Functional Studies Of Apple MdVQs And Their Interactors MdWRKY Transcription Factors In Response To Drought Stress

Apple?Malus domestica?cultivation area,production and consumption capacity in China rank first in the world,and the apple industry has played an important role in optimizing ecological environment,increasing farmer income and adjusting agricultural structure.However,abiotic stress is threatening the sustainable development of the apple industry in China.Various studies have shown that vitamin-glutamine motif containing proteins?VQ proteins?act as transcriptional regulators involved in many biological processes such as plant growth,development and stress.In this study,we conducted a genome-wide analysis and cloning of MdVQ gene family in apple.The phylogenetic tree of VQ proteins of multiple species was constructed.The expression patterns of MdVQs in different tissues and in response to water-deficit stress were analyzed.Further,the interactions among and between MdVQ proteins and MdWRKY transcription factors were examined,and some members were subjected to phenotypic analysis and functional identification under osmotic and drought stresses.These results provide important information about the evolution,structure,and function of apple MdVQ genes and lay a solid foundation for further studies about the role of WRKY-VQ interactions in regulating developmental and defense mechanisms in apple.The main results of this study are as follows:1.Using bioinformatics-based methods,RT-PCR,and qRT-PCR technologies,we identified 49 MdVQ genes and cloned 26 of them were in the v1.0 apple genome.Our evolutional analysis revealed that the proteins could be clustered into nine groups together with their homologues in 33 species.Historically,the main characteristics of proteins in Groups I,V,VI,VII,IX,and X were thought to have been generated before the monocot-dicot split,whereas those in Groups II,III+IV,and VIII were generated after that split.Results from sequence alignments and analyses of phylogenetics,genomics structure,and conserved domains indicated that apple MdVQs are highly and structurally conserved.Chromosomal localization analysis showed that 46 MdVQ genes were unevenly distributed on 17 chromosomes,MdVQ19/MdVQ20,MdVQ26/MdVQ35 and MdVQ33/MdVQ36 undergone tandem duplication,while MdVQ3/MdVQ4,MdVQ7/MdVQ8,MdVQ22/MdVQ23,MdVQ27/MdVQ28/MdVQ29,MdVQ38/MdVQ44and MdVQ40/MdVQ49 undergone fragment duplication.Comprehensive qRT-PCR analysis found that various expression patterns for MdVQs in different tissues and in plant responses to water-deficit stress.2.Using Y2H,BiFC and Pull-down assays,we systematically demonstrated that MdVQ5/6/7/10/11/15/17/18/19/20/21/25/30/34/35 proteins interacte with the Group I MdWRKY4/19 and Group IIc MdWRKY52/68 proteins while none interacte with the WRKY domains of Group IIa,IIb,IId,IIe or III MdWRKY proteins;The MdVQ1/27/28/31/33/36/37/41/42/47 proteins interacte with part of Group I or Group IIc MdWRKY proteins while none interacte with the WRKY domains of other Group MdWRKY proteins;MdVQ12 and MdVQ16 proteins fail to interact with any of the tested Group I or Group IIc MdWRKY proteins.Comprehensive Y2H and BiFC analysis found that MdVQ1 interacte with MdVQ10/15/36 proteins to form a heterodimer;MdVQ10interacte with MdVQ15/19/21/33/36/41/47 proteins to form a heterodimer;MdVQ15interacte with MdVQ16/18/19/36/41 proteins to form a heterodimer while MdVQ15interacte only with itself to form a homodimer;MdVQ36 interacte with MdVQ19/25/27 to form a heterodimer.Y2H analysis showed that the interactions of MdVQ10/MdWRKY52and MdVQ15/MdWRKY52 are required for the VQ motif and WRKY domain;the C-terminus domains of MdVQ10 and MdVQ15 are critical for their physical interactions.The functional analysis indicated that overexpression of MdVQ15,MdVQ25 and MdVQ37in Arabidopsis and tobacco plants effected their vegetative and reproductive growth.Overexpression of MdVQ37 transgenic apple plants had shorter internode lengths than wild type.3.The result of qRT-PCR analysis found that MdVQ36 expression was induced by drought stress.Subcellular localization analysis showed that MdVQ36 was located in the nucleus.MdVQ36-expressing transgenic Arabidopsis lines and transgenic apple callus were obtained by genetic transformation experiments.MdVQ36-expressing transgenic Arabidopsis lines reduced tolerance to osmotic and drought stresses during the seed germination stage,the seedling stage and mature plant stage,which was related to decreased seed germination and cotyledon greening rates,root length,fresh weight,proline and chlorophyll content of the seedlings,and survival rates of mature plants,increased relative electrolyte leakage and MDA level of the seedlings,compared with'Col'wild type.Overexpression of MdVQ36 transgenic apple callus reduced tolerance to osmotic stress,manifested by lower fresh weights than wild type callus.4.qRT-PCR results found that MdVQ37 and MdWRKY56 expression were induced by drought stress.Subcellular localization analysis showed that MdVQ37 and MdWRKY56were located in the nucleus.Yeast transcriptional activation analyses indicated that MdWRKY56 had key transcriptional activation domain at the N-terminal.Y2H assay confirmed that MdVQ36 and MdVQ37 interact with MdWRKY56,and the conversed WRKY domain,the VQ motif and their flanking sequences are responsible for their physical interaction.MdVQ37 and MdWRKY56-expressing transgenic Arabidopsis lines and transgenic apple callus and MdVQ37-expressing transgenic apple plants were obtained by genetic transformation experiments.MdVQ37 and MdWRKY56-expressing transgenic Arabidopsis lines enhanced tolerance to osmotic and drought stresses during the seed germination stage,the seedling stage and mature plant stage,which was related to increased seed germination and cotyledon greening rates,root length,fresh weight,proline and chlorophyll content of the seedlings,and survival rates of mature plants,decreased relative electrolyte leakage and MDA levels of the seedlings,compared with'Col'wild type.Overexpression of MdVQ37 and MdWRKY56 transgenic apple callus conferred tolerance to osmotic stress,manifested by higher fresh weights than wild type callus.Interestingly,we found that MdVQ37 overexpression reduced tolerance to drought stress in transgenic apple lines,compared with in transgenic Arabidopsis lines.This negative response was related to decrease Pn,gs,E,WUE,Fv/Fm,SOD,POD,CAT,survival rate,proline and RWC,increase relative electrolyte leakage,H₂O₂,and O₂·-levels compared with wild type plants.Taken together,these results suggested that MdVQ36 and MdVQ37 interact with MdWRKY56 to involve in response to osmotic and drought stresses.