Salicylic acid (SA) plays a crucial role in plant innate immunity. The deployment of SA-associated immune responses is primarily affected by SA concentration, which is determined by a balance between SA biosynthesis and catabolism. However, the mechanisms regulating SA homeostasis are poorly understood. In this study, we characterized a unique UDP-glycosyltransferase, UGT76D1, which plays an important role in SA homeostasis and associated immune responses in Arabidopsis thaliana. Expression of UGT76D1 was induced by treatment with both the pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 and SA. Overexpression of UGT76D1 resulted in high SA accumulation, significant up-regulation of pathogen-related genes, and a hypersensitive response (HR)-like lesion mimic phenotype. This HR-like phenotype was not observed following UGT76D1 overexpression in SA-deficient NahG transgenic or sid2 plants, suggesting that the phenotype is SA dependent. Biochemical assays showed that UGT76D1 glycosylated 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), the major catabolic forms of SA, to their glucose and xylose conjugates in vitro and in vivo. Moreover, in a mutant background blocked in the formation of 2,3-DHBA and 2,5-DHBA, UGT76D1 overexpression did not cause a HR-like lesion mimic phenotype. Following infection with Pst DC3000, UGT76D1 knockout mutants displayed a delayed immune response, with reduced levels of DHBA glycosides and SA, and down-regulated SA synthase expression. By contrast, UGT76D1 overexpression lines showed an enhanced immune response and increased SA biosynthesis before and after pathogen infection. Thus, we propose that UGT76D1 plays an important role in SA homeostasis and plant immune responses by facilitating glycosylation of dihydroxybenzoic acids.

UDP-glycosyltransferases (UGTs) play key roles in modulating plant development and responses to environmental challenges. Previous research reported that the Arabidopsis UDP-glucosyltransferase 74E2 (AtUGT74E2), which transfers glucose to indole-3-butyric acid (IBA), is involved in regulating plant architecture and stress responses. Here, we show novel and distinct roles of UGT74E2 in rice. We found that overexpression of AtUGT74E2 in rice could enhance seed germination. This effect was also observed in the presence of IBA and abscisic acid (ABA), as well as salt and drought stresses. Further investigation indicated that the overexpression lines had lower levels of free IBA and ABA compared to wild-type plants. Auxin signaling pathway gene expression such as for OsARF and OsGH3 genes, as well as ABA signaling pathway genes OsABI3 and OsABI5, was substantially downregulated in germinating seeds of UGT74E2 overexpression lines. Consistently, due to reduced IBA and ABA levels, the established seedlings were less tolerant to drought and salt stresses. The regulation of rice seed germination and stress tolerance could be attributed to IBA and ABA level alterations, as well as modulation of the auxin/ABA signaling pathways by UGT74E2. The distinct roles of UGT74E2 in rice implied that complex and different molecular regulation networks exist between Arabidopsis and rice.

The plant Di19 (drought-induced 19) protein belongs to zinc finger transcription factors, which play crucial roles in drought stress acclimatization. OsDi19-3, a drought-induced transcription factor in rice, has not been fully characterized for its biological role in stress acclimatization. In this study, transgenic rice overexpressing OsDi19-3 was generated. Water deprivation experiments showed that transgenic plants exhibited higher drought tolerance than wild-type (WT) plants, indicating that OsDi19-3 positively regulates drought stress acclimatization. Consistent with this, stomata in overexpression lines closed more significantly than those in WT under drought stress. To explore the molecular mechanism, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments identified two interacting proteins of OsDi19-3: OsCAMK1 and OsNEK6. Notably, these two proteins also interacted with each other. A transcriptome analysis of OsDi19-3 transgenic plants revealed 224 upregulated and 167 downregulated genes (log₂(OE/WT) > 1, p-value < 0.05), including multiple stress-responsive genes. Furthermore, a ChIP-PCR analysis confirmed that OsDi19-3 directly binds to three target genes. This study provides insights into the role of OsDi19-3 in drought acclimatization and its regulatory network in rice.