Overexpression of Populus euphratica PeHINT2 to enhance plant salt tolerance

Objective Populus euphratica, renowned for its exceptional salt tolerance, serves as a model for understanding salt stress mechanisms. However, the currently identified genes remain insufficient to fully elucidate the regulatory network underlying its salt tolerance. This study aims to identify and investigate the function of PeHINT2 gene of P. euphratica under salt stress, with the goal of providing a theoretical foundation for elucidating the molecular regulatory mechanisms of salt tolerance and advancing molecular breeding for stress resistance in forest.

Method The PeHINT2 gene was cloned from P. euphratica, and its protein sequence features were analyzed. A phylogenetic tree was constructed and subcellular localization was determined. Using Agrobacterium-mediated floral-dip and leaf-disc transformation, PeHINT2 was introduced separately into Arabidopsis thaliana and 84K poplar. The effects of PeHINT2 overexpression on plant salt tolerance were then comparatively analyzed at phenotypic, physiological, and molecular levels.

Result (1) PeHINT2 belongs to the HINT2 subfamily and carries the typical “His-X-His-X-His-XX” histidine triad motif, showing the closest phylogenetic relationship to HINT2 from Populus trichocarpa and Arabidopsis thaliana. (2) Subcellular localization revealed that PeHINT2 was targeted to chloroplast. (3) Under salt stress, the expression of PeHINT2 in tissue-culture seedlings of P. euphratica increased significantly, showing a rise-then-fall pattern as treatment time progressed. (4) Under salt stress, Arabidopsis thaliana lines overexpressing PeHINT2 exhibited significantly greater leaf growth, root length, and fresh mass than wild type, and the reductions in plant height and stem diameter of transgenic poplars were also markedly smaller than those of wild-type poplars. (5) Overexpression of PeHINT2 enhanced the efficiency of PSII electron transport, actual photochemical efficiency, photosynthetic performance, and light adaptability in poplar under salt stress, while alleviating oxidative damage by reducing reactive oxygen species accumulation and increasing the activities of antioxidant enzymes such as superoxide dismutase, peroxidase and catalase. (6) Expression levels of redox-related genes (CSD2, APX2, FNR, and NTRC) were significantly up-regulated in PeHINT2-overexpressing poplars under salt stress, indicating that PeHINT2 may regulate antioxidant capacity by influencing these genes.

Conclusion PeHINT2 responds to salt stress and may enhance plant salt tolerance by improving chloroplast photosynthetic efficiency and strengthening antioxidant regulation. This study provides important evidence for elucidating the function of PeHINT2 and offers a key genetic target for the breeding and improvement of salt-tolerant forest germplasm.