Transgenic Plants in Heat Stress Adaptation: Present Achievements and Prospects

Abstract

Global warming, which was rhetorical in the previous century, is a preeminent issue in multiple scientific areas today. Global warming has increased the frequency of extreme high temperature events all around the globe and expanded heat zones from tropic areas through both poles and even changed frigid poles to temperate zones. In the terrestrial earth, plants are the major CO2 consumers. The emergence and evolution of plants on earth decreased the global temperatures dramatically from mid-Devonian to mid-Carboniferous Era; however, the human factors as industrialization were not in equation. Today, plants are still main actors of the nature-based solutions to global warming through afforestation and reforestation solutions. However, high temperature is a major deleterious abiotic stress for plant growth and productivity. Plant heat stress adaptation has been a focus of research for both environmental and agricultural purposes. Plant heat stress adaptation requires utilization of complex physiological traits and molecular networks combined. The present chapter summarizes recent progress in transgenic approach through five main targets as heat shock proteins, osmoprotectants, antioxidants, transcription factors, and miRNAs. Additionally, miscellaneous novel transgenic attempts from photosynthetic machinery to signal transduction cascades are included to cover different physiological, transcriptional, and post-transcriptional regulation of the plant heat responses.

1. Introduction

Plants are subjected to various biotic (insects, parasites, nematodes, weeds, bacteria, fungi, viruses, etc.) and abiotic stress factors in their natural environment due to the stationary lifestyle. A major part of the abiotic stresses is caused by factors related to the physical and chemical composition of the soil, while the rest may be related to climate properties such as cold and heat, UV exposure, and light intensity. Among these, heat stress is particularly important since all the anabolism and catabolism reactions require particular cardinal temperatures for enzyme activities. Average surface temperature of the earth increased roughly 1°C since the beginning of pre-industrial era 120–140 years ago. In local terms, it may seem insignificant; however, globally accumulated heat has vast effects. Various independent research groups measure and calculate global average surface temperatures through absolute temperature observations and temperature anomalies from different locations [1]. According to the Annual Global Climate Report 2021 statistics of The National Oceanic and Atmospheric Administration (NOAA), 2021 was the sixth warmest recorded year of the earth since 1880 by the rate of 0.84°C higher than average of twentieth century. It was also the 45th consecutive year in which the average global temperature surpassed the average of twentieth century, which means it was never colder than average since 1977. Each year in the last decade takes place among the top ten warmest years. The average temperature increase per decade was in range of 0.08°C since 1880; however, the rate was increased 2.25-fold to rate of 0.18°C since 1981. Worse than this rate, the earth is expected to warm roughly 1.5°C within the next two decades [2]. Moreover, major crops in tropical and subtropical regions present 2.5–16% yield losses for every 1°C increase in seasonal temperatures. Global temperature rises also lead to reduction in land and sea, more frequent heavy rains, increase on habitat ranges of some plants and animals and decrease on some others, regionally [3].

Heat stress changes diverse molecular pathways and causes physiological and morphological alterations. Various stages of plant development such as germination, seedling emergence, tillering, floral initiation, pollination, fertilization, and consequently yield and grain quality are in range of heat effects. There are multiple factors which may divert the heat effects to a more dramatic or mild direction. Length, abruptness, and magnitude of heat are the major factors along with relatively minor factors as soil moisture and atmospheric CO₂ concentrations [4]. Anther and pollen development stages are considered as the most heat vulnerable stages; however, exposure during earlier stages may also lead to inadequate germination through reduced root and shoot growth. Heat exposure after the germination stage reduces green leaf area and the number of tillers per plant to ease the effects through reducing exposure surface. Prolonged exposure after anthesis may lead to flower abortion. Heat stress after flowering stage is referred as terminal heat stress which effects early meiosis to tetrad stages of pollen production and utterly reduce grain number, filling, and maturity. Terminal heat stress does not only reduce quantitative traits but also reduce qualitative traits such as dry matter accumulation and grain quality. Developmental stage-specific treatments and breeding strategies against various heat regimes are still under investigation [5, 6].

Physiological functions are mediated through enzymatic processes in all living organisms. Even though, there are thermophile organisms in lower evolutionary branches as archaea, bacteria, and fungus which all have resilient enzyme systems. Any deviations over the optimum temperature hamper enzymatic processes of plants. Photosynthesis is one of the most vital but fragile metabolic processes which is severely affected by heat stress. Some heat acclimation adaptations including reduced spiky leaf shape, altered leaf orientation, rolled leaves or small surface hairs, thick waxy cuticle, and stomatal crypts are present in high heat climate plants to reduce drastic effects; however, crop plants do not possess most of these structures. Symptoms as lower stomatal conductance, reduced CO₂ assimilation, and water loss utilize non-photorespiratory processes. Heat stress also directly alters enzyme and protein structure and cell membrane permeability leading to photochemical modifications in chloroplasts, damage on thylakoid membrane, and reduction of soluble proteins as Rubisco and Rubisco binding proteins. Damaged chloroplasts cripple the photosynthetic capacity of plants and lead to leaf senescence, while disturbed thylakoid membranes elevate cellular reactive oxygen species levels. Respiration is crucial for leaf surface cooling in trade-off water loss. Leaf water potential and transpiration pull is a driving force for nutritional uptake and transport of photosynthesis assimilates from leaves to the grains. Heat stress also disturbs nitrate and ammonium assimilation. Factors as decreased root mass, surface area, and/or a decrease in nutrient uptake per unit root or direct heat damage to roots are plausible for nutrient acquisition decrease. Uptake of most of the nutritional elements is mediated by specific influx or efflux protein activity. Therefore, reduced proteins per unit root rate directly affect the mineral content [7, 8].

Heat stress causes all the above-mentioned damage through direct (primary) and indirect (secondary) effects at different levels. Weakening and damaging bio membrane integrity, altering fluidity, leading to electrolyte leakage, denaturing and misfolding proteins are among the most deleterious direct damage effects. Indirect effects can be listed as oxidative stress, methylglyoxal (MG) stress, and osmotic stress. Therefore, plant heat stress tolerance and adaptation mechanisms include heat shock proteins (HSPs), antioxidant systems, osmolytes, fortification of membrane lipids, and MG detoxification, in general. The present chapter will summarize the current knowledge on heat stress tolerance/adaptation approaches and will discuss transgenic approach contribution to these mechanisms with the emphasis on prospects.

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 thermo-tolerance 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 ATP-binding 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, 12, 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].

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.

Gene	Gene action	Gene source	Transformed plant	Transfer method	Agrobacterium strain	Heat tolerance (°C)	Reference
Hsp90A2	Heat shock protein 90s	Glycine max	Glycine max	Cotyledonary node co-cultivation infection	Agrobacterium tumefaciens EHA101	50	[21]
Hsp90	Heat shock protein 90s	Dianthus caryophyllus	Arabidopsis thaliana	Floral dip	Agrobacterium tumefaciens GV3101	42	[22]
Hsp90.5	Heat shock protein 90s	Chrysanthemum morifolium	Arabidopsis thaliana	Floral dip	Agrobacterium tumefaciens	40	[23]
Hsp70	Heat shock protein 70s	Agave sisalana	Gossypium hirsutum	Embryos co-cultivation infection	Agrobacterium tumefaciens LBA4404	45	[24]
Hsp70	Heat shock protein 70s	Zostera japonica	Arabidopsis thaliana	Floral dip	Agrobacterium tumefaciens GV3101	40	[25]
Hsp40, J3	Heat shock protein 40s	Arabidopsis thaliana	Arabidopsis thaliana	Floral dip	Agrobacterium tumefaciens GV3101	37	[26]
Hsp17.2	Cytosolic class II small heat shock protein	Primula forrestii	Arabidopsis thaliana	Particle bombardment	—	42	[27]
Hsp23	Small heat shock protein	Medicago sativa	Medicago sativa	Hypocotyls segments co-cultivation infection	Agrobacterium tumefaciens EHA105	42	[28]
Hsp25.9	Small heat shock protein	Capsicum annuum (R9)	Arabidopsis thaliana (Col-0)	Infiltration	Agrobacterium tumefaciens CV3101	38	[29]

Table 1.

Recent HSP gene transfers to improve heat stress tolerance in 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, 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.

Gene	Gene source	Transformed plant	Transfer method	Agrobacterium strain	Heat tolerance (°C)	Reference
Apx1 (cytosolic ascorbate peroxidase)	Hordeum vulgare (Haruna-nijyo)	Arabidopsis thaliana (Col-0)	Floral dip	—	35	[36]
Apx	Cephalotaxus fortunei [37]	Arabidopsis thaliana (Col-0)	Floral dip	—	40	[38]
Grxs17 (glutaredoxin S17)	Arabidopsis thaliana	Zea mays (B104)	Zygotic embryos co-cultivation infection	Agrobacterium tumefaciens EHA101	37	[39]
Grxs17	Arabidopsis thaliana; (Col-0)	Chrysanthemum morifolium (Ramat)	Leaf disc infection	Agrobacterium tumefaciens GV3101	45	[40]
Grx5	Pteris vittata	Arabidopsis thaliana (Col-0)	Floral dip	Agrobacterium tumefaciens C58	36	[41]
Mdar (Monodehydro ascorbate reductase)	Lycopersicon esculentum (Zhongshu 4)	Lycopersicon esculentum	Leaf disc infection	Agrobacterium tumefaciens LBA4404	40	[42]
Noa1 (Nitric Oxide-Associated Protein 1)	Arabidopsis thaliana; (Col-0)	Arabidopsis thaliana (bohB/D Mutant)	Floral dip	—	45	[43]
Prx 2-Cys (2-Cys peroxiredoxin)	Arabidopsis thaliana	Festuca arundinacea	Callus co-cultivation infection	Agrobacterium tumefaciens EHA105	42	[44]
Sod (Superoxide dismutase)	Allium sativum (Daeseo)	Nicotiana benthamiana	Infiltration	Agrobacterium tumefaciens GV3101	45	[45]

Table 2.

Recent antioxidant gene transfers to improve heat stress tolerance in 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.

Gene	Gene action	Gene source	Transformed plant	Transfer method	Agrobacterium strain	Heat tolerance (°C)	Reference
codA	Choline oxidase, Glycine betaine accumulation	Arthrobacter globiformis	Solanum lycopersicum	Cotyledon and hypocotyl co-cultivation infection	Agrobacterium tumefaciens EHA101	42	[50]
codA and BADH	Choline oxidase- Glycine betaine accumulation,; Betaine aldehyde dehydrogenase	Arthrobacter globiformis for codA,; Spinacia oleracea for BADH	Solanum lycopersicum	Cotyledon and hypocotyl co-cultivation infection for codA,; Leaf disc infection for BADH	Agrobacterium tumefaciens EHA101 for codA,; Agrobacterium tumefaciens LBA4404 for BADH	42	[51]
MIPS2	Osmoprotectants, myo-inositol-1-phosphate synthase 2	Triticum aestivum	Arabidopsis	Floral dip	Agrobacterium tumefaciens	45	[52]
PRP6	Proline-rich proteins (PRPs), cell wall proteins	Malus domestica	Nicotiana nudicaulia	Leaf disc infection	Agrobacterium tumefaciens EHA105	48	[53]
SlSPS	Sucrose phosphate synthase, sucrose synthesis; pathway	Solanum lycopersicum	Solanum lycopersicum	Cotyledon explants co-cultivation infection	Agrobacterium tumefaciens GC3101	42	[54]
SSI	Soluble starch synthase gene, starch biosynthesis	Oryza sativa	Triticum aestivum,	Biolistic transformation	—	34	[55]
TPSP	Trehalose-6-phosphate synthase-phosphatase fusion gene, enhancing the level of trehalose	Escherichia coli	Solanum lycopersicum	Cotyledon explants co-cultivation infection	Agrobacterium tumefaciens LBA4404	55	[56]

Table 3.

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

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-turn-helix 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.

Gene	Gene source	Transformed plant	Transfer method	Agrobacterium strain	Heat Tolerance (°C)	Reference
AF1 and ANAC055	Arabidopsis thaliana (Col-0)	A. thaliana (knock-out mutants ataf1 and anac055)	Floral dip	Agrobacterium tumefaciens GV3101	44	[58]
bZIP60	Triticum aestivum (TAM107)	A. thaliana (Col-0)	Floral dip	A. tumefaciens GV3101	50	[59]
BZR1 (Brassinazole Resistant)	Solanum lycopersicum mutant (Condine Red)	Solanum lycopersicum (Condine Red)	CRISPR/Cas9	A. tumefaciens EHA105	42	[60]
CBF1 (C-Repeat Binding Factor)	A. thaliana (Col-0)	A. thaliana (cbf1 Mutant)	CRISPR/Cas9	A. tumefaciens GV3101	42	[61]
DREB20 (DRE-binding transcription factor)	Musa acuminata (Grand Nain and Hill Banana)	A. thaliana (Col-0)	Floral dlp	A. tumefaciens GV3101	44	[62]
DREB2A	Pennisetum glaucum	A. thaliana (Col-0)	Floral dip	A. tumefaciens GV3101	37	[63]
ERF110 (APETALA2/Ethylene-responsive factor)	Lilium longiflorum (White heaven)	A. thaliana (Col-0) and Nicotiana benthamiana	Floral dip	A. tumefaciens GV3101	45	[64]
HB4 (HomeoBox)	Helianthus annuus	Glycine max (Williams 82)	Agrobacterium-mediated	A. tumefaciens EHA101	25–30	[65]
Hsf05 (Heat shock factor)	Zea mays (H21)	A. thaliana (athsfa2)	Floral dip	A. tumefaciens GV3101	42–45	[66]
HsfA1	Amorphophallus albus	A. thaliana	Infiltration	A. tumefaciens GV3101	41	[67]
HsfA1d	A. thaliana	Pisum sativum	Cotyledons co-cultivation infection	A. tumefaciens GV3101	37	[68]
HsfA2	Populus euphratica	Populus tomentosa (YiXianCiZhu B385)	Leaf disc infection	A. tumefaciens EHA105	45	[69]
HsfA2c	Festuca arundinacea (Barlexas)	A. thaliana (Col-0)	Floral dip/PEG-mediated transformation	A. tumefaciens EHA105	45	[70]
HsfA4	Lilium Longiflorum (White heaven)	A. thaliana (Col-0)	Infiltration	A. tumefaciens GV3101	45	[71]
HsfA6b	Triticum aestivum (PBW343)	A. thaliana (Col-0)	Floral dip	A. tumefaciens GV3101	42	[72]
HsfA6b	Triticum aestivum	Hordeum vulgare	Particle bombardment	—	35	[73]
HsfB1	Solanum peruvianum,	Solanum lycopersicum	Leaf disc infection	A. tumefaciens GV3101	39	[74]
HsfC1b	Lolium perenne	A. thaliana	Floral dip	A. tumefaciens	42	[75]
JA2 (Jasmonic acid)	Solanum lycopersicum	Nicotiana tabacum (NC 89) and A. thaliana (Col-0)	Leaf disc infection	—	42	[76]
MADS114 and MADS115	Dactylis glomerata (Donata)	A. thaliana (Col-0)	Floral dip	A. tumefaciens EHA105	40	[77]
NAC56	Prunus persica (chunjie)	Solanum lycopersicum (Micro Tom)	Infiltration	A. tumefaciens GV3101	42	[78]
NAC074	Zea mays (B73)	A. thaliana (Col-0)	Floral dip	A. tumefaciens GV3101	42	[79]
NTL3 (Plasma Membrane-Associated NAC Transcription Factor)	Oryza Sativa (Nipponbare)	Oryza sativa (Nipponbare)	CRISPR/Cas9	—	—	[80]
SHN1	H. vulgare	N. tabacum	Leaf disc infection	A. tumefaciens GV3101	42	[81]
WRKY30	A. thaliana (Col-0)	Triticum aestivum; (Sakha-61)	Agrobacterium-mediated	A. tumefaciens EHA105	40	[82]
WRKY40	Capsicum annuum	N. tabacum (K326)	Leaf disc infection	A. tumefaciens LBA105	42	[83]
WRKY149	Brassica napus (ZS11)	A. thaliana (Col-0)	Agrobacterium-mediated	A. tumefaciens GV3101	40	[84]
ZnF	Triticum aestivum	A. thaliana (Col-0)	Floral dip	—	42	[85]

Table 4.

Recent TF gene transfers to improve heat stress tolerance in 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 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 post-transcriptional 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.

Gene	Gene source	Transformed plant	Transfer method	Agrobacterium strain	Heat Tolerance (°C)	Reference
miR156	Arabidopsis thaliana (Col-0)	A. thaliana (AGO1)	Floral dip	—	44	[88]
miR160	A. thaliana (Col-0)	A. thaliana (arf10, arf16, and arf17 Mutant)	Floral dip	Agrobacterium tumefaciens LBA4404	44–50	[89]
miR164	A. thaliana (Col-0)	A. thaliana (Overexpressors 164OE)	Floral dip	A. tumefaciens LBA4404	35	[90]
miR167	Vitis vinifera (Thompson Seedless)	A. thaliana (Col-0)	Floral dip	A. tumefaciens GV3101	45	[91]
miR172b-3p	Solanum tuberosum (Unica)	S. tuberosum (Russet Burbank)	Leaf and internodal co-cultivation infection	A. tumefaciens LBA4404	39	[92]
miR319d	Solanum habrochaites (LA1777)	Solanum lycopersicum (Micro-Tom)	Leaf disc infection	A. tumefaciens GV3101	40	[93]
miR398	A. thaliana (Col-0)	A. thaliana (csd2 Knockdown Mutant)	Floral dip	A. tumefaciens GV3101	37	[94]
miR447A	A. thaliana (Col-0)	A. thaliana Flower (Col-0)	Agrobacterium-mediated	A. tumefaciens GV3101	27	[95]
miR398a, miR398b, miR398c	A. thaliana (Col-0)	A. thaliana (Col-0)	—	A. tumefaciens GV3101	38	[96]
Novel_105 miRNA	S. tuberosum (Unica)	S. tuberosum (Russet Burbank)	Internode co-cultivation infection	A. tumefaciens LBA4404	39	[97]

Table 5.

Recent miRNA gene transfers to improve heat stress tolerance in 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, calcium-dependent 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.

Gene	Gene action	Gene source	Transformed plant	Transfer method	Agrobacterium strain	Heat Tolerance (°C)	Reference
ALA6	Aminophospholipid ATPase6 membrane systems	Arabidopsis thaliana	A. thaliana	Floral dip	Agrobacterium tumefaciens GV3101	37.5–43	[98]
ChiVI2	Chitin-binding proteins (CBP)	Capsicum annuum	A. thaliana	Floral dip	A. tumefaciens GV3101	45	[99]
CLPB1	Clp ATPases, energy-dependent proteases and; molecular chaperones.	Ziziphus nummularia	Nicotiana tabacum	Leaf disc; infection	A. tumefaciens EHA105	42	[100]
CDPK7	Calcium-dependent protein kinase, Ca2 + −mediated signaling pathways	Zea mays	Z. mays	Embryo co-cultivation infection	A. tumefaciens GV3101	28–42	[101]
CPL1	RNA polymerase II CTD phosphatase-like 1 enzyme, transcriptional regulator of the plant response to various; abiotic stresses	Chrysanthemum morifolium	C. morifolium	Leaf disc infection	Agrobacterium; tumefaciens EHA105	45	[102]
DJLP	DnaJ-like protein, molecular chaperone	Medicago sativa	N. tabacum	Leaf disc infection	A. tumefaciens EHA105	42	[103]
DOG1L-T	Delay of germination 1-like,	N. tabacum	N. tabacum	Infiltration	A. tumefaciens	45	[104]
F3H	flavanol 3-hydroxylase flavonoid biosynthesis	Oryza sativa	O. sativa	Calli co-cultivation infection	A. tumefaciens LBA4404	40	[105]
FBA1	F-Box protein gene, core component of the Skp1-Cullin-F-box (SCF) E3 ligase complex	Triticum aestivum	N. tabacum	Leaf disc infection	A. tumefaciens LBA440	45	[106]
FER-5B	Ferritin gene, iron storage protein, sequestering or releasing iron	T. aestivum	A. thaliana T. aestivum	Floral dip for Arabidopsis,; Immature embryos particle bombardment for T. aestivum	A. tumefaciens GV3101	40	[107]
FT-1	14–3-3 protein, molecular chaperone	Haloxylon ammodendron	A. thaliana	—	—	37–47	[108]
GLP	hydrogen peroxide-producing germin-like protein	Solanum tuberasum	Solanum tuberasum	Infiltration	A. tumefaciens GV3101	25–45	[109]
Golden SNP-Carrying Orange Gene	Carotenoid accumulation	Ipomoea batatas	I. batatas	Embryogenic calli co-cultivation infection	A. tumefaciens GV3101	47	[110]
HIRP1	E3 ligase, heat-induced RING finger protein 1	O. sativa	A. thaliana	Floral dip	A. tumefaciens GV3101	45	[111]
HVA1	Late embryogenesis abundant (Lea) protein, leading to the; accumulation of small Hsps	T. aestivum	T. aestivum	Anther culture-based approach	A. tumefaciens LBA4404	42	[112]
OEP16–2-5B	Wheat plastid outer envelope protein gene	T. aestivum	A. thaliana	Floral dip	A. tumefaciens GV3101	40	[113]
PhyCYS	Phytocystatins, proteinaceous inhibitors of the papain-like (C1A) and legumain (C13) families; of plant cysteine proteases (CPs)	A. thaliana	A. thaliana	Floral dip	A. tumefaciens GV3101	37–50	[114]
PRMT1	Protein arginine methyltransferases (PRMTs)	Z. mays	A. thaliana	Floral dip	Agrobacterium-mediated	42	[115]
Rca1β	Rubisco activase B,	T. aestivum,	O. sativa	Scutellum tissue co-cultivation infection	A. tumefaciens	42°C	[116]
Rubisco and Rubisco activase	Catalyzing the binding of CO2 to ribulose-1,-; 5-bisphosphate (RuBP) and; rubisco activation	Z. mays	O. sativa		Agrobacterium-mediated	36–40	[117]
SAMS	S- adenosyl methionine synthetase, Spd biosynthesis	Trifolium repens	A. thaliana	Floral dip	A. tumefaciens EHA105	33–38	[118]
SlEGY2	Metalloprotease	Solanum lycopersicum	S. lycopersicum (antisense transgenic tomato plant)	Leaf disc, anti-sens	A. tumefaciens LBA4404	42	[119]
PDI	Protein disulfide isomerase gene, chaperone; function and disulfide isomerase activity	Methanothermobacter thermautotrophicus	O. sativa	Tissues co-cultivationinfection	A. tumefaciens EHA105	42	[120]
TOGR1	DEAD-Box RNA helicase, RNA metabolism	O. sativa	Brassica rapa	Vacuum infiltration	A. tumefaciens EHA105	38–46	[121]

Table 6.

Miscellaneous recent gene transfers to improve heat stress tolerance in plants.

3. Conclusions

Proteins undertake important structural and functional properties in cells. Among all abiotic stress factors, heat affects biological activity of proteins more directly by leading to aggregation and/or misfolding. HSPs constitute the frontal zone of defense against heat stress-induced accumulation of aggregated/misfolded proteins which may induce heat shock responses (HSR) in plant cells. Hsps are main targets for gene transfer approaches due to their chaperone roles to co-operate functional networks as well as re-solubilization roles for the recovery phase of aggregated/misfolded proteins. Along with the definite evidence to succession of HSP gene transfer-related thermotolerance, osmolytes as members of non-enzymatic antioxidative system contribute to the process through habilitating cellular environment to more reductive state due to higher energy status. Hence, by binding to the cellular proteins, they protect them from denaturation/aggregation. Likewise, enzymatic antioxidant systems as cell detoxification components undertake the major role in regulation of reductive cellular environment and minimizing the loss of active proteins. Besides, classification and association of different HSFs and HSPs as functional candidates in heat stress tolerance and other developmental pathways are extremely crucial. Even though structural and functional association of Hsps/Hsfs have been widely established, they are still not mainstream targets in crop plant applications against heat stress. However, applicability is improving impetuously. On the other hand, transgenic approaches in heat stress tolerance through miRNAs in plants mainly involve model plants such as Arabidopsis or rice at present. Moreover, stress and species-specific miRNAs still require further discovery. A large number of miRNAs and their target genes related to heat stress have not been discovered yet. Since individual miRNAs may also play multiple roles in other various development regulatory pathways and biotic and/or abiotic stresses as well as heat, it is necessary to explore new miRNAs, reveal their target genes, and further evaluate the miRNA-mediated regulatory networks before announcing them as designated targets. Other than protecting protein stability, it is also a viable approach to sustain cellular membranes as their fluidity is vital to maintain cell volume. The physical state of the cellular membranes influences gene expression by initiating signal transduction. Altering membrane structures can also affect interactions of membrane lipids with proteins. Hereby, we can conclude transgenic approaches may still offer vast number of opportunities to heat stress tolerance area as in recent years we can follow miscellaneous novel targets from photosynthetic machinery to signal transduction cascades, despite the fact that there is still no biotech/GM crop events that have been approved for commercialization/planting and importation (food and feed) in International Service for the Acquisition of Agri-biotech Applications (ISAAA) GM Approval Database for heat stress tolerance.

Abbreviations

ABA	abscisic acid
ALA	aminophospholipid ATPase
AP2/ERF	APETALA2 (AP2)/ethylene responsive element binding factor (EREB)
APX	ascorbate peroxidase
AsA	ascorbic acid
ATP	adenosine triphosphate
BADH	betaine aldehyde dehydrogenase
bHLH	basic helix-loop-helix
bZIP	Basic Leucine Zipper
CAT	catalase
CBF	C-repeat binding factor
CBP	chitin-binding proteins
CDPK	calcium-dependent protein kinase
Cod	choline oxidase
Col-0	Columbia-0
CRISPR	clustered regularly interspaced short palindromic repeats
DHA	dehydroascorbate
DHAR	dehydroascorbate reductase
DJLP	DnaJ-like protein
DNA	deoxyribonucleic acid
DOG1L	delay of germination 1-like
DREB	dehydration-responsive element-binding protein
ER	endoplasmic reticulum
F3H	flavanol 3-hydroxylase
GABA	gamma-aminobutyric acid
GLP	Germin-like protein
GM	genetically modified
GPx	glutathione peroxidase
GPX	guaiacol peroxidase
GR	glutathione reductase
GSH	glutathione
GST	glutathione-S-transferase
HB	HomeoBox
HIRP	heat-induced RING finger protein
HOP	HSP90/HSP70-organizing protein
HS	heat stress
HSEs	heat shock elements
HSFs	heat shock transcription factors
HSPs	heat shock proteins
ISAAA	International Service for the Acquisition of Agri-biotech Applications
JA	jasmonic acid
kDa	kilodaltons
MDHA	monodehydroascorbate
MDHAR	monohydro ascorbate reductase
MG	methylglyoxal
MIPS2	Myo-inositol-1-phosphate synthase 2
miRNAs	micro RNAs
MYB	Myb-related protein B
NAC	NAM (No Apical Meristem), ATAF1/2 (Arabidopsis thaliana Transcription Activator Factor 1/2) and CUC2 (Cup-shaped Cotyledon 2)
Noa1	nitric oxide-associated protein 1
NOAA	The National Oceanic and Atmospheric Administration
nt	nucleotide
OEP	outer envelope protein
PDI	protein disulfide isomerase
PRMT	protein arginine methyltransferases
PRPs	proline-rich proteins
Prx	peroxiredoxine
Rca	rubisco activase
RISC	RNA-induced silencing complex
ROS	reactive oxygene species
Rubisco	ribulose-1,5-bisphosphate carboxylase/oxygenase
SAMS	S-adenosyl methionine synthetase
SISPS	sucrose phosphate synthase
SNP	single nucleotid polymorphism
SOD	super oxide dismutase
SSI	starch synthase I
TFs	transcription factors
TOGR	thermotolerant growth required
TPSP	trehalose-6-phosphate synthase
Trx	thioredoxine
UV	ultraviolet
ZnF	Zinc Finger