Molecular Cloning And Functional Characterization Of Cytokinin Oxidase/Dehydrogenases Gene MdCKX5.2 In Apple

Abiotic stress is one of the challenges facing the apple production,and screening for resistance-related genes is an important goal of molecular breeding.Cytokinins（CKs）are a series of adenine derivatives that play a major role in plant growth and development as well as in coping with environmental challenges.The fine-tuned control of CKs is properly maintained by their biosynthesis and metabolism.CKs synthesis is regulated by a series of enzymes,such as isopentenyl transferase and cytokinin-activating enzymes.The irreversible degradation of CKs is catalyzed by cytokinin oxidases/dehydrogenases（CKXs）,which are the only known enzymes specifically degrade CKs.CKXs are encoded in plants by a small gene family and have been reported to be involved in plant development such as root growth,senescence,flowering,and dealing with abiotic stress in model plants.In apple（Malus×domestica）,therefore,study on the mechanisms of the CKX gene family involved in root development and salt stress tolerance contribute to screen for resistance genes.In this study,we identified 10 genes encoding CKX protein by blasting the apple genome using Arabidopsis thaliana’s CKX proteins.The MdCKX5.2 gene,which was mainly expressed in roots,was selected for further study.Then,MdCKX5.2 was ectopically expressed in Arabidopsis to study its function in plant growth and development using Agrobacterium-mediated transformation.Finally,the role of MdCKX5.2 in response to environmental stress was tested under exogenous CKs and abiotic stress.Based on these results,the following conclusions were drawn:1.MdCKX5.2 is involved in root development and abiotic stresses response.Analysis of chromosomal localization information revealed large-scale gene duplication events.A phylogenetic analysis found that the apple CKX proteins were most similar to that found in peach（Prunus persica L.）.Tissue expression analysis of MdCKXs family genes revealed their diverse expression patterns,with MdCKX5.2 expressed mainly in roots.Finally,analysis of the promoter cis-acting elements showed this gene contained a high proportion of stress-related cis-elements.2.MdCKX5.2 affects root growth and development.The root phenotype of transgenic Arabidopsis suggested that MdCKX5.2 promoted primary root elongation and lateral roots development.Introducing the CKs and auxin reporter lines to MdCKX5.2-overexpression background showed that CKs signaling was inhibited,while auxin signaling was enhanced compared with wild-type（WT）.The expression levels of PIN genes were significantly upregulated in MdCKX5.2-overexpression lines.3.MdCKX5.2 affects plant flowering and senescence.Early flowering is found in MdCKX5.2 transgenic lines,the transcript of the genes associated with flowering was significantly upregulated in transgenic plants.Similarly,early senescence was found in transgenic lines,in which chlorophyll content was dramatically reduced.4.Transcript of MdCKX5.2 in response to CKs treatment.Transcript of MdCKX5.2 gene upregulated when treated with different types of CKs but was most strongly induced by trans-zeatin（ZT）in apple.The root growth of transgenic lines was diminished by different concentrations of ZT.The elongation of the primary roots was sensitive,but the lateral roots were less sensitive to ZT.5.MdCKX5.2 participates in abiotic stress tolerance.Transcript of MdCKX5.2 was significantly upregulated under drought and salt treatment in apple.MdCKX5.2-overexpression‘Orin’callus maintained high levels of fresh weight and reduced malondialdehyde content and relative electrolytic leakage under abiotic stress.Under drought conditions,the transgenic Arabidopsis exhibited higher survival rates compared with the WT.The transgenic Arabidopsis showed higher primary root length,lateral root number,fresh weight,and chlorophyll content than the WT under salt treatment.In addition,q RT-PCR revealed that transcript levels of genes involved in abscisic acid signaling,calcium signaling,Na⁺/H⁺transporter were significantly upregulated in the transgenic lines under salt treatment.