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.

NtCOMT1 responsible for phytomelatonin biosynthesis confers drought tolerance in Nicotiana tabacum.

Nicotiana tabacum (tobacco) is one of the most important industrial crops and its productivity is vulnerable to drought, particularly in Yunnan province, China due to the long water-deficit spring. Here, we aimed at identifying caffeic acid O-methyltransferase (COMT) in melatonin biosynthesis to provide genetic resources against drought tolerance of tobacco. The integration of the genome-wide identification, phylogenetic relationships, and conserved domain/motif analysis revealed that NtCOMT1 could be the probable functional COMT homolog for melatonin production. In vitro enzyme activity test approved that NtCOMT1 enabled the conversion of N-acetylserotonin into melatonin, occurring both in the cytoplasm and nucleus by subcellular localization analysis. The Km and Vmax values for NtCOMT1 at the optimum temperature (30 °C) were 266.0 µM and 2.155 nmol/min/mg protein. NtCOMT1 was significantly induced by drought stress; whereby if this gene functioned on promoting drought resistance was further conducted. Overexpression of NtCOMT1 resulted in decreased wilting in transgenic tobacco plants subjected to dehydration treatment. The combinatorial effects of NtCOMT1 in increasing melatonin content, inducing antioxidant system, and elevating the expression of drought-related genes could deliver the drought tolerance in tobacco. The characterization of NtCOMT1 may represent a solution to cope with the increasing drought stress in tobacco production in Yunnan province.