Genomic identification and expression pattern of copper-amine oxidase genes during peach fruit development and ripening

Abstract:【Objective】Peach (Prunus persica L.) is one of the important fruits worldwide. However, peach fruits are highly perishable and deteriorate after ripening. Many studies have shown that poly- amines (PAs) serve as endogenous anti-senescence agents. PAs are usually found at high levels during early stages of fruit development but at relatively low levels in mature fruits. Recent studies report that PA catabolism is associated with decreased PA content during fruit ripening. In addition, there is an increased interest in the function of PA catabolism during fruit ripening, but little is known about the role of PA catabolism during the ripening of peach Putrescine (Put), spermidine (Spd) and spermine (Spm) are the most common PAs in plants. PA catabolism is mediated by two kinds of amine oxidases, copper- amine oxidase (CuAO) and polyamine oxidase (PAO). It has been reported that CuAOs play important functions in plant growth, fruit development and ripening, as well as abiotic and biotic stress response. However, the potential role of CuAOs in peach fruit development and ripening are still unknown. This study aimed to identify the CuAO genes in peach genome and investigate the potential function of CuAO genes in PA catabolism as well as peach fruit development and ripening.【Methods】The free PA during Huangshuimi peach fruit development and ripening was ertracted by acetonitrile and its concen- tration was detected with HPLC-MS method. Genes encoding potential copper-containing amine oxidases (CuAOs), which are involved in PA catabolism, were identified across the peach genome. The expres- sion patterns of PpCuAOs were analyzed in different tissues and different development stages of peach fruit, as well as with the treatments of exogenous PAs. The relevance of PA concentration and the ex- pression of PpCuAOs was analyzed. Transient transformation system was used to investigate the func- tion of PpCuAO4 in PA catabolism. The expression level of PpCuAO4, NtCuAO1 and NtMPO1 were an- alyzed in control and tobacco transgenic lines. DAB staining and detection of PA contents were carried out for transgenic lines.【Results】The contents of free PAs including Put, Spd and Spm markedly de- creased as the peach fruit ripened. Four putative CuAO genes (PpCuAO1-PpCuAO4) were identified in peach using the recently released genome database. The PpCuAO genes exhibited tissue-specific expres- sion patterns. Specifically, PpCuAO1 and PpCuAO2 exhibited the highest expression level in old leaves and the lowest expression level in flowers. Conversely, PpCuAO3 and PpCuAO4 presented the highest transcription level in flowers and the lowest expression level in fruits. The PpCuAO genes also exhibit- ed different expression patterns during peach fruit development and ripening. The expression level ofPpCuAO1 decreased during fruit development and ripening, except a slight increase at S2/S3 stage. In- terestingly, the expression level of PpCuAO2 significantly increased from S1 to S3 stage and exhibited the highest expression level at S2/S3 stage, which was nearly 20 folds of S1 level. Subsequently, the ex- pression level of PpCuAO2 sharply decreased at S3/S4 stage and kept a low level up to S4 stage. The mRNA level of PpCuAO3 gradually increased during fruit development and ripening and exhibited two peaks at the stage of S3/S4 (5 folds of S1 level) and S4 (10 folds of S1 level), respectively. The tran- script level of PpCuAO4 gradually increased during fruit development and ripening except a slight de- crease at S2 stage. The transcription of PpCuAOs with exogenous PAs treatment was also investigated. The result showed PpCuAO3 was remarkably induced by Put treatment. The expression of PpCuAO4was dramatically induced by Spd treatment while obviously inhibited by Put and Spm treatments. To further investigate the function of the PpCuAOs in PA catabolism, PpCuAO4 was selected and transient- ly expressed in tobacco. The expression level of PpCuAO4 significantly increased in transgenic lines while the transcript level of NtCuAO1 and NtMPO1 exhibited no difference between control and trans- genic lines. The levels of free PAs markedly decreased in PpCuAO4 transgenic lines compared to the empty vector (EV) control line. In addition, the DAB staining of tobacco leaves showed PpCuAO4transgenic lines produced more H2O2 than control tobacco plants. These data indicated PpCuAO4 was probably involved in the terminal catabolism of Spd but not Spm.【Conclusion】In our study, the con- tents of free PAs markedly decreased as the peach fruit ripened. Four putative CuAO genes were identi- fied in peach genome. The PpCuAO1-4 genes exhibited obviously tissue-specific expression patterns. The expression level of PpCuAO4 increased during fruit development and ripening and was significantly induced by Spd treatment but inhibited by Put and Spm treatments. In addition, the levels of free PAs markedly decreased in PpCuAO4 transgenic lines. These results suggested PpCuAO4 was probably in- volved in Spd terminal catabolism. Furthermore, PpCuAO4- mediated PA catabolism was associated with decreased PA content during peach fruit development and ripening. Our study provides valuable knowledge for better understanding the roles of PA catabolism in peach development and ripening. However, the specific roles of PpCuAOs, especially PpCuAO4, in peach fruit ripening needs to be fur- ther investigated.