Chinta Sudhakar

The present day’s ongoing global warming and climate change adversely affect plants through imposing environmental (abiotic) stresses and disease pressure. The major abiotic factors such as drought, heat, cold, salinity, etc., hamper a plant’s innate growth and development, resulting in reduced yield and quality, with the possibility of undesired traits. In the 21st century, the advent of high-throughput sequencing tools, state-of-the-art biotechnological techniques and bioinformatic analyzing pipelines led to the easy characterization of plant traits for abiotic stress response and tolerance mechanisms by applying the ‘omics’ toolbox. Panomics pipeline including genomics, transcriptomics, proteomics, metabolomics, epigenomics, proteogenomics, interactomics, ionomics, phenomics, etc., have become very handy nowadays. This is important to produce climate-smart future crops with a proper understanding of the molecular mechanisms of abiotic stress responses by the plant’s genes, transcripts, proteins, epigenome, cellular metabolic circuits and resultant phenotype. Instead of mono-omics, two or more (hence ‘multi-omics’) integrated-omics approaches can decipher the plant’s abiotic stress tolerance response very well. Multi-omics-characterized plants can be used as potent genetic resources to incorporate into the future breeding program. For the practical utility of crop improvement, multi-omics approaches for particular abiotic stress tolerance can be combined with genome-assisted breeding (GAB) by being pyramided with improved crop yield, food quality and associated agronomic traits and can open a new era of omics-assisted breeding. Thus, multi-omics pipelines together are able to decipher molecular processes, biomarkers, targets for genetic engineering, regulatory networks and precision agriculture solutions for a crop’s variable abiotic stress tolerance to ensure food security under changing environmental circumstances.

Groundnut, cultivated under rain-fed conditions is prone to yield losses due to intermittent drought stress. Drought tolerance is a complex phenomenon and multiple gene expression required to maintain the cellular tolerance. Transcription factors (TFs) regulate many functional genes involved in tolerance mechanisms. In this study, three stress-responsive regulatory TFs cloned from horse gram, (Macrotyloma uniflorum (Lam) Verdc.), MuMYB96, involved in cuticular wax biosynthesis; MuWRKY3, associated with anti-oxidant defense mechanism and MuNAC4, tangled with lateral root development were simultaneously expressed to enhance drought stress resistance in groundnut (Arachis hypogaea L.). The multigene transgenic groundnut lines showed reduced ROS production, membrane damage, and increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzyme activity, evidencing improved antioxidative defense mechanisms under drought stress. Multigene transgenic plants showed lower proline content, increased soluble sugars, epicuticular wax content and higher relative water content suggesting higher maintenance of tissue water status compared to wildype and mock plants. The scanning electron microscopy (SEM) analysis showed a substantial increase in deposition of cuticular waxes and variation in stomatal number in multigene transgenic lines compared to wild type and mock plants. The multigene transgenic plants showed increased growth of lateral roots, chlorophyll content, and stay-green nature in drought stress compared to wild type and mock plants. Expression analysis of transgenes, MuMYB96, MuWRKY3, and MuNAC4 and their downstream target genes, KCS6, KCR1, APX3, CSD1, LBD16 and DBP using qRT-PCR showed a two- to four-fold increase in transcript levels in multigene transgenic groundnut plants over wild type and mock plants under drought stress. Our study demonstrate that introducing multiple genes with simultaneous expression of genes is a viable option to improve stress tolerance and productivity under drought stress.

R2R3 MYB transcription factors are involved in plant growth, plant development and abiotic and biotic stress responses. Several MYB genes are involved in salt tolerance in several plant species. Genotypic variation in the expression of a stress responsive regulatory gene,MYB94was observedin groundnut under drought and salinity stress conditions. In this study, a stress-responsive transcription factorAhMYB94was over-expressed in salt-susceptible groundnut cultivar K-6. Relative gene expression analysis revealed thatAhMYB94was significantly higher in groundnut cultivar K9 than in cultivar K6 due to salt treatments. Overexpression ofAhMYB94improved the salt tolerance of groundnut cultivar K6 as evidenced from better growth status, fresh and dry biomass of overexpression lines than wild type (WT) under salt stress conditions. Further, less reactive oxygen species (ROS) accumulated inAhMYB94overexpressing lines when exposed to salt stress, as estimated by diaminobenzidine (DAB) or nitroblue tetrazolium (NBT) histochemical staining. Furthermore, overexpression ofAhMYB94in groundnut confers significantly increased tolerance ability of plants to oxidative damage under salt stress, mainly by increasing the osmolyte content, the activities of antioxidative enzymes such as SOD, and APX, reducing malondialdehyde (MDA) content, and upregulation of the expression of stress-related genes. When exposed to salt stress, theAhMYB94transgenic lines demonstrated a higher Na⁺and K⁺content and a lower Na⁺/K⁺ratio than the wild type (WT). These results demonstrated thatAhMYB94plays a positive role in enhancing salt tolerance and could be a candidate for improved tolerance of plants to salt stress.

Cluster bean (Cyamopsis tetragonoloba L.) is one of the multipurpose underexplored crops grown as green vegetable and for gum production in dryland areas. Cluster bean is known as relatively tolerant to drought and salinity stress. To elucidate the molecular mechanisms involved in the drought tolerance of cluster bean cultivar RGC-1025, RNA sequencing (RNA-seq) of the drought-stressed and control samples was performed. De novo assembly of the reads resulted in 66,838 transcripts involving 203 pathways. Among these transcripts, differentially expressed gene (DEG) analysis resulted in some of the drought-responsive genes expressing alpha dioxygenase 2, low temperature-induced 65 kDa protein (LDI65), putative vacuolar amino acid transporter, and late embryogenesis abundant protein (LEA 3). The analysis also reported drought-responsive transcription factors (TFs), such as NAC, WRKY, GRAS, and MYB families. The relative expression of genes by qRT-PCR revealed consistency with the DEG analysis. Key genes involved in the wax biosynthesis pathway were mapped using the DEG data analysis. These results were positively correlated with epicuticular wax content and the wax depositions on the leaf surfaces, as evidenced by scanning electron microscope (SEM) image analysis. Further, these findings support the fact that enhanced wax deposits on the leaf surface had played a crucial role in combating the drought stress in cluster beans under drought stress conditions. In addition, this study provided a set of unknown genes and TFs that could be a source of engineering tolerance against drought stress in cluster beans.

R2R3 MYB transcription factors are involved in plant growth and development and abiotic and biotic stress responses. Several MYB genes are involved in salt tolerance in several plant species. In this study, salt-tolerant groundnut cultivar K-9's stress-responsive transcription factor AhMYB94 was overexpressed in salt-susceptible groundnut cultivar K-6. The AhMYB94 gene was activated under drought and salinity. Relative gene expression analysis revealed that AhMYB94 was significantly higher by salt stress treatments than drought stress in groundnut cultivar K-6. Overexpression of AhMYB94 in a salt susceptible groundnut cultivar improved the salt tolerance as evidenced by better growth status, fresh and dry biomass of overexpression lines than wild type (WT) under salt stress conditions. Further, less reactive oxygen species (ROS) accumulated in AhMYB94 overexpressing lines when exposed to salt stress, as estimated by diaminobenzidine (DAB) or nitroblue tetrazolium (NBT) histochemical staining. Further overexpression of AhMYB94 in groundnut confers significantly increased tolerance ability of plants to oxidative damage under salt stress, mainly by increasing the osmolyte content, the activities of antioxidative enzymes such as SOD, and APX, reducing malonaldehyde (MDA) content, and upregulation of the expression of stress-related genes. When exposed to salt stress, the AhMYB94 transgenic lines demonstrated a higher Na + and K + content and a lower Na + /K + ratio than the wild type (WT). These results demonstrated that AhMYB94 plays a positive role in enhancing salt tolerance and could be a candidate for improved tolerance of plants to salt stress.

Drought is one of the major environmental constraints affecting the crop productivity worldwide. One of the agricultural challenges today is to develop plants with minimized water utilization and reduced water loss in adverse environmental conditions. Epicuticular waxes play a major role in minimizing water loss. Epicuticular wax covers aerial plant parts and also prevents non-stomatal water loss by forming the outermost barrier from the surfaces. Epicuticular wax content (EWC) variation was found to be affiliated with drought tolerance of groundnut cultivars. In the current study, a fatty acid elongase gene, KCS1, which catalyzes a rate limiting step in the epicuticular wax biosynthesis was isolated from drought tolerant cultivar K-9 and overexpressed in drought sensitive groundnut cultivar (K-6) under the control of CaMV35S constitutive promoter. Transgenic groundnut plants overexpressing AhKCS1 exhibited normal growth and displaying greenish dark shiny appearance. Environmental scanning electron microscopy (ESEM) revealed the excess of epicuticular wax crystal depositions on the transgenic plant leaves compared to non-transgenic wild type plants. The findings were further supported by gas chromotography–mass spectroscopic analysis (GC-MS) that revealed enhanced levels of fatty acids, secondary alcohols, primary alcohols, aldehydes, alkanes, and ketones in transgenics compared to wild types. The AhKCS1 overexpressing transgenic groundnut plants exhibited increase in the cuticular wax content, reduction of water loss, lower membrane damage, decreased MDA content, and high proline content compared to that of non-transgenic groundnut plants. Our findings suggest that the AhKCS1 gene plays a major role in combating drought stress by preventing non-stomatal water loss in drought sensitive groundnut cultivar (K-6).

Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram (Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut (Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H₂O₂), and superoxide anion (O₂^•-), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD, and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants.

Polyamines are polycationic nitrogenous compounds that accumulate in plants exposed to abiotic stresses. In higher animals, they influence gene expression, brain development, and nerve growth and regeneration. Because of their known roles in plant stress responses, quantitative determination of polyamines is very important. Polyamines in their native form cannot be detected by optical and/or electrochemical methods as they do not show any structural features, and hence derivatization of polyamines is essential to make them to produce either chromophores or fluorescence. Here we describe various methods of derivatization using different labeling agents and suitable separation and detecting methods for a vast source of polyamines existing in living cells.

Polyamines can regulate the expression of antioxidant enzymes and impart plants tolerance to abiotic stresses. A comparative analysis of polyamines, their biosynthetic enzymes at kinetic and at transcriptional level, and their role in regulating the induction of antioxidant defense enzymes under salt stress condition in two foxtail millet (Setaria italica L.) cultivars, namely Prasad, a salt-tolerant, and Lepakshi, a salt-sensitive cultivar was conducted. Salt stress resulted in elevation of free polyamines due to increase in the activity of spermidine synthase and S-adenosyl methionine decarboxylase enzymes in cultivar Prasad compared to cultivar Lepakshi under different levels of NaCl stress. These enzyme activities were further confirmed at the transcript level via qRT-PCR analysis. The cultivar Prasad showed a greater decrease in diamine oxidase and polyamine oxidase activity, which results in the accumulation of polyamine pools over cultivar Lepakshi. Generation of free radicals, such as O ₂ ^·− and H₂O₂, was also analyzed quantitatively. A significant increase in O ₂ ^·− and H₂O₂ in the cultivar Lepakshi compared with cultivar Prasad was recorded in overall pool sizes. Further, histochemical staining showed lesser accumulation of O ₂ ^·− and of H₂O₂ in the leaves of cultivar Prasad than cultivar Lepakshi. Our results also suggest the ability of polyamine oxidation in regulating the induction of antioxidative defense enzymes, which involve in the elimination of toxic levels of O ₂ ^·− and H₂O₂, such as Mn-superoxide dismutase, catalase and ascorbate peroxidase. The contribution of polyamines in modulating antioxidative defense mechanism in NaCl stress tolerance is discussed.

The NAC family being the largest plant-specific transcription factors functions in diverse and vital physiological processes during development. NAC proteins are known to be crucial in imparting tolerance to plants against abiotic stresses, such as drought and salinity, but the functions of most of them are still elusive. In this study, we report for the first time expression of the MuNAC4, a member of NAC transcription factor from horsegram (Macrotyloma uniflorum) conferring drought tolerance. The groundnut (Arachis hypogaea) transgenics were generated using recombinant MuNAC4 binary vector transformation approach. Molecular analysis of these transgenic lines confirmed the stable gene integration and expression of the MuNAC4 gene. Twelve lines of T5 generation exhibited significantly enhanced tolerance to drought stress with proliferated lateral root growth as compared to wild types. Transgenics exposed to long-term desiccation stress assays showed increased lateral roots and greenish growth. The physiological parameters analysis also suggests that overexpression of MuNAC4 plays a significant role in improving the water stress tolerance of transgenic groundnut, reducing the damage to membrane structures and enhancing osmotic adjustment and antioxidative enzyme regulation under stress. This study validates MuNAC4 as an important candidate gene for future phytoengineering approaches for drought tolerance in crop plants.