Integrated analysis of metabolites and enzyme activities reveals the plasticity of central carbon metabolism in grape (Vitis vinifera cv. Cabernet Sauvignon) berries under carbon limitation

Abstract

High temperatures increase the sugar concentration of grape (Vitis vinifera L.) berries, which can negatively affect the composition and quality of wine, and global climate change is expected to exacerbate this problem. Modifying the source-to-sink ratio of grapevines by selective pruning is a potential strategy to mitigate this. To investigate the effects of low source-to-sink ratio (retaining three leaves per cluster) on carbon metabolism of grape (cv. Cabernet Sauvignon) berries, we conducted an analysis of 42 metabolites and 21 enzyme activities at nine berry developmental stages，as well as transcriptomes from berries grown under two leaves per cluster. The results revealed that the metabolic pathways were coordinately regulated to maintain homeostasis under low source-to-sink ratio conditions. Because of a delay between metabolites and enzyme activities, the metabolites were loosely correlated with enzyme activities, and a lower density of connectivity between them appeared in low source-to-sink conditions. Otherwise, transcripts of the carbohydrate and amino acid metabolism pathways were enriched by carbon limitation. In summary, this integrated analysis reveals a coordinated regulation of various metabolic pathways that maintains the balance of carbon metabolism and ensures survival in challenging environments, highlighting the high metabolic plasticity of grape berries.

Acknowledgements

This research was supported partly by National Natural Science Foundation of China (grant 32072519), the National Key R&D Program of China (2021YFE0109500), National Natural Science Foundation of China (U20A2041), the Frimouss (ANR-15-CE20-0009), Agricultural Breeding Project of Ningxia Hui Autonomous Region (NXNYYZ202101), CAS Youth Interdisciplinary Team (JCTD-2022-06), and CAS Project for Young Scientists in Basic Research (YSBR-093). Research conducted as part of the LIA INNOGRAPE International Associated Laboratory. We thank Christel Renaud (INRAE, France) for technical assistance during this work.

Author contributions

Conceptualization: Z.D.; methodology: Q.T., G.H., E.G., and Y.G.; software: Q.T., X.X., and Y.W.; validation: Q.T. and Z.D.; formal analysis: Q.T., X.X., Y.W., and J.K.; investigation: Q.T., Z.D., and Z.L.; resources: G.H., S.D., E.G., Y.G., and J.C.; writing—original draft preparation: Q.T.; writing—review and editing: Z.D., R.F., J.L., G.H., S.D., B.B., E.G., and Y.G.; All authors have read and agreed to the published version of the manuscript.

Data availability

All data supporting the findings of this study are available within the paper and within its supplementary materials. The RNA-seq data can be found in National Genomics Data Center under accession number CRA007714.

Conflict of interest statement

There is no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Carrari F, Baxter C, Usadel B. et al. Integrated analysis of metabo-lite and transcript levels reveals the metabolic shifts that under-lie tomato fruit development and highlight regulatory aspects of metabolic network behavior. Plant Physiol. 2006; 142:1380-96

[2] Dai ZW, Léon C, Feil R. et al. Metabolic profiling reveals coor-dinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit. JExp Bot. 2013; 64:1345-55

[3] Nardozza S, Boldingh HL, Kashuba MP. et al. Carbon starvation reduces carbohydrate and anthocyanin accumulation in red-fleshed fruit via trehalose 6-phosphate and MYB27. Plant Cell Environ. 2020; 43:819-35

[4] Centeno DC, Osorio S, Nunes-Nesi A. et al. Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening. Plant Cell. 2011; 23:162-84

[5] Jia HF, Wang Y, Sun M. et al. Sucrose functions as a signal involved in the regulation of strawberry fruit development and ripening. New Phytol. 2013; 198:453-65

[6] Monti LL, Bustamante CA, Budde CO. et al. Metabolomic and proteomic profiling of spring lady peach fruit with contrasting woolliness phenotype reveals carbon oxidative processes and proteome reconfiguration in chilling-injured fruit. Postharvest Biol Technol. 2019; 151:142-51

[7] Ali K, Maltese F, Fortes AM. et al. Monitoring biochemical changes during grape berry development in Portugese cultivars by NMR spectroscopy. Food Chem. 2011; 124:1760-9

[8] Kumari S, Bakshi P, Sharma A. et al. Use of plant growth regu-lators for improving fruit production in sub tropical crops. Int J Curr Microbiol App Sci. 2018; 7:659-68

[9] Fischer G, Almanza-Merchan PJ, Ramirez F. Source-sink relation-ships in fruit species. A review. Rev Colomb Cienc Hortic. 2012; 6: 238-53

[10] Pawar R, Rana V. Manipulation of source-sink relationship in pertinence to better fruit quality and yield in fruit crops: a review. Agric Rev. 2019; 40:200-7

[11] Feng H, Skinkis PA, Qian MC. Pinot noir wine volatile and antho-cyanin composition under different levels of vine fruit zone leaf removal. Food Chem. 2017; 214:736-44

[12] Diago MP, Ayestaran B, Guadalupe Z. et al. Impact of prebloom and fruit set basal leaf removal on the flavonol and anthocyanin composition of tempranillo grapes. Am J Enol Vitic. 2012; 63: 367-76

[13] Bairam E leMorvan C, Delaire M. et al. Fruit and leaf response to different source-sink ratios in apple, at the scale of the fruit-bearing branch. Forntiers in Plant Science. 2019; 10:1039-52

[14] Anthony BM, Chaparro JM, Prenni JE. et al. Early metabolic prim-ing under differing carbon sufficiency conditions influences peach fruit quality development. Plant Physiol Biochem. 2020; 157: 416-31

[15] Fukushima A, Hikosaka S, Kobayashi M. et al. A systems analysis with “simplified source-sink model” reveals metabolic repro-gramming in a pair of source-to-sink organs during early fruit development in tomato by LED light treatments. Front Plant Sci. 2018; 9:01439

[16] Aslani L, Gholami M, Mobli M. et al. Decreased sink/source ratio enhances hexose transport in the fruits of greenhouse tomatoes: integration of gene expression and biochemical analyses. Physiol Plant. 2020; 170:120-31

[17] Ulker T, Kamiloglu MU. Influences of girdling and potassium treatments on fruit quality and some physiological characters of ’Fremont’ mandarin variety. Folia Hortic. 2021; 33:195-202

[18] Bobeica N, Poni S, Hilbert G. et al. Differential responses of sugar, organic acids and anthocyanins to source-sink modulation in Cabernet Sauvignon and Sangiovese grapevines. Front Plant Sci. 2015; 6:00382

[19] Kubo T, Hohjo I, Hiratsuka S. Sucrose accumulation and its related enzyme activities in the juice sacs of Satsuma mandarin fruit from trees with different crop loads. Sci Hortic. 2001; 91: 215-25

[20] Do PT, Prudent M, Sulpice R. et al. The influence of fruit load on the tomato pericarp metabolome in a Solanum chmielewskii introgression line population. Plant Physiol. 2010; 154:1128-42

[21] Wang LN, Brouard E, Hilbert G. et al. Differential response of the accumulation of primary and secondary metabolites to leaf-to-fruit ratio and exogenous abscisic acid. Aust J Grape Wine Res. 2021; 27:527-39

[22] Wang LN, Brouard E, Prodhomme D. et al. Regulation of antho-cyanin and sugar accumulation in grape berry through car-bon limitation and exogenous ABA application. Food Res Int. 2022; 160:111478

[23] Fendt SM, Buescher JM, Rudroff F. et al. Tradeoff between enzyme and metabolite efficiency maintains metabolic homeostasis upon perturbations in enzyme capacity. Mol Syst Biol. 2010; 6: 356-66

[24] Wu Q, Ma X, Chen Q. et al. Dynamics of energy metabolism in carbon starvation-induced fruitlet abscission in litchi. Horticul-turae. 2021; 7:576-88

[25] Biais B, Bénard C, Beauvoit B. et al. Remarkable reproducibility of enzyme activity profiles in tomato fruits grown under con-trasting environments provides a roadmap for studies of fruit metabolism. Plant Physiol. 2014; 164:1204-21

[26] Tohge T, Scossa F, Fernie AR. Integrative approaches to enhance understanding of plant metabolic pathway structure and regu-lation. Plant Physiol. 2015; 169:1499-511

[27] Zhang WF, Gong Z-H, Wu M-B. et al. Integrative comparative analyses of metabolite and transcript profiles uncovers com-plex regulatory network in tomato (Solanum lycopersicum L.) fruit undergoing chilling injury. Sci Rep. 2019; 9:4470-82

[28] Sulpice R, Trenkamp S, Steinfath M. et al. Network analysis of enzyme activities and metabolite levels and their relationship to biomass in a large panel of Arabidopsis accessions. Plant Cell. 2010; 22:2872-93

[29] Steinhauser MC, Steinhauser D, Koehl K. et al. Enzyme activ-ity profiles during fruit development in tomato cultivars and Solanum pennellii. Plant Physiol. 2010; 153:80-98

[30] Li Q, Chai L, Tong N. et al. Potential carbohydrate regulation mechanism underlying starvation-induced abscission of tomato flower. Int J Mol Sci. 2022; 23:1952-65

[31] Belouah I, Nazaret C, Pétriacq P. et al. Modeling protein destiny in developing fruit. Plant Physiol. 2019; 180:1709-24

[32] Conde C, Silva P, Fontes N. et al. Biochemical changes throughout grape berry development and fruit and wine quality. Food. 2007; 1: 1-22

[33] Rienth M, Torregrosa L, Sarah G. et al. Temperature desynchro-nizes sugar and organic acid metabolism in ripening grapevine fruits and remodels their transcriptome. BMC Plant Biol. 2016; 16: 164-86

[34] Duchene E, Schneider C. Grapevine and climatic changes: a glance at the situation in Alsace. Agron Sustain Dev. 2005; 25:93-9

[35] Sweetman C, Sadras VO, Hancock RD. et al. Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit. JExp Bot. 2014; 65:5975-88

[36] Mack AL. Did fleshy fruit pulp evolve as a defence against seed loss rather than as a dispersal mechanism? J Biosci. 2000; 25:93-7

[37] Omer N, Razafimandimby D, Valenta K. et al. Signal and reward in wild fleshy fruits: does fruit scent predict nutrient content? Ecol Evol. 2019; 9:10534-43

[38] Ncama K, Aremu OA. Sithole NJ. In: GalanakisCM, Chilling, Heat.ed. Plant Adaptation to Environmental Stress:Drought, and Salinity in Environment and Climate-Smart Food Production. Cham: Springer International Publishing, 2022,151-79

[39] Dussarrat T, Prigent S, Latorre C. et al. Predictive metabolomics of multiple Atacama plant species unveils a core set of generic metabolites for extreme climate resilience. New Phytol. 2022; 234: 1614-28

[40] Patono DL, Said-Pullicino D, Eloi Alcatr ¯ao L. et al. Photosynthetic recovery in drought-rehydrated grapevines is associated with high demand from the sinks, maximizing the fruit-oriented performance. Plant J. 2022; 112:1098-111

[41] Intrieri C, Filippetti I, Allegro G. et al. Early defoliation (hand vs mechanical) for improved crop control and grape composition in Sangiovese (Vitis vinifera L.). Aust J Grape Wine Res. 2008; 14:25-32

[42] Bavaresco L, Gatti M, Pezzutto S. et al. Effect of leaf removal on grape yield, berry composition, and stilbene concentration. Am J Enol Vitic. 2008; 59:292-8

[43] Gatti M, Bernizzoni F, Civardi S. et al. Effects of cluster thinning and preflowering leaf removal on growth and grape composition in cv. Sangiovese. Am J Enol Vitic. 2012; 63:325-32

[44] Bogicevic M, Maras V, Mugoˇsa M. et al. The effects of early leaf removal and cluster thinning treatments on berry growth and grape composition in cultivars Vranac and Cabernet Sauvignon. Chem Biol Technol Agric. 2015; 2:13-20

[45] Tong Q, Liu L, Zhao Y. et al. Transcriptome remodeling in response to leaf removal and exogenous abscisic acid in berries of grapevine (Vitis vinifera L.) fruit cuttings. Horticulturae. 2022; 8: 905-20

[46] Rodriguez M, Parola R, Andreola S. et al. TOR and SnRK 1 sig-naling pathways in plant response to abiotic stresses: Do they always act according to the "yin-yang" model? Plant Sci. 2019; 288: 110220

[47] Gibon Y, Pyl E-T, Sulpice R. et al. Adjustment of growth, starch turnover, protein content and central metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short photoperiods. Plant Cell Environ. 2009; 32:859-74

[48] Osuna D, Usadel B, Morcuende R. et al. Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon-deprived Arabidopsis seedlings. Plant J. 2007; 49:463-91

[49] Hildebrandt TM. Synthesis versus degradation: directions of amino acid metabolism during Arabidopsis abiotic stress response. Plant Mol Biol. 2018; 98:121-35

[50] Forchhammer K, Selim KA. Carbon/nitrogen homeostasis con-trol in cyanobacteria. FEMS Microbiol Rev. 2020; 44:33-53

[51] Covarrubias MP, Lillo-Carmona V, Melet L. et al. Metabolite fruit profile is altered in response to source-sink imbalance and can be used as an early predictor of fruit quality in nectarine. Front Plant Sci. 2021; 11:4133-46

[52] Garde-Cerdan T, Lorenzo C, Lara JF. et al. Study of the evolution of nitrogen compounds during grape ripening. Application to differentiate grape varieties and cultivated systems. J Agric Food Chem. 2009; 57:2410-9

[53] Guan L, Wu B, Hilbert G. et al. Cluster shading modifies amino acids in grape (Vitis vinifera L.) berries in a genotype- and tissue-dependent manner. Food Res Int. 2017; 98:2-9

[54] Skopelitis DS, Paranychianakis NV, Paschalidis KA. et al. Abiotic stress generates ROS that signal expression of anionic gluta-mate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell. 2006; 18:2767-81

[55] Verbruggen N, Hermans C. Proline accumulation in plants: a review. Amino Acids. 2008; 35:753-9

[56] Smeekens S, Ma JK, Hanson J. et al. Sugar signals and molecular networks controlling plant growth. Curr Opin Plant Biol. 2010; 13: 274-9

[57] Eastmond PJ, Graham LA.Trehalose metabolism: a regulatory role for trehalose-6-phosphate? Curr Opin Plant Biol. 2003; 6: 231-5

[58] Fichtner F, Lunn JE. The role of trehalose 6-phosphate (Tre6P) in plant metabolism and development. Annu Rev Plant Biol. 2021; 72: 737-60

[59] Wingler A, Delatte TL, O’Hara LE. et al. Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability. Plant Physiol. 2012; 158:1241-51

[60] Lunn JE, Feil R, Hendriks JH. et al. Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch syn-thesis in Arabidopsis thaliana. Biochem J. 2006; 397:139-48

[61] Nardozza S, Boldingh HL, Osorio S. et al. Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism. JExp Bot. 2013; 64: 5049-63

[62] Gibon Y, Blaesing OE, Hannemann J. et al. A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell. 2004; 16:3304-25

[63] Beauvoit BP, Colombié S, Monier A. et al. Model-assisted analy-sis of sugar metabolism throughout tomato fruit development reveals enzyme and carrier properties in relation to vacuole expansion. Plant Cell. 2014; 26:3224-42

[64] Baldet P, Hernould M, Laporte F. et al. The expression of cell proliferation-related genes in early developing flowers is affected by a fruit load reduction in tomato plants. JExp Bot. 2006; 57:961-70

[65] Li YM, Forney C, Bondada B. et al. The molecular regulation of carbon sink strength in grapevine (Vitis vinifera L.). Front Plant Sci. 2021; 11:606918

[66] Cuadros-Inostroza A, Ruíz-Lara S, González E. et al. GC-MS metabolic profiling of Cabernet Sauvignon and Merlot culti-vars during grapevine berry development and network analysis reveals a stage- and cultivar-dependent connectivity of primary metabolites. Metabolomics. 2016; 12:39-55

[67] Gray JD, Coombe BG. Variation in Shiraz berry size originates before fruitset but harvest is a point of resynchronisation for berry development after flowering. Aust J Grape Wine Res. 2009; 15:156-65

[68] Shahood R, Torregrosa L, Savoi S. et al. First quantitative assess-ment of growth, sugar accumulation and malate breakdown in a single ripening berry. OENO One. 2020; 54:1077-92

[69] Savoi S, Torregrosa L, Romieu C. Transcripts switched off at the stop of phloem unloading highlight the energy efficiency of sugar import in the ripening V. vinifera fruit. Hortic Res. 2021; 8:193

[70] Tavernier F, Savoi S, Torregrosa L. et al. The single-berry metabolomic clock paradigm reveals new stages and metabolic switches during grapevine berry development. bioRxiv, 2024.07.06.602344

[71] Mullins MG, Rajasekaran K. Fruiting cutting: revised method for producing test plants of grapevine cultivars. Am J Enol Vitic. 1981; 32:35-40

[72] Figueroa CM, Feil R, Ishihara H. et al. Trehalose 6-phosphate coordinates organic and amino acid metabolism with carbon availability. Plant J. 2016; 85:410-23

[73] Pereira GE, Gaudillere JP, Pieri P. et al. Microclimate influence on mineral and metabolic profiles of grape berries. J Agric Food Chem. 2006; 54:6765-75

[74] Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30:2114-20

[75] Dobin A, Davis CA, Schlesinger F. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29:15-21

[76] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:550-70

[77] Chen CJ, Chen H, Zhang Y. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13:1194-202

[78] 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

[79] Moriya Y, Itoh M, Okuda S. et al. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007; 35:W182-5

[80] RCoreTeam. R: A Language and Environment for Statistical Com-puting. Vienna, Austria: R Foundation for Statistical Computing; 2021: