Abstract
Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata), an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1) contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93) was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.
Highlights
Soil salinity poses increasing threat to plant growth and agricultural productivity worldwide [1]
Transgenic 35S::VrNHX1 and RD29A::VrNHX1 lines exhibited 1.3 and 1.14 times higher Na+/K+ ratio, respectively, as compared to WT (Fig. 11 C, D). This is the first report on isolation and functional characterization of a vacuolar Na+/H+ antiporter (VrNHX1) from mungbean
Restored growth of AXTVrNHX1 cells in presence of high concentrations of Na+, K+, and Li+ and suppression of hygromycin sensitivity indicated the functional complementation of ScNHX1 by heterologous expression of VrNHX1
Summary
Soil salinity poses increasing threat to plant growth and agricultural productivity worldwide [1]. More than 20% of the cultivated area and nearly half of the world’s irrigated lands are adversely affected by salinity [2]. Enhanced crop production on salinity inflicted areas will rely on innovative agronomic practices coupled with use of genetically improved crop varieties [3]. Excess accumulation of Na+ in cytosol is detrimental to many metabolic and physiological processes, vital for plant growth and productivity, as it causes ion imbalance, hyper osmotic stress, and oxidative damage to plants [4]. To cope with salinity stress, plants have evolved sophisticated mechanisms, including restricted uptake/ exclusion of Na+ from cell, and compartmentalization of Na+ into vacuoles. Na+ efflux is catalyzed by a plasma membrane Na+/H+ antiporter (NHX) encoded by SOS1 [5,6] while, a vacuolar Na+/
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