Integrating physiological and proteomics signatures for understanding drought, salinity and alkalinity stress tolerance mechanisms in lentil

Abiotic stresses—such as drought, salinity, and alkalinity severely limit global lentil production by impairing physiological processes including osmolytes accumulation, chlorophyll synthesis, H 2 O 2 production and antioxidant activities. However, no proteomic signature studies have dissected the molecular mechanisms underlying tolerance to drought, salinity and alkalinity in lentil. In this study, proteomic profiling revealed differentially abundant proteins (DAPs) distinguishing tolerant from sensitive lines under multiple abiotic stress conditions including drought, salinity and alkalinity. Furthermore, the aforementioned physiological traits were phenotyped to compare the responses of tolerant versus sensitive genotypes toward these stresses. By integrating proteomics and physiological analyses, this study provides new insights into the physiological and molecular regulatory mechanisms of lentil in response to multiple abiotic stresses. Physiological analysis revealed genotypic differences in osmolyte regulation, chlorophyll content, H 2 O 2 production and antioxidant activities. Ultra performance liquid chromatography with tandem mass spectrometery (UPLC-MS/MS) analysis identified 707 differentially abundant proteins (DAPs) between tolerant v/s sensitive genotypes across stresses, with 283, 213 and 211 DAPs under drought, salinity and alkalinity, respectively. Ninety-five DAPs involved in protein biogenesis, cellular transport, photosynthesis, RNA regulation, defense and detoxification were common across stresses. Proteomic findings were validated by quantitative real-time polymerase chain reaction (qRT-PCR) of five DAPs at 1, 2 and 3 days post-stress. Functional annotation revealed enrichment in photosynthesis/energy metabolism, transcription/translational, protein folding/degradation, and antioxidative defense. Integrative transcriptomic and proteomic analysis highlighted superoxide dismutase and monodehydroascorbate reductase as central components of antioxidative defense. This represents the first proteomics study of lentil seedling response toward drought, salinity and alkalinity stresses. The differential abundance of proteins involved in varied regulatory pathways suggests a wide, yet complex network of proteins mediating lentil’s metabolic adaptation to drought, salinity and alkalinity stresses. Proteins identified in this study will provide new insight to lentil’s interconnected regulatory mechanisms against abiotic stresses.