Genome-Wide Identification and Calcium Stress-Responsive Expression of TaOSCA Genes in Triticum aestivum via Hydroponic System, with Localisation of TaOSCA.5 and TaOSCA.10

Abstract

Calcium (Ca²⁺) is both an essential structural macronutrient and a universal intracellular second messenger in plants. The OSCA (Osmosensitive Calcium-Permeable Channel) gene family encodes putative mechanosensitive Ca²⁺-permeable channels implicated in osmotic and ionic stress signalling; their expression under direct calcium stress in wheat has not been previously characterised by controlled experiment. Here we identify ten OSCA homologues (TaOSCA1–TaOSCA10) in Triticum aesitivum L. cv. Fakhr-e-Sarhad via BLAST screening of the IWGSC-URGI database, and report expression profiling of the seven genes for which reliable qRT-PCR assays were established (TaOSCA.1, .2, .4, .5, .7, .9, .10). Phylogenetic analysis using the LG+G model across 227 OSCA sequences from Poaceae, Brassicaceae, and Malvaceae resolved three conserved sub-functional clades. Thirty-six plants were grown hydroponically (Lombnaes medium; pH 6.0–6.2; 22º C) under calcium deficiency (0 mM), optimal control (2 mM), and toxicity (8 mM) with harvests at D0, D7, D14, and D21. Both stress conditions significantly reduced whole-plant biomass (42% and 69% reduction at D21 for deficiency and toxicity respectively; p < 0.01), with characteristic chlorosis and stunted root development. Reference gene stability was validated by NormFinder before Pfaffl-method qRT-PCR analysis. Seven TaOSCAs were significantly differentially expressed (FDR q < 0.05). An osmotic specificity control at D7 (18 mM mannitol and 9 mM NaCl, matched to the osmolality and ionic strength of 8 mM CaCl₂ respectively) confirmed that expression changes are Ca²⁺-specific rather than general osmotic or ionic-strength artefacts. Subcellular GFP-fusion localisation in Nicotiana benthamiana demonstrated exclusive plasma membrane localisation for TaOSCA.5 (Pearson’s r = 0.891 with AtPIP2;1) and dual plasma membrane plus tonoplast distribution for TaOSCA.10 (r = 0.714 and 0.823 with AtPIP2;1 and AtVAM3, respectively).

Keywords: Triticum aestivum; TaOSCA; calcium stress; expression profiling; hydroponic system; qRT-PCR; LG+G phylogeny; subcellular localisation; osmotic control; NormFinder; MIQE; abiotic stress.