Multiplexed Host-Induced Gene Silencing of Aspergillus flavus Genes Confers Aflatoxin Resistance in Groundnut.

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus, that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production (nsdC, veA, aflR, and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (<20 ppb). Comparative proteomic analysis of contrasting groundnut genotypes (WT and near-isogenic HIGS lines) supported a better understanding of the molecular processes underlying the induced resistance and identified several groundnut metabolites that might play a significant role in resistance to Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.

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.

CRISPR for accelerating genetic gains in under-utilized crops of the drylands: Progress and prospects.

Technologies and innovations are critical for addressing the future food system needs where genetic resources are an essential component of the change process. Advanced breeding tools like "genome editing" are vital for modernizing crop breeding to provide game-changing solutions to some of the "must needed" traits in agriculture. CRISPR/Cas-based tools have been rapidly repurposed for editing applications based on their improved efficiency, specificity and reduced off-target effects. Additionally, precise gene-editing tools such as base editing, prime editing, and multiplexing provide precision in stacking of multiple traits in an elite variety, and facilitating specific and targeted crop improvement. This has helped in advancing research and delivery of products in a short time span, thereby enhancing the rate of genetic gains. A special focus has been on food security in the drylands through crops including millets, teff, fonio, quinoa, Bambara groundnut, pigeonpea and cassava. While these crops contribute significantly to the agricultural economy and resilience of the dryland, improvement of several traits including increased stress tolerance, nutritional value, and yields are urgently required. Although CRISPR has potential to deliver disruptive innovations, prioritization of traits should consider breeding product profiles and market segments for designing and accelerating delivery of locally adapted and preferred crop varieties for the drylands. In this context, the scope of regulatory environment has been stated, implying the dire impacts of unreasonable scrutiny of genome-edited plants on the evolution and progress of much-needed technological advances.

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.

Bio-mimetic synthesis of catalytically active nano-silver using Bos taurus (A-2) urine.

Herein we have synthesized silver nanoparticles (Ag NPs) using liquid metabolic waste of Bos taurus (A-2 type) urine. Various bio-molecules present in cow urine, are effectively used to reduce silver (Ag) ions into silver nanoparticles in one step. This is bio-inspired electron transfer to Ag ion for the formation of base Ag metal and is fairly prompt and facile. These nanoparticles act as a positive catalyst for various organic transformation reactions. The structural, morphological, and optical properties of the as-synthesized Ag NPs are widely characterized by X-ray diffraction spectroscopy, ultraviolet-visible spectroscopy, scanning electron microscope, Fourier transmission infra-red spectroscopy, and atomic force microscopy. The as-synthesized bio-mimetic Ag NPs show potential activity for several reduction reactions of nitro groups. The Ag NPs were also used for degradation of hazardous dyes such as Methylene blue and Crystal violet with good degradation rate constant.

Functional characterization of late embryogenesis abundant genes and promoters in pearl millet (Pennisetum glaucum L.) for abiotic stress tolerance.

Late embryogenesis abundant (LEA) genes display distinct functions in response to abiotic stresses in plants. In pearl millet (Pennisetum glaucum L.), a total of 21 PgLEA genes were identified and classified into six groups including LEA1, LEA2, LEA3, LEA5, LEA7, and dehydrins (DHN). Open reading frames (ORFs) of PgLEAs range from 291 bp (PgLEA1-1) to 945 bp (PgLEA2-11) and distributed randomly among the seven chromosomes. Phylogenetic analysis revealed that all PgLEA proteins are closely related to sorghum LEA proteins. The PgLEAs were found to be expressed differentially under high progressive vapor pressure deficit (VPD), PgLEA7 was significantly expressed under high VPD and was selected for functional validation. In silico analysis of the PgLEA promoter regions revealed abiotic stress-specific cis-acting elements such as ABRE, CCAAT, MYBS, and LTRE. Based on the type of motifs, PgLEAPC promoter (758 bp), its deletion 1 (PgLpd1, 349 bp) and deletion 2 (PgLpd2, 125 bp) were cloned into the plant expression vector pMDC164 having the promoter-less uidA gene. All the three plant expression vectors were introduced into tobacco through Agrobacterium tumefaciens-mediated transformation to obtain T1 and T2 generations of transgenic plants. Based on expression of the uidA gene, tissue-specific expression was observed in mature stems, roots and seedlings of PgLEAPC and PgLpd1 carrying transgenics only. While the transgenic PgLEAPC plants displayed significantly higher uidA expression in the stem and root tissues under salt, drought, heat, and cold stresses, very low or no expression was observed in PgLpd1 and PgLpd2 transgenics under the tested stress conditions. The results of this study indicate that the complete promoter of PgLEAPC plays a role in developing abiotic stress tolerance in plants.

An update and perspectives on the use of promoters in plant genetic engineering.

Genetically engineered plants have varied applications in agriculture for enhancing the values of food and feed. Genetic engineering aims to introduce selected genetic regions with desirable traits into target plants for both spatial and temporal expressions. Promoters are the key elements responsible for regulating gene expressions by modulating the transcription factors (TFs) through recognition of RNA polymerases. Based on their recognition and expression, RNA polymerases were categorized into RNA pol II and pol III promoters. Promoter activity and specificity are the two prime parameters in regulating the transgene expression. Since the use of constitutive promoters like Cauliflower mosaic virus (CaMV) 35S may lead to adverse effects on nontarget organisms or ecosystem, inducible/tissue specific promoters and/or the RNA pol III promoters provide myriad opportunities for gene expressions with controlled regulation and with minimum adverse effects. Besides their role in transgene expression, their influence in synthetic biology and genome editing are also discussed. This review provides an update on the importance, current prospects, and insight into the advantages and disadvantages of promoters reported thus far would help to utilize them in the endeavour to develop nutritionally and agronomically improved transgenic crops for commercialization.

Functional characterization of the promoter of pearl millet heat shock protein 10 (PgHsp10) in response to abiotic stresses in transgenic tobacco plants.

In the present study, the promoter region of the pearl millet heat shock protein 10 (PgHsp10) gene was cloned and characterized. The PgHsp10 promoter (PgHsp10pro) sequence region has all the cis-motifs required for tissue and abiotic stress inducibility. The complete PgHsp10pro (PgHsp10PC) region and a series of 5' truncations of PgHsp10 (PgHsp10D1 and PgHsp10D2) and an antisense form of PgHsp10pro (PgHsp10AS) were cloned into a plant expression vector (pMDC164) through gateway cloning. All four constructs were separately transformed into tobacco through Agrobacterium-mediated genetic transformation, and PCR-confirmed transgenic plants progressed to T1 and T2 generations. The T2 transgenic tobacco plants comprising all PgHsp10pro fragments were used for GUS histochemical and qRT-PCR assays in different tissues under control and abiotic stresses. The PgHsp10PC pro expression was specific to stem and seedlings under control conditions. Under different abiotic stresses, particularly heat stress, PgHsp10PCpro had relatively higher activity than PgHsp10D1pro, PgHsp10D2pro and PgHsp10ASpro. PgHsp10pro from a stress resilient crop like pearl millet responds positively to a range of abiotic stresses, in particular heat, when expressed in heterologous plant systems such as tobacco. Hence, PgHsp10pro appears to be a potential promoter candidate for developing heat and drought stress-tolerant crop plants.

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.

Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)].

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, ß-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, ß-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, ß-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.

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.

Molecular insights into the functional role of nitric oxide (NO) as a signal for plant responses in chickpea.

The molecular mechanisms and targets of nitric oxide (NO) are not fully known in plants. Our study reports the first large-scale quantitative proteomic analysis of NO donor responsive proteins in chickpea. Dose response studies carried out using NO donors, sodium nitroprusside (SNP), diethylamine NONOate (DETA) and S-nitrosoglutathione (GSNO) in chickpea genotype ICCV1882, revealed a dose dependent positive impact on seed germination and seedling growth. SNP at 0.1mM concentration proved to be most appropriate following confirmation using four different chickpea genotypes. while SNP treatment enhanced the percentage of germination, chlorophyll and nitrogen contents in chickpea, addition of NO scavenger, cPTIO reverted its impact under abiotic stresses. Proteome profiling revealed 172 downregulated and 76 upregulated proteins, of which majority were involved in metabolic processes (118) by virtue of their catalytic (145) and binding (106) activity. A few crucial proteins such as S-adenosylmethionine synthase, dehydroascorbate reductase, pyruvate kinase fragment, 1-aminocyclopropane-1-carboxylic acid oxidase, 1-pyrroline-5-carboxylate synthetase were less abundant whereas Bowman-Birk type protease inhibitor, non-specific lipid transfer protein, chalcone synthase, ribulose-1-5-bisphosphate carboxylase oxygenase large subunit, PSII D2 protein were highly abundant in SNP treated samples. This study highlights the protein networks for a better understanding of possible NO induced regulatory mechanisms in plants.

Peanuts that keep aflatoxin at bay: a threshold that matters.

Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub-Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut-Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host-induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE-Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species-scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near-immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi-arid regions.

Molecular cloning and expression analysis of Aquaporin genes in pearl millet [Pennisetum glaucum (L) R. Br.] genotypes contrasting in their transpiration response to high vapour pressure deficits.

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 (1.2kPa) 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 (4.21kPa), 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.

Nitric Oxide (NO) in Plant Heat Stress Tolerance: Current Knowledge and Perspectives.

High temperature is one of the biggest abiotic stress challenges for agriculture. While, Nitric oxide (NO) is gaining increasing attention from plant science community due to its involvement in resistance to various plant stress conditions, its implications on heat stress tolerance is still unclear. Several lines of evidence indicate NO as a key signaling molecule in mediating various plant responses such as photosynthesis, oxidative defense, osmolyte accumulation, gene expression, and protein modifications under heat stress. Furthermore, the interactions of NO with other signaling molecules and phytohormones to attain heat tolerance have also been building up in recent years. Nevertheless, deep insights into the functional intermediaries or signal transduction components associated with NO-mediated heat stress signaling are imperative to uncover their involvement in plant hormone induced feed-back regulations, ROS/NO balance, and stress induced gene transcription. Although, progress is underway, much work remains to define the functional relevance of this molecule in plant heat tolerance. This review provides an overview on current status and discuss knowledge gaps in exploiting NO, thereby enhancing our understanding of the role of NO in plant heat tolerance.

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.

Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization.

Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

NO to drought-multifunctional role of nitric oxide in plant drought: Do we have all the answers?

Nitric oxide (NO) is a versatile gaseous signaling molecule with increasing significance in plant research due to its association with various stress responses. Although, improved drought tolerance by NO is associated greatly with its ability to reduce stomatal opening and oxidative stress, it can immensely influence other physiological processes such as photosynthesis, proline accumulation and seed germination under water deficit. NO as a free radical can directly alter proteins, enzyme activities, gene transcription, and post-translational modifications that benefit functional recovery from drought. The present drought-mitigating strategies have focused on exogenous application of NO donors for exploring the associated physiological and molecular events, transgenic and mutant studies, but are inadequate. Considering the biphasic effects of NO, a cautious deployment is necessary along with a systematic approach for deciphering positively regulated responses to avoid any cytotoxic effects. Identification of NO target molecules and in-depth analysis of its effects under realistic field drought conditions should be an upmost priority. This detailed synthesis on the role of NO offers new insights on its functions, signaling, regulation, interactions and co-existence with different drought-related events providing future directions for exploiting this molecule towards improving drought tolerance in crop plants.

Molecular Cloning and Differential Expression of Cytosolic Class I Small Hsp Gene Family in Pennisetum glaucum (L.).

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.

DREB1A overexpression in transgenic chickpea alters key traits influencing plant water budget across water regimes.

KEY MESSAGE: We demonstrate the role of DREB1A transcription factor in better root and shoot partitioning and higher transpiration efficiency in transgenic chickpea under drought stress Chickpea (Cicer arietinum L.) is mostly exposed to terminal drought stress which adversely influences its yield. Development of cultivars for suitable drought environments can offer sustainable solutions. We genetically engineered a desi-type chickpea variety to ectopically overexpress AtDREB1A, a transcription factor known to be involved in abiotic stress response, driven by the stress-inducible Atrd29A promoter. From several transgenic events of chickpea developed by Agrobacterium-mediated genetic transformation, four single copy events (RD2, RD7, RD9 and RD10) were characterized for DREB1A gene overexpression and evaluated under water stress in a biosafety greenhouse at T6 generation. Under progressive water stress, all transgenic events showed increased DREB1A gene expression before 50 % of soil moisture was lost (50 % FTSW or fraction of transpirable soil water), with a faster DREB1A transcript accumulation in RD2 at 85 % FTSW. Compared to the untransformed control, RD2 reduced its transpiration in drier soil and higher vapor pressure deficit (VPD) range (2.0-3.4 kPa). The assessment of terminal water stress response using lysimetric system that closely mimics the soil conditions in the field, showed that transgenic events RD7 and RD10 had increased biomass partitioning into shoot, denser rooting in deeper layers of soil profile and higher transpiration efficiency than the untransformed control. Also, RD9 with deeper roots and RD10 with higher root diameter showed that the transgenic events had altered rooting pattern compared to the untransformed control. These results indicate the implicit influence of rd29A::DREB1A on mechanisms underlying water uptake, stomatal response, transpiration efficiency and rooting architecture in water-stressed plants.