CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut.

The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

Genome-wide screening and characterization of phospholipase A (PLA)-like genes in sorghum (Sorghum bicolor L.).

MAIN CONCLUSION: The characterisation of PLA genes in the sorghum genome using in-silico methods revealed their essential roles in cellular processes, providing a foundation for further detailed studies. Sorghum bicolor (L.) Moench is the fifth most cultivated crop worldwide, and it is used in many ways, but it has always gained less popularity due to the yield, pest, and environmental constraints. Improving genetic background and developing better varieties is crucial for better sorghum production in semi-arid tropical regions. This study focuses on the phospholipase A (PLA) family within sorghum, comprehensively characterising PLA genes and their expression across different tissues. The investigation identified 32 PLA genes in the sorghum genome, offering insights into their chromosomal localization, molecular weight, isoelectric point, and subcellular distribution through bioinformatics tools. PLA-like family genes are classified into three groups, namely patatin-related phospholipase A (pPLA), phospholipase A1 (PLA1), and phospholipase A2 (PLA2). In-silico chromosome localization studies revealed that these genes are unevenly distributed in the sorghum genome. Cis-motif analysis revealed the presence of several developmental, tissue and hormone-specific elements in the promoter regions of the PLA genes. Expression studies in different tissues such as leaf, root, seedling, mature seed, immature seed, anther, and pollen showed differential expression patterns. Taken together, genome-wide analysis studies of PLA genes provide a better understanding and critical role of this gene family considering the metabolic processes involved in plant growth, defence and stress response.

Cytoplasmic male sterility-based hybrids: mechanistic insights.

MAIN CONCLUSION: A comprehensive understanding of the nucleocytoplasmic interactions that occur between genes related to the restoration of fertility and cytoplasmic male sterility (CMS) provides insight into the development of hybrids of important crop species. Modern biotechnological techniques allow this to be achieved in an efficient and quick manner. Heterosis is paramount for increasing the yield and quality of a crop. The development of hybrids for achieving heterosis has been well-studied and proven to be robust and efficient. Cytoplasmic male sterility (CMS) has been explored extensively in the production of hybrids. The underlying mechanisms of CMS include the role of cytotoxic proteins, PCD of tapetal cells, and improper RNA editing of restoration factors. On the other hand, the restoration of fertility is caused by the presence of restorer-of-fertility (Rf) genes or restorer genes, which inhibit the effects of sterility-causing genes. The interaction between mitochondria and the nuclear genome is crucial for several regulatory pathways, as observed in the CMS-Rf system and occurs at the genomic, transcriptional, post-transcriptional, translational, and post-translational levels. These CMS-Rf mechanisms have been validated in several crop systems. This review aims to summarize the nucleo-mitochondrial interaction mechanism of the CMS-Rf system. It also sheds light on biotechnological interventions, such as genetic engineering and genome editing, to achieve CMS-based hybrids.

Recent advancements in CRISPR/Cas technology for accelerated crop improvement.

MAIN CONCLUSION: 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.

Isolation and functional characterization of three abiotic stress-inducible (Apx, Dhn and Hsc70) promoters from pearl millet (Pennisetum glaucum L.).

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.

A novel mitochondrial orf147 causes cytoplasmic male sterility in pigeonpea by modulating aberrant anther dehiscence.

KEY MESSAGE: 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.

Genomic-based-breeding tools for tropical maize improvement.

Maize has traditionally been the main staple diet in the Southern Asia and Sub-Saharan Africa and widely grown by millions of resource poor small scale farmers. Approximately, 35.4 million hectares are sown to tropical maize, constituting around 59% of the developing worlds. Tropical maize encounters tremendous challenges besides poor agro-climatic situations with average yields recorded <3 tones/hectare that is far less than the average of developed countries. On the contrary to poor yields, the demand for maize as food, feed, and fuel is continuously increasing in these regions. Heterosis breeding introduced in early 90 s improved maize yields significantly, but genetic gains is still a mirage, particularly for crop growing under marginal environments. Application of molecular markers has accelerated the pace of maize breeding to some extent. The availability of array of sequencing and genotyping technologies offers unrivalled service to improve precision in maize-breeding programs through modern approaches such as genomic selection, genome-wide association studies, bulk segregant analysis-based sequencing approaches, etc. Superior alleles underlying complex traits can easily be identified and introgressed efficiently using these sequence-based approaches. Integration of genomic tools and techniques with advanced genetic resources such as nested association mapping and backcross nested association mapping could certainly address the genetic issues in maize improvement programs in developing countries. Huge diversity in tropical maize and its inherent capacity for doubled haploid technology offers advantage to apply the next generation genomic tools for accelerating production in marginal environments of tropical and subtropical world. Precision in phenotyping is the key for success of any molecular-breeding approach. This article reviews genomic technologies and their application to improve agronomic traits in tropical maize breeding has been reviewed in detail.

Molecular cloning, characterization and expression analysis of a heat shock protein 10 (Hsp10) from Pennisetum glaucum (L.), a C4 cereal plant from the semi-arid tropics.

Heat shock proteins (Hsp10) belong to the ubiquitous family of heat-shock molecular chaperones found in the organelles of both prokaryotes and eukaryotes. Chaperonins assist the folding of nascent and stress-destabilized proteins. A cDNA clone encoding a 10 kDa Hsp was isolated from pearl millet, Pennisetum glaucum (L.) by screening a heat stress cDNA library. The fulllength PgHsp10 cDNA consisted of 297 bp open reading frame (ORF) encoding a 98 amino acid polypeptide with a predicted molecular mass of 10.61 kDa and an estimated isoelectric point (pI) of 7.95. PgHsp10 shares 70-98 % sequence identity with other plant homologs. Phylogenetic analysis revealed that PgHsp10 is evolutionarily close to the maize Hsp10 homolog. The predicted 3D model confirmed a conserved eight-stranded ß-barrel with active site between the ß-barrel comprising of eight-strands, with conserved domain VLLPEYGG sandwiched between two ß-sheets. The gene consisted of 3 exons and 2 introns, while the position and phasing of these introns were conserved similar to other plant Hsp10 family genes. In silico analysis of the promoter region of PgHsp10 presented several distinct set of cis-elements and transcription factor binding sites. Quantitative RT-PCR analysis showed that PgHsp10 gene was differentially expressed in response to abiotic stresses with the highest level of expression under heat stress conditions. Results of this study provide useful information regarding the role of chaperonins in stress regulation and generated leads for further elucidation of their function in plant stress tolerance.

Genome-wide Scanning and Characterization of Sorghum bicolor L. Heat Shock Transcription Factors.

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.

Genome-wide identification and expression profiling of growth­regulating factor (GRF) and GRF­interacting factor (GIF) gene families in chickpea and pigeonpea.

The growth-regulating factor (GRF) and GRF-interacting factor (GIF) families encode plant-specific transcription factors and play vital roles in plant development and stress response processes. Although GRF and GIF genes have been identified in various plant species, there have been no reports of the analysis and identification of the GRF and GIF transcription factor families in chickpea (Cicer arietinum) and pigeonpea (Cajanus cajan). The present study identified seven CaGRFs, eleven CcGRFs, four CaGIFs, and four CcGIFs. The identified proteins were grouped into eight and three clades for GRFs and GIFs, respectively based on their phylogenetic relationships. A comprehensive in-silico analysis was performed to determine chromosomal location, sub-cellular localization, and types of regulatory elements present in the putative promoter region. Synteny analysis revealed that GRF and GIF genes showed diploid-polyploid topology in pigeonpea, but not in chickpea. Tissue-specific expression data at the vegetative and reproductive stages of the plant showed that GRFs and GIFs were strongly expressed in tissues like embryos, pods, and seeds, indicating that GRFs and GIFs play vital roles in plant growth and development. This research characterized GRF and GIF families and hints at their primary roles in the chickpea and pigeonpea growth and developmental process. Our findings provide potential gene resources and vital information on GRF and GIF gene families in chickpea and pigeonpea, which will help further understand the regulatory role of these gene families in plant growth and development.

Root and Leaf Anatomy, Ion Accumulation, and Transcriptome Pattern under Salt Stress Conditions in Contrasting Genotypes of Sorghum bicolor.

Roots from salt-susceptible ICSR-56 (SS) sorghum plants display metaxylem elements with thin cell walls and large diameter. On the other hand, roots with thick, lignified cell walls in the hypodermis and endodermis were noticed in salt-tolerant CSV-15 (ST) sorghum plants. The secondary wall thickness and number of lignified cells in the hypodermis have increased with the treatment of sodium chloride stress to the plants (STN). Lignin distribution in the secondary cell wall of sclerenchymatous cells beneath the lower epidermis was higher in ST leaves compared to the SS genotype. Casparian thickenings with homogenous lignin distribution were observed in STN roots, but inhomogeneous distribution was evident in SS seedlings treated with sodium chloride (SSN). Higher accumulation of K+ and lower Na+ levels were noticed in ST compared to the SS genotype. To identify the differentially expressed genes among SS and ST genotypes, transcriptomic analysis was carried out. Both the genotypes were exposed to 200 mM sodium chloride stress for 24 h and used for analysis. We obtained 70 and 162 differentially expressed genes (DEGs) exclusive to SS and SSN and 112 and 26 DEGs exclusive to ST and STN, respectively. Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis unlocked the changes in metabolic pathways in response to salt stress. qRT-PCR was performed to validate 20 DEGs in each SSN and STN sample, which confirms the transcriptomic results. These results surmise that anatomical changes and higher K+/Na+ ratios are essential for mitigating salt stress in sorghum apart from the genes that are differentially up- and downregulated in contrasting genotypes.

CRISPR/Cas9-Mediated Mutation in XSP10 and SlSAMT Genes Impart Genetic Tolerance to Fusarium Wilt Disease of Tomato (Solanum lycopersicum L.).

Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusarium wilt disease.

Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes.

Cotton is an important source of natural fibre used in the textile industry and the productivity of the crop is adversely affected by drought stress. High throughput transcriptomic analyses were used to identify genes involved in fibre development. However, not much information is available on cotton genome response in developing fibres under drought stress. In the present study a genome wide transcriptome analysis was carried out to identify differentially expressed genes at various stages of fibre growth under drought stress. Our study identified a number of genes differentially expressed during fibre elongation as compared to other stages. High level up-regulation of genes encoding for enzymes involved in pectin modification and cytoskeleton proteins was observed at fibre initiation stage. While a large number of genes encoding transcription factors (AP2-EREBP, WRKY, NAC and C2H2), osmoprotectants, ion transporters and heat shock proteins and pathways involved in hormone (ABA, ethylene and JA) biosynthesis and signal transduction were up-regulated and genes involved in phenylpropanoid and flavonoid biosynthesis, pentose and glucuronate interconversions and starch and sucrose metabolism pathways were down-regulated during fibre elongation. This study showed that drought has relatively less impact on fibre initiation but has profound effect on fibre elongation by down-regulating important genes involved in cell wall loosening and expansion process. The comprehensive transcriptome analysis under drought stress has provided valuable information on differentially expressed genes and pathways during fibre development that will be useful in developing drought tolerant cotton cultivars without compromising fibre quality.

Cloning and molecular characterization of a gene encoding late embryogenesis abundant protein from Pennisetum glaucum: protection against abiotic stresses.

Late embryogenesis abundant (LEA) protein family is a large protein family that protects other proteins from aggregation due to desiccation or osmotic stresses. A cDNA clone encoding a group 7 late embryogenesis abundant protein, termed PgLEA, was isolated from Pennisetum glaucum by screening a heat stress cDNA library. PgLEA cDNA encodes a 176 amino acid polypeptide with a predicted molecular mass of 19.21 kDa and an estimated isoelectric point of 7.77. PgLEA shares 70-74% sequence identity with other plant homologs. Phylogenetic analysis revealed that PgLEA is evolutionarily close to the LEA 7 group. Recombinant PgLEA protein expressed in Escherichia coli possessed in vitro chaperone activity and protected PgLEA-producing bacteria from damage caused by heat and salinity. Positive correlation existed between differentially up-regulated PgLEA transcript levels and the duration and intensity of different environmental stresses. In silico analysis of the promoter sequence of PgLEA revealed the presence of a distinct set of cis-elements and transcription factor binding sites. Transcript induction data, the presence of several putative stress-responsive transcription factor binding sites in the promoter region of PgLEA, the in vitro chaperone activity of this protein and its protective effect against heat and salt damage in E. coli suggest a role in conferring abiotic stress tolerance in plants.

Pearl Millet Aquaporin Gene PgPIP2;6 Improves Abiotic Stress Tolerance in Transgenic Tobacco.

Pearl millet [Pennisetum glaucum (L) R. Br.] is an important cereal crop of the semiarid tropics, which can withstand prolonged drought and heat stress. Considering an active involvement of the aquaporin (AQP) genes in water transport and desiccation tolerance besides several basic functions, their potential role in abiotic stress tolerance was systematically characterized and functionally validated. A total of 34 AQP genes from P. glaucum were identified and categorized into four subfamilies, viz., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs). Sequence analysis revealed that PgAQPs have conserved characters of AQP genes with a closer relationship to sorghum. The PgAQPs were expressed differentially under high vapor pressure deficit (VPD) and progressive drought stresses where the PgPIP2;6 gene showed significant expression under high VPD and drought stress. Transgenic tobacco plants were developed by heterologous expression of the PgPIP2;6 gene and functionally characterized under different abiotic stresses to further unravel their role. Transgenic tobacco plants in the T2 generations displayed restricted transpiration and low root exudation rates in low- and high-VPD conditions. Under progressive drought stress, wild-type (WT) plants showed a quick or faster decline of soil moisture than transgenics. While under heat stress, PgPIP2;6 transgenics showed better adaptation to heat (40°C) with high canopy temperature depression (CTD) and low transpiration; under low-temperature stress, they displayed lower transpiration than their non-transgenic counterparts. Cumulatively, lower transpiration rate (Tr), low root exudation rate, declined transpiration, elevated CTD, and lower transpiration indicate that PgPIP2;6 plays a role under abiotic stress tolerance. Since the PgPIP2;6 transgenic plants exhibited better adaptation against major abiotic stresses such as drought, high VPD, heat, and cold stresses by virtue of enhanced transpiration efficiency, it has the potential to engineer abiotic stress tolerance for sustained growth and productivity of crops.

Loss-of-function of triacylglycerol lipases are associated with low flour rancidity in pearl millet [Pennisetum glaucum (L.) R. Br.].

Pearl millet is an important cereal crop of semi-arid regions since it is highly nutritious and climate resilient. However, pearl millet is underutilized commercially due to the rapid onset of hydrolytic rancidity of seed lipids post-milling. We investigated the underlying biochemical and molecular mechanisms of rancidity development in the flour from contrasting inbred lines under accelerated aging conditions. The breakdown of storage lipids (triacylglycerols; TAG) was accompanied by free fatty acid accumulation over the time course for all lines. The high rancidity lines had the highest amount of FFA by day 21, suggesting that TAG lipases may be the cause of rancidity. Additionally, the high rancidity lines manifested substantial amounts of volatile aldehyde compounds, which are characteristic products of lipid oxidation. Lipases with expression in seed post-milling were sequenced from low and high rancidity lines. Polymorphisms were identified in two TAG lipase genes (PgTAGLip1 and PgTAGLip2) from the low rancidity line. Expression in a yeast model system confirmed these mutants were non-functional. We provide a direct mechanism to alleviate rancidity in pearl millet flour by identifying mutations in key TAG lipase genes that are associated with low rancidity. These genetic variations can be exploited through molecular breeding or precision genome technologies to develop elite pearl millet cultivars with improved flour shelf life.

ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought-stress conditions.

Drought is one of the most severe environmental stress factors limiting crop yield especially when occurring during anthesis and seed filling. This terminal drought is characterized by an excess production of the phytohormone abscisic acid (ABA) which plays an important role during seed development and dormancy. All the genes putatively involved in ABA biosynthesis and inactivation in barley were identified and their expression studied during plant ontogeny under standard and drought-stress conditions to learn more about ABA homeostasis and the possible mode of cross-talk between source and sink tissues. Out of 41 genes related to ABA biosynthesis and inactivation 19 were found to be differentially regulated under drought stress in both flag leaves and developing seed during seed filling. Transcripts of plastid-located enzymes are regulated similarly in flag leaf and seed under terminal drought whereas transcripts of cytosolic enzymes are differentially regulated in the two tissues. Detailed information on the expression of defined gene family members is supplemented by measurements of ABA and its degradation and conjugation products, respectively. Under drought stress, flag leaves in particular contain high concentrations of both ABA and the ABA degradation products phaseic acid (PA) and diphaseic acid (DPA); whereas, in seeds, besides ABA, DPA was mainly found. The measurements also revealed a positive correlation between ABA level and starch content in developing seeds for the following reasons: (i) genes of the ABA controlled SnRK2.6 and RCAR/PP2C-mediated signal transduction pathway to the ABF transcription factor HvABI5 are activated in the developing grain under drought, (ii) novel ABA- and dehydration-responsive cis-elements have been found in the promoters of key genes of starch biosynthesis (HvSUS1, HvAGP-L1) and degradation (HvBAM1) and these transcripts/activity are prominently induced in developing seeds during 12 and 16 DAF, (iii) spraying of fluridone (an ABA biosynthesis inhibitor) to drought-stressed plants results in severely impaired starch content and thousand grain weight of mature seeds.

Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses.

Molecular chaperones (Hsps) have been shown to facilitate protein folding or assembly under various developmental and adverse environmental conditions. The aim of this study was to unravel a possible role of heat-shock proteins in conferring abiotic stress tolerance to plants. We isolated a cDNA encoding a cytoplasmic Hsp70 (PgHsc70) from Pennisetum glaucum by screening heat-stress cDNA library. PgHsc70 cDNA encoding 649 amino acids represents all conserved signature motifs characteristic of Hsp70s. The predicted molecular model of PgHsc70 protein suggests that the N-terminus ATP-binding region is evolutionarily conserved, in comparison to C-terminus peptide-binding domains. A single intron in ATPase domain coding region of PgHsc70 exhibited a high degree of conservation with respect to its position and phasing among other plant Hsp70 genes. Recombinant PgHsc70 protein purified from E. coli possessed in vitro chaperone activity and protected PgHsc70 expressing bacteria from damage caused by heat and salinity stress. Nucleotide sequence analysis of 5' flanking promoter region of PgHsc70 gene revealed a potential heat-shock element (HSE) and other putative stress-responsive transcription factor binding sites. Positive correlation existed between differentially up-regulated PgHsc70 transcript levels and the duration and intensity of different environmental stresses. Molecular and biochemical analyses revealed that PgHsc70 gene was a member of the Hsp70 family and suggested that its origin was from duplication of a common ancestral gene. Transcript induction data, presence of several putative stress-responsive transcription factor-binding sites in the promoter region of PgHsc70 and the presence of a protective in vitro chaperone activity of this protein against damage caused by heat and salinity, when expressed in E. coli, suggest its probable role in conferring abiotic stress tolerance to this plant.

Genome-wide identification and transcriptional profiling of small heat shock protein gene family under diverse abiotic stress conditions in Sorghum bicolor (L.).

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.

Identification of salt-induced genes from Salicornia brachiata, an extreme halophyte through expressed sequence tags analysis.

Salinity severely affects plant growth and development causing crop loss worldwide. We have isolated a large number of salt-induced genes as well as unknown and hypothetical genes from Salicornia brachiata Roxb. (Amaranthaceae). This is the first description of identification of genes in response to salinity stress in this extreme halophyte plant. Salicornia accumulates salt in its pith and survives even at 2 M NaCl under field conditions. For isolating salt responsive genes, cDNA subtractive hybridization was performed between control and 500 mM NaCl treated plants. Out of the 1200 recombinant clones, 930 sequences were submitted to the NCBI database (GenBank accession: EB484528 to EB485289 and EC906125 to EC906292). 789 ESTs showed matching with different genes in NCBI database. 4.8% ESTs belonged to stress-tolerant gene category and approximately 29% ESTs showed no homology with known functional gene sequences, thus classified as unknown or hypothetical. The detection of a large number of ESTs with unknown putative function in this species makes it an interesting contribution. The 90 unknown and hypothetical genes were selected to study their differential regulation by reverse Northern analysis for identifying their role in salinity tolerance. Interestingly, both up and down regulation at 500 mM NaCl were observed (21 and 10 genes, respectively). Northern analysis of two important salt tolerant genes, ASR1 (Abscisic acid stress ripening gene) and plasma membrane H+ATPase, showed the basal level of transcripts in control condition and an increase with NaCl treatment. ASR1 gene is made full length using 5' RACE and its potential role in imparting salt tolerance is being studied.