A. Developing biotic- and abiotic-stress tolerant crop plants

  • Three viral RNAi suppresser proteins were characterized for their role in modulating defense responses: A) Groundnut bud necrosis virus-NSs protein is found to be localized in vacuoles and instigate necrotic reaction through vacuolar processing enzymes. B) Papaya ringspot virus-HcPro protein is cytoplasmic, nuclear and has reticulate presence in the cell. It modulates RNAi and proteosomal pathway. C) Tomato leaf curl virus-AC4 is cytoplasmic and aggregate around nucleus. It interacts with Argonaut 4 protein and modulates transcriptional gene silencing.

  • A considerable rise in phytosterols mainly campesterol, stigmasterol, and β-sitosterol as well as phytosteryl esters was observed with maturity and under water deficit stress in N22 (droght tolerant ) and IR 64 (drought susceptible) cultivars of rice. β-sitosterol was found to be more compared to the other two sterols. Activity and expression of a key rate limiting enzyme in the biosynthesis of phytosterols, HMGR was proportional to the severity of stress. A gene encoding Phospholipid: sterol acyl transferase (PSAT) was isolated from Oryza sativa ssp indica cvN22. Higher drought tolerance exhibited by N22 cultivar could be by virtue of its ability to accumulate more phytosterols, steryl esters, HMGR expression and activity and PSAT gene expression than IR64.
  • Abiotic stresses, such as heat, drought, salinity, radiation, etc. are posing serious threat to agriculturally important crops and food security. Plants employ diverse defence mechanisms to overcome the deleterious effects of the stress, and maintain growth and development. Heat stress (HS) is one of the major problems affecting the wheat productivity. We have identified more than 23000 stress-associated genes (SAGs) and 1500 stress-associated proteins (SAPs) through different approaches like SSH library screening, de novo transcriptome sequencing, and label-free proteomics. We also identified 6 heat-responsive miRNA and 7 heat-responsive SSRs in wheat. A thermostable RuBisCo activase enzyme was developed in our lab through protein engineering. The information about SAGs and SAPs can be utilised for the development of ‘climate smart’ wheat crop.

  • High-affinity Potassium Transporter (HKT) genes (HKT2;1 and HKT2;3) are involved in transport of Na+ and K + into the plants. The genes are differentially expressed in shoot and root of the contrasting wheat genotypes. Hypermethylation of the genes was found to down-regulate their expression.
  • An 11% increase in methylation (HD, shoot Vs Root) changes expression of HKT2;3 gene from 10-fold up-regulated in shoot to 1.5-fold down-regulated in root.
  • Progressive increase in total phenolics content (TPC) and total antioxidant activity (AO), while decrease in lipid peroxidation (LP), and protein oxidation (PO) were observed in  the rice seedlings (under drought stress) raised from primed seeds.
  • Bioelicitors- Methyle jasmonate (MJ), Salicylic acid (SA), Paclobutrazole (PBZ) at 100 µM concentration could mitigate the effects of drought stress by affecting  biochemical expressions. Among these, MJ  was found to be more efficient in mitigating the drought stress.
  • Estimation of global methylation revealed that drought stress increased 5-mC content in drought-tolerant genotypes (N-22 and IR-64-DTY1.1) while global methylation status was observed to be decreased in case of drought-sensitive genotypes (IR-64) on drought stress imposition.
  • After recovery from the stress, part of the increased methylation (25-60%) was retained in the drought-tolerant genotypes (N-22 and IR-64-DTY1.1), probably as stress memory.



B. Nutritional enhancement of crop plants

  • RNAi vector (pbin – IPK2) for silencing the expression of candidate gene IPK2 encoding inositol polyphosphate 6-/3-/5-kinase in the developing seeds was successfully generated using a seed specific vicilin promoter and transformed into soybean. Upto 55.7% reduction was observed in the phytate content in the transgenic soybean seeds (T3) transformed with vector. The low-phytate soybean seeds also showed improved mineral bioavailability with an increase in 22% iron, 14% zinc and 31% calcium levels.

  • IFS2 was identified as the major isoform of isoflavone synthase involved in isoflavone accumulation in soybean seeds. The 1566 bp long gene sequence encoding this prominent isoform was isolated and mobilized into pMDC32 expression vector under the control of seed-specific conglycinin promoter isolated from soybean itself. Besides, a RNAi construct for downregulation of Flavanone-3-hydroxylase (F-3-H) was prepared through gateway cloning utilizing pHELLSGATE 8 &12 vectors. These two constructs are now being used for the development of isoflavone-enriched soybeans.

  • To understand the molecular basis of differential accumulation of tocopherol in two contrasting genotypes of soybean, tocopherol pathway gene expression was analyzed by qPCR and observed highest gene expression of γ-TMT3 (Gamma tocopherol methyl transferase) in Bragg seed tissues as compared to DS2706 (Figure 1), and also observed highest antioxidant potential and lowest lipid peroxidation in Bragg seeds as compared to DS2706. Based on the differential expression of γ-TMT3 in contrasting genotypes, γ-TMT3 promoter was cloned, sequenced and analyzed. The promoter analysis showed variation in the most proximate positions of CAAT boxes i.e. -17bp and -635 bp from TSS (Transcriptional start) site suggesting the important role of nucleotide variation in differential expression of γ-TMT3 gene.