Proteome dynamics and transcriptome profiling in sorghum [ Sorghum bicolor (L.) Moench] under salt stress

Abstract

Sorghum is a C₄ cereal grain crop which is well adapted to harsh environment. It is a potential model for gaining better understanding of the molecular mechanism due to its wider adaptability to abiotic stresses. In this study, protein extraction was standardized using different methods to study the electrophoretic pattern of sorghum leaves under different salinity levels. The extraction of soluble protein with lysis buffer, followed by its clean-up was found to be the most effective method. The different profiles of salt-responsive proteins were analyzed in G-46 and CSV 44F sorghum genotypes based on their tolerance behavior towards salinity. The kafirin level also changed depending upon the concentration and exposure time to salts suggesting the stored proteins as energy source under stress conditions. The relative expression of salt-responsive genes was studied using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) which might be used as a molecular screening tool for identification of salt-tolerant genotypes in affected areas. The validated responses were examined in terms of metabolic changes and the expression of stress-induced proteins-viz. heat shock proteins (hsp) via immunoblotting assay. The results showed that the two sorghum genotypes adopted distinct approaches in response to salinity, with G-46 performing better in terms of leaf function. Also, we have standardized different protein extraction methods followed by their clean-up for electrophoretic profiling.

Keywords: Electrophoretic profile; Heat shock protein; Polyacrylamide gel electrophoresis; Salinity; Sorghum.

Fig. 1

Gel showing proteins extracted through different methods. Lane 1: Lysis buffer with 2D-clean-up; Lane 2: lysis buffer with acetone precipitation; Lane 3: phenol–sulfuric extraction with 2D-clean-up; Lane 4: Phenol–sulfuric extraction with acetone precipitation; Lane 5: 10% TCA containing 0.07% β-ME and 1 mM PMSF with acetone washing (0.07% β-ME + 1 mM PMSF); Lane 6: 10% TCA containing 0.07% β-ME and 1 mM PMSF with 2D-clean-up; lane 7: 10% TCA containing 0.07% β-ME with acetone washing (0.07% β-ME); Lane 8: 10% TCA containing 0.07% DTT with acetone washing (0.07% DTT)

Fig. 2

SDS-PAGE protein electrophoretic pattern of salt-responsive genes in G-46 genotype under salt stress. Lane 1: protein marker; Lane 2: 120 mM at 96 h*; Lane 3: 100 mM at 96 h; Lane 4: 120 mM at 48 h; Lane 5: 100 mM at 48 h; Lane 6: 120 mM at 24 h; Lane 7: 100 mM at 24 h; lane 8: control (without salt). *Leaves were harvested after time intervals

Fig. 3

SDS-PAGE protein electrophoretic pattern of salt-responsive genes in CSV 44F genotype under salt stress. Lane 1 and 2: control (without salt); Lane 3: 100 mM at 96 h*; Lane 4: 100 mM at 48 h; Lane 5: 100 mM at 24 h; Lane 6: 120 mM at 24 h; Lane 7: 120 mM at 48 h; 120 mM at 96 h. *Leaves were harvested after time intervals

Fig. 4

Kafirin spectra of sorghum genotypes at 100 mM salt concentration

Fig. 5

Expression of salt-responsive genes in sorghum under salinity. Act Actin, BADH1 betaine aldehyde dehydrogenase, H⁺-PPase Vacuolar hydrogen pyrophosphatase, P5CS1 Pyrroline-5 carboxylate synthetase 1, SOD Superoxide dismutase, Sb Sorghum bicolor

Fig. 6

Immunoblotting assay of heat shock protein (Hsp70) to validate the effect of salt concentration and its effect on protein expression