Vacuolar proteomic analysis reveals tonoplast transporters for accumulation of citric acid and sugar in citrus fruit

Abstract

Vacuole largely dictates the fruit taste and flavor, as most of the sugars and organic acids are stored in the vacuoles of the fruit. However, difficulties associated with vacuole separation severely hinder identification and characterization of vacuolar proteins in fruit species. In this study, we established an effective approach for separating vacuoles and successfully purified vacuolar protein from six types of citrus fruit with varying patterns of sugar and organic acid contents. By using label-free LC-MS/MS proteomic analysis, 1443 core proteins were found to be associated with the essential functions of vacuole in citrus fruit. Correlation analysis of metabolite concentration with proteomic data revealed a transporter system for the accumulation of organic acid and soluble sugars in citrus. Furthermore, we characterized the physiological roles of selected key tonoplast transporters, ABCG15, Dict2.1, TMT2, and STP7 in the accumulation of citric acid and sugars. These findings provide a novel perspective and practical solution for investigating the transporters underlying the formation of citrus taste and flavor.

Acknowledgements

We thank our colleague Prof. Pengwei Wang at Huazhong Agricul-tural University for providing tonoplast marker vector of AtTIP-mCherry.

This work was supported by National Key Research and Development Program of China (2022YFF1003100), the National Natural Science Foundation of China (32322073), Foundation of Hubei Hongshan Laboratory (2021hszd016), Fundamental Research Funds for the Central Universities (2662023PY011), and the Huazhong Agricultural University (start-up funding to C. L.).

Author contributions

C.L., J.-H.L., and Q. X. conceived and designed the research. Z.M. and Y. W. performed the experiments and analysed the data, M.L., S.Z., and Z.Z. assisted the experiments. Z.M. wrote the manuscript draft. C.L. and J.-H.L. finalized writing and revision of the manuscript.

Conflict of interest statement

The authors declare no conflict of interest.

Data availability

All data is available within manuscript and its supporting materials.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Butelli E, Licciardello C, Ramadugu C. et al. Noemi controls pro-duction of flavonoid pigments and fruit acidity and illustrates the domestication routes of modern citrus varieties. Curr Biol. 2019;29:158-164.e2

[2] Braidy N, Behzad S, Habtemariam S. et al. Neuroprotective effects of citrus fruit-derived flavonoids, nobiletin and tan-geretin in Alzheimer’s and Parkinson’s disease. CNS Neurol Disord Drug Targets. 2017;16:387-97

[3] Hwang SL, Shih PH, Yen GC. Neuroprotective effects of citrus flavonoids. J Agric Food Chem. 2012;60:877-85

[4] Wang L, Huang Y, Liu ZA. et al. Somatic variations led to the selection of acidic and acidless orange cultivars. Nat Plants. 2021;7:954-65

[5] Strazzer P, Spelt CE, Li S. et al. Hyperacidification of citrus fruits by a vacuolar proton-pumping P-ATPase complex. Nat Commun. 2019;10:744

[6] Wu GA, Terol J, Ibanez V. et al. Genomics of the origin and evolution of citrus. Nature. 2018;554:311-6

[7] Rouseff RL, Ruiz Perez-Cacho P, Jabalpurwala F. Historical review of citrus flavor research during the past 100 years. J Agric Food Chem. 2009;57:8115-24

[8] Ziyan Liu ZM, Li M, Cai C. et al. Vacuole: a repository to control fruit flavor quality. Fruit Research 3:12

[9] de Brito Francisco R, Martinoia E. The vacuolar transportome of plant specialized metabolites. Plant Cell Physiol. 2018;59:1326-36

[10] Ohnishi M, Anegawa A, Sugiyama Y. et al. Molecular components of Arabidopsis intact vacuoles clarified with metabolomic and proteomic analyses. Plant Cell Physiol. 2018;59:1353-62

[11] Furtauer L, Kustner L, Weckwerth W. et al. Resolving subcellular plant metabolism. Plant J. 2019;100:438-55

[12] Li SJ, Liu SC, Lin XH. et al. Citrus heat shock transcription factor CitHsfA7-mediated citric acid degradation in response to heat stress. Plant Cell Environ. 2022;45:95-104

[13] Ma M, Wang LB, Zhang SL. et al. Acid vacuolar invertase 1 (PbrAc-Inv1) and invertase inhibitor 5 (PbrII5) were involved in sucrose hydrolysis during postharvest pear storage. Food Chem. 2020;320:126635

[14] Wang Z, Ma B, Yang N. et al. Variation in the promoter of the sorbitol dehydrogenase gene MdSDH2 affects binding of the transcription factor MdABI3 and alters fructose content in apple fruit. Plant J. 2022;109:1183-98

[15] Feng GZ, Wu JX, Xu YH. et al. High-spatiotemporal-resolution transcriptomes provide insights into fruit development and ripening in Citrus sinensis. Plant Biotechnol J. 2021;19:1337-53

[16] Hussain SB, Shi CY, Guo LX. et al. Type I H+-pyrophosphatase regulates the vacuolar storage of sucrose in citrus fruit. JExp Bot. 2020;71:5935-47

[17] Shi CY, Hussain SB, Yang H. et al. CsPH8, a P-type proton pump gene, plays a key role in the diversity of citric acid accumulation in citrus fruits. Plant Sci. 2019;289:110288

[18] Etxeberria E, Pozueta-Romero J, Gonzalez P. In and out of the plant storage vacuole. Plant Sci. 2012;190:52-61

[19] Martinoia E. Vacuolar transporters - companions on a longtime journey. Plant Physiol. 2018;176:1384-407

[20] Davies KL, Stpiczynska M, Gregg A. Nectar-secreting floral stom-ata in Maxillaria anceps Ames & C. Schweinf. (Orchidaceae). Ann Bot-London. 2005;96:217-27

[21] Norikoshi R, Yamada K, Niki T. et al. Accumulation of mannitol in the cytoplasm and vacuole during the expansion of sepal cells associated with flower opening in delphinium x belladonna cv. Bellamosum. Planta. 2015;242:1467-77

[22] Daloso DM, Medeiros DB, dos Anjos L. et al. Metabolism within the specialized guard cells of plants. New Phytol. 2017;216: 1018-33

[23] Jung B, Ludewig F, Schulz A. et al. Identification of the transporter responsible for sucrose accumulation in sugar beet taproots. Nat Plants. 2015;1:14001

[24] Maeshima M. Tonoplast transporters: organization and func-tion. Annu Rev Plant Physiol. 2001;52:469-97

[25] Schulz A, Beyhl D, Marten I. et al. Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2. Plant J. 2011;68:129-36

[26] Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 2008;54:733-49

[27] Faraco M, Spelt C, Bliek M. et al. Hyperacidification of vacuoles by the combined action of two different P-ATPases in the tonoplast determines flower color. Cell Rep. 2014;6:32-43

[28] Gao M, Zhao H, Zheng L. et al. Overexpression of apple Ma12, a mitochondrial pyrophosphatase pump gene, leads to malic acid accumulation and the upregulation of malate dehydrogenase in tomato and apple calli. Hortic Res. 2022;9:uhab053

[29] Hedrich R, Sauer N, Neuhaus HE. Sugar transport across the plant vacuolar membrane: nature and regulation of carrier proteins. Curr Opin Plant Biol. 2015;25:63-70

[30] Shi CY, Hussain SB, Han H. et al. Reduced expression of CsPH8, a P-type ATPase gene, is the major factor leading to the low cit-rate accumulation in citrus leaves. Plant Physiol Bioch. 2021;160: 211-7

[31] Zhu LC, Li B, Wu L. et al. MdERDL6-mediated glucose efflux to the cytosol promotes sugar accumulation in the vacuole through up-regulating TSTs in apple and tomato. PNAS Nexus. 2021;118:e2022788

[32] De Angeli A, Baetz U, Francisco R. et al. The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera. Planta. 2013;238:283-91

[33] Li CL, Dougherty L, Coluccio AE. et al. Apple ALMT9 requires a conserved C-terminal domain for malate transport underlying fruit acidity. Plant Physiol. 2020;182:992-1006

[34] Shimada T, Nakano R, Shulaev V. et al. Vacuolar citrate/H+ symporter of citrus juice cells. Planta. 2006;224:472-80

[35] Ren Y, Guo S, Zhang J. et al. A tonoplast sugar transporter under-lies a sugar accumulation QTL in watermelon. Plant Physiol. 2018;176:836-50

[36] Peng Q, Wang L, Ogutu C. et al. Functional analysis reveals the regulatory role of PpTST1 encoding tonoplast sugar transporter in sugar accumulation of peach fruit. Int J Mol Sci. 2020;21:1112

[37] Leach KA, Tran TM, Slewinski TL. et al. Sucrose transporter2 contributes to maize growth, development, and crop yield. J Integr Plant Biol. 2017;59:390-408

[38] Martinoia E, Maeshima M, Neuhaus HE. Vacuolar transporters and their essential role in plant metabolism. JExp Bot. 2006;58: 83-102

[39] Ma BQ, Liao L, Fang T. et al. A Ma10 gene encoding P-type ATPase is involved in fruit organic acid accumulation in apple. Plant Biotechnol J. 2019;17:674-86

[40] Martinoia E, Meyer S, De Angeli A. et al. Vacuolar transporters in their physiological context. Annu Rev Plant Biol. 2012;63:183-213

[41] Albertini MV, Carcouet E, Pailly O. et al. Changes in organic acids and sugars during early stages of development of acidic and acidless citrus fruit. J Agric Food Chem. 2006;54:8335-9

[42] Mahmood T, Khalid S, Abdullah M. et al. Insights into drought stress signaling in plants and the molecular genetic basis of cotton drought tolerance. Cell. 2020;9:105

[43] Urrutia M, Blein-Nicolas M, Prigent S. et al. Maize metabolome and proteome responses to controlled cold stress partly mimic early-sowing effects in the field and differ from those of Ara-bidopsis. Plant Cell Environ. 2021;44:1504-21

[44] Li JM, Huang XS, Li LT. et al. Proteome analysis of pear reveals key genes associated with fruit development and quality. Planta. 2015;241:1363-79

[45] Li MJ, Li D, Feng F. et al. Proteomic analysis reveals dynamic reg-ulation of fruit development and sugar and acid accumulation in apple. JExp Bot. 2016;67:5145-57

[46] Lv JH, Zheng T, Song Z. et al. Strawberry proteome responses to controlled hot and cold stress partly mimic post-harvest storage temperature effects on fruit quality. Front Nutr. 2022;8:812666

[47] Carter C, Pan S, Zouhar J. et al. The vegetative vacuole proteorne of Arabidopsis thaliana reveals predicted and unexpected pro-teins. Plant Cell. 2004;16:3285-303

[48] Schulze WX, Schneider T, Starck S. et al. Cold acclimation induces changes in Arabidopsis tonoplast protein abundance and activity and alters phosphorylation of tonoplast monosac-charide transporters. Plant J. 2012;69:529-41

[49] Endler A, Meyer S, Schelbert S. et al. Identification of a vac-uolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach. Plant Physiol. 2006;141: 196-207

[50] Schmidt UG, Endler A, Schelbert S. et al. Novel tonoplast trans-porters identified using a proteomic approach with vacuoles isolated from cauliflower buds. Plant Physiol. 2007;145:216-29

[51] Kuang LQ, Chen S, Guo Y. et al. Proteome analysis of vacuoles isolated from fig (Ficus carica L.) flesh during fruit development. Plant Cell Physiol. 2022;63:785-801

[52] Kuang L, Chen S, Guo Y. et al. Quantitative proteome analysis reveals changes in the protein landscape during grape berry development with a focus on vacuolar transport proteins. Front Plant Sci. 2019;10:641

[53] Sadka A, Shlizerman L, Kamara I. et al. Primary metabolism in citrus fruit as affected by its unique structure. Front Plant Sci. 2019;10:1167

[54] Bain JM. Morphological, anatomical, and physiological changes in the developing fruit of the Valencia orange, Citrus sinensis (L) Osbeck. J Appl Anim Res. 1958;40:154-62

[55] Guo LX, Shi CY, Liu X. et al. Citrate accumulation-related gene expression and/or enzyme activity analysis combined with metabolomics provide a novel insight for an orange mutant. Sci Rep. 2016;6:29343

[56] Wang J, Yu X, Ding ZJ. et al. Structural basis of ALMT1-mediated aluminum resistance in Arabidopsis. Cell Res. 2022;32: 89-98

[57] Wen SY, Neuhaus HE, Cheng JT. et al. Contributions of sugar transporters to crop yield and fruit quality. JExp Bot. 2022;73: 2275-89

[58] Chen LQ, Cheung LS, Feng L. et al. Transport of sugars. Annu Rev Biochem. 2015;84:865-94

[59] Yang B, Wang J, Yu M. et al. The sugar transporter ZmSUGCAR1 of the nitrate transporter 1/peptide transporter family is critical for maize grain filling. Plant Cell. 2022;34:4232-54

[60] Li YB, Provenzano S, Bliek M. et al. Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification. New Phy-tol. 2016;211:1092-107

[61] Moskowitz AH, Hrazdina G. Vacuolar contents of fruit subepi-dermal cells from vitis species. Plant Physiol. 1981;68:686-92

[62] Kamireddy K, Sonbarse PP, Mishra SK. et al. Proteomic approach to identify the differentially abundant proteins during flavour development in tuberous roots of Decalepis hamiltonii Wight & Arn. 3Biotech. 2021;11:173

[63] Shimada T, Takagi J, Ichino T. et al. Plant vacuoles. Annu Rev Plant Biol. 2018;69:123-45

[64] Zhu L, Li Y, Wang C. et al. The SnRK2.3-AREB1-TST1/ 2 cas-cade activated by cytosolic glucose regulates sugar accumula-tion across tonoplasts in apple and tomato. Nat Plants. 2023;9: 951-64

[65] Huai BY, Yuan P, Ma X. et al. Sugar transporter TaSTP3 activa-tion by TaWRKY19/61/82 enhances stripe rust susceptibility in wheat. New Phytol. 2022;236:266-82

[66] Buttner M. The Arabidopsis sugar transporter (AtSTP) family: an update. Plant Biol. 2010;12:35-41

[67] Lee CP, Elsässer M, Fuchs P. et al. The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate-citrate exchange. Plant Cell. 2021;33:3700-20

[68] Etienne A, Genard M, Lobit P. et al. What controls fleshy fruit acidity? A review of malate and citrate accumulation in fruit cells. JExp Bot. 2013;64:1451-69

[69] Ratajczak R, Luttge U, Gonzalez P. et al. Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent cit-rate transport across the tonoplast of citrus juice cells. JPlant Physiol. 2003;160:1313-7

[70] Kim J, Cater RJ, Choy BC. et al. Structural insights into transporter-mediated drug resistance in infectious diseases. J Mol Biol. 2021;433:167005

[71] Lee M, Choi Y, Burla B. et al. The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2. Nat Cell Biol. 2008;10:1217-23

[72] Pizzio GA, Paez-Valencia J, Khadilkar AS. et al. Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning. Plant Physiol. 2015;167:1541-53

[73] Gaxiola RA, Palmgren MG, Schumacher K. Plant proton pumps. FEBS Lett. 2007;581:2204-14

[74] Muller ML, Taiz L. Regulation of the lemon-fruit V-ATPase by variable stoichiometry and organic acids. J Membrane Biol. 2002;185:209-20

[75] Li C, Meng D, Piñeros MA. et al. A sugar transporter takes up both hexose and sucrose for sorbitol-modulated in vitro pollen tube growth in apple. Plant Cell. 2020;32:449-69

[76] Gillette MA, Satpathy S, Cao S. et al. Proteogenomic characteriza-tion reveals therapeutic vulnerabilities in lung adenocarcinoma. Cell. 2020;182:200-225.e35

[77] Kachuk C, Stephen K, Doucette A. Comparison of sodium dode-cyl sulfate depletion techniques for proteome analysis by mass spectrometry. JChromatogrA. 2015;1418:158-66

[78] Wisniewski JR, Zougman A, Nagaraj N. et al. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6: 359-62

[79] Jaquinod M, Villiers F, Kieffer-Jaquinod S. et al. A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture. Mol Cell Proteomics. 2007;6:394-412

[80] Tian T, Liu Y, Yan H. et al. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res. 2017;45:W122-9

[81] Mao X, Cai T, Olyarchuk JG. et al. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics. 2005;21:3787-93

[82] Chou KC, Shen HB. Cell-PLoc: a package of web servers for pre-dicting subcellular localization of proteins in various organisms. Nat Protoc. 2008;3:153-62

[83] Chen T, Zhang H, Liu Y. et al. EVenn: Easy to create repeatable and editable Venn diagrams and Venn networks online. J Genet Genomics. 2021;48:863-6

[84] Nelson BK, Cai X, Nebenfuhr A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007;51:1126-36

[85] Sparkes IA, Runions J, Kearns A. et al. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2006;1:2019-25

[86] Ku YS. How sweet! Transcription factor CitZAT5 regulates Cit-SUS5 and CitSWEET6 to promote sugar accumulation in citrus fruit. Plant Physiol. 2023;192:1669-70