STUDY OF ROOT ARCHITECTURE MODIFICATION IN WHEAT: BIOINFORMATIC AND MOLECULAR APPROACHES

Abstract

Root architecture is considered as an important agronomic trait for yield development in crops. Despite the global importance of wheat, little information is available on the molecular mechanism of root architecture modification in response to nitrate stress. To understand the molecular mechanism of root architecture in response to nitrate stress, the root architecture traits of eight commercial wheat cultivars at various concentrations of nitrate were tested. Two cultivars, WH1021 and PBW343, were selected as a nitrate stress tolerant cultivar and a nitrate stress sensitive cultivar, respectively for further studies. The specific activities of two antioxidant enzymes, namely, peroxidase and superoxide dismutase, were found increased in the nitrate stress tolerant cultivar WH1021 under nitrate stress. By designing the primers from 4 root architecture related genes, namely, TaEXP, TaGLU3, TaNRT2 and TaMnSOD, root architecture related genes in wheat were amplified by polymerase chain reaction (PCR) technique. The reverse transcriptase (RT) PCR results revealed that except TaMnSOD, the expression of all the other gene was downregulated in the roots of nitrate tolerant cultivar WH1021 seedlings under nitrate stress. Identification and functional characterization of rootless concerning crown and seminal roots (RTCS) gene in wheat was also done. The amino acid conservation of RTCS protein was studied in six monocot (Zea mays, Sorghum bicolour, Setaria italica, Oryza sativa, Panicum virgatum and Triticum aestivum,) and six dicot (Arabidopsis thaliana, Solanum tuberosum, Solanum lycopersicum, Glycine max, Capsella rubella and Brassica rapa) species of plants. The results revealed that RTCS protein consists of conserved LOB domain in all the 12 plant species. It was also found that all monocot gene sequences of RTCS carried CpG islands at both 5’ and 3’ ends. The in-silico physical mapping results indicated the presence of TaRTCS gene on chromosomes 4A, 4B and 4D of wheat. The absence of PCR amplification in the deletions lines of chromosome 4 of wheat confirmed the results obtained by the in-silico physical mapping. Semi-quantitative PCR analysis showed the expression of TaRTCS gene to be highest in seedling roots (6.5 folds) followed by germinating roots (5 folds) and crown (3.8 folds) normalized to actin. Virus induced gene silencing (VIGS) of TaRTCS gene using barley stripe mosaic virus (BSMV) was done to functionally characterize this gene in Chinese spring cultivar of wheat. The phenotype obtained after the inoculation with infectious virus containing γ.TaRTCS construct showed 30% decrease in the root length as compared to the control plants. The real time PCR results showed 66% decrease in RTCS transcripts level in the seedling roots inoculated with infectious virus ii containing γ.TaRTCS construct as compared to the control plants. The expression of TaRTCS gene was found to be maximum (6.2 folds) after the 6 h exposure with indole 3-acetic acid. These findings are expected to be helpful in understanding the molecular mechanism of root architecture modification in wheat.