Cloning And Functional Characterization Of Polyamine Oxidase Genes In Citrus Sinensis

Citrus is one of the most important fruit crops in the world. However, biotic and abiotic stresses cause great adverse effects on the production and quality. Polyamines(PAs) are low-molecular mass aliphatic polycations that are ubiquitously distributed in higher plants. PA catabolism plays an important role in regulation of the PA homoeostasis. PA catabolism is catalyzed by two classes of amine oxidases, the copper-containing amine oxidases(CuAOs) and the FAD-dependent polyamine oxidases(PAOs). In the present study, the CuAO and PAO family members were explored with the recently released sweet orange genome sequence. In addition,expression patterns of the CsCuAOs and CsPAOs were examined. At last, transgenic and biochemical technologies were applied to further clarify the functions of some CsPAOs. The main results are as follows:1. In this study, eight putative CuAO genes(CsCuAO1-CsCuAO8) were identified in sweet orange(Citrus sinensis) genome. The chromosomal distribution analysis suggested that the eight CsCuAOs were anchored on three chromosomes. The coding sequence(CDS) sizes of eight CsCuAO genes ranged from 1389 bp to 2328 bp. The pI of CsCuAOs varied from 6.11 to 8.77, while molecular weight(MW) ranged from52.1 kDa to 82.8 kDa. CsCuAOs can be divided into two major groups according to the phylogenetic analysis. The 33 amino acid residues typically conserved in plant CuAOs were present in the CsCuAOs. They contained either a C-terminal peroxisomal targeting or an N-terminal signal peptide. Transcript levels of four CsCuAO genes(CsCuAO1-3, CsCuAO5) were abundantly detected in leaf, stem,cotyledon, and root, while the rest genes(CsCuAO6, CsCuAO7 and CsCuAO8) were only weakly expressed. In addition, the CsCuAOs were significantly induced by exogenous polyamines, including putrescine(Put), spermidine(Spd) and spermine(Spm). However, most of the CsCuAOs were down-regulated by ABA and abiotic stresses(cold, salt, and osmotic shock), whereas CsCuAO3 and CsCuAO7 were up-regulated under these circumstances.2. In the present work, six putative PAO genes(CsPAO1-CsPAO6) were unraveledin sweet orange(Citrus sinensis) using the released citrus genome sequences. The CsPAOs can be divided into four major groups according to the phylogenetic analysis.Transcripts of CsPAOs were detected in leaf, stem, cotyledon, and root, with the highest levels in the roots. According to the expression patterns of CsPAOs under exogenous polyamine treatment, we concluded that CsPAO4 probably functions in polyamine terminal catabolism whereas the other CsPAOs were involved in polyamine back conversion reactions. Overexpression of CsPAO3 in tobacco demonstrated that Spd and Spm were decreased in the transgenic line, while Put was significantly enhanced, implying a potential role of this gene in polyamine back conversion. In addition, the CsPAOs displayed various responses to ABA and abiotic stresses(cold, salt and mannitol), indicating their potential roles in hormone and abiotic stresses response. A total of 203 putative cis-regulatory elements involved in hormone and stress response were predicted in promoter regions at the upstream of CsPAOs.3. In this work, we analyzed the function of CsPAO4 in polyamine catabolism and abiotic stress response with transgenic and biochemical technologies. CsPAO4 was localized to the apoplast of onion epidermal cells. HPLC analysis showed that when Spd and Spm were incubated in the CsPAO4 reaction system, a noticeable increase in Dap, concomitant with a decrease of Spd and Spm, suggesting that CsPAO4 was involved in PAs terminal catabolism reactions. Transgenic tobacco overexpressing CsPAO4 displayed prominent increase in PAO activity, concurrent with marked decrease of Spm and Spd and elevation of Dap and H2O2. Seeds of transgenic lines displayed much faster germination and presented higher germination rate when compared with wild type(WT) upon exposure to salt stress. By contrast, both vegetative growth and root elongation of the transgenic lines were prominently inhibited under salt stress, accompanied by higher level of H2O2 and more conspicuous programmed cell death(PCD) in these lines. Exogenous supply of catalase(CAT), a H2O2 scavenger, partially recovered the vegetative growth and root elongation. In addition, root growth of the transgenic plants was significantly inhibited in the presence of Spm supplementation.