Proteomic analysis of ageing in black gram (Vigna mungo L.) seeds and its relation to seed viability

Abstract

The Indian minimum seed certification standards recommend that black gram seeds should have a minimum germination of at least 75% in order to pass the germination standard. The aim of this study is to investigate, using 2D-PAGE analysis, the possible proteome changes in black gram seeds when their germination rate falls below 75% and thereby, identify proteins that correlate with loss of seed viability during seed ageing. Firstly, in order to obtain seed samples with germination rate less than 75%, for 2D-PAGE analysis, we analyzed the proteomic (using SDS-PAGE) and physiological changes in seeds, which were artificially aged for 1 to 10 days at 98 ± 2% relative humidity and 40 ± 1° C temperature. The results revealed the major physiological and protein changes occurs from sixth day onwards and germination rate falls below 75% at sixth day, suggesting that 2D-PAGE seed samples can be obtained from 6-day-artificially aged seeds. Secondly, we carried out 2D-PAGE analysis using fresh and 6-day-artificially aged seeds and characterized 16 differentially expressed proteins in aged seeds. These proteins were identified using MALDI-TOF-MS and classified into 8 functional groups. Out of 16 proteins, 4 were up-regulated and 12 were down-regulated, indicating that these proteins play an important role in seed viability. Seven down-regulated proteins were functionally related to cell structure, transporters, metabolism and transcription, and one up-regulated protein was related to defense. In summary, this study has identified proteins that correlate with seed deterioration and loss of viability during ageing in black gram seeds.