Identification Of Citrus Vacuolar H~+-Pyrophosphatase Genes And Their Function In The Regulation Of Sucrose And Citrate Accumulation

Citrus fruits are globally appreciated and consumed due to providing an excellent source of many health-beneficial phytochemicals.The fruit taste is an important factor of organoleptic quality and hence the marketability of citrus fruits.It is principally governed by the levels of soluble sugars(including sucrose)and organic acids(citrate),and their relative ratio.Vacuolar compartmentation has significance importance in regulating the fruit taste quality.On the vacuolar membrane,there are three distinct proton pumps,namely vacuolar-type H~+-ATPase(V-ATPase),plasma membrane-type H~+-ATPase(P-type V-ATPase)and vacuolar-type H~+-pyrophosphatase(V-PPase)that generate proton electrochemical gradients across the lumen of the vacuole to power the secondary energy-dependent transporters.Previous studies suggested that the type I V-PPase genes play a key role in plant adaptation to adverse environmental conditions and the solutes accumulation such as organic acids and anthocyanin in a given tissue.However,their function in vacuolar storage of sucrose and citrate,the main determinants of taste in citrus fruit,remains unclear.In this study,we took an integrated approach to identify,functionally characterize,and explore the role of V-PPase proton pump in the vacuolar storage of sucrose and citrate in citrus fruit through co-analysis of sugars and organic acids content,gene expression,genetic transformation,and under normal condition,drought stress or ABA treatment.On the other hand,we also investigated the possible role of V-PPase genes in the low accumulation of sucrose in‘Hong Anliu'(HAL,Citrus sinensis)orange.The main results are as following:1. Three candidate genes were identified for the citrus V-PPase,designated Cs VPP-1,Cs VPP-2,and Cs VPP-4.Subcellular localization assay revealed that citrus type I V-PPase genes(Cs VPP-1 and Cs VPP-2)are targeted to the tonoplast,whereas the citrus type II V-PPase gene(Cs VPP-4)is located in the Golgi bodies.A gene structure analysis showed that both Cs VPP-1 and Cs VPP-2 contain 8 exons and 7 introns,while Cs VPP-4contains 14 exons and 13 introns.Moreover,the tissue-specific transcript analysis revealed that type I V-PPase genes have higher transcript levels in juice sacs tissues.2. The transcript levels of Cs VPP-1 and Cs VPP-2 were highly consistent with the change of sucrose content in the fruits of‘Guoqing No.1'satsuma mandarin(GQ No.1,C.unshiu cv.Guoqing No.1)and‘Eureka'lemon(EL,C.limonia cv.Eureka).In detail,the transcript levels of Cs VPP-1 and Cs VPP-2 increased rapidly with the accumulation of sucrose in‘GQ No.1'fruit but decreased gradually with the reduction of sucrose in‘EL'fruit.Moreover,correlation analysis in another three citrus cultivars[‘Huagan No.2'ponkan(C.reticulata cv.Huagan No.2),‘Nanfeng'tangerine(C.reticulata cv.Nanfeng)and‘Newhall'navel orange(C.sinensis cv.Newhall)]showed a strong positive correlation between sucrose content and transcript level of type I V-PPase genes.However,the change in Cs VPP-1 and Cs VPP-2 transcript levels didn't display a consistently significant correlation with citrate content in these cultivars.3. When drought and ABA treatments were independently applied to‘GQ No.1'fruits,Cs VPP-1 and Cs VPP-2 transcript levels were significantly induced alongside a notable increase in sugar content.Moreover,citric acid content was significantly increased under drought stress,while there was no significant change due to ABA treatment as compared to their respective controls.4. Overexpression of type I V-PPase genes or V-PPase inhibitors treatment significantly decreased or increased pyrophosphate content and significantly increased or decreased sucrose content,respectively,in both citrus fruits and leaves,while no change occurred in citrate content.Moreover,altering the expression levels of type I V-PPase genes significantly influenced the expression levels of sucrose transporter genes.5. A spontaneous sweet orange mutant‘Hong Anliu'accumulates low soluble sugar content in fruit juice sacs than its wild type,‘Anliu'(AL)orange.In both cultivars,fructose and glucose displayed the increasing trends with significantly lower contents in‘HAL'than‘AL'after 160 DAF;moreover,sucrose had a declining trend in‘HAL'and increasing trend in‘AL'with fruit development.On the other hand,transcript levels of VINV,CWINV1,CWINV2,SUS4,SUS5,SPS1,SPS2,VPP-1,VPP-2,and some sugar transporter genes were significantly decreased in‘HAL'compared with‘AL'after 100DAF or 160 DAF.Interestingly,the transcript levels of SPS2 and SUT2 exhibited a similar trend as it was found for sucrose content in both cultivars.Taken together,these results demonstrated that Cs VPP-1 and Cs VPP-2 play key roles in sucrose storage in the vacuole,at least through regulating the pyrophosphate homeostasis and sucrose transport.Moreover,the citrus type I V-PPase genes play a role in the regulation of sugar and acid accumulation in the cell vacuole at least under drought stress conditions.By comparing a sweet orange mutant‘HAL'with its wild type‘AL',results further suggested that the reduction of Cs VPP-1 and Cs VPP-2 transcripts possibly play a key role in the low sucrose accumulation in the mutant juice sacs.