ALA up-regulated PpWRKY18 to enhance freezing tolerance of nectarine pistils

5-Aminolevulinic acid (ALA) is a natural plant growth regulator that promotes plant freezing tolerance. The WRKY family consists of plant-specific transcription factors (TFs) associated with abiotic stress responses. Up to now, whether WRKYs are involved in ALA-induced plant freezing tolerance and the underlying mechanism is not clear. In this study, we found that pretreatment with 50 mg·L−1 ALA one week earlier significantly increased the freezing tolerance of nectarine (Prunus persica var. nectarina) pistils with higher antioxidant enzyme activity and osmotic solutes when the floral twigs were stressed by −3 °C for 6 h. ALA also enhanced the expression of PpWRKY18, PpCBF1, PpCOR1, and several genes encoding antioxidant enzymes (such as superoxide dismutase, peroxidase, and catalase) and pyrroline-5-carboxylate synthase (P5CS). When PpWRKY18 was overexpressed in tobacco, the transgenic plants exhibited greater freezing tolerance, which was further promoted by exogenous ALA. Y2H, Pull-down, BiFC, and LCI analyses revealed that PpWRKY18 interacts with PpCBF1, promoting the latter transcriptional activity. Additionally, Y1H experiments showed that PpWRKY18 directly binds to the promoter of PpPOD41 while PpCBF1 binds to the promoters of PpP5CS1 and PpCOR1, activating the target gene expressions. Furthermore, we established a yeast library using the promoter of PpWRKY18 as the bait to screen the upstream regulatory factors. By library screening, Y1H, DLR, and EMSA, we found that PpC3H37, a zinc finger protein, was responsive to chilling and ALA treatment, and as a transcription factor, it activated PpWRKY18 expression by directly binding to the promoter. Taken together, we reveal a regulatory network where ALA induces upregulation of PpC3H37 expression, which positively regulates PpWRKY18 expression. Subsequently, the regulatory pathway diverges into two branches. The first is CBF-dependent, where PpCBF1 interacts with PpWRKY18, binding the promoters of PpP5CS1 and PpCOR1. The second is CBF-independent, where PpWRKY18 directly binds the promoter of PpPOD41 to upregulate the gene expression and increase the antioxidant enzyme activity and freezing tolerance. These findings provide a novel insight of the mechanism of ALA in regulating the cold hardiness of nectarine pistil.