Polyphenol oxidases regulate pollen development through modulating flavonoids homeostasis in tobacco.

Pollen development is critical in plant reproduction. Polyphenol oxidases (PPOs) genes encode defense-related enzymes, but the role of PPOs in pollen development remains largely unexplored. Here, we characterized NtPPO genes, and then investigated their function in pollen via creating NtPPO9/10 double knockout mutant (cas-1), overexpression 35S::NtPPO10 (cosp) line and RNAi lines against all NtPPOs in Nicotiana tabacum. NtPPOs were abundantly expressed in the anther and pollen (especially NtPPO9/10). The pollen germination, polarity ratio and fruit weights were significantly reduced in the NtPPO-RNAi and cosp lines, while they were normal in cas-1 likely due to compensation by other NtPPO isoforms. Comparisons of metabolites and transcripts between the pollen of WT and NtPPO-RNAi, or cosp showed that decreased enzymatic activity of NtPPOs led to hyper-accumulation of flavonoids. This accumulation might reduce the content of ROS. Ca2+ and actin levels also decreased in pollen of the transgenic lines.Thus, the NtPPOs regulate pollen germination through the flavonoid homeostasis and ROS signal pathway. This finding provides novel insights into the native physiological functions of PPOs in pollen during reproduction.