Identification and molecular characterization of genes involved in ubiquitin proteasome system in high amylose wheat

Abstract

The present study identified 1,255 RING proteins in wheat through in silico approaches. Comparative expression and correlation analysis demonstrated that RING E3 ligases might play a potential role in the amylose biosynthesis as positive or negative regulators, hence would impart great knowledge for the grain quality enhancement. The two RING protein genes TaRNF1 and TaRNF2 considered as potential regulators of starch biosynthesis from expression and correlation studies were further characterized for functional validation. Conserved domain analysis confirmed the presence of RING-H2 domain in TaRNF1 and TaRNF2 protein sequences. Comparative analysis of TaRNF1 and TaRNF2 transcripts and protein abundance showed significant difference in high amylose line ( TAC 75 ) compared to the parent variety ( C 306 ). Subcellular localization studies by confocal microscopy revealed the stomatal guard cell localization of TaRNF1::GFP and TaRNF2::GFP proteins in tobacco leaves. TaRNF1-OX and TaRNF2- OX transgenic Arabidopsis plants were found more drought tolerant than WT and indicated the overall increased ABA sensitivity of transgenic lines than WT. Amylose distribution studies in TaRNF1-OX and TaRNF2-OX transgenic Arabidopsis plants suggested the role of TaRNF1-OX in amylose biosynthesis. Hence, this whole study would be helpful to reduce the study gap of ubiquitin mediated post translational modifications of starch biosynthesis enzymes and ABA mediated drought responses in cereal crops. newline