Heterologous Expression of Heliotropium thermophilum XTH Genes Improves Tomato Tolerance to Combined Heat and Drought Stress

High temperature and drought are among the primary abiotic stresses restricting crop growth. Under the progression of global climate change, the incidence of their combined occurrence is anticipated to intensify. Elucidating the mechanisms underlying thermotolerance is critical for breeding climate-resilient crops. Heliotropium thermophilum, native to geothermal regions characterized by extreme soil temperatures, provides an ideal model for investigating plant responses to high-temperature stress. Xyloglucan endotransglucosylase/hydrolases (XTHs) are key enzymes involved in cell wall remodeling, and their critical functions in cell expansion and morphogenesis indicate a pivotal role in mediating plant stress responses. In this study, we identified and overexpressed HtXTH-7 and HtXTH-15 in tomato to examine their roles in tolerance to heat, drought, and combined heat-drought stress. Analysis of the XTH protein domains showed that all sequences possessed both GH16XET and BglS domains, except for HtXTH15, which contained only the GH16XET domain. Under all stress conditions, HtXTH7. 4 (OE-1) and HtXTH15. 7 (OE-2) lines showed significantly elevated levels of chlorophyll a, total chlorophyll, and total carotenoids compared to wild-type plants. Chlorophyll b, however, increased significantly only in OE-1 under combined stress and OE-2 under heat and combined stress. Moreover, both transgenic lines showed the most pronounced reduction in TBARS under heat stress relative to other treatments, while OE-2 consistently exhibited the highest proline accumulation across all stress conditions. Regarding antioxidant enzyme activity, both SOD and APX were elevated in response to stress, exhibiting an inverse correlation with H₂O₂ levels. RT-PCR confirmed HtXTH expression in all transgenic lines, with HtXTH7 showing 5–50 fold and HtXTH15 8–62 fold under control condtion. The expression profiles of HtXTH7 and HtXTH15 were consistent with those of established abiotic stress marker genes, including RD22 (Responsive to Dehydration 22), HSFA (Heat Shock Transcription Factor A), HSP90, HSP70, and NCED (9-cis-epoxycarotenoid dioxygenase). Our findings highlight the roles of HtXTH7 and HtXTH15 in tomato responses to heat and drought stress, providing a basis for future functional and mechanistic investigations of HtXTHs.