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Figure 1. 

Salt secretion in Z. matrella and Z. japonica under in vitro conditions with ambient NaCl. (a) Adaxial leaf surface after 24 h of salt treatment, illustrating the quantity of salt secreted. Boxplots comparing salt secretion rates per unit area for the two species measured at (b) 24 h, and (c) 48 h of salt treatment. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2. 

Phylogenetic analysis of (a) HKT, and (b) NHX gene families. Protein sequences were aligned using the MUSCLE alignment tool, and Neighbor-Joining (NJ) phylogenetic trees were constructed with 1,000 bootstrap replicates using MEGA-11 software. The sequences used in the analysis are listed in Supplementary Tables S2 and S3. Species abbreviations: Zmd, Zoysia matrella cv Diamond; Zjn, Zoysia japonica cv Nagirizaki; At, Arabidopsis thaliana; Os, Oryza sativa; Ta, Triticum aestivum; Zem, Zea mays.

Figure 3. 

Genome collinearity analysis between Z. matrella cv. Diamond and O. sativa subsp. japonica, highlighting the relationships of HKT and NHX genes between the two species. Genes located on unanchored contigs in Z. matrella were excluded from the analysis.

Figure 4. 

Dynamics of relative expression levels of HKT and NHX genes in zoysia leaf blades under in vitro conditions treated with varying NaCl concentrations. (a)–(d) Expression profiles of Z. matrella genes: ZmdHKT1;4-A, ZmdHKT1;4-B, ZmdHKT2;4, and ZmdNHX6-D. (e), (f) Expression profiles of Z. japonica genes: ZjnHKT1;4-A, ZjnHKT1;4-B, ZjnHKT2;4, and ZjnNHX6. Statistical differences among NaCl treatments were analyzed using Tukey's HSD test (p < 0.05).

Figure 5. 

Proposed model of zoysiagrass response to salinity. Under salinity stress, apoplastic Na⁺ enters leaf cell through class I HKT transporters or other inward transporters. At high salinity stress levels, the cytosol may act as a temporary sink for Na⁺. The accumulated Na⁺ can be loaded to vascular system (not depicted in the figure), transported into endomembrane system via endosomal NHX transporters, or sequestered into the vacuole by vacuolar NHXs (option II). Excess Na⁺ within endomembrane system may be transported to salt glands via plasmodesmata (option I), to the vacuole (option III) or excreted back into the apoplast (option IV). In Z. matrella, the upregulation of endosomal NHXs under high levels of salinity stress suggests that options III and IV may serve as alternative responses to salt stress.