Nitric oxide delays tomato fruit carotenoid accumulation by inhibiting the transcriptional activation of the SlbZIP16-SlPol η module

The massive accumulation of carotenoids concomitant with the degradation of chlorophyll constitutes a feature of tomato fruit ripening. Nitric oxide (NO) significantly inhibits carotenoid synthesis, yet the underlying molecular regulatory mechanism remains unclear. Here, we identified a DNA polymerase, SlPol eta (SlPol η), whose expression is positively correlated with carotenoid accumulation. Transient overexpression of SlPol η in tomato significantly upregulated the expression of carotenoid biosynthesis genes and accelerated chlorophyll degradation; Virus-induced gene silencing (VIGS) of SlPol η markedly inhibited the fruit coloring process. A bZIP family transcription factor, SlbZIP16, was identified through yeast one-hybrid screening as specifically binding to the promoter of SlPol η and activating its expression, as collectively confirmed by electrophoretic mobility shift assay (EMSA), dual-luciferase reporter, and β-glucuronidase (GUS) analysis. Further biochemical analysis revealed that exogenous NO significantly suppresses the transcriptional activity of SlbZIP16, and this inhibitory effect directly targeted the SlbZIP16-SlPol η module, as manifested by a marked delay in fruit coloring. Density functional theory (DFT) calculations were used to simulate the structural modification of SlbZIP16 by NO, indicating that Tyr173 was a high-confidence target for NO-mediated nitration. This residue is located within a structural domain of SlbZIP16, and modification at this site, if it occurred, was highly likely to induce local conformational disturbances in the protein, thereby affecting its stability and function. In summary, our study preliminarily revealed a previously unreported molecular regulatory mechanism by which NO inhibits carotenoid synthesis in fruit, providing potential molecular targets for improving fruit appearance and nutritional value.