Identification And Characterization Of ATP/ADP PpIPT Genes In Peach

ATP/ADP isopentenyltransferases(IPTs) genes encoding key enzymes involved in cytokinin synthesis. In this study, based on peach genome database GDR, the sequences of ATP/ADP PpIPT family genes were identified and numbered in peach. By using transgenic technology, the functions of ATP/ADP PpIPTs in cytokinin synthesis and regulating growth and development in peach were investigated. By using qRT-PCR, we have comprehensive studied the expression characteristic of ATP/ADP PpIPTs in response to nitrogen and different phytohormones in various peach tissues. To better understand the molecular mechanisms of plant hormonal control of apical dominance in peach, we focused on the expression of ATP/ADP IPTs. The effect of auxin and strigolactone on ATP/ADP IPT gene expression was analyzed to uncover the relationship and possible links between auxin, strigolactone, and cytokinin in the control of shoot branching in peach. We examined the changes in cytokinins content and expression of ATP/ADP PpIPT genes in pulp and seeds during peach fruit development to analyze the fruit’s ability to synthesize cytokinins.The main results are shown as follows:1. Identification, bioinformatics and expression analysis of the ATP/ADP PpIPT gene family in peachAccording to the GDR, there are 5 members in peach ATP/ADPIPT gene family, but based on our results, there are only 4 members in the LuXing nectarine. These 4 members were named PpIPT1, PpIPT3, PpIPT5 a, and PpIPT5 b respectively, they all have no intron, and have a common ATP/GTP binding domain of [(A, G)-X4-G-K-(S, T)]. Bioinformatics analysis revealed that there are multiple putative cis-acting elements in their promoter sequences, suggesting that their expression can been regulated by multifarious signals from inside and outside of the plant. Sublocation and tissue expression of PpATP/ADPIPTs showed quite different results, indicating their different roles in cytokinin biosynthesis in peach. The results showed that the levels of cytokinins(sum of tZ-type and i P-type) in transgenic Arabidopsis over expressing 35S::PpIPT1, 35S::PpIPT3, 35S::PpIPT5a, and 35S::Pp IPT5 b were significantly higher than those observed in wild-type plants. Various phenotypes typical of cytokinin overproduction were observed in the transgenic plants, such as vigorous growth, unique root structure, and enhanced stress resistance; PpIPT5 a over expressing Arabidopsis was chosen to compare with the wild-type plants. These results suggest that the enzymes encoded by these genes produce cytokinins that are active in vivo.2. Characterization of ATP/ADP PpIPT gene expression during peach fruit developmentHere, we examined their expression in pulp and seeds during fruit development. ATP/ADP PpIPT expression levels decreased significantly in pulp within two weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time, suggesting that the cytokinin required for pulp cell division does not originate solely in the pulp. PpIPT1, 3 and 5b expression in seeds also decreased during early fruit development, while PpIPT5 a expression in seed increased significantly within two weeks after flowering. The expression of PpIPT5 a in seed was consistent with cytokinin levels, which increased significantly during early fruit development; moreover PpIPT5 a expression was significantly higher in seeds than in other tissues, suggesting that PpIPT5 a in seed is the key gene for cytokinin biosynthesis during early fruit development. In seeds, ATP/ADP PpIPT expression also increased significantly during later fruit development, which is consistent with the higher cytokinin contents in seeds during these stages. In pulp, high cytokinin levels were observed during early fruit development, suggesting that cytokinin mainly promotes flesh cell division during this time. In seeds, high cytokinins levels were observed during later fruit development, which is consistent with rapid seed development. The tZ content exhibited a second peak during stone hardening, and exogenous tZ treatment significantly increased POD activity in endocarp during early stone hardening, suggesting that tZ-type cytokinins promote lignin biosynthesis during stone hardening.3. Relationship between cytokinin and nitrogen nutrition in peachRelationship between cytokinin and nitrogen nutrition in peach was in-depth analyzed in this study. Expression patterns of ATP/ADP PpIPT genes in different peach tissues after different treatments were detected to analysis the role of nitrogen on regulating cytokinins biosynthesis. Only when NO3- was applied to the N-deprived peach seedlings, PpIPT3 was upregulated within 2 h. and the increase was resistant to two kinds of specific nitrate metabolism inhibitor(Tungstate and L-methionine sulfoximine), indicating that PpIPT3 is rapidly induced by nitrate, and the induction does not require nitrogen assimilation and de novo protein synthesis. Cytokinins are a class of phytohormones implicated in many aspects of plant growth and development. Roles of cytokinins on regulating nitrogen metabolism have also been studied in both roots and leaves. After treatments of cytokinins, expression of genes involved in nitrogen metabolism in peach roots showed that cytokinins could inhibit the uptake and promote the assimilation and the export of nitrate in peach roots. Pretreatment of Lovastatin(a specific inhibitor of cytokinins synthesis) could relieve the negative feedback of nitrate uptake in peach roots. These results indicating that cytokinins in roots could act as satiety signal of nitrogen. Changes of the activity of enzymes involved in the nitrogen metabolism and expression of their encoding genes in leaves from different positions(upper, middle, and lower) on the stems after exogenous cytokinin treatments showed that cytokinins could regulate the nitrogen metabolism in leaves. And the roles of cytokinins in regulating nitrogen metabolism in leaves from different positions are different. Exogenous cytokinin treatment can promote the absorption of nitrogen in leaves, does not affect the distribution of N in different tissues, but can promote the N transport to the lower part of branches.4. Effect of exogenous cytokinins and other phytohormones on ATP/ADP PpIPTs gene expression in different peach tissuesThe expression of ATP/ADP PpIPTs was regulated by exogenous cytokinins and other phytohormones in various peach tissues, and the response was tissue-dependent. Expression of ATP/ADP PpIPTs was downregulated by cytokinins in roots and leaves, but upregulated in early stage fruits, indicating feedback regulation of cytokinin biosynthesis only in peach roots and leaves. In addition to exogenous auxin, gibberellin, abscisic acid and ethylene also affected the expression of ATP/ADP PpIPTs in distinct ways.5. Effect of Auxin, and Strigolactone on ATP/ADP PpIPTs expression in Apical Dominance in PeachIn this study, we analyzed the effect of auxin and strigolactone on the expression of ATP/ADP ISOPENTENYLTRANSFERASE(IPT) genes(key cytokinin biosynthesis genes) and the regulation of apical dominance in peach. After decapitation, the expression levels of PpIPT1, PpIPT3, and PpIPT5 a in nodal stems sharply increased. This observation is consistent with the changes in tZ-type and iP-type cytokinin levels in nodal stems and axillary buds observed after treatment; these changes are required to promote the outgrowth of axillary buds in peach. These results suggest that ATP/ADP PpIPT genes in nodal stems are key genes for cytokinin biosynthesis, as they promote the outgrowth of axillary buds. We also found that auxin and strigolactone inhibited the outgrowth of axillary buds. After decapitation, IAA treatment inhibited the expression of ATP/ADP PpIPTs in nodal stems to impede the increase in cytokinin levels. By contrast, after GR24(strigolactone) treatment, the expression of ATP/ADP IPT genes and cytokinin levels still increased markedly, but the rate of increase in gene expression was markedly lower than that observed after decapitation in the absence of IAA treatment. In addition, GR24 inhibited basipetal auxin transport at the nodes(by limiting the expression of PpPIN1 a in nodal stems), thereby inhibiting ATP/ADP PpIPT expression in nodal stems. Therefore, strigolactone inhibits the outgrowth of axillary buds in peach only when terminal buds are present.