**2. Plant heat stress adaptation and tolerance targets**

#### **2.1 Heat shock proteins (HSPs)**

HSPs are a highly conserved group of proteins which are expressed abundantly following the sudden increase of temperature in wide variety of evolutionary branches as bacteria, fungi, plants, and animals. Even though plants are more responsive to temperature changes and react to fluctuation as small as 1°C, HSPs are expressed in response to sudden 8–10°C temperature increases. HSPs expression may increase within a few seconds following the temperature increase and reach the maximum level of transcripts within one to two hours of exposure. In high temperatures, protein synthesis is reduced to prevent misfolded protein production and protein denaturation which may present toxic properties for cells. Likewise, HSP expression is reduced following the cooldown of environment to optimum temperatures. Expressed HSPs are detectable for approximately 20 hours and generate thermotolerance for further temperature increases. Plant HSPs can be categorized under five conserved families based on their molecular weights as HSP100, HSP90, HSP70, HSP60, and small HSPs (sHSPs) [9].

HSP100 family members which are found in prokaryotes as well as eukaryotes are 75–100 kDa proteins. They can be further divided into two classes based on their ATPbinding sites as class I contains two while class II contains one site. HSP100 family protein takes part in acquisition of thermotolerance through preventing and unfolding of protein aggregations in association with chaperons by ATP-dependent manner. Their expression increases in different developmental stages as well as in response to heat shock. High salt, desiccation, abscisic acid (ABA), and cold stress-induced

expressions are also reported. HSP100 protein accumulation initiates as soon as heat stress begins and is retained for prolonged durations during recovery. Hence, the crucial role of Hsp100 family is generally speculated for recovery instead of prevention. However, early accumulation of these proteins as stress initiates suggests that HSP100 members may play important during stress as well [10].

HSP90s are evolutionarily conserved essential molecular chaperones in eukaryotic cells, undertaking key functions in signal transduction networks, cell-cycle control, folding of newly synthesized proteins as well as re-folding and stabilizing tertiary structures of already folded proteins, and protein trafficking. HSP90s, which are constitutively expressed and abundant as 1–2% of total proteins in cell, are induced during stress conditions particularly in response to heat. They involve root, hypocotyl, shoot apical meristem, and stomatal development as well as fertilization and embryo formation. HSP90 is an ATP-dependent chaperone, which constitutes HSP90 chaperon complex in cooperation with other chaperons and co-chaperons to maintain its function. For an instance, proteins which require HSP90 chaperon activity to re-gain their functional conformation called client proteins as newly synthesized or misfolded proteins, initially bind to general protein folding chaperones such as HSP40 and HSP70 which can recognize unfolded proteins. Then, HSP90/ HSP70-organizing protein (HOP) mediates binding of the client protein to HSP90. Role of the HSP40/HSP70 chaperone machinery during abiotic stress response is well documented. Acute heat shock temporarily reduces the cytoplasmic HSP90 activity, as it is recruited to stress-labile proteins hence releasing inhibition on stress response induction [11–13].

HSP70s are the most structurally and functionally conserved members of the whole protein family. Hsp70s are the most ubiquitous class of ATP-dependent chaperone proteins which are present in the cytosol of all eubacteria and eukaryotes, and some archaea, as well as within mitochondria, ER, and plastids of eukaryotic cells. In plants and other higher eukaryotes, they have constitutive expression for undertaking the cellular protein quality control and degradation system roles. In other organisms, they are stress-inducible for cyto-protective functions under several different conditions. As the most abundant HSPs, Hsp70 holds hydrophobic regions of misfolded proteins and prevents protein aggregation that can present toxicity to cells. They utilize ubiquitin-mediated proteasomal degradation pathway. Under heat shock and other abiotic stress conditions, heat shock transcription factors are triggered by the signal transduction from misfolded or unfolded outer membrane proteins to inner targets. One of the most notorious trans-acting elements are heat shock transcription factors (HSFs) which are associated with cis-acting heat shock elements (HSEs) in promoter regions of heat stress responsive genes [3, 14, 15].

HSP60s are ATP-dependent mitochondrial chaperones which are involved in importing mitochondrial proteins and macromolecule assembly. They can be categorized into structurally similar two groups which differ in amino acid sequences. Group I HSP60s are found in mitochondria and chloroplasts as well as prokaryotes. This group includes chaperonin 60 and its co-chaperon chaperonin 10. Chloroplast chaperonins have effects on growth, embryo development, flowering, and chlorosis of plants. In unstressed conditions, HSP60s utilize appropriate folding of the key proteins, while under heat stress they take part in prevention of protein misfolding and promote re-assembling and refolding of mitochondrial matrix proteins. Group II chaperonins are found in archaea and eukaryote cytosols, in general [16, 17].

#### *Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*

Small HSPs are in the range of 15–42 kDa. They have highly conserved sequences in C-terminal; hence they are found in all domains of life. They interact with higher HSPs as co-chaperons in response to heat stress. Individually, they constitute the first line of maintenance for misfolding of proteins. Contrary to the higher HSPs, sHSPs are not ATP-dependent and have high specificity and capacity to bind disordered proteins during primarily as heat, oxidative, and salinity stress conditions. They do not possess the ability to fold unfolded proteins; however, they can prevent irreversible unfolding and protein aggregations by re-folding denaturated or already folded proteins to some extent. This large protein family consists of six classes based on their cellular localizations, immunological properties, and sequence alignments. Cytoplasmic and nuclear groups are clustered in classes I, II, and III, while classes IV, V, and VI are the groups found in chloroplast, ER, and mitochondria, respectively [18, 19].

In past decade, substantial knowledge has been accumulated on mechanism of HSPs and chaperones as they are regulatory molecules that participation in stress sensing, signal transduction, and transcription activation of stress responsive genes in heat stress management. Therefore, transgenic plant approach is widespread among the studies which aim to improve crop productivity during consistently increasing heat stress worldwide [20]. **Table 1** summarizes the recent progress of transgenic approach regarding HSPs to improve heat stress tolerance in crop and model plants.

### **2.2 Antioxidants**

Different plants present variations in temperature response depending on species, organs, and developmental stages. Disturbance in equilibrium between ROS scavenging capacity and ROS production during heat stress leads to major indirect effects in plants [30]. Perception of heat is a crucial step for induction of stress responsive gene expression. Beside its deleterious cellular effects, ROS has significant intra- and inter-cellular signaling properties for local and holistic control in plants. Through signal transduction, they contribute to the acquisition of thermotolerance along with HSPs, molecular chaperones, and phytohormones. Hyper-activation of the ROS scavenging components is also a viable strategy since it prevents cellular damage caused by ROS to membranes, organelles, and critical biomolecules as DNA, proteins, lipids, and more.

Enzymatic antioxidant defense in plants is composed of super oxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monohydro ascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST), guaiacol peroxidase (GPX), peroxiredoxine (Prx), and thioredoxine (Trx). On the other hand, non-enzymatic antioxidant defense is divided into two categories as water solubles as ascorbic acid (AsA), glutathione (GSH), polyphenol, and lipid solubles as α-tocopherols, carotenoids, flavonoids, and retinoids [31].

In cellular processes, superoxide ions (O2 − ) are converted to H2O2 by SOD in chlorophyll, cytosol, apoplast, mitochondria, and peroxisomes. H2O2 is detoxified into H2O by CAT in peroxisomes, chlorophyll, and mitochondria, APX in chlorophyll, cytosol, apoplast, mitochondria, peroxisomes, and GPX in mitochondria and cytosol. GST also contributes to the process in chlorophyll, cytosol, and mitochondria. The oxidized form of GSH is produced through the DHAR activity in chlorophyll,


**Table 1.**

*Recent HSP gene transfers to improve heat stress tolerance in plants.*

*Abiotic Stress in Plants – Adaptations to Climate Change*

**128**

#### *Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*

cytosol, mitochondria, and GPx activity in mitochondria and cytosol. GR reduced this by-product back into the reduced form of GSH. Monodehydroascorbate (MDHA) and dehydroascorbate (DHA) are produced as a result of APX activity, and both are reduced to AsA by MDHAR and DHAR, respectively, in chlorophyll, cytosol, and mitochondria [32]. Beside the notorious antioxidant enzymes as CAT and GPX, kinetic studies point out that Prxs reduce more than 90% of cellular peroxides [33]. Trxs function as cysteine reductases in plants. Eukaryotic cells utilize sensor proteins with redox-sensitive cysteine residues that function as signaling switches. Cysteines also provide signaling complexity through allowing reversible redox-based modifications such as S-nitrosylation, S-sulfenation, S-thiolation, and S-glutathionylation [34]. Therefore, redox sensors as Prx and redox transmitters as Trx take part crucial roles in posttranscriptional/translational regulation and initiation of signaling cascades during stress conditions [35].

Basal heat tolerance is significantly stronger in enhanced ROS scavenger species since their ROS scavenging gene expression is rapidly induced during heat stress. Therefore, fortification of antioxidant machinery is preferable option for reverse genetic applications as transgenic approaches. **Table 2** summarizes the recent progress of transgenic approach regarding antioxidants to improve heat stress tolerance in crops and model plants.

#### **2.3 Osmolytes**

Osmolytes, also known as cytoprotectants, osmoprotectants, or compatible solutes, are low molecular weight (LMW) compounds or metabolites that play important roles in balancing cellular redox, maintaining membrane integrity and protein stability, scavenging ROS, defending antioxidant compounds, and easing toxicity, and protecting cellular components in total. There are numerous samples which can be categorized as sugars, polyamines, secondary metabolites, amino acids, and polyols as proline, glycine betaine, trehalose, sorbitol, gamma-aminobutyric acid (GABA) which are widely used in bioengineering applications named as osmolyte induced stress tolerance [46]. Polyols as mannitol, D-ononitol, trehalose, sucrose, and fructane have been proven to accumulate in distinct evolutionary groups in response to various osmotic stress factors. They interact with the glutathione-ascorbate cycle enzymes which were mentioned earlier in this chapter to protect cellular membranes and enzyme complexes [47]. Proline, as one of the most studied amino acid type compatible solutes, has high water solubility and stable structure. Besides its essential structural roles, it plays well-known osmotic adjustment roles in plant cells. By these fundamental properties, its accumulation is observed in different kingdoms from bacteria to marine invertebrates. Most of the osmoprotectants are localized in cytoplasm during osmotic stress as it initiates. Osmoprotectants are suggested to ease osmotic imbalance through regulating osmotic potential within the cell. Reduced osmotic pressure maintains turgor pressure under heat stress conditions in which water potential is low as well as the conditions of high ionic strength. They also stabilize protein complexes and cellular membranes by protecting the hydration shell of proteins [48]. Genetic transformation technologies allow deliberate transfer of genes precisely in predictable manner. Therefore, transgenic approach is a viable option to manipulate the osmoprotectant biosynthesis pathways for enhanced accumulation of such molecules [49]. **Table 3** summarizes the recent improvements in osmolytes overexpressing transgenic plant approaches to provide protection by the osmotic action alone.


*Recent antioxidant gene transfers to improve heat stress tolerance in plants.*


**Table 3.**

*Recent osmoprotectant gene transfers to improve heat stress tolerance in plants.*

#### *Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*

### **2.4 Transcription factors (TFs)**

Heat stress adversely affects the vegetative and reproductive stages of crop plants and leads to vast yield losses. Response to heat stress requires alterations on regular metabolic pathways through changes in gene expression profiles which are mainly regulated by various types of transcription factors (TFs). TFs are trans-acting elements which interact with cis-acting elements in promoter region of target stress responsive genes through genome. They are important signal transducers which convert perceived stress signals to stress specific responses. Many TFs including WRKY, MYB, NAC, bZIP, zinc finger protein, AP2/ERF, DREB, ERF, bHLH, and brassinosteroids transcription factors are associated with families of heat stress transcription factors or heat shock factors (HSFs). Approximately 7% of protein coding sequences of plant genomes consist of TFs. HSFs are among the largest gene families in plants compared to the other eukaryotes. The multiplicity of HSFs in plants is suggested to be related to the gene duplications and whole-genome duplications at different stages of evolution [57]. Plant HSFs have highly conserved modular structure. Their N-terminal domain has DNA binding properties. Promoter sequences of heat stress responsive genes include heat stress elements (HSEs) and are specific targets for central helix-turnhelix motif of HSPs. The C-terminal is an activation domain for plant HSFs. It contains short peptide motifs which play important roles in transcription activation of stress-inducible genes. Depending on hydrophobic amino acid residues linked to the DNA binding domain, plant HSFs are classified into three classes as HSFA, B, and C. HSFBs present common properties to the HSFs of other domains of life. On the other hand, HSFAs have 21 additional amino acid residues, while HSFCs have seven amino acid residue extensions [37]. HSFs are responsive to various abiotic stresses as drought, heat, and salinity. In nature, plants are constantly subjected to combination of different biotic and abiotic stresses. Therefore, it is considerably challenging to extrapolate the tolerance contribution of individual HSFs directly. Nevertheless, each TFs regulates many genes and thus are good candidates for engineering crop plants with enhanced heat stress tolerance due to their regulatory role. **Table 4** summarizes the recent progress of transgenic approach regarding TFs to improve heat stress tolerance in crop and model plants.

#### **2.5 MicroRNAs (miRNAs)**

In recent years, as an inevitable result of global climate change, there has been a significant increase in the number and severity of abiotic stress factors that plants are exposed to. Plants are vulnerable to the effects of heat, drought, salinity, cold, heavy metals, diseases, and pests due to their sessile nature. Therefore, the importance of developing plants tolerant to stress is increasing day by day. One of the most powerful methods for producing tolerant plants stands out as transgenic plants. MicroRNA (miRNA) transfer to plants is used as an important tool for thermotolerance.

miRNAs are RNA molecules with a length of 19–24 nucleotides (nt), not encoded by genes and involved in the regulation of gene expression. miRNAs are synthesized in the nucleus by RNA polymerase II as pri-miRNAs called precursor miRNAs. These pri-miRNAs are in hairpin structure and contain the mature miRNA sequence. The pri-miRNA structure is cleaved by the RNAase III enzyme to form the pre-miRNA


#### *Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*


*Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*


**Table 4.**

*Recent TF gene transfers to improve heat stress tolerance in plants.*

molecule. The resulting pre-miRNA is transported to the cytoplasm via Exportin-5 (XPO5). 19–24 nt long duplex miRNA is formed by being cut again by Dicer, a ribonuclease enzyme in the cytoplasm. Argonaute in the RNA-induced silencing complex (RISC) complex, which will form the mature miRNA sequence, is loaded. The miRNA-loaded RISC complex regulates transcriptional repression or degradation of mRNA. miRNAs play a role in the regulation of many biological processes in the cell, such as plant growth, development, stress responses, and control of the correct folding of proteins [86, 87].

High temperatures reduce the efficiency of photosynthetic activity in plants, cause negative effects on growth, damage to cell membranes, cell death due to senescence, protein misfolding, decrease in germination percentage, and release of weak pollen by preventing decomposition of anthers. Transfer of miRNAs to plants for tolerance to abiotic stresses is an important tool for plant tolerance. miRNAs increase the tolerance to stress factors by acting on the expressed genes at the transcriptional and posttranscriptional levels, inhibiting or regulating them. Temperature-sensitive miRNAs provide refolding of proteins, regulation of flowering, protection of reproductive tissues, repair of photosynthetic damage and regulating the antioxidant defense mechanism to alleviate the effects of stress. **Table 5** summarizes the recent progress of transgenic approach regarding miRNAs to improve heat stress tolerance in crop and model plants.

#### **2.6 Other approaches**

For producing temperature-tolerant genetically modified plants, it is a prerequisite to figure out how plants respond and adapt to heat stress and to characterize and identify novel heat stress-related genes. Heat stress (HS) can affect almost all aspects of plant processes such as germination, growth, development, reproduction, and yield, particularly by disturbing metabolic homeostasis, protein folding and processing capacity. In response to this challenge, plants utilize pathways/molecular mechanisms in complex and diverse systems, including photosynthetic metabolism, chaperones, signal transduction, epigenetic regulation, hormone signaling, lipid biosynthesis, plant growth regulation and additional intracellular actions. This radius of influence has allowed the development of a wide variety of strategies for the improvement of thermotolerance enhanced crop plants using genetic engineering approaches. Previous parts of the chapter presented that heat stress proteins (HSPs), heat stress factors (HSFs), transcription factors, osmoprotectants, ROS scavenging enzymes, and miRNAs are vital players in the plant's response to heat stress. In addition to all these responses, numerous studies have been reported to increase thermotolerance of plants by transferring genes that play a key role in plant metabolism to heat sensitive plants. Among these genes involved in stress management, genes encoding energy-dependent proteases, intramembrane proteases, calciumdependent protein kinases, methyltransferases responsible for histone methylation, rubisco-related enzymes involved in carbon assimilation, enzymes involved in RNA metabolism, proteins acting as transcriptional regulators, molecular chaperones such as disulfide isomerases, 14-3-3 and DnaJ-like proteins, phytohormones, proteins participated in metal hemostasis, the ubiquitin-proteasome system, carotenoid and flavonoid accumulation, and late embryogenesis abundant proteins come to the forefront as an effective targets. **Table 6** summarizes the recent progress of transgenic approach regarding miscellaneous targets to improve heat stress tolerance in crop and model plants.


**Table 5.**

*Recent miRNA gene transfers to improve heat stress tolerance in plants.*

## *Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*


*Abiotic Stress in Plants – Adaptations to Climate Change*


*Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*


**Table 6.**

 *Miscellaneous recent gene transfers to improve heat stress tolerance in plants.* *Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects DOI: http://dx.doi.org/10.5772/intechopen.111791*
