Isolation And Functional Characterization Of Sugar Transporter Genes During Fruit Sugar Accumulation Of Sweet Orange

Soluble sugar, mainly including glucose, fructose and sucrose, is key component to determine the nutrition quality of sweet orange fruits. Soluble sugar in fruits is transporting from mature leaves and along the vascular bundle phloem. Sugar transporter, acting as carrier protein that located in the cell membrane or organelles membrane and mediated sugar translocation across membrane, is widely participated in phloem loading in source leaves, phloem unloading in sink tissues, sugar uptake by sink tissue cells and sugar transportation across tonoplast, and thus regulated sugar partition and accumulation. Therefore, it is of great significance to understand sugar partition and transportation in sweet orange and regulate fruit sugar accumulation by studying sugar transporter genes. In the present study, aimed to soluble sugar accumulation in sweet orange fruits, using phylogenetic analysis, gene expression patterns, subcelluar localization analysis, yeast growth complementation test, isotope and fluorescence labeled sugar uptake assay, we isolated gene families encoding sweet orange sugar transporters, identified their functions in fruit sugar accumulation and discussed related regulatory mechanism. We expected that obtaining key genes that controlled fruit sugar accumulation, and further understanding the mechanism of fruit sugar accumulation. The main results were as follows:(1) Isolation and expression profiling of sugar transporter genes in sweet orangeSeventy-seven sugar transporter genes encoding 3 SUT, 58 MST and 16 SWEET transporters were isolated in the sweet orange genome. The 3 SUTs were distributed in the SUT1, SUT2 and SUT4 clade. The 58 MST were divided into 7 subfamilies, and tandem replication occurred in the STP and ERD6 L subfamily, leading to a massive increase of family members. The STP subfamily members were responsive to ABA and low temperature treatments, reflecting the tandem replication may be related to stress defense. A large number of sugar transporter genes were expressed in leaves, flowers, fruits and calluses, indicating that they widely participated in sugar transportation in source and sink tissues. Most sugar transporter genes that expressed in orange fruits were up-regulated with fruit development, which suggested they played important roles in fruit sugar accumulation.(2) Function characterization of sweet orange SUT family and roles in high sucrose accumulation in ‘Hong Anliu’ bud mutant orange fruitsGrowth complementation assay in yeast cells demonstrated that Cs SUT1, Cs SUT2 and Cs SUT4 were all functional sucrose transporters. Using sucrose analogues fluorescent Esculin uptake assay, it proved that Cs SUT1 was a sucrose proton symporter, and with a maximum activity at p H 5.0. Using C14-labeled sucrose uptake assay, it also demonstrated that Cs SUT4 was a sucrose proton symporter and specially transported sucrose and maltose, with a maximum activity for sucrose at p H 4.0. The protonophore CCCP and plasma membrane proton pump inhibitor Vanadate inhibited Cs SUT4 activity. Subcelluar localization analysis in the rice mesophyll protoplast, it suggested that Cs SUT4 was located in the tonoplast. From the view of sucrose transportation and allocation, we explored the mechanism of high levels of sugar accumulation in the sweet orange bud mutation ‘Hong Anliu’ fruits. Comparing with its parent ‘Anliu’, the bud mutated fruits contained higher level sucrose and lower level starch. In source leaves of the bud mutation, Cs SUT1, Cs SUT2 and Cs SUT4 expression showed higher levels than in parent, which suggested that they promoted phloem loading of sucrose. Accordingly, sucrose that transported in the phloem of bud mutation was higher than in parent. In developmental fruits of the bud mutation, Cs SUT2 exhibited a higher expression level, indicating it promoted phloem unloading of sucrose in fruits. The juice sacs p H value reached to 5.8, and was obviously higher than p H 3.8 in its parent, indicating the sucrose transport activity mediated by Cs SUT4 from vacuoles to the cytosol was reduced in bud mutated fruits, and more sucrose was stored in vacuoles. The oxidative stress induced Cs SUT2 expression,which suggested that the higher expression level of Cs SUT2 in the bud mutation ‘Hong Anliu’ fruit was possiblely related to that ‘Hong Anliu’ fruit was suffering from high level oxidative stress.(3) Roles in fruit sugar accumulation and regulatory mechanism of a tonoplast-located glucose transporter Cs ERD6LGrowth complementation test in yeast cells demonstrated that a monosaccharide transporter Cs STP13 that expressed in sweet orange fruits was a glucose and fructose transporter, and mediated the apoplastic monosaccharide uptake during fruit development and ripening. We then developed a method for testing the uptake function of monosaccharide transporter by using fluorescent labeled glucose 2NBDG. Cs ERD6 L belonging to the ERD6 L subfamily was isolated. Cs ERD6 L showed the highest expression in flowers, and weakly expressed in leaves. Subcelluar localization analysis in tobacco leaves demonstrated Cs ERD6 L was located in the tonoplast. By over-expressing in the glucose uptake deficient yeast strain EBY.VW4000, Cs ERD6 L could not rescue the yeast growth. Using 2NBDG uptake assay, it demonstrated that Cs ERD6 L omitting the potential tonoplast-located signal sequence was a functional glucose transporter, and the transport process did not depend on the p H value. Over-expression of Cs ERD6 L in yeast cells reduced glucose level, indicating that Cs ERD6 L promoted vacuolar glucose efflux to the cytosol. Cs ERD6 L, together with vacuole acid invertase gene Csb Fruct1 that catalyzed vacuolar sucrose into glucose and fructose, were upregulated during fruit development, suggesting that they coordinately transported the generated glucose from vacuoles into the cytosol during fruit development. Using fruit treatments, reporter gene expression driven by Cs ERD6 L promoter and microarray data retrieval, it demonstrated that ABA, oxidative stress and sugar starvation induced Cs ERD6 L expression. Using reporter gene assay driven by Cs ERD6 L promoter and transiently expressed in tobacco leaves, the Cs ERD6 L expression regulated by transcription factors was studied. An ASR transcription factor Cs ASR and MYB transcription factor Cs8g12680 did not activate the expression of reporter gene, indicating that they did not regulate Cs ERD6 L expression. Cs ERD6 L and Csb Fruct1-2 showed higher expression levels in the bud mutation ‘Hong Anliu’ fruits, which indicted that mass sucrose that accumulated in the vacuole was catalyzed into monosaccharide and transported into the cytosol for stress defense under the higher level oxidative stress.In summary, we isolated gene families encoding sugar transporters from whole genome-wide level, confirmed the functions of key candidate genes controlling fruit sugar accumulation, and discussed their regulatory mechanism. From the view of sugar transportation, we discussed the roles of orange SUT family in high sucrose content of the bud mutation ‘Hong Anliu’ fruit. For the first time in perennial woody fruit crops, we isolated and identified a tonoplast-located monosaccharide transporter Cs ERD6 L that mediated the transportation of vacuolar glucose into the cytosol during fruit development and stress conditions. This study would provide important basis for further understanding fruit sugar accumulation process of sweet orange and improving fruit quality.