Fraxinus mandshurica SPL2 enhances tolerance to drought stress by activating the phenylpropanoid pathway

Drought is a major abiotic stressor that severely threatens forest tree growth and survival worldwide. The SQUAMOSA promoter binding protein-like (SPL) family is a group of plant-specific transcription factors that play important roles in plant growth, development, and environmental adaptation. Our study showed that overexpression of Fraxinus mandshurica SPL2 (FmSPL2) promotes lignin biosynthesis and enhances drought tolerance. Phenotypic analysis indicated that the stomatal aperture of FmSPL2-OE1 and FmSPL2-OE2 was reduced by 12.05% and 19.23%, respectively, compared to the wild type (WT). Additionally, the xylem width of the sixth internode in these lines increased by 26.07% and 25.93% relative to WT. Correspondingly, lignin content was elevated by 19.87% and 18.48% compared with WT, respectively. Under drought stress, the lignin content in FmSPL2-OE plants remained elevated compared to WT. Furthermore, the overexpression lines exhibited increased SOD and POD activities, accompanied by reduced levels of MDA, H₂O₂, and superoxide anion. Transcriptome analysis indicated significant upregulation of multiple genes involved in phenylpropanoid biosynthesis in FmSPL2-OE plants, including phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and 4-coumarate-CoA ligase (4CL). Moreover, the yeast one-hybrid and dual-luciferase reporter assays demonstrated that FmSPL2 directly binds to the promoter regions of phenylpropanoid-related genes like PAL, CAD, and PRX, thereby activating the expression of these genes and enhancing lignin biosynthesis. These results elucidate a novel function of FmSPL2 in enhancing drought tolerance through the regulation of phenylpropanoid pathway-mediated lignin accumulation.