Dehydration responsive proteomics analysis of endoplasmic reticulum in chickpea (Cicer arietinum L.) for...

Dehydration stress is a significant environmental factor that adversely impacts the growth, development, and yield of chickpea (Cicer arietinum L.). In this proteomics study, we isolated the endoplasmic reticulum (ER) from chickpea shoots using ultracentrifugation and validated ER purity through enzymatic assays, immunoblotting, and biochemical analyses. To investigate ER-associated protein responses under mannitol-induced dehydration stress, 28-day-old chickpea plants were treated with mannitol, and ER proteins were subsequently analyzed using Easy-nlc LC-MS/MS. A total of 296 differentially expressed proteins (DEPs) were identified, comprising 190 upregulated and 106 downregulated proteins. Upregulated proteins were mainly associated with ER stress response, transport, metabolism, cell organization, and biogenesis. Notably, the calcium-transporting ATPases, which mediate the re-pumped of calcium ions into the ER, were highly upregulated. Sphingosine-1-phosphate lyase, a critical signaling enzyme that regulates cellular migration, immune response, and growth. It is regulate sphingosine-1-phosphate (S1P) homeostasis, through the balance between the SIP production and degradation within the cells, was also upregulated. The elevated expression of both genes was confirmed by qRT-PCR. Among the downregulated proteins, the ubiquitin-conjugating enzyme E2 variant, which mediates ubiquitin transfer and the degradation of misfolded proteins through interactions with other ligases, was significantly suppressed. Additionally, ERD-14, one of the identified DEPs, was cloned and analyzed using bioinformatic and confocal microscopy approaches. These analyses revealed its predominant localization in the nucleus, suggesting a potential novel role in the plant's response to dehydration stress. This study presents the first comprehensive proteomic characterization of ER-associated responses to dehydration stress in chickpea, offering valuable insights into the molecular mechanisms underlying stress resilience. Collectively, these findings may inform future strategies for developing dehydration-tolerant chickpea cultivars.