HO-generating polyamine oxidases (PAOs) are modulated during sweet pepper ripening: Spermine oxidase (SpmOX) as a case study of post-translational modification regulation.

Polyamine oxidases (PAOs) are flavin-containing enzymes involved in the catabolism of polyamines through the oxidative deamination of spermine (Spm) and spermidine (Spd). Transcriptome analysis of pepper (Capsicum annuum) fruit revealed six PAO genes (CaPAO1 to CaPAO6) expressed during ripening. Time-course expression profiling across three developmental stages, green immature (G), breaking point (BP), and red ripe (R), showed differential regulation: CaPAO5 and CaPAO6 were upregulated during ripening, while CaPAO1 and CaPAO3 were downregulated. CaPAO4 expression remained relatively stable. Exposure to exogenous nitric oxide (NO) gas induced upregulation of CaPAO1 to CaPAO3 and downregulation of CaPAO4 and CaPAO6, with CaPAO5 unaffected. Non-denaturing PAGE assays identified four PAO isozymes (CaSpmOX and CaSpdOX I-IV), using Spm or Spd as substrates. CaSpmOX IV and CaSpdOX IV exhibited the highest activities, while CaSpmOX III and CaSpdOX III were progressively inhibited during ripening, with CaSpdOX III showing complete inhibition at the red stage. To assess the influence of signaling molecules, in vitro assays were performed using green fruit extracts treated with nitric oxide (NO) donors (GSNO, CysNO), peroxynitrite (SIN-1), hydrogen sulfide (HS) donor (NaHS), hydrogen peroxide, and reducing agents (GSH, L-cysteine). CaSpmOX III emerged as the most sensitive isozyme, displaying 85-100 % inhibition under these treatments. This suggests susceptibility to post-translational modifications (PTMs) such as nitration, S-nitrosation, and persulfidation. Overall, these results demonstrate that HO-producing CaPAOs are tightly regulated at both the gene and activity levels during fruit ripening, and that NO and HS contribute to their modulation, integrating them into the broader redox and signaling network of ripening pepper fruit.