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.

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.

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.

Diversity of Linum genetic resources in global genebanks: from agro-morphological characterisation to novel genomic technologies - a review.

Linseed or flaxseed is a well-recognized nutritional food with nutraceutical properties owing to high omega-3 fatty acid (α-Linolenic acid), dietary fiber, quality protein, and lignan content. Currently, linseed enjoys the status of a 'superfood' and its integration in the food chain as a functional food is evolving continuously as seed constituents are associated with lowering the risk of chronic ailments, such as heart diseases, cancer, diabetes, and rheumatoid arthritis. This crop also receives much attention in the handloom and textile sectors as the world's coolest fabric linen is made up of its stem fibers which are endowed with unique qualities such as luster, tensile strength, density, bio-degradability, and non-hazardous nature. Worldwide, major linseed growing areas are facing erratic rainfall and temperature patterns affecting flax yield, quality, and response to biotic stresses. Amid such changing climatic regimes and associated future threats, diverse linseed genetic resources would be crucial for developing cultivars with a broad genetic base for sustainable production. Furthermore, linseed is grown across the world in varied agro-climatic conditions; therefore it is vital to develop niche-specific cultivars to cater to diverse needs and keep pace with rising demands globally. Linseed genetic diversity conserved in global genebanks in the form of germplasm collection from natural diversity rich areas is expected to harbor genetic variants and thus form crucial resources for breeding tailored crops to specific culinary and industrial uses. Global genebank collections thus potentially play an important role in supporting sustainable agriculture and food security. Currently, approximately 61,000 germplasm accessions of linseed including 1,127 wild accessions are conserved in genebanks/institutes worldwide. This review analyzes the current status of Linum genetic resources in global genebanks, evaluation for agro-morphological traits, stress tolerance, and nutritional profiling to promote their effective use for sustainable production and nutrition enhancement in our modern diets.

A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables.

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.

Integrated omics approaches for flax improvement under abiotic and biotic stress: Current status and future prospects.

Flax (Linum usitatissimum L.) or linseed is one of the important industrial crops grown all over the world for seed oil and fiber. Besides oil and fiber, flax offers a wide range of nutritional and therapeutic applications as a feed and food source owing to high amount of α-linolenic acid (omega-3 fatty acid), lignans, protein, minerals, and vitamins. Periodic losses caused by unpredictable environmental stresses such as drought, heat, salinity-alkalinity, and diseases pose a threat to meet the rising market demand. Furthermore, these abiotic and biotic stressors have a negative impact on biological diversity and quality of oil/fiber. Therefore, understanding the interaction of genetic and environmental factors in stress tolerance mechanism and identification of underlying genes for economically important traits is critical for flax improvement and sustainability. In recent technological era, numerous omics techniques such as genomics, transcriptomics, metabolomics, proteomics, phenomics, and ionomics have evolved. The advancements in sequencing technologies accelerated development of genomic resources which facilitated finer genetic mapping, quantitative trait loci (QTL) mapping, genome-wide association studies (GWAS), and genomic selection in major cereal and oilseed crops including flax. Extensive studies in the area of genomics and transcriptomics have been conducted post flax genome sequencing. Interestingly, research has been focused more for abiotic stresses tolerance compared to disease resistance in flax through transcriptomics, while the other areas of omics such as metabolomics, proteomics, ionomics, and phenomics are in the initial stages in flax and several key questions remain unanswered. Little has been explored in the integration of omic-scale data to explain complex genetic, physiological and biochemical basis of stress tolerance in flax. In this review, the current status of various omics approaches for elucidation of molecular pathways underlying abiotic and biotic stress tolerance in flax have been presented and the importance of integrated omics technologies in future research and breeding have been emphasized to ensure sustainable yield in challenging environments.

Overexpression of Arabidopsis ICE1 enhances yield and multiple abiotic stress tolerance in indica rice.

ICE1 (Inducer of CBF Expression 1), a MYC-type bHLH transcription factor, is a regulator of cold tolerance in Arabidopsis. Indica rice, which occupies the major rice cultivated area, is highly sensitive to cold stress. Hence in this study, Arabidopsis ICE1 (AtICE1) was overexpressed in indica rice to analyze its role in reproductive stage cold and other abiotic stress tolerance to indica rice. AtICE1 was overexpressed by using stress inducible AtRD29A promoter in mega rice cv. MTU1010. Under cold stress conditions, AtICE1 overexpression lines showed lower accumulation of MDA and H2O2, higher membrane stability, and thus higher seedling survival rate than the WT plants. Expression levels of OsDREB1A, OsMYB3R2, and OsTPP1 were significantly higher in transgenics as compared with WT under cold stress conditions. AtICE1 transgenic rice plants produced 44-60% higher grain yield as compared with WT plants under control conditions in three independent experiments. Of the three AtICE1 overexpression lines, two lines produced significantly higher grain yield as compared with WT plants after recovery from cold, salt and drought stresses. AtICE1 overexpression lines showed significantly higher stomatal density and conductance under non-stress conditions. qRT-PCR analysis showed that expression levels of stomatal pathway genes viz., OsSPCH1, OsSPCH2, OsSCR1, OsSCRM1, OsSCRM2 and OsMUTE were significantly higher in AtICE1 transgenics as compared with WT plants. The components of water use viz., stomatal conductance, photosynthesis, and instantaneous WUE were higher in transgenics as compared with WT plants. The results showed that AtICE1 confers multiple stress tolerance to indica rice, and the role of ICE1 in stress tolerance and stomatal development is conserved across species.

Overexpression of ABA Receptor PYL10 Gene Confers Drought and Cold Tolerance to Indica Rice.

Abscisic acid (ABA) plays versatile functions in regulating plant development and tolerance to various biotic and abiotic stresses. Towards elucidating the functions of one of the ABA receptors (ABARs) in rice, OsPYL10 was cloned from drought tolerant rice cv. Nagina 22 and was overexpressed under stress inducible RD29A promoter in a mega rice variety MTU1010 by using Agrobacterium mediated genetic transformation. Four single copy transgenic lines selected based on Southern blot analysis were used for physiological and molecular analysis. PYL10 receptor appears to regulate its ligand ABA accumulation as PYL10 overexpressing transgenics accumulated 2-3.3-fold higher levels of ABA than that of WT in flag leaf at anthesis under non-stress conditions. The enhanced accumulation of ABA was associated with enhanced expression of genes for ABA biosynthesis viz., ZEP1, NCED1, NCED2, NCED3, and NCED4 in transgenics than in WT plants. At seedling stage, PYL10 transgenics showed significantly higher survival rate under cold stress as compared with WT plants. qRT-PCR analysis showed that expression levels of cold responsive genes viz., DREB1F, MYB3R2, TPP1, COR410, DEHYDRIN, and LEA3 were significantly higher in PYL10 overexpressing transgenic lines as compared to WT plants under cold stress. PYL10 transgenic and WT plants grown in the same pot were subjected to -80 kPa drought stress and recovery treatments at vegetative and reproductive stages. At vegetative stage drought stress, three overexpressing lines showed significantly higher grain yield (40-58%) and at reproductive stage drought stress one of these overexpression lines showed two-fold higher grain yield than that of WT plants. Excised leaf water loss analysis showed that PYL10 transgenic lost about 20% less water than WT plants. At reproductive stage, OsPYL10 transgenic maintained higher RWC, membrane stability index, chlorophyll content, and accumulated lower amount of MDA and H2O2 as compared with WT plants. qRT-PCR analysis showed that expression levels of RAB16, Dehydrin, LEA3, and ABA45 were higher in PYL10 transgenics as compared with WT plants under drought stress. Thus, overall results showed that OsPYL10 overexpression has potential to improve both drought and cold stress tolerance of indica rice.