Xin Zhong Zhang

Isolation and functional characterization of MxCS1: a gene encoding a citrate synthase in Malus xiaojinensis

Iron is one of the essential micronutrients required by all living organisms. In this study, we isolated a gene encoding putative citrate synthase (CS) from Malus xiaojinensis, designated as MxCS1. The MxCS1 gene encodes a protein of 473 amino acid residues with a predicted molecular mass of 52.5 kDa and a theoretical isoelectric point of 8.67. The expression of MxCS1 was enriched in the leaf rather than in phloem and root, however, its expression was hardly detected in the xylem. The gene expression was strongly induced by Fe stress treatment in the M. xiaojinensis seedlings. Over-expression of MxCS1 improved Fe deficiency tolerance in transgenic Arabidopsis. We argued that MxCS1 is a new member of the CS genes, and it may function as a regulator in response to iron stress in plants.

Possible roles of auxin and zeatin for initiating the dwarfing effect of M9 used as apple rootstock or interstock

Endogenous indole-3-acid (IAA) and zeatin (t-Z) may play important roles in the dwarfing mechanisms of rootstocks. The concentrations of IAA and t-Z, as well as the expression of the genes PIN1 and IPT3 were measured in leaves, barks, and roots from nine treatments: M9, Malus X micromalus Makino, Red Fuji/M9/Malus X micromalus, Red Fuji/M9, Red Fuji/Malus X micromalus, M9 rootstock substitution above and below the original graft union, interstock bridging, and interstock bark substitution of M9. The results show that there were greater amounts of t-Z and expressions of IPT3 in invigorated trees (Malus X micromalus and Red Fuji/Malus X micromalus) when compared with dwarfing trees (M9, Red Fuji/M9/Malus X micromalus and Red Fuji/M9) during the period of fast shoot growth (early June to mid-August). Moreover, the variation of IAA contents and PIN1 expressions shared the same pattern with t-Z contents and IPT3 expressions in all the tested locations. IAA content was extremely high in the bark of M9 interstock when compared with that of the scion and the rootstock, yet PIN1 expression in corresponding tissues was very low. After M9 rootstock were substituted above and below the original graft union, contents of t-Z, IAA, and PIN1 expressions in leaves and branch-barks recovered gradually to standard tree levels. However, there is no significant difference between the two treatments. We conclude graft union has no obvious influence on hormone transport. After M9 interstock and its bark were substituted, the hormone measurement of every index was consistent with that of rootstock substitution.

Differences in Gene Expression and Regulation during Ontogenetic Phase Change in Apple Seedlings

A woody perennial plant has to undergo a gradual and continuous process of transition from juvenility to reproductive maturity. To better understand the underlying mechanism of ontogenesis, our aim in this study was to identify differentially expressed genes between juvenile phase and adult phase not only among different seedlings but also among different hybrid crosses. Two reciprocal subtracted cDNA libraries of juvenile versus adult phases were constructed using suppression subtractive hybridization (SSH) in an apple (Malus domestica Borkh.) hybrid seedling (Jonathan × Golden Delicious). The expression uniformity of genes between the two ontogenetic phases was reconfirmed by real-time PCR (RT-qPCR) in three seedlings from the same population and also in three seedlings from different hybrid populations. Some potential post-transcriptional regulated genes were confirmed by semi-quantitative PCR. Eighty-five expressed sequence tags (ESTs) were up-regulated in the juvenile phase, and 103 ESTs were up-regulated in the adult phase. The transcription of genes associated with lipid metabolism, chloroplast protein metabolism and secondary metabolism was up-regulated in the adult phase; however, the expression of some phytohormone responsive genes was up-regulated during the juvenile phase. Several ontogenetic differential genes, such as rbcS, differed in sequences or elements in mRNA 3′ untranslated region (3′ UTR). In summary, the ontogenetic variations are probably under both transcriptional and post transcriptional regulation. The expression of redox related genes differed between juvenile and adult phase, indicating that redox homeostasis may play a role in vegetative phase change and floral transition.

miR156 switches on vegetative phase change under the regulation of redox signals in apple seedlings

In higher plants, miR156 regulates the vegetative phase change via the target SBP/SPL genes. The regulation of miR156 during ontogenetic processes is not fully understood. In the apple genome, of 31 putative MdMIR156 genes that encode pre-miR156, seven were dominantly expressed. However, the transcript levels of only MdMIR156a5 and MdMIR156a12 decreased significantly during the vegetative phase change, which was consistent with the mature miR156 level, indicating that miR156 is under transcriptional regulation. Leaf H2O2 content was higher in the adult phase than in the juvenile phase because of excess H2O2 accumulation in chloroplasts. When in vitro shoots were treated with menadione, diphenyleneiodonium, L-2-oxothiazolidine-4-carboxylic acid or buthionine sulphoximine, the expressions of MdMIR156a5, MdMIR156a12, and as well miR156 were coordinated with reduced glutathione (GSH) contents and glutathione/glutathione disulfide ratio but not H2O2 contents. Alteration of miR156 expression level by MdMIR156a6-overexpressing or miR156-mimetic transgenic Nicotiana benthamiana did not cause a corresponding change in reactive oxygen species or GSH status. Collectively, the results indicate that the vegetative phase change in apple is controlled by the MdMIR156a5 and MdMIR156a12 transcriptional regulatory network in response to the plastid–nucleus redox signals, such as GSH.

Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

BackgroundAlthough protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms.MethodsPhosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry.ResultsA total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases.ConclusionsProtein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations.