Transgenic sugarcane overexpressing Glyoxalase III improved germination and biomass production at formative stage under salinity and water-deficit stress conditions.

The glyoxalase system, involving Glyoxalase I (GlyI) and Glyoxalase II (Gly II), plays a vital role in abiotic stress tolerance in plants. A novel enzyme Glyoxalase III (Gly III) was found recently from bacteria, yeast, and plant species. This enzyme provides a new way to detoxify Methylglyoxal (MG), a cytotoxic α-oxoaldehyde, which, in excess, can cause complete cell destruction by forming Reactive Oxygen Species (ROS) and Advanced Glycation End products (AGEs) or DNA/RNA mutation. In this background, the current study examined sugarcane transgenic events that exhibit an increase in expression of EaGly III, to assess their performance in terms of germination and biomass production during formative stage under stress conditions. Southern blot analysis outcomes confirmed the integration of transgene in the transgenic plants. The results from quantitative RT-PCR analyses confirmed high expression levels of EaGly III in transgenic events compared to wild type (WT) under salinity (100 and 200 mM NaCl) and drought (withholding watering) conditions. Transgenic events exhibited enhanced biomass productivity ranged between 0.141 Kg/pot and 0.395 Kg/pot under 200 mM salinity and 0.262 Kg/pot and 0.666 Kg/pot under drought stress. Further, transgenic events observed significantly higher germination rates under salinity and drought conditions compared to that of WT. Subcellular localization prediction by EaGlyIII-GFP fusion expression in sugarcane callus showed that it is distributed across the cytoplasm, thus indicating its widespread activity within the cell. These results strongly suggest that enhancing EaGly III activity is a useful strategy to improve the salinity and drought-tolerance in sugarcane as well as other crops.

Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis.

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

Comparative RNA-Seq analysis unravels molecular mechanisms regulating therapeutic properties in the grains of traditional rice Kavuni.

Developing rice varieties with enhanced levels of functional bioactives is an important intervention for achieving food and nutritional security in Asia where rice is the staple food and Type II diabetes incidences are higher. The present study was aimed at dissecting out the molecular events underlying the accumulation of bio active compounds in pigmented traditional rice Kavuni. Comparative transcriptome profiling in the developing grains of Kavuni and a white rice variety ASD 16 generated 37.7 and 29.8 million reads respectively. Statistical analysis identified a total of 9177 exhibiting significant differential expression (DEGs) between the grains of Kavuni and ASD 16. Pathway mapping of DEGs revealed the preferential up-regulation of genes involved in the biosynthesis of amylose and dietary fibres in Kavuni accounting for its low glycemic index (GI). Transcripts involved in the biosynthesis of carotenoids, flavonoids, anthocyanins, phenolic acids and phenylpropanoids were also found to be up-regulated in the grains of Kavuni. This study identified up-regulation of key transcripts involved in the accumulation of phenolic acids having potential for inhibiting major hydrolytic enzymes α-amylase and α-glucosidase and thus accounting for the slow digestibility leading to low GI. Overall, this study has identified molecular targets for the genetic manipulation of anti-diabetic and anti-oxidant traits in rice.

Comparative transcriptome and metabolome profiling in the maturing seeds of contrasting cluster bean (Cyamopsis tetragonoloba L. Taub) cultivars identified key molecular variations leading to increased gum accumulation.

Cluster bean (Guar) is the major source of industrial gum. Knowledge on the molecular events regulating galactomannan gum accumulation in guar will pave way for accelerated development of gummy guar genotypes. RNA Seq analysis in the immature seeds of contrasting cluster bean genotypes HGS 563 (gum type) and Pusa Navbahar (vegetable type) resulted in the generation of 19,855,490 and 21,488,472 quality reads. Data analysis identified 4938 differentially expressed genes between the gummy vs vegetable genotypes. A set of 2241 genes were up-regulated and 2587 genes were down-regulated in gummy guar. Significant up-regulation of genes involved in the biosynthesis of galactomannan and cell wall storage polysaccharides was observed in the gummy HGS 563. Genes involved in carotenoids, flavonoids, non mevalonic acid, terpenoids, and wax metabolism were also up-regulated in HGS 563. Mannose and galactose were the major nucleotide sugars in Pusa Navbahar and HGS 563 immature seeds. Immature seeds of HGS 563 showed high concentration of mannose and galactose accumulation compared to Pusa Navbahar. qRT-PCR analysis of selected genes confirmed the findings of transcriptome data.

Creation of novel alleles of fragrance gene OsBADH2 in rice through CRISPR/Cas9 mediated gene editing.

Fragrance in rice grains is a key quality trait determining its acceptability and marketability. Intensive research on rice aroma identified mutations in betaine aldehyde dehydrogenase (OsBADH2) leading to production of aroma in rice. Gene editing technologies like CRISPR/Cas9 system has opened new avenues for accelerated improvement of rice grain quality through targeted mutagenesis. In this study, we have employed CRISPR/Cas9 tool to create novel alleles of OsBADH2 leading to introduction of aroma into an elite non-aromatic rice variety ASD16. PCR analysis of putative transformants using primers targeting the flanking regions of sgRNA in the 7th exon of OsBADH2 identified 37.5% potential multi-allelic mutations in T0 generation. Sensory evaluation test in the leaves of T0 lines identified thirteen lines belonging to five independent events producing aroma. Sequence analysis of these aromatic T0 lines identified 22 different types of mutations located within -17 bp to +15bp of sgRNA region. The -1/-2 bp deletion in the line # 8-19 and -8/-5 bp deletion in the line # 2-16 produced strong aroma and the phenotype was stably inherited in the T1 generation. Comparative volatile profiling detected novel aromatic compounds viz., pyrrolidine, pyridine, pyrazine, pyradazine and pyrozole in the grains of T1 progenies of line # 8-19. This study has demonstrated the use of CRISPR/Cas9 in creating novel alleles of OsBADH2 to introduce aroma into any non-aromatic rice varieties.

Isolation, characterization and expression analysis of stress responsive plant nuclear transcriptional factor subunit (NF-YB2) from commercial Saccharum hybrid and wild relative Erianthus arundinaceus.

Plant nuclear factor (NF-Y) is a transcription activating factor, consisting of three subunits, and plays a key regulatory role in many stress-responsive mechanisms including drought and salinity stresses. NF-Ys function both as complex and individual subunits. Considering the importance of sugarcane as a commercial crop with high socio-economic importance and the crop being affected mostly by water deficit stress and salinity stress causing significant yield loss, nuclear transcriptional factor NF-YB2 was focused in this study. Plant nuclear factor subunit B2 from Erianthus arundinaceus (EaNF-YB2), a wild relative of sugarcane which is known for its drought and salinity stress tolerance, and commercial Saccharum hybrid Co 86032 (ShNF-YB2) was isolated and characterized. Both EaNF-YB2 and ShNF-YB2 genes are 543 bp long that encodes for a polypeptide of 180 amino acid residues. Comparison of EaNF-YB2 and ShNF-YB2 gene sequences revealed nucleotide substitutions at nine positions corresponding to three synonymous and six nonsynonymous amino acid substitutions that resulted in variations in physiochemical properties. However, multiple sequence alignment (MSA) of NF-YB2 proteins showed conservation of functionally important amino acid residues. In silico analysis revealed NF-YB2 to be a hydrophilic and intracellular protein, and EaNF-YB2 is thermally more stable than that of ShNF-YB2. Phylogenetic analysis suggested the lower rate of evolution of NF-YB2. Subcellular localization in sugarcane callus revealed NF-YB2 localization at nucleus that further evidenced it to be a transcription activation factor. Comparative RT-qPCR experiments showed a significantly higher level of NF-YB2 expression in E. arundinaceus when compared to that in the commercial Saccharum hybrid Co 86032 under drought and salinity stresses. Hence, EaNF-YB2 could be an ideal candidate gene, and its overexpression in sugarcane through genetic engineering approach might enhance tolerance to drought and salinity stresses.

OsARD4 encoding an acireductone dioxygenase improves root architecture in rice by promoting development of secondary roots.

This study was aimed at unravelling the molecular basis of root growth behavior in a drought-tolerant upland rice genotype, Nootripathu. Root tips of Nootripathu were found to possess shorter root caps and a greater number of dividing cells, favoring faster elongation compared to shallow-rooted IR20. Width and length of cortical cells in the roots of rapidly growing Nootripathu were found to be two to three times higher than IR20. Evaluation of shallow-rooted IR20, deep-rooted Nootripathu and their Recombinant Inbred Lines (RILs) for root characteristics revealed the presence of genetic variation for root traits among RILs. 2D-PAGE analysis of proteins in roots of IR20, Nootripathu and bulks of extreme RILs differing in root traits resulted in the identification of proteins co-segregating with root growth behavior and co-localized with QTLs for root traits. A putative candidate gene, OsARD4, encoding an "acireductone dioxygenase" was validated for its role in modulating the root growth pattern through genetic transformation. Transgenic ASD16 rice plants engineered for the overexpression of OsARD4 exhibited root growth characteristics similar to those of Nootripathu, including faster radical emergence, more rapid elongation of primary roots, early initiation of crown/lateral roots, and higher root biomass than the non-transgenic plants.

Over-expression of a NAC 67 transcription factor from finger millet (Eleusine coracana L.) confers tolerance against salinity and drought stress in rice.

BACKGROUND: NAC proteins (NAM (No apical meristem), ATAF (Arabidopsis transcription activation factor) and CUC (cup-shaped cotyledon)) are plant-specific transcription factors reported to be involved in regulating growth, development and stress responses. Salinity responsive transcriptome profiling in a set of contrasting finger millet genotypes through RNA-sequencing resulted in the identification of a NAC homolog (EcNAC 67) exhibiting differential salinity responsive expression pattern. METHODS: Full length cDNA of EcNAC67 was isolated, characterized and validated for its role in abiotic stress tolerance through agrobacterium mediated genetic transformation in a rice cultivar ASD16. RESULTS: Bioinformatics analysis of putative NAC transcription factor (TF) isolated from a salinity tolerant finger millet showed its genetic relatedness to NAC67 family TFs in related cereals. Putative transgenic lines of rice over-expressing EcNAC67 were generated through Agrobacterium mediated transformation and presence/integration of transgene was confirmed through PCR and southern hybridization analysis. Transgenic rice plants harboring EcNAC67 showed enhanced tolerance against drought and salinity under greenhouse conditions. Transgenic rice plants were found to possess higher root and shoot biomass during stress and showed better revival ability upon relief from salinity stress. Upon drought stress, transgenic lines were found to maintain higher relative water content and lesser reduction in grain yield when compared to non-transgenic ASD16 plants. Drought induced spikelet sterility was found to be much lower in the transgenic lines than the non-transgenic ASD16. CONCLUSION: Results revealed the significant role of EcNAC67 in modulating responses against dehydration stress in rice. No detectable abnormalities in the phenotypic traits were observed in the transgenic plants under normal growth conditions. Results indicate that EcNAC67 can be used as a novel source for engineering tolerance against drought and salinity stress in rice and other crop plants.