Response Of Sectretory Protein EPFL Gene From Apple To Drought Stress And Characterization Of The MdEPF2

Apple?Malus domestica?is one of the most important fruit crops grown in temperate regions,and China is the largest producer and consumer of apples.The Loess Plateau area is the largest superior production region of apple in northwestern China.Less rainfall,scarcity of water resource and lack of irrigation are becoming main limiting factors for apple cultivation in this area.An effective and targeted approach to alleviating water stress is to cultivate apple cultivars with higher water use-efficiency?WUE?and drought resistance using molecular breeding.Epidermal patterning factor like?EPFL?are secretory peptides conserved among land plants.Two founding members of this family,EPF1 and EPF2,are stomatal lineage-specific factors and they repress stomatal development in specific stages and affected plant WUE.Little is known about EPFL family in apple.We identified EPFL family members in apple using the published apple genome database.The main results of this study are documented as follows:1.Four cultivars of 'Fuji','Honeycrisp','Qinguan'and'Pacific rose'were treated with long-term moderate drought.The results showed that the WUE of'Qinguan'plants was obviously increased after drought treatment,whereas the WUE of other 3 cultivars was not significantly changed after drought stress.Drought stress had obvious impacts on growth parametres.The plant height,biomass,total leaf area?TLA?,stomatal length and stomatal aperture in four cultivars were all significantly decreased under drought stress,and root:shoot ratio?RSR?of four cultivars were obviously increased.The changes of stomatal density of four cultivars were not conformity.Stomatal density significantly increased in'Qinguan'and'Pacific rose',and did not show obvious difference in'Fuji'and'Honeycrisp'cultivars under drought stress.Under drought treatments,some parameters related to photosynthesis,such as RWC,chlorophyll contents,net photosynthetic rates,transpiration and stomatal conductance,were obviously reduced.Photosynthetic rates,chlorophyll contents and RWC reduced less severly in'Qinguan'compared to in other 3cultivars after drought treatments,whereas transpiration and stomatal conductance reduced greater than other 3 cultivars under drought stress.The injuries in four cultivars were various under drought treatment.The electrolyte leakage,malonyldialdehyde and H₂O₂contents were significantly increased after drought treatments,and the increment of these parameters was less in'Qinguan'than other 3 cultivars after drought stress.Conversely,the antioxidant enzymes,such as SOD,POD and CAT were increased higher in'Qinguan'compared to other 3cultivars.These antioxidant parameters contributed to less injuries and higher tolerance in'Qinguan'under drought treatments.We identified corresponding EPFL members in apple because the stomatal density of'Qinguan'changed remarkable under drought stress,and several EPFL members in Arabidopsis were related to stomatal patterning.The expression profiles of MdEPFLs in 4 cultivars under long-term drought showed that MdEPF1,MdEPF2 and MdEPFL2 were obviously induced by drought and peaked at 45 d.The expression level of MdEPFL9 was gradually decreased before starting rising in'Pacific rose',whereas MdEPFL9 decreased continuously in other 3 cultivars under drought stress.The expression tendency of MdEPF2 was opposite to MdEPFL9under drought stress.2.11 members of EPFL family were identified in apple genome,which are distributed on 8 chromesomes of apple.The C terminal ends of these members are conserved region,containing 6 or 8 cycsteines that forming 3 or 4 pairs of disulfide bonds.It is predicted that a conserved scaffold for activity is contained in the three dimensional structure.According motif analysis,there are two conserved motifs in the last exon of each gene.The phylogenetic tree divided the plant EPFL proteins into four clades.The Spatiotemporal expression pattern of MdEPFL genes exhibited different expression levels in different apple tissues.The expression levels of MdEPF1,MdEPF2?MdEPFL2,MdEPFL7,MdEPFL8 and MdEPFL9 were higher in leaves;and the expression level of MdEPFL6 was higher in flower buds.The expression profiles of MdEPFLs under cold,heat,natural drought and saline treatments were analyzed by q RT-PCR.The transcription of MdEPF2was up regulated about 12 folds,whereas the expression levels of MdEPFL2,MdEPFL8and MdEPFL9 was reduced significantly under natural dought stress.Responses of some of MdEPFLs to cold treatments,temperature stress and saline stress were various.These results indicated that these genes had close relationship with abiotic stresses.3.To investigate its function in drought tolerance,we ectopically expressed MdEPF2in Solanum lycopersicum cultivar'Micro-Tom'.When compared with the wild type?WT?,stomatal density was significantly lower in the leaves from the transgenics,which enabled those plants to avoid dehydration.Under drought stress,transgenic plants had higher values for relative leaf water content,chlorophyll,photosynthetic rates,and WUE than did WT.The former also had lower accumulations of reactive oxygen species and malonyldialdehyde but greater activities of antioxidant enzymes,thereby leading to less oxidative damage and higher drought tolerance.4.Transition expression of MdEPF2-eGFP in tobacco leaf showed that MdEPF2located in apoplast.Bimolecular fluorescence complement analysis indicated that MdEPF2could intereact with MdER and MdERL2,respectively.Stomatal density of apple leaves was reduced after treatment with refolded synthesized peptide m EPF2 that is the putative mature region of MdEPF2.The expression levels of Md YODA,Md MAPKK,Md MAPK3,Md MAPK6-1 and Md MAPK6-2 in MAPK signaling transduction pathway increased after m EPF2 treatment.Expression level of Md SPCH declined through m EPF2 treatment,however,there is no visible difference in the expression level of Md MUTE and Md FAMA.Under long term drought,plants with m EPF2 treatment had higher values for relative leaf water content,chlorophyll,photosynthesis rates,and WUE than did plants treated with blank.The former also had lower accumulations of reactive oxygen species,relative electrolyte leakage and malonydialdehyde but greater activities of antioxidant enzymes,thereby leading to less oxidative damage and higher drought tolerance.