Real-time expression and in silico characterization of pea genes involved in salt and water-deficit stress

Background: To tolerate salt and water-deficit stress, the plant adapts to the adverse environment by regulating its metabolism and expressing certain stress-induced metabolic pathways. This research analyzed the relative expression of four pea genes (P5CR, PAL1, SOD, and POX) in three pea varieties (Climax, Green grass, and Meteor) under different levels of salt and water-deficit stress.

Methods and results: The experiments on salt stress and water-deficit stress were carried out within greenhouse settings under controlled environment. The saturation percentage was employed to create artificial salinity conditions: Control without NaCl treatment, Treatment 1: 50 mM NaCl treatment, Treatment 2: 75 mM NaCl treatment, and Treatment 3: 100 mM NaCl treatment. Field capacity (FC) was used for the development of artificial water-deficit treatments in the pots, i.e., Treatment 1 (Control; water application 100% of FC), Treatment 2 (water application 75% of FC), and Treatment 3 (water application 50% of FC). Pea genes involved in biosynthetic pathways of proline, flavonoids, and enzymatic antioxidant enzymes including P5CR, PAL1, SOD, and POX were selected based on literature. Quantitative real-time PCR using cDNA as a template was used to analyze the gene expression. Pea genes were analyzed for phylogenetic analysis in closely related crops having similarity percent identity 80% and above. In silico characterization of selected proteins including the family classification was done by the NCBI CDD and INTERPRO online servers. Results from RT-qPCR analysis showed increased expression of P5CR, PAL1, and POX genes, while SOD gene expression decreased under both stresses. Climax exhibited superior stress tolerance with elevated expression of P5CR and PAL1, while Meteor showed better tolerance through increased POX expression. Phylogenetic analysis revealed common ancestry with other species like chickpea, red clover, mung bean, and barrel clover, suggesting the cross relationship among these plant species. Conserved domain analysis of respective proteins revealed that these proteins contain PLNO 2688, PLN02457, Cu-Zn Superoxide dismutase, and secretory peroxidase conserved domains. Furthermore, protein family classification indicated that the oxidation-reduction process is the most common chemical process involved in these stresses given to pea plant which validates the relationship of these proteins.

Conclusions: Salt and water-deficit stresses trigger distinct metabolic pathways, leading to the up-regulation of specific genes and the synthesis of corresponding proteins. These findings further emphasize the conservation of stress-tolerance-related genes and proteins across various plant species. This knowledge enhances our understanding of plant adaptation to stress and offers opportunities for developing strategies to improve stress resilience in crops, thereby addressing global food security challenges.