Transcriptional Regulation Mechanism Of Citrus SWEET11d On Fruit Sucrose Accumulation

Sucrose is the primary product of photosynthesis and the main carbon form of carbohydrate transportation.For fruit,sucrose contributes to sweetness and favor and is crucial for fruit quality.Sucrose produced in photosynthetic leaves enters fruit tissue through phloem loading,long-distance transportation and phloem unloading,which requires multiple transporters involved.As a novel sugar transporter family,SWEETs have been reported to be widely implicated in plant growth and development process.However,the functions of SWEETs in citrus fruit are largely unknown,understanding the physiological role of SWEET genes during citrus fruit development and ripening is of great significance for improving fruit quality.In the present study,Satsuma mandarin fruits are used to investigate the functions of SWEET genes and transcriptional regulation.The main results are as follows:1.A total of 18 citrus SWEET members with Mt N3_-slv domain were identified by BLASTP search using the protein sequences of Arabidopsis and rice SWEETs in citrus genome database and NCBI database.According to the phylogenetic tree analysis,SWEET genes in citrus could be clustered into four subfamilies,the numbers of members of which are 6,3,4 and 5,respectively.The exon/intron distribution result displayed that most SWEET genes in citrus had five introns and a few SWEETs have lost introns.There existed seven different conserved motifs in the protein structure of Cit SWEETs,which is possibly related to the formation of protein trans-membrane structures.The expression patterns of SWEET gene family in citrus displayed tissue-specific.All SWEET genes were expressed in floral organ,and 7genes even had higher expression levels in flowers.During fruit development,most SWEET genes had very low expression levels,and Cit SWEET2b,Cit SWEET5,Cit SWEET11c and Cit SWEET11e were not expressed in fruit,the expression abundance of Cit SWEET1a,Cit SWEET3,Cit SWEET7,Cit SWEET9 and Cit SWEET11a gradually decreased during fruit development and ripening,in contrast,the transcripts of Cit SWEET11d gradually increased and reached a peak at later stage.2.Based on the correlation between the transcript level of Cit SWEET11d and sucrose content during fruit development,a significant positive correlation was found between Cit SWEET11d transcripts and sucrose content,which was further verified in different citrus varieties fruits.The subcellular localization assay showed that Cit SWEET11d located at the tonoplast.And overexpression of Cit SWEET11d in citrus callus and tomato fruit significantly increased sucrose content.3.Two transcription factors Cit ERF16 and Citb ZIP1 were obtained by yeast one-hybrid library screening using the promoter of Cit SWEET11d as bait,and yeast one-hybrid assay further demonstrated that both Cit ERF16 and Citb ZIP1 could directly bind to the promoter of Cit SWEET11d.Dual-luciferase experiments showed that Citb ZIP1 had no significant regulatory effect on the promoter of Cit SWEET11d,while Cit ERF16 could significantly activate the promoter of Cit SWEET11d,and the regulatory effect was about 10-fold.EMSA analysis and binding element mutation experiments showed that Cit ERF16 regulated Cit SWEET11d by directly binding to the DRE(GTCGGT)element in the promoter of Cit SWEET11d.The expression pattern of Cit ERF16 during fruit development was similar with that of Cit SWEET11d,and positively correlated with sucrose content,which has been confirmed in different citrus varieties fruits.Moreover,overexpression of Cit ERF16 in citrus callus significantly induced Cit SWEET11d transcripts and elevated sucrose content.Collectively,Cit ERF16 could promote sucrose accumulation by regulating the expression of Cit SWEET11d.