Cellular Redox Homeostasis Regulates The Transcription Of MdMIR156 In Apple Seedlings

Perennial plants undergo the transition from the juvenile to adult phase after the seed germination during the life cycle.The vegetative phase change is targeted by plant breeders to shorten the breeding cycle,whereas rejuvenation is critical for plant nurseries and propagators of many perennial woody plants to achieve rapid vegetative growth,to improve rooting ability and to realize efficient proliferation.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.We used the young leaves and unlignified shoot tips of ten-year-old trees raised from three hybrid seeds of the cross Malus asiatica 'Zisai Pearl' × M.domestica 'Red Fuji'(07-07-115,07-07-119,07-18-094)and developmentally de-differentiated suspension cells derived from the leaf of M.domestica cultivar 'Orin',The regulation mechanism of miR156 and the relationship between the redox and miR 156 were analysed by exogenous chemicals treatment,transgene and RNA-seq,BS-seq.The main results were as follows:1.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 MdMIR156al2 decreased significantly during the vegetative phase change,which was consistent with the mature miR156 level,and confirmed that MdMIRl56a5 and MdMIR156a12 were The precursor gene of miR156 by transient transformation of tobacco,indicating that miR156 is under transcriptional regulation.2.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 MdMIR156-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 miR156 is downstream of GSH.3.Subcellular H2O2 compartmentation was visualized using CeCl3 staining and transmission electron microscopy.Accumulation of a large amount of H2O2 was clearly observed in the chloroplast in leaf samples of the adult phase rather than in the juvenile phase.No obvious differences in CeCl3 deposition were observed in the cytosol,nucleus,and other subcellular compartments between leaf samples of the juvenile and adult phases.The NOX activity and MdRbohs expression were significantly higher in the juvenile phase than in the adult phase.These data indicated that the phase-related H2O2 are generated and accumulated in plastids.4.In order to analyse the regulatory pathways of MdMIR156a5 and MdMIR156a12,we used the leaves from different nodes of 07-07-115 seedlings to perform RNA-seq and BS-seq.During the phase transition,there was no change in the DNA-methylation of MIR156s gene.With the treatment of 5-AzaC,the relative expression of miR156 was significantly increased in adult plantlets,and the relative expression of MdMIR156a5 was processed.There was no change in MdMIR156a5 between juvenile and adult,but the relative expression of MdMIR156a12 increased significantly in adult,indicating that the upstream regulatory mechanisms of MdMIR156a5 and MdMIR156a12 are different.Co-expressing genes with MdMIR156a5 and MdMIR156a12 were screened to cluster the common cis-elements in the upstream sequence of the co-expressed gene.The upstream transcription factor CCA1 and WRKY40 of MdMIR156a5 and MdMIR156a12 were validated by yeast one-hybrid assay.