<i>In vitro</i> assessment of salt secretion and its correlation with transporter gene expression in zoysiagrass (<i>Zoysia</i> spp.)

Soil salinity is a major threat to global agriculture. The recretohalophytic turfgrass species <italic>Zoysia matrella</italic> (L.) Merr. and <italic>Zoysia japonica</italic> Steud. possess unique salt glands that enable them to secrete excess salt. Understanding the molecular mechanisms underlying salt secretion could enable the introduction of salt secretion in other grasses, thereby improving their salt tolerance. In this study, we developed an <italic>in vitro</italic> leaf assay to investigate salt secretion patterns and the expression of key sodium/potassium transporters, including HKTs and endosomal NHXs. The present study revealed that <italic>Z. matrella</italic> consistently exhibited a higher salt secretion rate than <italic>Z. japonica</italic>. In both species, salt secretion increased with rising salinity, peaking at 300 mM NaCl. Beyond this threshold, further increases in salinity did not significantly enhance secretion, suggesting a maximum capacity for the salt glands. Interestingly, at 900 mM NaCl, salt secretion was completely inhibited in <italic>Z. japonica</italic>, whereas <italic>Z. matrella</italic> maintained a low level of secretion. Among the <italic>HKT</italic> genes, <italic>HKT1;4</italic> was the most highly expressed in the leaves of both species, and its expression was negatively correlated with salt secretion. Additionally, <italic>NHX6</italic> expression in <italic>Z. matrella</italic> was negatively associated with secretion, a pattern not observed in <italic>Z. japonica</italic>. The contrasting expression patterns of endosomal <italic>NHX</italic> genes may contribute to the differential salt tolerance between the two species. The <italic>in vitro</italic> leaf assay developed in this study provides an efficient tool for evaluating salt secretion in breeding programs. The present findings offer valuable insights into salt secretion and tolerance mechanisms in zoysiagrass, paving the way for the development of new salt-tolerant varieties.