Adventitious root (AR) formation is a critical factor in the vegetative propagation of forestry and horticultural plants. Competence for AR formation declines in many species during the miR156/SPL-mediated vegetative phase change. Auxin also plays a regulatory role in AR formation. In apple rootstock, both high miR156 expression and exogenous auxin application are prerequisites for AR formation. However, the mechanism by which the miR156/SPL module interacts with auxin in controlling AR formation is unclear. In this paper, leafy cuttings of juvenile (Mx-J) and adult (Mx-A) phase Malus xiaojinensis were used in an RNAsequencing experiment. The results revealed that numerous genes involved in phytohormone signaling, carbohydrate metabolism, cell dedifferentiation, and reactivation were downregulated in Mx-A cuttings in response to indole butyric acid treatment. Among the differentially expressed genes, an HD-ZIP transcription factor gene, MxHB13, was found to be under negative regulation of MdSPL26 by directly binding to MxHB13 promoter. MxTIFY9 interacts with MxSPL26 and may play a role in co-repressing the expression of MxHB13. The expression of MxTIFY9 was induced by exogenous indole butyric acid. MxHB13 binds to the promoter of MxABCB19-2 and positively affects the expression. A model is proposed in which MxHB13 links juvenility-limited and auxin-limited AR recalcitrance mechanisms in Mx-A.

The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of miR156 are not well understood. The glutathione/glutathione disulfide (GSH/GSSG) ratios and GSH levels decline significantly during phase change, which is consistent with miR156 expression in apple ( Malus domestica Borkh.). Here, we found that the content of protein conjugated glutathione was remarkably higher in chloroplasts and nuclei of adult than juvenile phase apple hybrids. The decrease in miR156 expression was most relevant to the activities of serine acetyltransferase (SAT) and soluble γ-glutamyl transpeptidase (GGT), and the expressions of MdGGT1 or MdSATs . Transgenic apples over-expressing MdMIR156 or miR156-mimetic (MIM156) did not alter MdGGT1 expression or the soluble GGT activity. Inhibition of GGT activity with serine-borate complex or acivicin led to significant reduction in GSH content, the GSH/GSSG ratio, and the expressions of MdMIR156a5 , MdMIR156a12 , and miR156. Depletion of GSH with diethyl maleate without altering GGT activity caused a dramatic decrease in the expression of MdMIR156a5 , MdMIR156a12 , and miR156. Manipulating GGT activity and GSH homeostasis by transgenic over-expressing or RNAi MdGGT1 increased or decreased MdMIR156a5 and MdMIR156a12 levels, respectively. These data provided novel evidence that MdGGT1 participates in transcriptional level of transcription regulation of miR156 precursors during ontogenesis. HIGHLIGHTS - MdGGT1 affects thiol redox status and indirectly participates in the regulation of miR156 expression during vegetative phase change.

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