Molecular and physiological evidence of HvCaLB1 regulates calcium homeostasis and salt tolerance in Barley

The loss of crop productivity due to soil salinity is an increasing threat to agriculture worldwide. Calcium (Ca²⁺) plays vital roles in salt-responsive signaling through the perception of various calcium-binding proteins, such as Ca²⁺-dependent lipid-binding proteins (CaLBs). Here, CaLBs from barley (Hordeum vulgare), a moderate salt-tolerant crop, and other green plants were selected for evolutionary and bioinformatics analysis. The emergence of the CaLB1 and C2 domains could be traced back to green algae, such as the chlorophyte alga Uronema belka (Uronemataceae). The physiological roles of HvCaLB1 in the salt-tolerant barley accession CM72 were investigated through gene silencing induced by barley stripe mosaic virus. Knockdown of HvCaLB1 significantly and differentially impaired the performance of plant growth, photosynthetic, and chlorophyll fluorescence parameters under the treatments of 200 and 400 mM NaCl. Moreover, the knockdown of HvCaLB1 disrupted the homeostasis of essential elements, particularly in the significant decrease of root potassium (K⁺) and Ca²⁺ contents in HvCaLB1 silencing plants compared to the control plants in response to salt stress. Significantly increased accumulation of reactive oxygen species (ROS), reduced cytosolic Ca²⁺ levels, as well as the decreased expression of HvHVP10 (Vacuolar H⁺-pyrophosphatase 10) and HvCaM1 (Calmodulin 1), were observed in the roots of the HvCaLB1-silencing plants subjected to 400 mM NaCl treatment compared to those of control plants. Taken together, CaLBs represent an ancient group of Ca²⁺-binding domain-containing proteins, and HvCaLB1 regulates NaCl-induced ion, ROS homeostasis, and gene expression in barley roots, demonstrating the potential application of CaLBs for crop improvement with increased tolerance to salt stress.