LIST OF TABLES
2. INTRODUCTION
2.4 Potential strategies to combat the climatic challenges
density of Na+ compared to K+ makes it a weaker kosmotrope, affecting the biochemistry of enzymes, other proteins, and DNA. Na+ cannot compensate for the biochemical requirement of K+ in enzymatic and polysome activity, which can also lead to K+ associated programmed cell death (PCD) [278]. Salinity also transiently arrests cell-cycle by diminishing the activity of cyclins and cyclin-dependent kinases (CDKs) to conserve energy and augment defense and repair cellular systems [279]. Despite osmotic stress and ion toxicity, salinity also imposes oxidative damage, photosynthetic degradation, nutrient deficiency, and deteriorates the water- uptake ability to induce water deficiency. High salt levels upset the nutrient balance by interfering with the uptake of nitrogen, phosphorus, calcium, potassium, etc. [280, 281].
Salinity declines photosynthesis efficiency mainly through shrinking leaf area, chlorophyll content, and stomatal conductance [282]. Salinity adversely affects reproductive development by inhibiting microsporogenesis, ovule abortion, and senescence of fertilized embryos [283, 284].
2.3.4 Nutrition deficiency and oxidative stress inhibit growth and survival
Dehydration and salt stress often impose nutrition deficiency in the stressed plants. The physiological damage caused by the stress degrades the plant’s ability to assimilate and transport the available sources of nutrition. Besides channeling of plant energy towards defense and repair, nutrition starvation can also be one of the factors leading to growth retardation during stress. Moreover, oxidative stress may also occur as a concomitant effect, resulting in the generation of reactive oxygen species (ROS) such as superoxide anion radicals (O2-), hydroxyl radicals (OH-), hydrogen peroxide (H2O2), alkoxy radicals (RO), and singlet oxygen (O21) [285]. Chloroplasts are the primary source of strong oxidants generated due to excited pigments in thylakoid membranes. Their excess accumulation can react with cellular biomolecules, leading to lipid peroxidation, membrane injuries, protein degradation, and enzyme inactivation.
2.4.1 Life-span and phenotypic flexibility
Crop stress tolerance is defined as maintaining the shoot growth and yield under prolonged terminal stress. When the phenological development is compatible with the available soil- moisture and seasonal temperature, the plant may escape drought and heat by cutting its life- cycle short and induce early flowering and seed-setting, facilitating the completion of reproductive span, or even delaying the reproductive phase until the unfavorable conditions.
Adjusting flowering time is helpful for short-term stress mitigation in crops with indeterminate growth habits like cowpea and chickpea. However, under terminal stress conditions, the plants employ avoidance strategies involving adjustment in morphological features of shoot and root, such as controlling water loss through stomatal transpiration, increasing water-use efficiency (WUE) by developing glacousness and waxy bloom on leaves, and improving water uptake through a deeper rooting system. Plants generally limit water consumption through xeromorphic traits like adjusting leaf number, area, and pubescence. To retain the water potential and improve water-use efficiency, plants undergo leaf shedding and produce smaller leaves. The production of leaf hairs and trichomes protects plants from transpiration, radiation, and excessive heat. Although tolerance is defined as the plant’s ability to rescue the shoot growth, the root is the only medium that can acquire available water to maintain a favorable water status [286]. Developing an extensive, prolific, and thicker root architecture improves drought adaption by constitutive water uptake. Drought-induced rhizogenesis occurring in Brassicaceae and related families is associated with the formation of short, tuberized, hairless roots capable of retaining turgor pressure to withstand prolonged drought. As crop yield is linearly related to the duration of crop, growth, and biomass production, most of the above- stated adaptive strategies mitigate stress, but at the cost of yield penalty.
2.4.2 Osmotic adjustment, osmoprotectant, and maintenance of membrane-integrity A more progressive drought tolerance mechanism includes physiological mechanisms such as osmotic adjustment, production of osmolytes, and strengthening of the cell membrane [287].
Although cell or tissue water potential is not a defining feature for drought sensitivity, osmotic adjustment and cell wall elasticity can elevate the tissue water status [288]. Osmotic adjustment can be achieved by the active accumulation of various compatible solutes such as proline, glutamate, carnitine, glycine betaine, organic acids, sugar derivatives like sorbitol, mannitol, trehalose, Ca+, Na+, and Cl- ions, etc. The compatible solutes are highly soluble, non-toxic at high concentrations, and do not interfere with cellular macromolecules. Their accumulation
decreases the internal water-potential, allowing the water-influx to maintain the high turgor and water-status for a prolonged period. This can delay dehydrative and osmotic damage and support cell-turgor, photosynthesis, nutrient translocation, and other physiological functioning crucial for growth.
Besides, the compatible solutes also serve as osmoprotectants to detoxify ROS, stabilize membranes, and retain the native conformation of cellular enzymes and biomolecules. Free proline serves as cytosolute to lower cytosolic water potential, a ROS scavenger, molecular chaperone, pH buffer, and store for carbon and nitrogen assimilation [289]. Similarly, glycinebetaine also plays a vital role in multiple stress tolerance. Citrulline is the most effective OH- scavenger, also guarding DNA and enzymes against oxidative injuries. Trehalose, a non- reducing sugar, functions by stabilizing dehydrated biomolecules under desiccation, even at a small amount. Apart from stress adaptation, the compatible solutes also participate in stress- induced signal transduction pathways. In grain legumes, osmotic adjustment is achieved by increasing the sugar alcohols like mannitol, sorbitol, and inositol with a parallel decline in sugar.
Biological membrane integrity is a primary physiological index to measure drought, salt, and heat tolerance. The cell and organelle membrane may be injured by dehydration, salt- induced ionic imbalance, or temperature-induced deformation of membrane proteins. The increased permeability of the membrane leaves them exposed to the risk of membrane fusion, protein denaturation, and solute leakage. The membrane stability may be strengthened by osmotic adjustment by accumulating compatible solutes. Another way of adapting is increasing polar lipids to refine the biochemical composition of the membrane.
2.4.3 Increased production of anti-oxidants and ROS scavengers
To mitigate the deleterious effects, plants employ a ROS scavenging system consisting of the non-enzymatic components like cysteine, reduced glutathione, reduced ascorbate, carotenoids, tocopherols, flavonoids, di-terpenes, etc., and the enzymatic components such as ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (PRX) [285]. In the photosynthetic tissue, the ROS scavengers are crucial for sustaining photochemical processes and chloroplast functioning. As stress tolerance is itself a cost-intensive phenomenon, it involves spending a considerable amount of energy on root growth, maintenance, and dry matter production,
resulting in ATP depletion. ATP synthesis accompanies ROS generation. Plants use alternative oxidase (AOX) as the terminal electron acceptor to circumvent the mitochondrial ROS level.
Boosting the synthesis of antioxidants and ROS scavenging molecules can mitigate oxidative damage, and supporting the cellular energy pool, can be a more efficient strategy.
2.4.4 Tuning the balance between growth regulators and stress hormones
Plant growth regulators (PGRs), also referred to as phytohormones, such as auxins, gibberellins (GAs), cytokinins (CKs), ethylene, and abscisic acid (ABA), jasmonic acid (JA), that control diverse aspects of plant growth and development, endogenously as well as when applied externally. They act both under normal and stressed conditions, although differentially.
Generally, under drought stress, the endogenous concentration of growth hormones (auxin and cytokinin) decreases, while the content of stress hormones such as ABA and ethylene elevates [290]. Auxin, CKs, and GAs are mandatory for the induction of cell division to regulate growth and flower development. Auxins induce new root formation by breaking the root apical dominance induced by CKs. Besides, the two hormones cross-talk between the growth and ROS signaling [291], and regulate stomatal opening by counteracting the action of ABA, a key mediator of stress signaling [43]. Under water-deficit conditions, ABA biosynthesis is induced, and its catabolism is suppressed. ABA regulates stomatal aperture to control water loss, enhances anti-oxidant enzymatic activities, orchestrates cascades of stress signaling (mainly desiccation), and expression of ABA-induced genes, usually in coordination with JA [292, 293]. Nevertheless, being a growth inhibitor, ABA retards growth by increasing seed dormancy, limiting the formation of leaf area and deep root system. Though, its effect can be antagonized by GAs and CKs [44]. Similarly, ethylene signal the onset of senescence and optimizes the vegetative growth to survive abiotic stresses [294]. However, a balance in stress and growth hormone signaling such as ABA/GA ratio and its homeostasis is required to avoid growth retardation under stress.
2.4.5 Overexpressing the molecular regulators of stress tolerance
Plants employ ubiquitously available functional and regulatory proteins to mitigate stress.
The functional stress molecules include water channels/transporters, protection factors (LEA, chaperones), antioxidants, osmolytes, biosynthetic enzymes, and proteases. In contrast, transcription factors (TFs), protein kinases, hormones, and other signaling molecules, are the regulatory molecules.
2.4.5.1 Upregulating stress-mitigating functional proteins
Aquaporins are transmembrane intrinsic proteins (IPs), forming channels in plasma membranes (PIPs), tonoplast (TIPs), or nodulin (NIPs) to facilitate the passive movement of water in either direction across the water channel pore. In addition to water, some major intrinsic proteins (MIPs) can also transport glycerol, CO2, urea, ammonia, hydrogen peroxide, boron, lactic acid, and O2 [295]. Several reports are available that support overexpression of aquaporins to improve hydraulic conductivity of membranes and enhance the water uptake by more than 10-fold, resulting in increased plant vigor, water-use efficiency, and water retention, to cope with dehydration, ion toxicity, and osmotic stress [296-298]. For instance, the expression of VfPIP1 in Arabidopsis improved drought resistance by reducing transpiration rates. Over-expression of RWC3 in rice showed increased resistance to osmotic stress by enhancing root hydraulic conductivity [296]. In tobacco, overexpression of AtPIP1b increased stomatal density and transpiration rates. Synthesis of stress proteins, molecular chaperones, and antioxidant enzymes is exclusively implicated in abiotic stress tolerance [297, 298]. Late embryogenesis abundant (LEA)/dehydrin-type proteins and heat-shock proteins are induced in drought, extreme temperatures, and even at hypoxia conditions to stabilize and protect the vital cellular enzymes and proteins from denaturation. Dehydrins confer stress tolerance by increasing water-binding capacity through hydrophobic interactions and sequestering ions concentrated under a desiccated environment [297]. Heat-shock proteins (chaperones) prevent irreversible misfolding and aggregation of proteins and guide the correct refolding of denatured proteins [298].
2.4.5.2 Transcriptional reprogramming of stress-responsive genes
TFs monitors stress-specific gene expression by binding to cis-regulatory elements (TFBS) in the promoter of their target genes to mediate ABA-dependent and ABA-independent stress transduction. AREB/ABF family regulates ABA-mediated drought and salt stress signal signaling by binding to the ABRE motif (ACGTGGC) present in bZIP proteins encoded by genes, such as RD29A, RD20A, RD22, RD26, etc [299]. Another drought-responsive family of MYB/MYC/MYBR proteins like MYB2 and MYC2 recognizes stress marker genes like RD22 via MYBRS/MYCRS motifs (YAACR/CANNTG) to confer ABA-dependent tolerance [300- 302]. In contrast, DREB1/CBF family uses the DRE motif ((A/G)CCGACNT) to effectively improve drought, cold, and salinity tolerance ability in several crops, including groundnut and rice, by an ABA-independent mechanism [303, 304]. Another gene, ERD1, induced by dehydration and senescence, but not by cold or ABA, is also a crucial part of ABA-independent
stress response, involving interaction with novel transcriptional proteins, later identified as ANAC019 and ANAC055 [140].
2.4.5.3 Amplifying the stress-signal perception
The sensing of stress signal and resultant activation of acclimation pathways is mediated by complex signaling events orchestrated by ROS, Ca2+-regulated proteins, a cascade of mitogen-activated protein kinases (MAPKs), and cross-talking TF-mediated signaling.
Increased cytosolic Ca2+ has been established as a ubiquitous secondary messenger in plants during various abiotic stresses, which interacts with downstream Ca2+-dependent sensors comprising calcium-dependent protein kinases (CDPKs), salt-overly sensitive proteins (SOS), voltage-gated calcium channels, etc. A group of SNF1-related protein kinase (SnRKs) serves as an essential bridge between abiotic stress and metabolic responses to govern the energy balance. Some lipid-signaling mechanisms are also activated by stress to generate an array of secondary messengers through the phosphoinositide pathway. Additionally, stress signals are mediated by DNA-damage induced repair processes and cell-cycle checkpoints.
Overexpressing the key signal mediators improve stress tolerance.