electrical potential. The energy stored in this way is cheap and dense as compared to expensive battery storage [3]. Besides these APS is more environmentally attractive than solar panels as it absorbs excess carbon dioxide from the environment and releases oxygen back into the environment and can revolutionize the world of solar power [4]. This chapter includes a comprehensive view of the artificial photosynthesis system, its limitation, challenges and the future scope of APS as an alternate source of energy.

## **2. Photosynthesis: nature's marvel**

Green plants, algae and photosynthetic bacteria are photosynthesizing for more than a billion years without any significant change. Photosynthesis is an integral part of simulation models to safeguard the future of our planet. The electron transport system of photosynthesis transports electrons faster than the photons of solar energy reaching light-harvesting complex present in the chloroplast membrane. The light reaction takes place in three distinct protein complexes which are an integral part of the chloroplast membrane of higher plants viz., two light-harvesting complexes or photosystem (LHC I/PS I and LHCII/PSII) or antennae and cytochrome b6f connected by different electron carriers. Both the photosystems or light-harvesting complexes transfer photons to the reaction centers (RCs) in the form of resonance energy and create charge differences across the membrane. A strong oxidant complex commonly referred to as oxygen-evolving complex (OEC), is present on the donor side of PSII which does photolysis of water into molecular oxygen, protons and electrons. This electron moves to the plastoquinone pool to cyt b6f followed by plastocyanin to PSI and finally via ferrodoxin reduces NADP<sup>+</sup> to NADPH. The electron movement is coupled with proton pumping from one side of the membrane to the other side of the membrane creating a potential and pH difference across the membrane ultimately leading to the formation of ATP [5]. This assimilatory power trapped in the light reaction is utilized for the unique process of fixation of atmospheric carbon dioxide by the enzyme Rubisco, the most abundant protein on this earth. The enzyme Rubisco has a very slow catalytic rate (1–3 cycles per second) creating a major bottleneck in increasing photosynthetic efficiency. The unique feature of this fundamental process of life is the structural features of RCs complexes constituting a network of molecular cofactors held by the membrane at an appropriate distance and orientation to capture maximum light and perform the movement of electrons (**Figure 2**). This mosaic structure is such a unique nanoscale complex working perfectly for billions of years and providing energy to all the heterotrophs residing on this planet. To exactly mimic the complete process of photosynthesis is definitely an undaunted task.

## **3. Artificial photosynthesis**

The concept of artificial photosynthesis was given by an Italian chemist in 1912 but no remarkable research has been done till 1972 when Kenichi Honda and his student

#### **Figure 2.**

*The diagrammatic representation of different complexes involved in electron transfer in the Z scheme present on the membrane of thylakoid in the chloroplast. The photosystems PSI and PS II and intermediate carrier of the electron, cytochrome b6f, sand witched between them is the key structural organization of RCs. The complete structure is present in the membrane protein scaffold at an appropriate distance to facilitate electron transfer and convert solar energy to chemical energy.*

Akira Fujishima for the first time reported the successful designing of water splitting device powered by light [6], which was named as "Honda-Fujishima effect". The device includes a photoelectrochemical cell comprised of a photoanode made of TiO2 and a black cathode made up of platinum (Pt) black cathode. Both anode and cathode were completely submerged during exposure to light, an electron was released from TiO2 leading to the formation of an "electron hole", or positive charge on the Ti atom which was filled by an electron released from a water molecule, oxidizing the water to produce oxygen. The released electron was donated to a proton derived from water, thus reducing the proton to form hydrogen. The light of wavelength more than

#### **Figure 3.**

*The diagram representing the fundamental process of artificial photosynthesis systems having an efficient light absorber to trap sunlight and efficient water oxidation and proton reduction catalysts.*

*Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

400nm was given to the photoanode and the device was able to generate oxygen at the anode while hydrogen was generated at the cathode by photolysis of water and ultimately releasing oxygen and hydrogen [7] as summarized in **Figure 3**. The major bottleneck of using the molecule TiO2 was that it only absorbed ultraviolet wavelengths and is inactive in visible light wavelength [8].

In 1978, M. Halmann used a p-type phosphide semiconductor made of gallium phosphide as the photocathode suspended in an aqueous solution and achieved the reduction of carbon dioxide into hydrocarbons such as formic acid (HCOOH), formaldehyde (CH2O) and methanol (CH3OH) [9]. In 1983, William Ayers designed and patented the first visible light water-splitting device named as "artificial leaf" made up of silicon with a nafion membrane for ion transport above the cell. The entire structure is immersed in water and when illuminated results in the release of oxygen from the back of the metal surface, while hydrogen evolved on the silicon surface [10].

## **4. Artificial photosynthesis: principle**

As mentioned above the three principle steps involved in artificial photosynthesis, similar to natural photosynthesis, are the absorption of light causing excitation, charge generation followed by charge separation and finally chemical conversion leading to the production of fuel.

### **4.1 Light absorption**

In natural photosynthesis, chlorophylls and carotenoids are arranged in antennae to capture the maximum red and blue wavelength of light and bring about the excitation of electrons. These pigment molecules can absorb only a limited range of wavelengths ranging from about 400 to 700 nm which makes less than 50% of the sunlight that reaches earth [11]. The first major task is to design photosensitizers that can optimally use photons on exposure and are capable of aggregating light energy. In addition, the materials used should be capable of absorbing a wider wavelength of the solar spectrum so as to extract maximum energy falling on the earth.

Inspired by the "Honda-Fujishima effect", many materials such as TiO2 photoanode semiconductors like silicon, metal oxides such as ZnO, Fe2O3 and BiVO4, metal nitrides such as Ta3N5, metal phosphides such as GaP, metal oxynitrides such as TaON etc., have been tried by a different group of researchers [12]. Silicon, an abundant and cheap source, can absorb a wider spectrum of light. Another semiconductor Gallium Nitride has been used to produce formic acid and ethanol using thin film technology [13].

### **4.2 Lysis of water**

Water oxidation is a thermodynamically uphill process and requires free energy of ΔG ≈ 237 kJ mol<sup>1</sup> and potential E0 ≈ 1.23 V to transfer 4H<sup>+</sup> and 4e. In natural photosynthesis, water splitting is achieved through the involvement of an oxygenevolving complex (OEC) which has manganese (Mn), a tetrameric high valent oxo species, that catalyzes oxygen-oxygen bond formation. Semiconductor nanowires are used to absorb light resulting in the oxidation of water producing oxygen, proton and electrons. The electrons move toward other ends while protons move through a proton-conducting membrane made up of Nafion, ultimately reduced to hydrogen.

Thus, photolysis of water in APS is achieved through the combination of two different customized systems for their respective purpose [12].

The redox equations involved in water splitting are as follows:

$$\text{Oxidation reaction}: 2\text{H}\_2\text{O} \to 4\text{e}^- + 4\text{H}^+ + \text{O}\_2 \tag{1}$$

$$\text{Reduction reaction}: 4\text{H}^+ + 4\text{e}^- \to 2\text{H}\_2\tag{2}$$

$$\text{Redox reaction}: 2\text{H}\_2\text{O} \to 2\text{H}\_2 + \text{O}\_2 \tag{3}$$

The splitting of water needs an energy of approximately 2.5 V, hence a catalyst is essential to absorb photons of sunlight and set off the reactions [14]. The bioinspired approach of using manganese as a catalyst resulted in instability due to short-term and inefficient function [15]. In comparison, cobalt oxide (CoO) found to be stable, efficient and is easily available [16].

Different materials tested show efficiency in some steps and inefficiency in other steps leading to the use of coupled materials, which are customized for their respective reactivity. Recently, molecular water-oxidation catalysts are designed for splitting water and evolution of oxygen. These catalysts generally comprise a metal complex with wide open coordination sites as well as an electronic structure to stabilize a metal-hydride intermediate. The most common materials used are noble metals such as rhodium and platinum-based complexes. To develop catalysts from earth-abundant metals such as cobalt, iron, molybdenum, and nickel have also been tried by various scientists. The most stable and efficient catalyst out of many tried and tested is Nickel complexes. Ruthenium and iridium-based catalysts showed good reactivity and stability but are scarce and expensive [17]. The transition metal family such as copper, nickel and iron-based were tested in order to improve the catalytic function. Cobalt and Zirconium heterobimetallic on porous silica separated by nanotube separation membranes was also used by researchers [18]. However, the search and optimization of materials to be used in APS are still under investigation.

#### **4.3 Reduction of carbon dioxide**

Carbon dioxide is a linear, highly stable molecule having very low electron affinity. Conversion of carbon dioxide is an uphill reaction and requires a nucleophilic attack on the carbon atom, the dissociation bond energy of C]O is �750 kJ/mol. The carbon atom has the highest valence due to which different fuels can be created in addition to the production of oxygen and hydrogen derived from water. The different fuel compounds that can be generated are formic acid (HCOOH), methanol (CH3OH), carbon monoxide (CO) and methane (CH4) as given in these equations:

$$\text{CH}\_2 + 2\text{H}^+ + 2\text{e}^- \rightarrow \text{HCOOH} \tag{4}$$

$$\text{CO}\_2 + 2\text{H}^+ + 2\text{e}^- \rightarrow \text{CO} + \text{H}\_2\text{O} \tag{5}$$

$$\rm{CH}\_2 + \rm{6H}^+ + \rm{6e}^- \rightarrow \rm{CH}\_3\rm{OH} + \rm{H}\_2\rm{O} \tag{6}$$

$$\rm{CO\_2 + 8H^+ + 8e^- \to CH\_4 + 2H\_2O} \tag{7}$$

The generation of these liquid hydrocarbons has added advantage of being easily integrated into energy infrastructure. Nevertheless, the greatest scientific challenge due to carbon's multi-electron nature imposes additional complexity [19]. The exact *Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

mechanism is not clearly established but it is predicted that the reduction of carbon dioxide is similar to water splitting during the process of photosynthesis. In APS, carbon dioxide interacts with catalyst along with the transfer of electrons resulting in a reduction reaction followed by proton coupling creating an electron hole in the photosensitizer which is filled by an electron leading to the end of the process [20]. A co-catalysts and photoelectrode material break carbon and oxygen bonds and form carbon and hydrogen bond i.e., CdH bond. The choice of catalyst is very critical in APS which includes low cost and durability. Researchers have tried various combinations of complexes viz., rhenium-based, cobalt-based, nickel-based, iron-based and zinc-based complexes [21]. With the use of innovative porous materials adsorption of intermediates at the surface of the electrode is significantly enhanced. In another trial, co-catalyst such as copper, silver and gold were found to be very effective in enhancing conserving efficiency. However, an ideal catalyst is yet to design which can improve the performance of chemical conversion (**Figure 4**).

## **5. Natural photosynthesis vs artificial photosynthesis**

Photosynthesis involves two reactions viz, light-dependent (light reaction) harnessing light photons and converting it in assimilatory powers in the form NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate) which are utilized to fix carbon in light-independent reaction (dark reaction). The light reaction involves two photosystems, viz., PSI and PSII (photosystem I and II) consisting of light-absorbing pigments in the form of antennae system involving chlorophylls and carotenoids and have a reaction center having chlorophyll a molecule. The two photosystems are present in the thylakoid membrane along with other electron carriers, absorb light photons and transfer the energy to the reaction center through resonance energy transfer and release an electron from chlorophyll molecule

#### **Figure 4.**

*The formation of different end products due to the reduction of carbon dioxide catalyzed by different catalysts in artificial photosynthesis.*

from the reaction center. This electron moves through a series of electron carriers and finally reduces NADP into NADPH and the formation of ATP. The removal of electrons from chlorophyll results in the formation of "electron hole" in the chlorophyll pigment which is filled by photolysis of water by oxygen-evolving complex present next to PSII releasing an electron and oxygen as a waste product for the plants and the most valuable by-product for the survival of life on this biosphere. This lightdependent reaction also involves a complex array of enzymes such as the photosystems themselves, as well as hydrogenases which interact with hydrogen derived from water molecules, is known as photophosphorylation; a unique system of generation of chemical energy present only in photosynthesizing organisms [4, 5].

The assimilatory powers being generated during light reaction then bring about a reduction of carbon through the light-independent reaction or dark reaction involving the Calvin cycle. The dark reaction, discovered by Malvin Calvin, occurs in the stroma of chloroplast through a series of reactions catalyzed by different enzymes to transform carbon dioxide from the atmosphere into carbohydrates. The carbon dioxide is absorbed by the stomata present on the leaves and ultimately converted into carbohydrates, the overall reaction of photosynthesis is

$$6\text{ H}\_2\text{O} + 6\text{ CO}\_2 \frac{\text{Sunlight}}{\text{Chlorophyll}} \rightarrow \text{C}\_6\text{H}\_{12}\text{O}\_6 + 6\text{O}\_2\tag{8}$$

Thus, photosynthesis is ingeniously designed through series evolution to provide autotrophic organisms to produce their own food by converting solar energy into chemical energy stored in the energy-rich bonds of a carbohydrate.

Three major components of natural photosynthesis that need to be imitated by artificial photosynthesis are (i) light capture and electron transport (ii) water splitting (into hydrogen and oxygen) and (iii) reduction of carbon dioxide. Two types of fuel are being generated through APS viz., hydrocarbons (methanol and formic acid) and pure hydrogen. Hydrogen is a clean option generated via APS and can be used directly as liquid fuel, consumed in the fuel cell, thermal processes, electrolysis, biological processes and other application to substitute fossil fuel [2, 22].

## **6. Solar harvesting devices vs artificial photosynthesis**

Various efforts have been made to design the devices with the aim to convert solar energy into chemical energy which is stored in chemical bonds. The two most powerful devices designed by researchers are the photoelectrochemical cell and photovoltaic-coupled electrolyzer. Both the systems are designed on certain similarities and differences as well as having advantages and limitations which are discussed here.

#### **6.1 Photoelectrochemical cell**

The photoelectrochemical cells (PEC) consist of two electrodes, a photoanode and a photocathode immersed in an electrolyte and an external wire [19, 22]. One of the variants designed comprises a single electrode evolving mixture of oxygen and hydrogen which entails contamination which is an additional step of separating hydrogen and oxygen. The reduction of carbon dioxide by PEC is the most efficient method involving the amalgamation of photo and electrocatalysis, an addition of

## *Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

biocatalysts enhanced its efficiency further. The photoelectrodes are constructed using either a molecular electrode or a light-absorbing semiconductor [23]. At the photoanode, water splits to get oxidized to form oxygen while it is reduced to form hydrogen at the photocathode. On exposure to solar energy excitation at the photoanode releases an electron which is donated to water leading to the reduction of hydrogen molecules at the photocathode. The "electron hole" created at the photoanode is filled by an electron donated by water molecule oxidation ultimately leading to the production of oxygen (**Figure 5**).

The material of the photoanode has a tremendous effect on water-splitting efficiency, stability and absorption of visible light in an aqueous solution (**Figure 6**). A membrane composed of Nafion, having proton-conductive and separation properties, has also been tried in some photoelectrochemical cells. Even semiconductors have also been tried but found to be having low efficiency, instability and only narrow visible light can be adsorbed. Therefore, the criteria of selection shifted toward carbon-based, transition metals, and nanostructured photoanodes e.g., carbon-based materials, graphene, carbon nanotubes, carbon dots and carbon quantum dots all demonstrated stability as well as photocurrent generation. In addition, transition metals such as germanium-doped hematite, cadmium sulfide, zinc oxide, copper sulfide and molybdenum exhibited high performance due to their high electrical conduction and electrochemical stability. Nanomaterials such as nanowires, nanotapers and nanorods also exhibited excellent performance in terms of high hydrogen evolution at photocathode [24]. A serially coupled PSII and PSI along with an Au electrode has been developed recently where semi-artificial PECs produced photocurrent and imitated Z scheme of natural photosynthesis, interfaced by two types of redox osmium complex hydrogel for the transfer of electrons [23].

#### **Figure 5.**

*The working principle of a photoelectrochemical cell where the charge separation is induced by light which leads to water splitting releasing molecular oxygen, proton and electrons. The protons move to the cathode where it is reduced to molecular hydrogen and electrons.*

**Figure 6.**

*A modified photoelectrochemical cell having a molecular chromophore and catalyst for water splitting. C\* is excited chromophore C which transfers electrons and leads to water splitting.*

## **6.2 Photovoltaic-coupled electrolyzer**

A photovoltaic-coupled electrolyzer working principle is based on the working of both solar and electrochemical cells [13, 25]. The photovoltaic cell includes absorption of light as well as charge separation. The energy potential generated by the photovoltaic cell is transferred to an electrolyzer for redox reaction [26]. Thus, in the first step solar radiation is converted into electricity, which is further used for the oxidation and reduction of water or carbon dioxide in the electrolyzer cell. This system has the more efficient strategy and has remarkable solar-to-hydrogen efficiencies of 10–15%, reaching as high as 30%. This ultra-efficient photovoltaic-coupled electrolyzer system utilizes a triplejunction solar cell which is made of three subcells having indium gallium phosphide (InGaP), gallium arsenide (GaAs) and gallium indium nitrogen arsenic antimonideGaInNAs(Sb), respectively. This solar cell involved two polymer electrolyte membranes consisting of a Nafion membrane coated with Pt black catalyst at the cathode and an Ir black catalyst at the anode [27]. The power generated at the triple-junction cell pumped water into the anode of the first electrolyzer and effluent having water and oxygen is further pumped into the second electrolyzer where hydrogen is transferred from the first electrolyzer cathode to the second electrolyzer cathode. As a result, hydrogen and oxygen are collected from the cathode and anode, respectively, at the second electrolyzer, the leftover water is recycled back for the next cycle without any disruption for almost 48 hours with solar to hydrogen conversion efficiency of 30% [27].

## **7. Semi-artificial photosynthesis**

It involves the merging of unique features of both natural and artificial photosynthesis such as high quantum efficiency (100%), selectivity, specificity and self-repair mechanisms of natural photosynthesis [28]. At the same time, the ability to use synthetic material with a wider light absorption spectrum as well as modified molecular chemistries thus minimizing the limitations. The photocatalytic properties of two photosystems PSI and PSII viz., generation of reducing power and photolysis of water, respectively are used for the creation of semi-artificial devices which convert light energy into molecular hydrogen and carbon-based fuels [29].

*Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

## **7.1 Enzyme hybrids**

Enzymes have high molecular, chiral and substrate specificity along with high turnover and biodegradability. Enzymes utilization is limited due to their thermal instability, limited optimum pH range, denaturation by organic solvents and inhibition by metal ions. To fully utilize their potentiality as biocatalyst researchers have used porous matrices to elevate their stability *in vivo* (**Figure 7**). Different network materials such as metal-organic frameworks (MOFs) and metal-phenolic networks (MPNs) have been utilized to hold biocatalysts for enhancing their stability. A hundred percent efficiency has been achieved using artificial light absorbers and biological catalysts where photocurrents through electrode-wired enzymes generate kinetically and thermodynamically efficient products at very fast rates. Photosystem II enzymes along with semiconductors and mesoporous opal indium tin oxide (ITO) electrodes are well-studied examples of an inorganic collector with a biological catalyst creating efficient systems [30].

## **7.2 Cell hybrids**

Biological unicellular systems are more efficient in terms of specificity as compared to isolated enzymes from living organisms. The use of microorganisms along with inorganic semiconductors or metal nanoparticles has been investigated by researchers (**Figure 8**) [31]. In one of the studies, the anaerobe *Methanosarcina barkeri*, bacteria, was used along with a materials catalyst, nickel sulfide electrode, a material inspired by naturally occurring nickel-dependent hydrogenases. The bacterial culture was added to the cathode and was exposed to visible light which resulted in the reduction of carbon dioxide to methane with no net loss in performance efficiency [32]. In another study, an acetogenic bacterium *Moorella thermoacetica* was treated with water-soluble gold nanoclusters AuNCs (mainly Au22(SG)18) and illuminated with light of 532 nm wavelength. The cytoplasm mediators in bacteria selectively pick up photo-generated electrons (bypassing cell membrane) and produce acetic acid from carbon dioxide for six days [20]. Another hybrid system was reported where light harvesting indium phosphide (InP) nanoparticles were introduced in genetically engineered yeast cells where photoexcited electrons produced from InP activate nicotinamide adenine dinucleotide phosphate regeneration [33].

#### **Figure 7.**

*An outline diagram of a hybrid of photoelectrochemical and biological approach where the biological catalyst is utilized to catalyze the reaction for the production of high-value products.*

**Figure 8.**

*A biohybrid made by PS-I photosystem I from a red microalga* Cyanidioschyzon merolae *nanostructured present on multilayer hematite/FTO electrode.*

## **7.3 Artificial leaf**

The imitation of the Z-scheme and designing of the artificial leaf is another important benchmark in the quest for foolproof operational systems using a biohybrid approach. American chemist Daniel G. Nocera and colleagues (2011) developed a

## **Figure 9.**

*The artificial leaf designed with a silicon chip coated with a chemical catalyst to speed up the water-splitting reaction on exposure to sunlight. The separated electrons and protons are captured to form hydrogen gas which can be used for the generation of electricity.*

## *Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

silicon-based device that can produce hydrogen and oxygen using solar energy without adding any pollutants was named as artificial leaf [34]. The artificial leaf has a silicon chip coated with a chemical catalyst which on exposure to sunlight speed up the water-splitting reaction and captures separated electrons and protons to form hydrogen gas which can be used for the generation of electricity (**Figure 9**). Artificial leaf still faces significant challenges and focused research is needed to increase hydrogen fuel efficiencies which is very low at about 5% of the total possible fuel availability in solar energy. The technology is highly expensive and the safety of hydrogen fuel generated is negligible limiting the use of artificial leaves for commercial purposes. An artificial leaf was designed using cuprous oxide to produce methanol and oxygen from carbon dioxide and sunlight [35].

## **8. Improvement strategies**

The advancement in nanotechnology (in the area of imaging and modification of nanomaterial) and molecular manipulation such as adding impurities to semiconductors resulted in an increase in light absorption capability, efficient catalyst performance and selectivity [36]. Researchers have created highly efficient antennae complex mimicking light-harvesting complex involved in natural photosynthesis. The antennae reaction center has a hexad nanoparticle having four zinc tetraarylporphyrin molecules, (PZP)3-PZC coupled to a free-base porphyrin-fullerene molecule, P-C60 to form a hexad structured nanoparticle (PZP)3PZCPC60 [37]. The nanoscale materials and devices have been developed by "bottom-up" nanofabrication creating "molecular-lego" which are then used in manufacturing new devices. The creation of supramolecular and their usage in the construction of devices based on molecular components have enhanced the efficiency of catalysts by creating supramolecular cages and hence preventing degradation [17]. The development of single photoelectrode, photovoltaic-coupled electrolyzer, use of nanostructure materials can influence the functioning and efficiency of the device to a greater extent [13, 30]. The physical factors such as temperature, pressure and ion concentration of the environment hold significant leeway.

Wastewater treatment (WWT) leads to the emission of a huge amount of carbon dioxide which is harvested as a source of renewable energy. To tackle the wasteenergy-carbon challenge, an integrated approach has been adopted which involves hybrid microbial photoelectrochemical (MPEC) integrated with microbial electrochemical system WWT with artificial photosynthesis. The energy released during WWT aids in achieving carbon neutrality goal side by side assist in solar harvesting, conversion and storage [38].

## **9. Limitations and challenges**

Natural photosynthesis has high quantum efficiency leading to efficient charge separation but the overall conversion of solar energy to chemical energy is quite low nearly about 1%. Using APS, efficiency up to 10% or even higher is demonstrated [12]. One of the most significant bottlenecks in APS is attaining a cost-effective, efficient and stable catalyst material. The organic-based catalyst has the tendency to lose its stability on multiple uses and shows corroding or obstructing the working of system equipment. Many metal-based catalysts have been tried and the search for cost-

#### **Figure 10.**

*The comparison of the processes in the natural and artificial photosynthetic system at the leaf, chloroplast and molecular levels.*

effective and stability for at least ten years is still under process. To mimic a complex process like photosynthesis is very challenging.

Another notable challenge within the area of mimicking a natural process is the complex molecular geometry found in photosynthesizing organisms. Researchers are having a great deal of trouble replicating the level of intricacy that it entails (**Figure 10**). Many catalysts have been synthesized in the past few decades; however, these catalysts are unstable. Nevertheless, with the help of supramolecular strategies and nanotechnology, scientists are able to easily manipulate the workings of their devices through structural and molecular composition. Studies related to molecular catalyst heterogeneity are limited as it is difficult to match the details present in natural photosynthesis. The development of efficient molecular catalysts will allow the field of APS to advance toward a viable system [15].

## **10. Future impact and concluding remarks**

The top priority of researchers is to search sources of renewable energy that can be used to get some relief from the current state of crisis all over the world [28]. The airplane was created inspired by the flight of the bird, in a similar manner natural photosynthesis serves as a model to mimic the functions of self-sustaining photoautotroph organisms which hopefully one day create a self-sustaining world. APS already working efficiently and outperforms natural catalytic systems with respect to simplicity, charge transport and light absorption spectral range. Moreover, just as solar panels can be installed onto roofs, providing a secondary source of electricity,

## *Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

future artificial photosynthesis devices can also be applied to power homes as it offers a way to store energy for later use. More than 60% of oil depletion globally is because of its usage in transportation. Electric cars are an excellent alternative but recently new models of cars powered by hydrogen, the byproduct of APS claimed to revolutionize the vehicle industry. These hydrogen-powered vehicles require a very short refueling time and are environmentally friendly.

Natural photosynthesis is solely responsible for all the energy that is required to survive on this planet. In addition, photosynthesis adds energy stored in fossil fuels. It took a billions of years for evolution to make protobiont to evolve into multicellular photosynthesizing systems. To mimic, natural photosynthesis may take more than a decade of extensive research before APS is fully equipped for industrial utilization [11]. Therefore, it is imperative to try and extract energy from a biomimetic approach to this natural process. The endeavor for creating a self-sustained system using APS is still in its infancy. Many successful versions of APS has been devised but not all models are infallible and have drawback related to efficiency, instability or financial expenses. The search for a cost-effective, robust and scalable APS continues in organizations viz., Liquid Sunlight Alliance (LiSA) and Center for Hybrid Approaches in Solar Energy to Liquid Fuels (CHASE). The various attempts to practically apply APS fall short of many efficiencies but still solar fuel production by natural photosynthesis is achievable in the laboratory. The scientific community is well versed in terms and working principles of solar fuel, artificial leaf and artificial photosynthesis and working hard to provide energy using clean, green alternatives globally.

## **Author details**

Renu Kathpalia<sup>1</sup> and Anita Kamra Verma<sup>2</sup> \*

1 Department of Botany, Kirori Mal College, University of Delhi, Delhi, India

2 Nano-Biotech Lab, Kirori Mal College, University of Delhi, Delhi, India

\*Address all correspondence to: akverma@kmc.du.ac.in

© 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.

## **References**

[1] Ciamician G. The photochemistry of the future. Science. 1912;**36**(926): 385-394. DOI: 10.1126/ science.36.926.385

[2] Schmidt J, Gruber K, Klingler M, Klöckl C, Camargo LR, Regner P, et al. A new perspective on global renewable energy systems: Why trade in energy carriers matters. Energy & Environmental Science. 2019;**12**: 2022-2029. DOI: 10.1039/C9EE00223E

[3] Tachibana Y, Vayssieres L, Durrant J. Artificial photosynthesis for solar watersplitting. Nature Photonics. 2012;**6**: 511-518. DOI: 10.1038/nphoton.2012.175

[4] Davey T. Artificial photosynthesis: Can we harness the energy of the sun as well as plants? [Internet] 2016. Available from: https://futureoflife.org/2016/09/ 30/artificial-photosynthesis/ [Accessed: December 15, 2022]

[5] Lal M. Photosynthesis. In: Bhatla SC, Lal M, editors. Plant Physiology, Development and Metabolism. Singapore: Springer; 2018. pp. 159-226. Available from: https://link.springer.c om/book/10.1007/978-981-13-2023-1

[6] Fujishima A, Honda K. Electrochemical photolysis of water at a semiconductor electrode. Nature. 1972; **238**:37-38. DOI: 10.1038/238037a0

[7] Maeda K. Photocatalytic water splitting using semiconductor particles: History and recent developments. Journal of Photochemistry and Photobiology. 2011; **12**(4):237-268. DOI: 10.1016/j. jphotochemrev.2011.07.001

[8] Dette C, Pérez-Osorio MA, Kley CS, Punke P, Patrick CE, Jacobson P, et al. TiO2 anatase with a bandgap in the visible region. Nano Letters. 2014;

**14**(11):6533-6538. DOI: 10.1021/ nl503131s

[9] Halmann M. Photoelectrochemical reduction of aqueous carbon dioxide on p-type gallium phosphide in liquid junction solar cells. Nature. 1978;**275**: 115-116. DOI: 10.1038/275115a0

[10] William A. US Patent 4,466,869 Photolytic Production of Hydrogen [Internet]. 1984. Available from: https:// patents.google.com/patent/US4466869A/ en Accessed: December 11, 2022]

[11] Artificial Photosynthesis. Future Ideas—The Green Age [Internet]. 2017. Available from: https://www.thegree nage.co.uk/tech/artificial-photosynthesis [Accessed: November 15, 2022]

[12] Poudyal RS, Tiwari I, Koirala AR, Masukawa H, Inoue K, Tomo T, et al. Hydrogen production using photobiological methods. In: Subramani V, Basile A, Veziroğlu TN, editors. Compendium of Hydrogen Energy. Volume 1. Hydrogen Production and Purification. Woodhead Publishing Series of Energy, Elsevier; 2015. pp. 289-317. Available from: https:// www.sae.org/images/books/toc\_pdfs/ BELS126.pdf

[13] Yotsuhashi S, Deguchi M, Hashiba H, Zenitani Y, Hinogami R, Yamada Y, et al. Enhanced CO2 reduction capability in an AlGaN/GaN photoelectrode. Applied Physics Letters. 2012;**100**:243904. DOI: 10.1063/ 1.4729298

[14] Layton J. How artificial photosynthesis works [Internet]. How Stuff Works Science. HowStuffWorks. 2020. Available from: https://science. howstuffworks.com/environmental/ green-tech/energy-production/artificial*Artificial Photosynthesis as an Alternative Source of Renewable Energy: Potential… DOI: http://dx.doi.org/10.5772/intechopen.111501*

photosynthesis.htm [Accessed: November 17, 2022]

[15] Balamurugan M, Saravanan N, Ha H, Lee YH, Nam KT. Involvement of highvalent manganese-oxo intermediates in oxidation reactions: Realisation in nature, nano and molecular systems. Nano Convergence. 2018;**5**:18. DOI: 10.1186/s40580-018-0150-5

[16] Paul B, Bhanja P, Sharma S, Yamauchi Y, Alothman ZA, Wang Z, et al. Morphologically controlled cobalt oxide nanoparticles for efficient oxygen evolution reaction. Journal of Colloid and Interface Science. 2021;**582**(A): 322-332. DOI: 10.1016/j.jcis.2020.08.029

[17] Yu F, Poole D, Mathew S, Yan N, Hessels J, Orth N, et al. Control over electrochemical water oxidation catalysis by preorganization of molecular ruthenium catalysts in self-assembled nanospheres. Angewandte Chemie (International Ed. in English). 2018; **57**(35):11247-11251. DOI: 10.1002/ anie.201805244

[18] Liu J, Goetjen TA, Wang Q, Knapp JG, Wasson MC, Yang Y, et al. MOF-enabled confinement and related effects for chemical catalyst presentation and utilization. Chemical Society Reviews. 2022;**51**:1045. Available from: https://www.researchgate.net/ publication/357731261

[19] Barber J, Tran PD. From natural to artificial photosynthesis. Journal of the Royal Society Interface. 2013;**10**(81):20120984. DOI: 10.1098/ rsif.2012.0984

[20] Kumaravel V, Bartlett J, Pillai SC. Photoelectrochemical conversion of carbon dioxide (CO2) into fuels and value-added products. ACS Energy Letters. 2020;**5**(2):486-519. DOI: 10.1021/acsenergylett.9b02585

[21] Kathpalia R, Verma AK. Bio-inspired nanoparticles for artificial photosynthesis. Materials Today Proceedings. 2021;**45**:3825-3832. DOI: 10.1016/j.matpr.2021.03.214

[22] Styring S. Artificial photosynthesis for solar fuels. Faraday Discussions. 2012;**155**:357-376. Available form: https://pubs.rsc.org/en/content/articlela nding/2012/fd/c1fd00113b

[23] Olmos JDJ, Kargu J. Oxygenic photosynthesis: Translation to solar fuel technologies. Acta Societatis Botanicorum Poloniae. 2014;**83**(4): 423-440. DOI: 10.5586/asbp.2014.037

[24] Braach-Maksvytis V. Nanotechnology and artificial photosynthesis: Go smart, mimic nature. Energy & Environment. 2001;**12**(4): 331-333. DOI: 10.1260/ 0958305011500814

[25] Zhang B, Sun L. Artificial photosynthesis: Opportunities and challenges of molecular catalysts. Chemical Society Reviews. 2019;**2019**:7. Available from: https://pubs.rsc.org/en/ content/articlelanding/2019/CS/ C8CS00897C#!divCitation

[26] Gust D, Moore TA, Moore AL. Solar fuels via artificial photosynthesis. Accounts of Chemical Research. 2009; **42**(12):1890-1898. DOI: 10.1021/ ar900209b

[27] Jia J, Seitz L, Benck J, Huo Y, Chen Y, Ng JWD, et al. Solar water splitting by photovoltaic-electrolysis with a solar-tohydrogen efficiency over 30%. Nature Communications. 2016;**7**:13237. DOI: 10.1038/ncomms13237

[28] Xiao K, Liang J, Wang X, Hou T, Ren X, Yin P, et al. Panoramic insights into semi-artificial photosynthesis: Origin, development, and future

perspective. Energy & Environmental Science. 2022;**15**:529-549. Available from: https://pubs.rsc.org/en/journals/ journal/ee

[29] Brown KA, King PW. Coupling biology to synthetic nanomaterials for semi-artificial photosynthesis. Photosynthesis Research. 2020;**143**: 193-203. DOI: 10.1007/s11120-019- 00670-5

[30] Sokol K, Mersch D, Hartmann V, Zhang J, Nowaczyk MM, Rögner M, et al. rational wiring of photosystem II to hierarchical indium tin oxide electrodes using redox polymers. Energy & Environmental Science. 2016;**9**(12): 3698-3709. DOI: 10.1039/C6EE01363E

[31] Li X, Xu H, Chen Z, Chen G. Biosynthesis of nanoparticles by microorganisms and their applications. Journal of Nanomaterials. 2011;**2011**:1- 16. DOI: 10.1155/2011/270974

[32] Paulo LM, Ramiro-Garcia JR, van Mourik S, Stams AJM, Sousa DZ. Effect of Nickeland cobalt on methanogenic enrichment cultures and role of biogenic sulfide in metal toxicity attenuation. Frontiers in Microbiology. 2017;**8**:1341. Available from: https://www. researchgate.net/deref/https%3A%2F% 2Fdoi.org%2F10.3389% 2Ffmicb.2017.01341

[33] Guo J, Suástegui M, Sakimoto KK, Moody VM, Xiao G, Nocera DG, et al. Light-driven fine chemical production in yeast biohybrids. Science. 2018;**362**: 813-816. DOI: 10.1126/science.aat9777

[34] McAlpin JG, Stich TA, André Ohlin C, Surendranath Y, Nocera DG, Casey WH, et al. Electronic structure description of a [Co(III)3Co(IV)O4] cluster: A model for the paramagnetic intermediate in cobalt-catalyzed water oxidation. Journal of the American

Chemical Society. 2011;**133**(39): 15444-15452. DOI: 10.1021/ja202320q

[35] Kim JH, Hansora D, Sharma P, Jang J, Le JS. Toward practical solar hydrogen production – an artificial photosynthetic leaf-to-farm challenge. Chemical Society Reviews. 2019;**48**: 1908-1971. DOI: 10.1039/C8CS00699G

[36] Yang Y, Ajmal S, Zhenga X, Zhang L. Efficient nanomaterials for harvesting clean fuels from electrochemical and photoelectrochemical CO2 reduction. Sustainable Energy & Fuels. 2018;**2**(3): 510-537. Available from: https://pubs.rsc. org/en/content/articlelanding/2018/se/ c7se00371d

[37] Kodis G, Liddell PA, de la Garza L, Clausen PC, Lindsey JS, Moore AL, et al. Efficient energy transfer and electron transfer in an artificial photosynthetic antenna-reaction center complex. The Journal of Physical Chemistry A. 2002; **106**:2036-2048. DOI: 10.1021/jp012133s

[38] Li Z, Lu L. Wastewater treatment meets artificial photosynthesis: Solar to green fuel production, water remediation and carbon emission reduction. Frontiers of Environmental Science & Engineering. 2022;**16**:53. DOI: 10.1007/s11783-022-1536-5

## **Chapter 4**
