Cucumber malate decarboxylase, CsNADP-ME2, functions in the balance of carbon and amino acid metabolism in fruit

Abstract

Central metabolism produces carbohydrates and amino acids that are tightly correlated to plant growth and thereby crop productivity. Malate is reported to link mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. Although the function of malate metabolism-related enzymes in providing carbon has been characterized in some plants, evidence for this role in the fleshy fruit of cucumber is lacking. Here, radiolabeled bicarbonate fed into the xylem stream from the cucumber roots was incorporated into amino acids, soluble sugars, and organic acids in the exocarp and vasculature of fruits. The activities of decarboxylases, especially decarboxylation from NADP-dependent malic enzyme (NADP-ME), were higher in cucumber fruit than in the leaf lamina. Histochemical localization revealed that CsNADP-ME2 was mainly located in the exocarp and vascular bundle system of fruit. Radiotracer and gas-exchange analysis indicated that overexpression of CsNADP-ME2 could promote carbon flux into soluble sugars and starch in fruits. Further studies combined with metabolic profiling revealed that the downregulation of CsNADP-ME2 in RNA interference (RNAi) lines caused the accumulation of its substrate, malate, in the exocarp. In addition to inhibition of glycolysis-related gene expression and reduction of the activities of the corresponding enzymes, increased amino acid synthesis and decreased sugar abundance were also observed in these lines. The opposite effect was found in CsNADP-ME2-overexpressing lines, suggesting that there may be a continuous bottom-up feedback regulation of glycolysis in cucumber fruits. Overall, our studies indicate that CsNADP-ME2 may play potential roles in both central carbon reactions and amino acid metabolism in cucumber fruits.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32272695 and 31972398 to X.S.), the National Key Research and Development Program of China (2019YFD1000300), the National Natural Science Foundation of China (31960591 to N.S.), the Max-Planck Society and European Union’s Horizon 2020 research and innovation programme, project PlantaSYST (SGA-CSA No. 664621 and No. 739582 under FPA No. 664620), the China Agriculture Research System of MOF and MARA (CARS-23), and the 111 Project of Ministry of Education of P.R.C. (B17043).

Author contributions

X.S. and N.S. conceived the project and designed the experiments; N.S., Y.Z., Y.G., W.Z., and J.N. performed the experiments; Y.Z., W.Z., and A.R.F. provided technical assistance to X.S., N.S., and Y.G.; N.S., Y.Z., Y.G., and X.S. analyzed the data. N.S., X.S., and Y.Z. wrote the article, and A.R.F revised the article. All authors read and approved the final draft of the manuscript.

Data availability

The accession numbers of all genes used in this paper were obtained from the Cucurbit Genomics Database (Cucumber Chinese Long Genome v2 http://cucurbitgenomics.org/organism/2) and are listed in Supplementary Data Table S3.

Conflict of interest

The authors declare that they have no conflicts of interest.

References

[1] Baslam M, Mitsui T, Sueyoshi K, et al. Recent advances in carbon and nitrogen metabolism in C₃ plants . Int J Mol Sci. 2021; 22: 318

[2] Kinoshita H, Nagasaki J, Yoshikawa N, et al. The chloroplastic 2-oxoglutarate/malate transporter has dual function as the malate valve and in carbon/nitrogen metabolism. Plant J. 2011; 65: 15-26

[3] Riebeseel E, Häusler RE, Radchuk R, et al. The 2-oxoglutarate/malate translocator mediates amino acid and storage protein biosynthesis in pea embryos. Plant J. 2010; 61: 350-63

[4] Morley SA, Ma F, Alazem M, et al. Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans. New Phytol. 2023; 239: 1834-51

[5] Nunes-Nesi A, Carrari F, Lytovchenko A, et al. Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiol. 2005; 137: 611-22

[6] Tronconi MA, Fahnenstich H, Gerrard Weehler MC, et al. Arabidopsis NAD-malic enzyme functions as a homodimer and heterodimer and has a major impact on nocturnal metabolism . Plant Physiol. 2008; 146: 1540-52

[7] Shi J, Yi K, Liu Y, et al. Phosphoenolpyruvate carboxylase in Arabidopsis leaves plays a crucial role in carbon and nitrogen metabolism . Plant Physiol. 2015; 167: 671-81

[8] 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

[9] Meyer S, De Angeli A, Fernie AR, et al. Intra- and extra-cellular excretion of carboxylates. Trends Plant Sci. 2010; 15: 40-7

[10] Sweetlove LJ, Beard KFM, Nunes-Nesi A, et al. Not just a circle: flux modes in the plant TCA cycle. Trends Plant Sci. 2010; 15: 462-70

[11] Chen Q, Wang B, Ding H, et al. Review: the role of NADP-malic enzyme in plants under stress. Plant Sci. 2019; 281: 206-12

[12] Dong H, Bai L, Zhang Y, et al. Modulation of guard cell turgor and drought tolerance by a peroxisomal acetate-malate shunt. Mol Plant. 2018; 11: 1278-91

[13] 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

[14] Gerrard Wheeler MC, Arias CL, Tronconi MA, et al. Arabidopsis thaliana NADP-malic enzyme isoforms: high degree of identity but clearly distinct properties . Plant Mol Biol. 2008; 67: 231-42

[15] Gerrard Wheeler MC, Tronconi MA, Drincovich MF, et al. A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis. Plant Physiol. 2005; 139: 39-51

[16] Maurino VG, Gerrard Wheeler MC, Andreo CS, et al. Redundancy is sometimes seen only by the uncritical: does Arabidopsis need six malic enzyme isoforms? Plant Sci. 2009; 176: 715-21

[17] Detarsio E, Maurino VG, Alvarez CE, et al. Maize cytosolic NADP-malic enzyme (ZmCytNADP-ME): a phylogenetically distant isoform specifically expressed in embryo and emerging roots. Plant Mol Biol. 2008; 68: 355-67

[18] Maier A, Zell MB, Maurino VG . Malate decarboxylases: evolution and roles of NAD(P)-ME isoforms in species performing C₄ and C₃ photosynthesis . J Exp Bot. 2011; 62: 3061-9

[19] Arias CL, Pavlovic T, Torcolese G, et al. NADP-dependent malic enzyme 1 participates in the abscisic acid response in Arabidopsis thaliana. Front Plant Sci. 2018; 9: 1637

[20] Yazdanpanah F, Maurino VG, Mettler-Altmann T, et al. NADP−MALIC ENZYME 1 affects germination after seed storage in Arabidopsis thaliana. Plant Cell Physiol. 2019; 60: 318-28

[21] Brown NJ, Palmer BG, Stanley S, et al. C₄ acid decarboxylases required for C₄ photosynthesis are active in the mid-vein of the C₃ species Arabidopsis thaliana, and are important in sugar and amino acid metabolism . Plant J. 2010; 61: 122-33

[22] Voll LM, Zell MB, Engelsdorf T, et al. Loss of cytosolic NADP-malic enzyme 2 in Arabidopsis thaliana is associated with enhanced susceptibility to Colletotrichum higginsianum. New Phytol. 2012; 195: 189-202

[23] Badia MB, Arias CL, Tronconi MA, et al. Enhanced cytosolic NADP-ME2 activity in A. thaliana affects plant development, stress tolerance and specific diurnal and nocturnal cellular processes . Plant Sci. 2015; 240: 193-203

[24] Hibberd J, Quick W . Characteristics of C₄ photosynthesis in stems and petioles of C₃ flowering plants . Nature. 2002; 415: 451-4

[25] Schaaf J, Walter MH, Hess D . Primary metabolism in plant defense (regulation of a bean malic enzyme gene promoter in transgenic tobacco by developmental and environmental cues). Plant Physiol. 1995; 108: 949-60

[26] Borba AR, Serra TS, Górska A, et al. Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C₄ photosynthesis is based on an ancient code found in the ancestral C₃ state . Mol Biol Evol. 2018; 35: 1690-705

[27] Wu B, Li P, Hong X, et al. The receptor-like cytosolic kinase RIPK activates NADP-malic enzyme 2 to generate NADPH for fueling ROS production. Mol Plant. 2022; 15: 887-903

[28] Hu L, Meng FZ, Wang SH, et al. Changes in carbohydrate levels and their metabolic enzymes in leaves, phloem sap and mesocarp during cucumber (Cucumis sativus L.) fruit development . Sci Hortic. 2009; 121: 131-7

[29] Li Y, Liu H, Yao X, et al. Hexose transporter CsSWEET7a in cucumber mediates phloem unloading in companion cells for fruit development. Plant Physiol. 2021; 186: 640-54

[30] Sui X, Shan N, Hu L, et al. The complex character of photosynthesis in cucumber fruit. J Exp Bot. 2017; 68: 1625-37

[31] Sui X, Nie J, Li X, et al. Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development . Plant J. 2018; 96: 982-96

[32] Sui X, Nie J, Liu H, et al. Complexity untwined: the structure and function of cucumber (Cucumis sativus L.) shoot phloem . Plant J. 2021; 106: 1163-76

[33] Janacek SH, Trenkamp S, Palmer B, et al. Photosynthesis in cells around veins of the C₃ plant Arabidopsis thaliana is important for both the shikimate pathway and leaf senescence as well as contributing to plant fitness . Plant J. 2009; 59: 329-43

[34] Shen WJ, Ye L, Ma J, et al. The existence of C₄-bundle-sheath-like photosynthesis in the mid-vein of C₃ rice . Rice. 2016; 9: 20

[35] Alvarez CE, Bovdilova A, Höppner A, et al. Molecular adaptations of NADP-malic enzyme for its function in C₄ photosynthesis in grasses . Nature Plants. 2019; 5: 755-65

[36] Yao K, Wu YY . Phosphofructokinase and glucose-6-phosphate dehydrogenase in response to drought and bicarbonate stress at transcriptional and functional levels in mulberry. Russ J Plant Physiol. 2016; 63: 235-42

[37] Taiz L, Zeiger E . Plant Physiology. Sunderland: Sinauer Associates; 2010

[38] Berveiller D, Damesin C . Carbon assimilation by tree stems: potential involvement of phosphoenolpyruvate carboxylase. Trees. 2008; 22: 149-57

[39] Chen ZH, Walker RP, Tecsi LI, et al. Phosphoenolpyruvate carboxykinase in cucumber plants is increased both by ammonium and by acidification, and is present in the phloem. Planta. 2004; 219: 48-58

[40] Brown NJ, Newell CA, Stanley S, et al. Independent and parallel recruitment of preexisting mechanisms underlying C₄ photosynthesis . Science. 2011; 331: 1436-9

[41] Burgess SJ, Reyna-Llorens I, Stevenson SR, et al. Genome-wide transcription factor binding in leaves from C₃ and C₄ grasses . Plant Cell. 2019; 31: 2297-314

[42] Wang J, Liu J, Guo Z . Natural uORF variation in plants. Trends Plant Sci. 2023

[43] Reis RS, Deforges J, Sokoloff T, et al. Modulation of shoot phosphate level and growth by PHOSPHATE1 upstream open reading frame. Plant Physiol. 2020; 183: 1145-56

[44] Salazar-Díaz K, Dong Y, Papdi C, et al. TOR senses and regulates spermidine metabolism during seedling establishment and growth in maize and Arabidopsis. iScience. 2021; 24: 103260

[45] Alatorre-Cobos F, Cruz-Ramírez A, Hayden CA, et al. Translational regulation of Arabidopsis XIPOTL1 is modulated by phosphocholine levels via the phylogenetically conserved upstream open reading frame 30 . J Exp Bot. 2012; 63: 5203-21

[46] Laing WA, Martínez-Sánchez M, Wright MA, et al. An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. Plant Cell. 2015; 27: 772-86

[47] Jorgensen R, Dorantes-Acosta A . Conserved peptide upstream open reading frames are associated with regulatory genes in angiosperms. Front Plant Sci. 2012; 3: 191

[48] Palmieri L, Picault N, Arrigoni R, et al. Molecular identification of three Arabidopsis thaliana mitochondrial dicarboxylate carrier isoforms: organ distribution, bacterial expression, reconstitution into liposomes and functional characterization . Biochem J. 2008; 410: 621-9

[49] Cheng J, Wang Z, Yao F, et al. Down-regulating CsHT1, a cucumber pollen-specific hexose transporter, inhibits pollen germination, tube growth, and seed development. Plant Physiol. 2015; 168: 635-47

[50] Bovdilova A, Alexandre BM, Höppner A, et al. Posttranslational modification of the NADP-malic enzyme involved in C₄ photosynthesis modulates the enzymatic activity during the day . Plant Cell. 2019; 31: 2525-39

[51] Pikart FC, Matiz A, Alves FRR, et al. Diurnal modulation of PEPCK decarboxylation activity impacts photosystem II light-energy use in a drought-induced CAM species. Environ Exp Bot. 2020; 173: 104003

[52] Fankhauser N, Aubry S . Post-transcriptional regulation of photosynthetic genes is a key driver of C₄ leaf ontogeny . J Exp Bot. 2017; 68: 137-46

[53] Shen Z, Dong XM, Gao ZF, et al. Phylogenic and phosphorylation regulation difference of phosphoenolpyruvate carboxykinase of C₃ and C₄ plants . J Plant Physiol. 2017; 213: 16-22

[54] Walker RP, Paoletti A, Leegood RC, et al. Phosphorylation of phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) in the flesh of fruits. Plant Physiol Biochem. 2016; 108: 323-7

[55] Chastain CJ, Heck JW, Colquhoun TA, et al. Posttranslational regulation of pyruvate, orthophosphate dikinase in developing rice (Oryza sativa) seeds . Planta. 2006; 224: 924-34

[56] Wang Z, Li H, Liu X, et al. Reduction of pyruvate orthophosphate dikinase activity is associated with high temperature-induced chalkiness in rice grains. Plant Physiol Biochem. 2015; 89: 76-84

[57] Chen YB, Lu TC, Wang HX, et al. Posttranslational modification of maize chloroplast pyruvate orthophosphate dikinase reveals the precise regulatory mechanism of its enzymatic activity. Plant Physiol. 2014; 165: 534-49

[58] Rademacher T, Häusler RE, Hirsch HJ, et al. An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants. Plant J. 2002; 32: 25-39

[59] Osorio S, Vallarino JG, Szecowka M, et al. Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic acid, metabolism, and transitory starch accumulation. Plant Physiol. 2013; 161: 628-43

[60] Nunes-Nesi A, Fernie AR, Stitt M . Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Mol Plant. 2010; 3: 973-96

[61] Pinto H, Powell JR, Sharwood RE, et al. Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C₄ grasses at inter-glacial CO₂. Plant Cell Environ. 2016; 39: 514-26

[62] Arp JJ, Kambhampati S, Chu KL, et al. Developmental effects on relative use of PEPCK and NADP-ME pathways of C₄ photosynthesis in maize . bioRxiv. 2021.06.25.449949

[63] Kumar S, Stecher G, Li M, et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35: 1547-9

[64] Ma S, Sun L, Sui X, et al. Phloem loading in cucumber: combined symplastic and apoplastic strategies. Plant J. 2019; 98: 391-404

[65] Dever LV, Boxall SF, Kneˇrová J, et al. Transgenic perturbation of the decarboxylation phase of crassulacean acid metabolism alters physiology and metabolism but has only a small effect on growth. Plant Physiol. 2015; 167: 44-59

[66] Hebbelmann I, Selinski J, Wehmeyer C, et al. Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase . J Exp Bot. 2012; 63: 1445-59

[67] Liu X, Wei W, Zhu W, et al. Histone deacetylase AtSRT1 links metabolic flux and stress response in Arabidopsis. Mol Plant. 2017; 10: 1510-22

[68] Jackson DP . In-situ hybridisation in plants. In: Bowles DJ, Gurr SJ, McPherson MJ, eds. Molecular Plant Pathology: A Practical Approach. Oxford, UK: Oxford University Press, 1992, 163-74

[69] Schauer N, Semel Y, Roessner U, et al. Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement. Nat Biotechnol. 2006; 24: 447-54

[70] Luedemann A, Strassburg K, Erban A, et al. TagFinder for the quantitative analysis of gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling experiments. Bioinformatics. 2008; 24: 732-7