Abstract
Polyamine oxidase (PAO) is a key enzyme catalyzing polyamine catabolism leading to H2O2 production. We previously demonstrated that Citrus sinensis contains six putative PAO genes, but their functions are not well understood. In this work, we reported functional elucidation of CsPAO4 in polyamine catabolism and salt stress response. CsPAO4 was localized to the apoplast and used both spermidine (Spd) and spermine (Spm) as substrates for terminal catabolism. Transgenic plants overexpressing CsPAO4 displayed prominent increase in PAO activity, concurrent with marked decrease of Spm and Spd and elevation of H2O2. Seeds of transgenic lines displayed better germination when compared with wild type (WT) under salt stress. However, 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). Exogenous supply of catalase (CAT), a H2O2 scavenger, partially recovered the vegetative growth and root elongation. In addition, spermine inhibited root growth of transgenic plants. Taken together, these data demonstrated that CsPAO4 accounts for production of H2O2 causing oxidative damages under salt stress and that down-regulation of a PAO gene involved in polyamine terminal catabolism may be an alternative approach for improving salt stress tolerance.
Highlights
Downregulating the expression level of SAMDC could reduce PAs synthesis in tobacco and make the plants more sensitive to salt stress[13]
PAs catabolism is catalyzed by polyamine oxidases (PAOs) that are involved in either terminal catabolism or back-conversion reactions depending on the end products; these two groups of PAOs have been demonstrated to exist in different plant species up to now
Amino acid sequences alignment showed that CsPAO4 was different from other PAO genes implicated in PA terminal catabolism, as it lacks the signal peptide (SP) that is crucial for protein localization[20]
Summary
Alignment of CsPAO4 with other PAOs involved in PAs terminal catabolism. Our previous work suggested that CsPAO4 probably catalyzes the PAs terminal catabolism. After treatment with 1 mM Spd or Spm, H2O2 was significantly increased in transgenic lines, as shown by deeper staining of the leaves, compare to WT This is obvious in the case of application of exogenous Spm. By contrast, when the transgenic plants were supplied with 20 μM guazatine, a PAO specific inhibitor, H2O2 was dramatically decreased (Fig. 5a,b). Efforts were made to investigate whether growth inhibition of the CsPAO4-overexpressing transgenic lines under salt stress was associated with ROS accumulation. In order to know whether the PAs oxidation differed between the transgenic lines and WT, we analyzed PAO activity and the PAs levels in the plants grown with or without salt stress. Consistent with the increased PAO activity in the transgenic lines, Spd and Spm contents in these lines were dramatically decreased compared to WT. No significant difference in Put level was observed among the tested lines before salt stress, but the putrescine level of WT was slightly higher than those of transgenic lines after imposition of salt stress
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