Comparative analysis of HKT genes in Ipomoea pes-caprae unveils conserved Na+/K+ symporter functions within the gene family

The HKT protein family plays a vital role in plant responses to salt stress by mediating sodium (Na⁺) and potassium (K⁺) transport and maintaining Na⁺-K⁺ balance. <i>Ipomoea pes-caprae</i> (<i>IPC</i>), a pantropical creeping plant distributed along coastal regions in tropical and subtropical zones, exhibits exceptional salt tolerance. Understanding its salt tolerance mechanisms provides valuable insights for developing salt-tolerant crops and identifying candidate genes for genetic engineering. In this study, we identified two HKT genes, <i>IpcHKT1;1</i> and <i>IpcHKT1;2</i>, in <i>IPC</i>. Phylogenetic analysis with HKT genes from other <i>Ipomoea</i> species revealed that all analyzed species contain two HKT genes located adjacently on the same chromosome. Comparative analysis of conserved motifs and intron-exon structures indicated that, despite their close evolutionary relationship, the HKT genes in <i>IPC</i> may exhibit functional divergence. Promoter analysis showed that their regulatory regions are enriched with cis-elements associated with responses to biotic and abiotic stresses, hormonal signaling, and growth, highlighting functional diversity within the HKT family. Subcellular localization experiments demonstrated that <i>IpcHKT1;1</i> and <i>IpcHKT1;2</i> are ion transporters localized to the plasma membrane. Heterologous expression in yeast confirmed their role in Na⁺/K⁺ symporter. Furthermore, RT-qPCR analysis revealed distinct expression patterns under salt stress: <i>IpcHKT1;2</i> was significantly upregulated in roots, while <i>IpcHKT1;1</i> expression was transitionally downregulated at 400 mM NaCl treatment. Prolonged high expression of <i>IpcHKT1;2</i> in roots suggests its critical role in sustained salt stress tolerance. These findings provide new insights into the molecular mechanisms of salt tolerance in <i>IPC</i>. The identification of <i>IpcHKT1;1</i> and <i>IpcHKT1;2</i> as key players in salt stress responses offers promising genetic resources for enhancing crop resilience to soil salinity, addressing challenges associated with global salinization.