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Precise genome engineering approaches could be perceived as a second paradigm for targeted trait improvement in crop plants, with the potential to overcome the constraints imposed by conventional CRISPR/Cas technology. The likelihood of reduced agricultural production due to highly turbulent climatic conditions increases as the global population expands. The second paradigm of stress-resilient crops with enhanced tolerance and increased productivity against various stresses is paramount to support global production and consumption equilibrium. Although traditional breeding approaches have substantially increased crop production and yield, effective strategies are anticipated to restore crop productivity even further in meeting the world's increasing food demands. CRISPR/Cas, which originated in prokaryotes, has surfaced as a coveted genome editing tool in recent decades, reshaping plant molecular biology in unprecedented ways and paving the way for engineering stress-tolerant crops. CRISPR/Cas is distinguished by its efficiency, high target specificity, and modularity, enables precise genetic modification of crop plants, allowing for the creation of allelic variations in the germplasm and the development of novel and more productive agricultural practices. Additionally, a slew of advanced biotechnologies premised on the CRISPR/Cas methodologies have augmented fundamental research and plant synthetic biology toolkits. Here, we describe gene editing tools, including CRISPR/Cas and its imitative tools, such as base and prime editing, multiplex genome editing, chromosome engineering followed by their implications in crop genetic improvement. Further, we comprehensively discuss the latest developments of CRISPR/Cas technology including CRISPR-mediated gene drive, tissue-specific genome editing, dCas9 mediated epigenetic modification and programmed self-elimination of transgenes in plants. Finally, we highlight the applicability and scope of advanced CRISPR-based techniques in crop genetic improvement.

Commercialization and utilization of pearl millet (Pennisetum glaucum L.) by consumers and processing industry is constrained due to rapid onset of rancidity in its milled flour. We studied the underlying biochemical and molecular mechanisms to flour rancidity in contrasting inbreds under 21-day accelerated storage. Rapid TAG decrease was accompanied by FFA increase in high rancidity genotype compared to the low rancidity line, that maintained lower FFA and high TAG levels, besides lower headspace aldehydes. DNA sequence polymorphisms observed in two lipase genes revealed loss-of-function mutations that were functionally confirmed in yeast system. We outline a direct mechanism for mutations in these key TAG lipases in pearl millet and the protection of TAG and fatty acids from hydrolytic and oxidative rancidity respectively,. Natural variation in the PgTAGLip1 and PgTAGLip2 genes may be selected through marker assisted breeding or by precision genetics methods to develop hybrids with improved flour shelf life.

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The small heat shock proteins (sHsps/Hsp20s) are the molecular chaperones that maintain proper folding, trafficking and disaggregation of proteins under diverse abiotic stress conditions. In the present investigation, a genome-wide scan revealed the presence of a total of 47 sHsps in Sorghum bicolor (SbsHsps), distributed across 10 subfamilies, the major subfamily being P (plastid) group with 17 genes. Chromosomes 1 and 3 appear as the hot spot regions for SbsHsps, and majority of them were found acidic, hydrophilic, unstable and intron less. Interestingly, promoter analysis indicated that they are associated with both biotic and abiotic stresses, as well as plant development. Sorghum sHsps exhibited 15 paralogous and 20 orthologous duplications. Expression analysis of 15 genes selected from different subfamilies showed high transcript levels in roots and leaves implying that they are likely to participate in the developmental processes. SbsHsp genes were highly induced by diverse abiotic stresses inferring their critical role in mediating the environmental stress responses. Gene expression data revealed that SbsHsp-02 is a candidate gene expressed in all the tissues under varied stress conditions tested. Our results contribute to the understanding of the complexity of SbsHsp genes and help to analyse them further for functional validation.

Pearl millet is a C4 cereal crop that grows in arid and semi-arid climatic conditions with the remarkable abiotic stress tolerance. It contributed to the understanding of stress tolerance not only at the physiological level but also at the genetic level. In the present study, we functionally cloned and characterized three abiotic stress-inducible promoters namely cytoplasmic Apx1 (Ascorbate peroxidase), Dhn (Dehydrin), and Hsc70 (Heat shock cognate) from pearl millet. Sequence analysis revealed that all three promoters have several cis-acting elements specific for temporal and spatial expression. PgApx pro, PgDhn pro and PgHsc70 pro were fused with uidA gene in Gateway-based plant transformation pMDC164 vector and transferred into tobacco through leaf-disc method. While PgApx pro and PgDhn pro were active in seedling stages, PgHsc70 pro was active in stem and root tissues of the T2 transgenic tobacco plants under control conditions. Higher activity was observed under high temperature and drought, and less in salt and cold stress conditions. Further, all three promoters displayed higher GUS gene expression in the stem, moderate expression in roots, and less expression in leaves under similar conditions. While RT-qPCR data showed that PgApx pro and PgDhn pro were expressed highly in high temperature, salt and drought, PgHsc70 pro was fairly expressed during high temperature stress only. Histochemical and RT-qPCR assays showed that all three promoters are inducible under abiotic stress conditions. Thus, these promoters appear to be immediate candidates for developing abiotic stress tolerant crops as these promoter-driven transgenics confer high degree of tolerance in comparison with the wild-type (WT) plants.

Aquaporin (AQP) discovery allowed the understanding of a new notion about the dynamics of rapid and controlled water transport across membranes at the rate exceeding that of water diffusion. The small basic intrinsic proteins define the fourth plant AQP subgroup that was first uncovered from genome sequence analysis. Plant AQPs expressions are often triggered or suppressed by the symbiotic association of micro-organisms. A powerful tool in elucidating the AQP function is given by reverse genetics that can also reveal unexpected functions of water channel proteins, which benefit our understanding of sequence–structure and structure–function relationships in plants. The AQP family is a set of genes whose functions are intuitively perceived as important; much isolated information has been accumulated, yet their function is far from being understood in living plants, and we still have a long way to go to fully understand the significance of these proteins.

A novel open reading frame (ORF) identified and cloned from the A4 cytoplasm of Cajanus cajanifolius induced partial to complete male sterility when introduced into Arabidopsis and tobacco. Pigeonpea (Cajanus cajan L. Millsp.) is the only legume known to have commercial hybrid seed technology based on cytoplasmic male sterility (CMS). We identified a novel ORF (orf147) from the A4 cytoplasm of C. cajanifolius that was created via rearrangements in the CMS line and co-transcribes with the known and unknown sequences. The bi/poly-cistronic transcripts cause gain-of-function variants in the mitochondrial genome of CMS pigeonpea lines having distinct processing mechanisms and transcription start sites. In presence of orf147, significant repression of Escherichia coli growth indicated its toxicity to the host cells and induced partial to complete male sterility in transgenic progenies of Arabidopsis thaliana and Nicotiana tabacum where phenotype co-segregated with the transgene. The male sterile plants showed aberrant floral development and reduced lignin content in the anthers. Gene expression studies in male sterile pigeonpea, Arabidopsis and tobacco plants confirmed down-regulation of several anther biogenesis genes and key genes involved in monolignol biosynthesis, indicative of regulation of retrograde signaling. Besides providing evidence for the involvement of orf147 in pigeonpea CMS, this study provides valuable insights into its function. Cytotoxicity and aberrant programmed cell death induced by orf147 could be important for mechanism underlying male sterility that offers opportunities for possible translation for these findings for exploiting hybrid vigor in other recalcitrant crops as well.

Dehydrins (DHNs) are highly hydrophilic, thermo stable, calcium dependent chaperons involved in plant developmental processes as well as in diverse abiotic stresses. A systematic survey resulted in the identification of 7 dehydrins (DHNs) in Setaria italica and Zea mays, but 6 in Sorghum bicolor. They are classified into 5 sub-groups, namely YnSKn, SKn, KnS, S, and YnS. DHNs of Sorghum exhibit 1 ortholog with Oryza sativa and Z. mays and 3 with S. italica. Unlike other DHNs, SbDHN5 has been found as an ordered protein with many phosphorylation sites. Network analyses of novel YnS subgroup showed interaction with HSP70 and FKBP genes. In silico promoter analysis revealed the presence of abscisic acid (ABA), drought, salt, low temperature stress-responsive elements. The miRNA target analysis revealed DHNs are targeted by 51 miRNAs responsive to abiotic stresses. High transcript expressions of DHNs were observed in root, stem and leaf compared to inflorescence in S. bicolor. All DHN genes exhibited high levels of expression in stem under cold, heat, salt, and drought stresses. In contrast to other DHNs, the SbDHN2 of YnS subgroup, exhibited the highest expression, under multiple stresses in all the tissues indicating its involvement against a wide array of abiotic stresses.

Plasma membrane proteolipid 3 (Pmp3) is a gene family involved in abiotic stress response and cellular protection. Here we report cloning of two genes PgPmp3-1 and PgPmp3-2 from Pennisetum glaucum, and characterization with respect to their functions and responsiveness to various abiotic stresses. Both PgPmp3-1 and PgPmp3-2 genes are 171 bp long and encode for 56 amino acid long peptides. PgPmp3 sequences share 70–99% sequence identity with their homologs. Protein secondary structure prediction revealed membrane-spanning regions containing a membrane potential modulator domain in both PgPmp3 proteins. In silico network analyses revealed Pmp3 co-expression and association with proteins conferring abiotic stress tolerance in plants. Expression profiles of PgPmp3-1 and PgPmp3-2 revealed their up-regulation in P. glaucum under cold and salt stresses, but showed reduced expression in response to heat stress. These findings provide insight into the role of P. glaucum Pmp3 in abiotic stress amelioration.

Major abiotic stress factors such as salt and drought adversely affect important physiological processes and biochemical mechanisms and cause severe loss in crop productivity worldwide. Plants develop various strategies to stand healthy against these stress factors. The accumulation of proline (Pro) is one of the striking metabolic responses of plants to salt and drought stress. Pro biosynthesis and signalling contribute to the redox balance of cell under normal and stressful conditions. However, literature is meager on the sustainable strategies potentially fit for modulating Pro biosynthesis and production in stressed plants. Considering the recent literature, this paper in its first part overviews Pro biosynthesis and transport in plants and also briefly highlights the significance of Pro in plant responses to salt and drought stress. Secondly, this paper discusses mechanisms underlying the regulation of Pro metabolism in salt and drought-exposed plant via phytohormones, mineral nutrients and transgenic approaches. The outcome of the studies may give new opportunities in modulating Pro metabolism for improving plant tolerance to salt and drought stress and benefit sustainable agriculture.

Pearl millet is a C

Abiotic stress conditions lead to the defects in plant growth and development and also reduction in flowering and fertility. Under prolonged stresses, imminent death of the plants has been observed. To cope with such stress conditions, plants accumulate a wide variety of organic solutes called osmolytes. Osmolytes are accumulated in bacteria, lower, and higher plants as a response primarily to abiotic stress. They encompass amino acids such as proline, tertiary sulfonium, and quaternary ammonium compounds like beatines, sugars (trehalose), and polyhydric alcohols (mannitol, sorbitol, pinitol, etc.). Osmolytes are accumulated in the cytoplasm as well as in chloroplasts in certain cases for osmotic adjustment under stress conditions. This enables the plants to absorb water and survive under stress. Out of the many phytohormones that play diverse roles during abiotic stress, abscisic acid (ABA) is an important one and perceived by plants by a core signaling module. As an integral part of signal transduction during stress conditions, ABA and other hormones regulate not only stomatal closure, but also a wide array of gene expressions including osmolyte biosynthetic pathway genes. Many signal molecules like nitric oxide, carbon monoxide, and hydrogen sulfide also play a vital role in osmolyte biosynthesis. Osmolytes appear to have multiple functions during stress such as osmotic adjustment and scavenging of reactive oxygen species (ROS). Thus, generation of ROS and osmolyte accumulation are linked together. This review summarizes the role played by ABA in signal transduction, the role of hormones to regulate osmolyte biosynthesis, and various functions carried out by them.

The perception of abiotic stress and signal transduction to switch on adaptive innate responses are crucial steps in plant persistence under adverse environmental conditions. The ability of plants to respond to different stresses by maintaining protein in functional active form is of high importance and essential for cell survival. This is possible with the functional activity of heat shock proteins (Hsps), which play a role in maintaining the cellular homeostasis, and preserving the nonnative protein in a competent state for further remodeling as well as in signal transduction. Hsps mediate the stress signals either directly or indirectly, and play an important role in maintaining the homeostasis of plant cells under adverse stress conditions. Much research has been conducted to understand the complex mechanisms of the Hsps including Hsp70/90, ATPase-coupled conformational modifications, and interactions with cochaperones. In addition to this, several signaling proteins including MAP kinases, Ca2+ regulated proteins, Hsfs, reactive oxygen species are seen to be interacting with Hsps. In this study, we discuss the signal transduction of different Hsps, and their role in different signaling mechanisms.

Late embryogenesis abundant (LEA) proteins, the space fillers or molecular shields, are the hydrophilic protective proteins which play an important role during plant development and abiotic stress. The systematic survey and characterization revealed a total of 68 LEA genes, belonging to 8 families in Sorghum bicolor. The LEA-2, a typical hydrophobic family is the most abundant family. All of them are evenly distributed on all 10 chromosomes and chromosomes 1, 2, and 3 appear to be the hot spots. Majority of the S. bicolor LEA (SbLEA) genes are intron less or have fewer introns. A total of 22 paralogous events were observed and majority of them appear to be segmental duplications. Segmental duplication played an important role in SbLEA-2 family expansion. A total of 12 orthologs were observed with Arabidopsis and 13 with Oryza sativa. Majority of them are basic in nature, and targeted by chloroplast subcellular localization. Fifteen miRNAs targeted to 25 SbLEAs appear to participate in development, as well as in abiotic stress tolerance. Promoter analysis revealed the presence of abiotic stress-responsive DRE, MYB, MYC, and GT1, biotic stress-responsive W-Box, hormone-responsive ABA, ERE, and TGA, and development-responsive SKn cis-elements. This reveals that LEA proteins play a vital role during stress tolerance and developmental processes. Using microarray data, 65 SbLEA genes were analyzed in different tissues (roots, pith, rind, internode, shoot, and leaf) which show clear tissue specific expression. qRT-PCR analysis of 23 SbLEA genes revealed their abundant expression in various tissues like roots, stems and leaves. Higher expression was noticed in stems compared to roots and leaves. Majority of the SbLEA family members were up-regulated at least in one tissue under different stress conditions. The SbLEA3-2 is the regulator, which showed abundant expression under diverse stress conditions. Present study provides new insights into the formation of LEAs in S. bicolor and to understand their role in developmental processes under stress conditions, which may be a valuable source for future research.

Na+ transporters play an important role during salt stress and development. The present study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na+/H+ exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein–protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)- CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.

Dehydrins (DHNs) or group 2 LEA (late embryogenesis abundant) proteins play a protective role in plants under different abiotic stress conditions like drought, salinity, cold and heat stress. DHNs are expressed in late embryogenesis and accumulate in vegetative tissues in response to desiccation stress in all photosynthetic organisms. Here we report the cloning and characterization of a PgDHN gene from the C4 plant Pennisetum glaucum. The PgDHN cDNA encoded for a polypeptide of 133 amino acids with an estimated molecular weight of 13.87 kDa and isoelectric point of 6.81. The protein sequence analysis of PgDHN classified it into the YnSKn subgroup of dehydrins. Phylogenetic analysis revealed that PgDHN is evolutionarily related to a Setaria italica DHN. In silico sequence analysis of the PgDHN promoter identified a distinct set of cis-elements and transcription factor binding sites. PgDHN mRNA accumulated in leaves of P. glaucum upon treatment with NaCl stress. Recombinant PgDHN transformed E. coli cells showed improved tolerance and exhibited better growth rate under high salt concentration (750 mM) and heat stress in comparison to their respective controls. Heterologous expression of PgDHN in transgenic yeast showed increased tolerance to multiple abiotic stresses. This study provides a possible role of PgDHN in stress adaptation and stress tolerance in pearl millet.

A genome-wide scanning of Sorghum bicolor resulted in the identification of 25 SbHsf genes. Phylogenetic analysis shows the ortholog genes that are clustered with only rice, representing a common ancestor. Promoter analysis revealed the identification of different cis-acting elements that are responsible for abiotic as well as biotic stresses. Hsf domains like DBD, NLS, NES, and AHA have been analyzed for their sequence similarity and functional characterization. Tissue specific expression patterns of Hsfs in different tissues like mature embryo, seedling, root, and panicle were studied using real-time PCR. While Hsfs4 and 22 are highly expressed in panicle, 4 and 9 are expressed in seedlings. Sorghum plants were exposed to different abiotic stress treatments but no expression of any Hsf was observed when seedlings were treated with ABA. High level expression of Hsf1 was noticed during high temperature as well as cold stresses, 4 and 6 during salt and 5, 6, 10, 13, 19, 23 and 25 during drought stress. This comprehensive analysis of SbHsf genes will provide an insight on how these genes are regulated in different tissues and also under different abiotic stresses and help to determine the functions of Hsfs during drought and temperature stress tolerance.

Small heat shock protein (Hsp) family genes have been reported in several plant species that function as molecular chaperones to protect proteins from being denatured in extreme conditions. As a first step towards the isolation and characterization of genes that contribute to combating abiotic stresses particularly heat stress, construction and screening of the subtracted complementary DNA (cDNA) library is reported here. In this study, a subtractive heat stress cDNA library was constructed that was used to isolate members of small Hsps (sHsps) using PgsHsp17.9A gene as a probe. As a result, a total of 150 cDNA clones were isolated from the subtracted cDNA library screening, leading to 121 high-quality expressed sequence tags (ESTs), with an average size of 450 bp, comprising of 15 contigs, and majority of these isolated sHsp genes belong to cytosolic class I (CI) family. In silico sequence analysis of CI-sHsp family genes revealed that the length of sHsp proteins varied from 151 to 159 amino acids and showed large variation in isoelectric point value (5.03 to 10.05) and a narrow range of molecular weight (16.09 to 17.94 kDa). The real-time PCR results demonstrated that CI-sHsp genes are differentially expressed in Pennisetum leaves under different abiotic stress conditions particularly at high temperature. The results presented in this study provide basic information on PgCI-sHsp family genes and form the foundation for future functional studies of these genes.

Na+ transporters play an important role during salt stress and development. The present study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na+/H+ exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein–protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)- CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.

Finger millet (Eleusine coracana L.) is an annual herbaceous self-pollinating C4 cereal crop of the arid and semi-arid regions of the world. Finger millet is a food security crop proven to have resilience to changing climate and scores very high in nutrition. In the current study, we have assessed sixteen candidate reference genes for their appropriateness for the normalization studies in finger millet subjected to experimental regimes and treatments. Ten candidate reference genes (GAPDH, β-TUB, CYP, EIF4α, TIP41, UBC, G6PD, S24, MACP and MDH) were cloned and six (ACT, ELF1α, PP2A, PT, S21 and TFIID) were mined from the NCBI database as well as from the literature. Expression stability ranking of the finger millet reference genes was validated using four different statistical tools i.e., geNorm, NormFinder, BestKeeper, ΔCt and RefFinder. From the study, we endorse MACP, CYP, EIF4α to be most stable candidate reference genes in all 'tissues', whereas PT, TFIID, MACP ranked high across genotypes, β-TUB, CYP, ELF1α were found to be best under abiotic stresses and 'all samples set'. The study recommends using minimum of two reference genes for RT-qPCR data normalizations in finger millet. All in all, CYP, β-TUB, and EF1α, in combination were found to be best for robust normalizations under most experimental conditions. The best and the least stable genes were validated for confirmation by assessing their appropriateness for normalization studies using EcNAC1 gene. The report provides the first comprehensive list of suitable stable candidate reference genes for nutritional rich cereal finger millet that will be advantageous to gene expression studies in this crop.

To facilitate gene expression studies in pearl millet (Pennisetum glaucum (L.) R. Br.), the key reference genes including ACP, ACT, TUB, CYP, EF-1α, EIF4A, GAPDH, MDH, PP2C, UBC and S24 were selected based on the available literature, and their expression stabilities were studied to determine their suitability for normalizing gene expression in pearl millet. Sequence information of the reference genes were obtained from the closely related species and cloned from pearl millet using homology based cloning strategy. Further, expression stabilities were validated for their accurate expression in different tissues, genotypes and abiotic stress treatments using three statistical algorithms including geNorm, NormFinder and RefFinder. Analysis showed that while the expression of EF-1α and EIF4A was most stable in different plant tissues, MDH and EIF4A were stable under different abiotic stress conditions. Amongst the different genotypes of pearl millet tested, while UBC and MDH genes exhibited most stable expression, MDH and ACP showed greater stability in all samples set. Interestingly, the widely used reference genes S24 and TUB were found to be least stable across all the tested samples. Pair-wise analysis showed that two reference genes were sufficient for proper normalization, except when analyzing the gene expression studies in all samples set. Results of this study can help in the selection of reference genes for quantitative real time PCR (qRT-PCR) normalization in pearl millet that will contribute towards more accurate and reliable quantification of transcripts in this important crop of the drylands.

Pearl millet is a crop of the semi-arid tropics having high degree of genetic diversity and variable tolerance to drought stress. To investigate drought tolerance mechanism that possibly accounts for differences in drought tolerance, four recombinant inbred lines from a high resolution cross (HRC) were selected for variability in their transpiration rate (Tr) response to vapour pressure deficit (VPD) conditions. The differential Tr response of the genotypes to increased VPD conditions was used to classify the genotypes as sensitive or insensitive to high VPD. Aquaporin (AQP) genes PgPIP1;1, PgPIP1;2 , PgPIP2;1 , PgPIP2;3 , PgPIP2;6 , PgTIP1;1 and PgTIP2;2 were cloned. Phylogenetic analysis revealed that the cloned PgAQPs were evolutionarily closer to maize AQPs than to rice. PgAQP genes, including PgPIP1;1 and PgPIP2;6 in root tissue showed a significant expression pattern with higher expression in VPD-insensitive genotypes than VPD-sensitive genotypes under low VPD conditions (1.2 kPa) i.e when there is no high evaporative demand from the atmosphere. PgAQP genes (PgPIP2;1 in leaf and root tissues; PgPIP1;2 and PgTIP2;2 in leaf and PgPIP2;6 in root) followed a diurnal rhythm in leaves and roots that have either higher or lower expression levels at different time intervals. Under high VPD conditions (4.21 kPa), PgPIP2;3 showed higher transcript abundance in VPD-insensitive genotypes, and PgPIP2;1 in VPD-sensitive genotypes, while rest of the PgAQPs showed differential expression. Our current hypothesis is that these differences in the expression of AQP genes under different VPDs suggests a role of the AQPs in tuning the water transport pathways with variation between genotypes.

Eukaryotic translational initiation factors belong to family of helicases, involved in multifunctional activities during stress and non-stress conditions. An eukaryotic translational initiation factor 4A (eIF4A) gene was isolated and cloned from semi-arid cereal crop of Pennisetum glaucum. In the present study, the PgeIF4A gene was expressed under the regulation of stress inducible Arabidopsis rd29A promoter in groundnut (cv JL-24) with bar as a selectable marker. The de-embryonated cotyledons were infected with Agrobacterium (LBA4404) carrying gene construct and generated high frequency of multiple shoots in phosphinothricin medium. Twenty- four T0 plants showed integration of both nos-bar and rd29A-PgeIF4A gene cassettes in genome with expected amplification products of 429 and 654 bps, respectively. Transgene copy number integration was observed in five T0 transgenic plants through Southern blotting analysis. Predicted Mendelian ratio of segregation (3:1) was noted in single gene integrated plants at T1 generation. The T2 homozygous lines (L1-5, L8-2 and L16-2,) expressing PgeIF4A gene were exhibited superior growth performance with respect to phenotypic parameters like shoot length, tap root length and lateral root formation under simulated drought and salinity stresses compared to the wild type. In addition, the chlorophyll retention was found to be higher in these plants compared to the control plants. Semi quantitative RT-PCR results confirmed the accumulation PgeIF4A transcript in L1-5, L8-3 and L16-2 plants imposed drought/salt stress. Further, the salt stress tolerance was associated with increase in oxidative stress markers, such as superoxide dismutase accumulation, reactive oxygen species scavenging and membrane stability in transgenic plants. Taken together our results confirmed that the PgeIF4A gene expressing transgenic groundnut plants exhibited better adaptation to stress conditions.

Abscisic acid (ABA) is a central player in plant responses to drought stress. How variable levels of ABA under short-term versus long-term drought stress impact assimilation and growth in crops is unclear. We addressed this through comparative analysis, using two elite breeding lines of barley (Hordeum vulgare) that show senescence or stay-green phenotype under terminal drought stress and by making use of transgenic barley lines that express Arabidopsis (Arabidopsis thaliana) 9-cis-epoxycarotenoid dioxygenase (AtNCED6) coding sequence or an RNA interference (RNAi) sequence of ABA 8'-hydroxylase under the control of a drought-inducible barley promoter. The high levels of ABA and its catabolites in the senescing breeding line under long-term stress were detrimental for assimilate productivity, whereas these levels were not perturbed in the stay-green type that performed better. In transgenic barley, drought-inducible AtNCED expression afforded temporal control in ABA levels such that the ABA levels rose sooner than in wild-type plants but also subsided, unlike as in the wild type , to near-basal levels upon prolonged stress treatment due to down-regulation of endogenous HvNCED genes. Suppressing of ABA catabolism with the RNA interference approach of ABA 8'-hydroxylase caused ABA flux during the entire period of stress. These transgenic plants performed better than the wild type under stress to maintain a favorable instantaneous water use efficiency and better assimilation. Gene expression analysis, protein structural modeling, and protein-protein interaction analyses of the members of the PYRABACTIN RESISTANCE1/PYRABACTIN RESISTANCE1-LIKE/REGULATORY COMPONENT OF ABA RECEPTORS, TYPE 2C PROTEIN PHOSPHATASE Sucrose non-fermenting1-related protein kinase2, and ABA-INSENSITIVE5/ABA-responsive element binding factor family identified specific members that could potentially impact ABA metabolism and stress adaptation in barley.