CRISPR/Cas9-induced knockout of an amino acid permease gene (AAP6) reduced Arabidopsis thaliana susceptibility to Meloidogyne incognita.

BACKGROUND: Plant-parasitic root-knot nematode (Meloidogyne incognita) causes global yield loss in agri- and horticultural crops. Nematode management options rely on chemical method. However, only a handful of nematicides are commercially available. Resistance breeding efforts are not sustainable because R gene sources are limited and nematodes have developed resistance-breaking populations against the commercially available Mi-1.2 gene-expressing tomatoes. RNAi crops that manage nematode infection are yet to be commercialized because of the regulatory hurdles associated with transgenic crops. The deployment of the CRISPR/Cas9 system to improve nematode tolerance (by knocking out the susceptibility factors) in plants has emerged as a feasible alternative lately. RESULTS: In the present study, a M. incognita-responsive susceptibility (S) gene, amino acid permease (AAP6), was characterized from the model plant Arabidodpsis thaliana by generating the AtAAP6 overexpression line, followed by performing the GUS reporter assay by fusing the promoter of AtAAP6 with the ß-glucuronidase (GUS) gene. Upon challenge inoculation with M. incognita, overexpression lines supported greater nematode multiplication, and AtAAP6 expression was inducible to the early stage of nematode infection. Next, using CRISPR/Cas9, AtAAP6 was selectively knocked out without incurring any growth penalty in the host plant. The 'Cas9-free' homozygous T3 line was challenge inoculated with M. incognita, and CRISPR-edited A. thaliana plants exhibited considerably reduced susceptibility to nematode infection compared to the non-edited plants. Additionally, host defense response genes were unaltered between edited and non-edited plants, implicating the direct role of AtAAP6 towards nematode susceptibility. CONCLUSION: The present findings enrich the existing literature on CRISPR/Cas9 research in plant-nematode interactions, which is quite limited currently while compared with the other plant-pathogen interaction systems.

Tetraspanin 5 orchestrates resilience to salt stress through the regulation of ion and reactive oxygen species homeostasis in rice.

Tetraspanins (TETs) are integral membrane proteins, characterized by four transmembrane domains and a unique signature motif in their large extracellular loop. They form dynamic supramolecular complexes called tetraspanin-enriched microdomains (TEMs), through interactions with partner proteins. In plants, TETs are involved in development, reproduction and immune responses, but their role in defining abiotic stress responses is largely underexplored. We focused on OsTET5, which is differentially expressed under various abiotic stresses and localizes to both plasma membrane and endoplasmic reticulum. Using overexpression and underexpression transgenic lines we demonstrate that OsTET5 contributes to salinity and drought stress tolerance in rice. OsTET5 can interact with itself in yeast, suggesting homomer formation. Immunoblotting of native PAGE of microsomal fraction enriched from OsTET5-Myc transgenic rice lines revealed multimeric complexes containing OsTET5, suggesting the potential formation of TEM complexes. Transcriptome analysis, coupled with quantitative PCR-based validation, of OsTET5-altered transgenic lines unveiled the differential expression patterns of several stress-responsive genes, as well as those coding for transporters under salt stress. Notably, OsTET5 plays a crucial role in maintaining the ionic equilibrium during salinity stress, particularly by preserving an elevated potassium-to-sodium (K+/Na+) ratio. OsTET5 also regulates reactive oxygen species homeostasis, primarily by modulating the gene expression and activities of antioxidant pathway enzymes and proline accumulation. Our comprehensive investigation underscores the multifaceted role of OsTET5 in rice, accentuating its significance in developmental processes and abiotic stress tolerance. These findings open new avenues for potential strategies aimed at enhancing stress resilience and making valuable contributions to global food security.

Rhizobacteria Bacillus spp. mitigate osmotic stress and improve seed germination in mustard by regulating osmolyte and plant hormone signaling.

Drought, a widespread abiotic stressor, exerts a profound impact on agriculture, impeding germination and plant growth, and reducing crop yields. In the present investigation, the osmotolerant rhizobacteria Bacillus casamancensis strain MKS-6 and Bacillus sp. strain MRD-17 were assessed for their effects on molecular processes involved in mustard germination under osmotic stress conditions. Enhancement in germination was evidenced by improved germination percentages, plumule and radicle lengths, and seedling vigor upon rhizobacterial inoculation under no stress and osmotic stress conditions. Under osmotic stress, rhizobacteria stimulated the production of gibberellins and reserve hydrolytic enzymes (lipases, isocitrate lyase, and malate synthase), bolstering germination. Furthermore, these rhizobacteria influenced the plant hormones such as gibberellins and abscisic acid (ABA), as well as signalling pathways, thereby promoting germination under osmotic stress. Reduced proline and glycine betaine accumulation, and down-regulation of transcription factors BjDREB1_2 and BjDREB2 (linked to ABA-independent signalling) in rhizobacteria-inoculated seedlings indicated that bacterial treatment mitigated water deficit stress during germination, independently of these pathways. Hence, the advantageous attributes exhibited by these rhizobacterial strains can be effectively harnessed to alleviate drought-induced stress in mustard crops, potentially through the development of targeted bio-formulations.

Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy.

BACKGROUND: Plant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease. RESULTS: Initially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1-3 bp deletions and 1 bp insertions adjacent to the PAM site, a long deletion of approximately 161 bp was documented in the T0 generation. Phenotypic analysis of homozygous, 'transgene-free' T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants. CONCLUSION: Our results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.

Targeting Induced Local Lesions in Genomes (TILLING): advances and opportunities for fast tracking crop breeding.

The intensification of food production via conventional crop breeding alone is inadequate to cater for global hunger. The development of precise and expeditious high throughput reverse genetics approaches has hugely benefited modern plant breeding programs. Targeting Induced Local Lesions in Genomes (TILLING) is one such reverse genetics approach which employs chemical/physical mutagenesis to create new genetic sources and identifies superior/novel alleles. Owing to technical limitations and sectional applicability of the original TILLING protocol, it has been timely modified. Successions include: EcoTILLING, Double stranded EcoTILLING (DEcoTILLING), Self-EcoTILLING, Individualized TILLING (iTILLING), Deletion-TILLING (De-TILLING), PolyTILLING, and VeggieTILLING. This has widened its application to a variety of crops and needs. They can characterize mutations in coding as well as non-coding regions and can overcome complexities associated with the large genomes. Combining next generation sequencing tools with the existing TILLING protocols has enabled screening of huge germplasm collections and mutant populations for the target genes. In silico TILLING platforms have transformed TILLING into an exciting breeding approach. The present review outlines these multifarious TILLING modifications for precise mutation detection and their application in advance breeding programmes together with relevant case studies. Appropriate use of these protocols will open up new avenues for crop improvement in the twenty first century.

Influence of Thermotolerant Rhizobacteria Bacillus spp. on Biochemical Attributes and Antioxidant Status of Mustard Under High Temperature Stress.

Due to global warming, increasing incidences of higher-than-normal temperatures have been observed, which adversely affect seed germination, crop growth, and productivity. Several reports are available on the effect of inoculation with rhizobacteria on plant growth and biochemical attributes; however, information on their influence on seed germination and plant stress levels is lacking. In the present study, under heat stress, we studied the effect of three thermotolerant rhizobacterial strains on mustard seed germination, seedling vigor, and plant growth. Effect of inoculation with the rhizobacterial strains on the plant stress levels, biochemical attributes and antioxidant activity was also determined. Under heat stress, inoculation with the rhizobacterial strains improved seed germination and seedling fresh weight and plumule length; while only Bacillus licheniformis SSA 61 inoculated plants showed better radicle length. There was a concomitant decrease in the plant ethylene levels in the inoculated treatments. Inoculated plants showed higher shoot fresh weight, however, Bacillus sp. MRD-17 inoculated plants only improved root growth. There was significant increase in most of the plant biochemical parameters and activities of antioxidant enzymes superoxide dismutase, catalase, and ascorbate peroxidase. Significant reduction in proline and total sugar content was noted in the inoculated treatments; while increase in the amino acid and phenolics content was observed. A further increase in the antioxidant enzyme activity was recorded in most of the inoculated treatments compared with no stress. Thus, our study indicated that thermotolerant rhizobacterial strains reduced plant stress levels; enhanced seed germination, seedling vigor, plant biomass, and thermotolerance of mustard.

Evolution of an intermediate C4 photosynthesis in the non-foliar tissues of the Poaceae.

Carbon concentrating mechanisms (CCMs) in plants are abaptive features that have evolved to sustain plant growth in unfavorable environments, especially at low atmospheric carbon levels and high temperatures. Uptake of CO2 and its storage in the aerenchyma tissues of Lycopsids and diurnal acidity fluctuation in aquatic plants during the Palaeozoic era (ca. 300 Ma.) would represent the earliest evolution of a CCM. The CCM parts of the dark reactions of photosynthesis have evolved many times, while the light reactions are conserved across plant lineages. A C4 type CCM, leaf C4 photosynthesis is evolved in the PACMAD clade of the Poaceae family. The evolution of C4 photosynthesis from C3 photosynthesis was an abaptation. Photosynthesis in reproductive tissues of sorghum and maize (PACMAD clade) has been shown to be of a weaker C4 type (high CO2 compensation point, low carbon isotope discrimination, and lack of Rubisco compartmentalization, when compared to the normal C4 types) than that in the leaves (normal C4 type). However, this does not fit well with the character polarity concept from an evolutionary perspective. In a recent model proposed for CCM evolution, the development of a rudimentary CCM prior to the evolution of a more efficient CCM (features contrasting to a weaker C4 type, leading to greater biomass production rate) has been suggested. An intermediate crassulacean acid metabolism (CAM) type of CCM (rudimentary) was reported in the genera, Brassia, Coryanthes, Eriopsis, Peristeria, of the orchids (well-known group of plants that display the CAM pathway). Similarly, we propose here the evolution of a rudimentary CCM (C4-like type pathway) in the non-foliar tissues of the Poaceae, prior to the evolution of the C4 pathway as identified in the leaves of the C4 species of the PACMAD clade.

Walking through crossroads-rice responses to heat and biotic stress interactions.

Rice, the most important source of calories for humans is prone to severe yield loss due to changing climate including heat stress. Additionally, rice encounters biotic stresses in conjunction with heat stress, which exacerbates the adverse effects, and exponentially increase such losses. Several investigations have identified biotic and heat stress-related quantitative trait loci (QTLs) that may contribute to improved tolerance to these stresses. However, a significant knowledge gap exists in identifying the genomic regions imparting tolerance against combined biotic and heat stress. Hereby, we are presenting a conceptual meta-analysis identifying genomic regions that may be promising candidates for enhancing combined biotic and heat stress tolerance in rice. Fourteen common genomic regions were identified along chromosomes 1, 2, 3, 4, 6, 10 and 12, which harbored 1265 genes related to heat stress and defense responses in rice. Further, the meta expression analysis revealed 24 differentially expressed genes (DEGs) involved in calcium-mediated stress signaling including transcription factors Myb, bHLH, ROS signaling, molecular chaperones HSP110 and pathogenesis related proteins. Additionally, we also proposed a hypothetical model based on GO and MapMan analysis representing the pathways intersecting heat and biotic stresses. These DEGs can be potential candidate genes for improving tolerance to combined biotic and heat stress in rice. We present a framework highlighting plausible connecting links (QTLs/genes) between rice response to heat stress and different biotic factors associated with yield, that can be extended to other crops.

Plant growth-promoting rhizobacteria Shewanella putrefaciens and Cronobacter dublinensis enhance drought tolerance of pearl millet by modulating hormones and stress-responsive genes.

Drought is a major abiotic stress that affects crop productivity. Endophytic bacteria have been found to alleviate the adverse effects of drought on plants. In the present study, we evaluated the effects of two endophytic bacteria Shewanella putrefaciens strain MCL-1 and Cronobacter dublinensis strain MKS-1 on pearl millet (Pennisetum glaucum (L.) R. Br.) under drought stress conditions. Pearl millet plants were grown under three water levels: field capacity (FC), mild drought stress (MD), and severe drought stress (SD). The effects of inoculation on plant growth, physiological attributes, phytohormone content, and drought stress-responsive genes were assessed. The inoculation of pearl millet seeds with endophytes significantly improved shoot and root dry weight and root architecture of plants grown under FC and drought stress conditions. There was a significant increase in relative water content and proline accumulation in the inoculated plants. Among the phytohormones analyzed, the content of ABA and IAA was significantly higher in endophyte-treated plants under all moisture regimes than in uninoculated plants. C. dublinensis-inoculated plants had higher GA content than uninoculated plants under all moisture regimes. The expression level of genes involved in phytohormone biosynthesis (SbNCED, SbGA20oX, and SbYUC) and coding drought-responsive transcription factors (SbAP2, SbSNAC1 and PgDREB2A) was significantly higher under SD in endophyte-inoculated plants than in uninoculated plants. Thus, these endophytic bacteria presumably enhanced the tolerance of pearl millet to drought stress by modulating root growth, plant hormones, physiology and the expression of genes involved in drought tolerance.

Upregulation of genes encoding plastidic isoforms of antioxidant enzymes and osmolyte synthesis impart tissue tolerance to salinity stress in bread wheat.

Wheat genotype Kharchia is a donor for salt tolerance in wheat breeding programs worldwide; however, the tolerance mechanism in Kharchia is yet to be deciphered completely. To avoid spending energy on accumulating organic osmolytes and to conserve resources for maintaining growth, plants deploy sodium (Na+) ions to maintain turgor. The enhanced ability to tolerate excess ion accumulation and ion toxicity is designated as tissue tolerance. In this study, salt-tolerant wheat genotype (Kharchia 65) and sensitive cultivars (HD2687, HD2009, WL711) were exposed to vegetative stage salinity stress (for four weeks). Kharchia 65 showed better tissue tolerance to salinity than the other genotypes based on different physiological parameters. Gene expression and abundance of chloroplast localized antioxidant enzymes and compatible osmolyte synthesis were upregulated by salinity in Kharchia 65. In Kharchia 65, the higher abundance of NADPH Oxidase (RBOH) transcripts and localization of reactive oxygen species (ROS) suggested an apoplastic ROS burst. Expression of calcium signaling genes of SOS pathway, MAPK6, bZIP6 and NAC4 were also upregulated by salinity in Kharchia 65. Considering that Kharchia local is the donor of salt tolerance trait in Kharchia 65, the publically available Kharchia local transcriptome data were analyzed. Our results and the in-silico transcriptome analysis also confirmed that higher basal levels and the stress-induced rise in the expression of plastidic isoforms of antioxidant enzymes and osmolyte biosynthesis genes provide tissue tolerance in Kharchia 65. Thus, in salinity tolerant genotype Kharchia 65, ROS burst mediated triggering of calcium signaling improves Na+ exclusion and tissue tolerance to Na+. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01237-w.

Plant epigenomics for extenuation of abiotic stresses: challenges and future perspectives.

Climate change has escalated abiotic stresses, leading to adverse effects on plant growth and development, eventually having deleterious consequences on crop productivity. Environmental stresses induce epigenetic changes, namely cytosine DNA methylation and histone post-translational modifications, thus altering chromatin structure and gene expression. Stable epigenetic changes are inheritable across generations and this enables plants to adapt to environmental changes (epipriming). Hence, epigenomes serve as a good source of additional tier of variability for development of climate-smart crops. Epigenetic resources such as epialleles, epigenetic recombinant inbred lines (epiRILs), epigenetic quantitative trait loci (epiQTLs), and epigenetic hybrids (epihybrids) can be utilized in epibreeding for improving stress tolerance of crops. Epigenome engineering is also gaining momentum for developing sustainable epimarks associated with important agronomic traits. Different epigenome editing tools are available for creating, erasing, and reading such epigenetic codes in plant genomes. However, epigenome editing is still understudied in plants due to its complex nature. Epigenetic interventions such as epi-fingerprinting can be exploited in the near future for health and quality assessment of crops under stress conditions. Keeping in view the challenges and opportunities associated with this important technology, the present review intends to enhance understanding of stress-induced epigenetic changes in plants and its prospects for development of climate-ready crops.

The abscisic acid receptor OsPYL6 confers drought tolerance to indica rice through dehydration avoidance and tolerance mechanisms.

Abscisic acid (ABA) is a key regulator of plant development and stress tolerance. Here we report functional validation of the ABA receptor OsPYL6 by constitutive and stress-inducible overexpression and RNAi silencing, in an indica rice cultivar 'Pusa Sugandh 2'. Overexpression of OsPYL6 conferred ABA hypersensitivity during germination and promoted total root length. Overexpression and RNAi silencing of OsPYL6 resulted in enhanced accumulation of ABA in seedlings under non-stress conditions, at least, in part through up-regulation of different 9-cis epoxycarotenoid dioxygenase (NCED )genes. This suggests that PYL6 expression is crucial for ABA homeostasis. Analysis of drought tolerance of OsPYL6 transgenic and wild type plants showed that OsPYL6 overexpression enhanced the expression of stress-responsive genes and dehydration tolerance. Transgenic rice plants overexpressing OsPYL6 with AtRD29A (Arabidopsis thaliana Responsive to Dehydration 29A) promoter also exhibited about 25% less whole plant transpiration, compared with wild type plants under drought, confirming its role in activation of dehydration avoidance mechanisms. However, overexpression of PYL6 reduced grain yield under non-stress conditions due to reduction in height, biomass, panicle branching and spikelet fertility. RNAi silencing of OsPYL6 also reduced grain yield under drought. These results showed that rice OsPYL6 is a key regulator of plant development and drought tolerance, and fine-tuning of its expression is critical for improving yield and stress tolerance.

Development of a QuEChERS-LCMS/MS method for simultaneous estimation of tebuconazole and chlormequat chloride in wheat crop.

Tebuconazole (TBZ) and Chlormequat chloride (CCC) combination has been established as highly effective in reducing plant height of lodging prone wheat varieties. In this work, a novel analytical method employing the quick, easy, cheap, effective, rugged and safe (QuEChERS) cleanup technique and LC-MS/MS (liquid chromatography-tandem mass spectroscopy) was developed for simultaneous estimation of TBZ and CCC in wheat grains and harvest stage plant leaves. A total of 10 mL of acetonitrile and 50 mg of primary secondary amine (PSA) sorbent was consumed in the optimized QuEChERS process for leaves and grain samples. The LC-MS/MS analysis was performed using a C-18 column operating under electrospray ionization in positive mode. The QuEChERS approach achieved extraction recoveries in the acceptable range of 70%-120%, for both the compounds and was validated in terms of accuracy, precision, sensitivity and linearity. Persistence study was conducted using Lihocin (CCC 50% SL), Folicur (TBZ 25.9% EC) and their combination tank mix (Lihocin + Folicur-50% SL + 25.9% EC) applied as foliar spray twice in wheat crop (tester tall variety C-306). The results demonstrated that the developed QuEChERS-LCMS/MS is rapid and confirmatory for simultaneous quantification of both the test analytes in wheat crop.

Genome-wide identification and characterization of ABA receptor PYL gene family in rice.

BACKGROUND: Abscisic acid (ABA), a key phytohormone that controls plant growth and stress responses, is sensed by the pyrabactin resistance 1(PYR1)/PYR1-like (PYL)/regulatory components of the ABA receptor (RCAR) family of proteins. Comprehensive information on evolution and function of PYL gene family in rice (Oryza sativa) needs further investigation. This study made detailed analysis on evolutionary relationship between PYL family members, collinearity, synteny, gene structure, protein motifs, cis-regulatory elements (CREs), SNP variations, miRNAs targeting PYLs and expression profiles in different tissues and stress responses. RESULTS: Based on sequence homology with Arabidopsis PYL proteins, we identified a total of 13 PYLs in rice (BOP clade) and maize (PACCMAD clade), while other members of BOP (wheat - each diploid genome, barley and Brachypodium) and PACCMAD (sorghum and foxtail millet) have 8-9 PYLs. The phylogenetic analysis divided PYLs into three subfamilies that are structurally and functionally conserved across species. Gene structure and motif analysis of OsPYLs revealed that members of each subfamily have similar gene and motif structure. Segmental duplication appears be the driving force for the expansion of PYLs, and the majority of the PYLs underwent evolution under purifying selection in rice. 32 unique potential miRNAs that might target PYLs were identified in rice. Thus, the predicted regulation of PYLs through miRNAs in rice is more elaborate as compared with B. napus. Further, the miRNAs identified to in this study were also regulated by stresses, which adds additional layer of regulation of PYLs. The frequency of SAPs identified was higher in indica cultivars and were predominantly located in START domain that participate in ABA binding. The promoters of most of the OsPYLs have cis-regulatory elements involved in imparting abiotic stress responsive expression. In silico and q-RT-PCR expression analyses of PYL genes revealed multifaceted role of ABARs in shaping plant development as well as abiotic stress responses. CONCLUSION: The predicted miRNA mediated regulation of OsPYLs and stress regulated expression of all OsPYLs, at least, under one stress, lays foundation for further validation and fine tuning ABA receptors for stress tolerance without yield penalty in rice.

Gene network modules associated with abiotic stress response in tolerant rice genotypes identified by transcriptome meta-analysis.

Abiotic stress tolerance is a complex trait regulated by multiple genes and gene networks in plants. A range of abiotic stresses are known to limit rice productivity. Meta-transcriptomics has emerged as a powerful approach to decipher stress-associated molecular network in model crops. However, retaining specificity of gene expression in tolerant and susceptible genotypes during meta-transcriptome analysis is important for understanding genotype-dependent stress tolerance mechanisms. Addressing this aspect, we describe here "abiotic stress tolerant" (ASTR) genes and networks specifically and differentially expressing in tolerant rice genotypes in response to different abiotic stress conditions. We identified 6,956 ASTR genes, key hub regulatory genes, transcription factors, and functional modules having significant association with abiotic stress-related ontologies and cis-motifs. Out of the 6956 ASTR genes, 73 were co-located within the boundary of previously identified abiotic stress trait-related quantitative trait loci. Functional annotation of 14 uncharacterized ASTR genes is proposed using multiple computational methods. Around 65% of the top ASTR genes were found to be differentially expressed in at least one of the tolerant genotypes under different stress conditions (cold, salt, drought, or heat) from publicly available RNAseq data comparison. The candidate ASTR genes specifically associated with tolerance could be utilized for engineering rice and possibly other crops for broad-spectrum tolerance to abiotic stresses.

Leaf mass area determines water use efficiency through its influence on carbon gain in rice mutants.

Saving water and enhancing rice productivity are consensually the most important research goals globally. While increasing canopy cover would enhance growth rates by higher photosynthetic carbon gain, an accompanied increase in transpiration would have a negative impact on saving water as well as for sustainability under water-limited conditions. Increased water use efficiency (WUE) by virtue of higher carbon assimilatory capacity can significantly circumvent this trade-off. Here, we report leaf mass area (LMA) has an important canopy architecture trait which when combined with superior carboxylation efficiency (CE) would achieve higher water productivity in rice. A set of 130 ethyl methanesulfonate induced mutants of an upland cultivar Nagina-22 (N22), was screened for leaf morphological traits leading to the identification of mutants differing in LMA. The wild-type, N22, along with a selected low-LMA (380-4-3) and two high-LMA mutants (392-9-1 and 457-1-3), all with comparable total leaf area, were raised under well-watered (100% Field Capacity (FC)) and water-limited (60% FC) conditions. Low Δ13 C and a higher RuBisCO content in high-LMA mutants indicated higher carboxylation efficiency, leading to increased carbon gain. Single parent backcross populations developed by crossing high and the low-LMA mutants with N22, separately, were screened for LMA, Δ13 C and growth traits. Comparison of dry matter accumulation per unit leaf area among the progenies differing in LMA and Δ13 C reiterated the association of LMA with CE. Results illustrated that high-LMA when combined with higher CE (low Δ13 C) lead to increased WUE and growth rates.

CRISPR-Cas9 mediated genome editing of drought and salt tolerance (OsDST) gene in indica mega rice cultivar MTU1010.

Development of abiotic stress tolerant rice cultivars is necessary for sustainable rice production under the scenario of global climate change, dwindling fresh water resources and increase in salt affected areas. Several genes from rice have been functionally validated by using EMS mutants and transgenics. Often, many of these desirable alleles are not available indica rice which is mainly cultivated, and where available, introgression of these alleles into elite cultivars is a time and labour intensive process, in addition to the potential introgression of non-desirable genes due to linkage. CRISPR-Cas technology helps development of elite cultivars with desirable alleles by precision gene editing. Hence, this study was carried out to create mutant alleles of drought and salt tolerance (DST) gene by using CRISPR-Cas9 gene editing in indica rice cv. MTU1010. We used two different gRNAs to target regions of DST protein that might be involved in protein-protein interaction and successfully generated different mutant alleles of DST gene. We selected homozygous dst mutant with 366 bp deletion between the two gRNAs for phenotypic analysis. This 366 bp deletion led to the deletion of amino acid residues from 184 to 305 in frame, and hence the mutant was named as dst ∆184-305 . The dst ∆184-305 mutation induced by CRISPR-Cas9 method in DST gene in indica rice cv. MTU1010 phenocopied EMS-induced dst (N69D) mutation reported earlier in japonica cultivar. The dst ∆184-305 mutant produced leaves with broader width and reduced stomatal density, and thus enhanced leaf water retention under dehydration stress. Our study showed that the reduction in stomatal density in loss of function mutants of dst is, at least, in part due to downregulation of stomatal developmental genes SPCH1, MUTE and ICE1. The Cas9-free dst ∆184-305 mutant exhibited moderate level tolerance to osmotic stress and high level of salt stress in seedling stage. Thus, dst mutant alleles generated in this study will be useful for improving drought and salt tolerance and grain yield in indica rice cultivars.

Quantitative proteomic analysis reveals novel stress-associated active proteins (SAAPs) and pathways involved in modulating tolerance of wheat under terminal heat.

Terminal heat stress has detrimental effect on the growth and yield of wheat. Very limited information is available on heat stress-associated active proteins (SAAPs) in wheat. Here, we have identified 159 protein groups with 4271 SAAPs in control (22 ± 3 °C) and HS-treated (38 °C, 2 h) wheat cvs. HD2985 and HD2329 using iTRAQ. We identified 3600 proteins to be upregulated and 5825 proteins to be downregulated in both the wheat cvs. under HS. We observed 60.3% of the common SAAPs showing upregulation in HD2985 (thermotolerant) and downregulation in HD2329 (thermosusceptible) under HS. GO analysis showed proton transport (molecular), photosynthesis (biological), and ATP binding (cellular) to be most altered under HS. Most of the SAAPs identified were observed to be chloroplast localized and involved in photosynthesis. Carboxylase enzyme was observed most abundant active enzymes in wheat under HS. An increase in the degradative isoenzymes (α/ß-amylases) was observed, as compared to biosynthesis enzymes (ADP-glucophosphorylase, soluble starch synthase, etc.) under HS. Transcript profiling showed very high relative fold expression of HSP17, CDPK, Cu/Zn SOD, whereas downregulation of AGPase, SSS under HS. The identified SAAPs can be used for targeted protein-based precision wheat-breeding program for the development of 'climate-smart' wheat.

Allele-specific analysis of single parent backcross population identifies HOX10 transcription factor as a candidate gene regulating rice root growth.

Understanding the molecular and physiological mechanisms of trait diversity is crucial for crop improvement to achieve drought adaptation. Root traits such as high biomass and/or deep rootedness are undoubtedly important drought adaptive traits. The major aim of this investigation was to functionally characterize a set of ethyl methane sulfonate-induced rice mutants for root traits. We report the identification of a high-root biomass mutant through a novel screening strategy for yield and Δ13 C measurements. The high-root mutant (392-9-1) thus identified, had a 66% higher root biomass compared to wild-type (Nagina-22). Better maintenance of leaf turgor and carbon assimilation rates resulted in lower drought susceptibility index in 392-9-1. Targeted resequencing revealed three non-synonymous single nucleotide variations in 392-9-1 for the genes HOX10, CITRATE SYNTHASE and ZEAXANTHIN EPOXIDASE. Segregation pattern of phenotype and mutant alleles in a single parent backcross F2 population revealed a typical 3:1 segregation for each of the mutant alleles. The number of F2 progeny with root biomass equal to or greater than that of 392-9-1 represented approximately one-third of the population indicating a major role played by HOX10 gene in regulating root growth in rice. Allele-specific Sanger sequencing in contrasting F2 progenies confirmed the co-segregation of HOX10 allele with the root biomass. The non-synonymous mutations in the other two genes did not reveal any specific pattern of co-segregation with root phenotype, indicating a strong role of HOX10, an upstream transcription factor, in regulating root biomass in rice.

Exploring the heat-responsive chaperones and microsatellite markers associated with terminal heat stress tolerance in developing wheat.

Global warming is a major threat for agriculture and food security, and in many cases the negative impacts are already apparent. Wheat is one of the most important staple food crops and is highly sensitive to the heat stress (HS) during reproductive and grain-filling stages. Here, whole transcriptome analysis of thermotolerant wheat cv. HD2985 was carried out at the post-anthesis stage under control (22 ± 3 °C) and HS-treated (42 °C, 2 h) conditions using Illumina Hiseq and Roche GS-FLX 454 platforms. We assembled ~24 million (control) and ~23 million (HS-treated) high-quality trimmed reads using different assemblers with optimal parameters. De novo assembly yielded 52,567 (control) and 59,658 (HS-treated) unigenes. We observed 785 transcripts to be upregulated and 431 transcripts to be downregulated under HS; 78 transcripts showed >10-fold upregulation such as HSPs, metabolic pathway-related genes, etc. Maximum number of upregulated genes was observed to be associated with processes such as HS-response, protein-folding, oxidation-reduction and photosynthesis. We identified 2008 and 2483 simple sequence repeats (SSRs) markers from control and HS-treated samples; 243 SSRs were observed to be overlying on stress-associated genes. Polymorphic study validated four SSRs to be heat-responsive in nature. Expression analysis of identified differentially expressed transcripts (DETs) showed very high fold increase in the expression of catalytic chaperones (HSP26, HSP17, and Rca) in contrasting wheat cvs. HD2985 and HD2329 under HS. We observed positive correlation between RNA-seq and qRT-PCR expression data. The present study culminated in greater understanding of the heat-response of tolerant genotype and has provided good candidate genes for the marker development and screening of wheat germplasm for thermotolerance.