| Apple(Malus domestica Borkh.)is a major horticultural crop worldwide.The apple industry is an important way to increase growers’ income and improve agricultural efficiency.‘Hanfu’ apples have strong cold resistance,large fruit size,and good quality,which are widely grown in cold regions of northern China.Phloem unloading is the determinant of crop yield and quality.The phloem unloading of apple fruit assimilates is an apoplastic unloading pathway,in which sugar transporters play important roles.In this study,27 SWEETs were identified from the apple genome,and then the expression patterns of these 27 SWEETs in different tissues/organs of ‘Hanfu’ apples were analyzed.The correlation between the expression levels of MdSWEETs and sugar accumulation during fruit development and maturity was also analyzed.Based on these research results,two SWEET family members,MdSWEET23 from the clade III and MdSWEET4 from the clade I,which are significantly positively correlated with the sugar content of ‘Hanfu’ fruit,were identified and the further functional analysis of the two genes were carried out to reveal roles of them in sugar accumulation in ’Hanfu’ apple fruit.The main results are as follows:1.In this study,27 MdSWEETs were identified from the apple genome and their expression patterns in different tissues/organs of the ’Hanfu’ apples were analyzed using quantitative real-time PCR(q RT-PCR).The results showed that MdSWEET5,7,and 21 highly expressed in mature leaves,MdSWEET4 highly expressed in young leaves,MdSWEET2,12,13,14,17,and 22 highly expressed in peduncles,MdSWEET3,11,15 16,19,24,and 25 highly expressed in flowers,and MdSWEET1,6,8,9,10,18,20,23,and 26 highly expressed in fruits.Correlation analysis showed that the expression levels of MdSWEET4,6,9,20,23,and 26 were significant positive correlated with sugar accumulation during fruit development and maturation.2.The MdSWEET23 gene(clade III)was cloned from fruits of ‘Hanfu’ apples.The expression level of MdSWEET23 was extremely positively correlated with fruit sugar accumulation of ’Hanfu’ apples(0.897).Yeast functional complementation and subcellular localization showed that MdSWEET23 is a plasma membrane-localized sucrose transporter.q RT-PCR analysis and GUS activity assays showed that MdSWEET23 was mainly expressed in the regions of sepal vascular bundle and carpel vascular bundle of fruits.Transiently silence of MdSWEET23 in apple fruits of rapid sugar accumulation period by the virus-induced gene silencing(VIGS)technique significantly reduced the contents of sucrose and sorbitol in fruits.Overexpression of MdSWEET23 in ’Orin’ apple callus significantly increased the soluble sugar content.Heterologous overexpression of MdSWEET23 in tomato increased the sucrose content in mature green fruits of tomato and the contents of sucrose,fructose,and glucose in breaking color fruits.In addition,overexpression of MdSWEET23 in tomato significantly increased the sugar and starch content in leaves,resulting in the decrease of photosynthesis and plant dwarfing,and enhanced cold stress tolerance.3.The MdSWEET4 gene(clade I)was cloned from ’Hanfu’ apple fruits.The expression level of MdSWEET23 was extremely positively correlated with fruit sugar accumulation of’Hanfu’ apples(0.612).Yeast functional complementation showed that MdSWEET4 can transport glucose,and subcellular localization showed that MdSWEET4 was localized on the plasma membrane.q RT-PCR analysis showed that MdSWEET4 was mainly expressed in the regions of fleshy paremchyma cell.GUS activity assays showed that MdSWEET4 was mainly expressed in the vascular bundle regions of fruits.Transient silencing of MdSWEET4 using the VIGS significantly reduced the contents of glucose and fructose in ’Hanfu’ apple fruits,while significantly increasing the sucrose content.Heterologous overexpression of MdSWEET4 in tomato significantly increased the sugar content in red ripe fruit,especially the contents of glucose and fructose.Tomato plants overexpressing MdSWEET4 had shortened seed germination time,earlier flowering and fruit coloring.4.The results of protein interaction prediction by ZDOCK and GRAMM and yeast two-hybrid assay indicated that MdSWEET23 interacts with MdSWEET4.The results demonstrate that MdSWEET23 and MdSWEET4 have significant roles in the accumulation of sugar in ’Hanfu’ apple fruits.Specifically,MdSWEET23 functions as a sucrose transporter,while MdSWEET4 functions as a glucose transporter.Furthermore,an interaction exists between MdSWEET23 and MdSWEET4. |
