Malate metabolism mediated by the cytoplasmic malate dehydrogenase gene MdcyMDH affects sucrose synthesis in apple fruit

Abstract

The types and proportions of soluble sugar and organic acid in fruit significantly affect flavor quality. However, there are few reports on the crosstalk regulation between metabolism of organic acid and sugar in fruit. Here, we found that the overexpression of cytoplasmic malate dehydrogenase genes (MdcyMDHs) not only increased the malate content but also increased the sucrose concentration in transgenic apple calli and mature fruit. Enzyme activity assays indicated that the overexpression of MdcyMDH1 and MdcyMDH5 enhanced sucrose phosphate synthase (SPS) activity in transgenic materials. RNA-seq and expression analysis showed that the expression levels of SPS genes were up-regulated in MdcyMDH1-overexpressed apple fruit and MdcyMDH5-overexpressed apple calli. Further study showed that the inhibition of MdSPSB2 or MdSPSC2 expression in MdcyMDH1 transgenic fruit could reduce or eliminate, respectively, the positive effect of MdcyMDH1 on sucrose accumulation. Moreover, some starch cleavage-related genes (MdBAM6.1/6.2, MdBMY8.1/8.2, MdISA1) and the key gluconeogenesis-related phosphoenolpyruvate carboxykinase MdPEPCK1 gene were significantly up-regulated in the transcriptome differentially expressed genes of mature fruit overexpressing MdcyMDH1. These results indicate that alteration of malate metabolism mediated by MdcyMDH might regulate the expression of MdSPSs and SPS activity via affecting starch metabolism or gluconeogenesis, and thus accelerate sucrose synthesis and accumulation in fruit.

References

[1] Jaeger SR, Andani Z, Wakeling IN et al. Consumer preferences for fresh and aged apples: a cross-cultural comparison. Food Qual Prefer. 1998; 9: 355-66.

[2] Ruffner HP, Hawker JS . Control of glycolysis in ripening berries of Vitis vinifera. Phytochemistry. 1977; 16: 1171-5.

[3] Sweetman C, Deluc LG, Cramer GR et al. Regulation of malate metabolism in grape berry and other developing fruits. Phytochemistry. 2009; 70: 1329-44.

[4] Berüter J . Carbohydrate metabolism in two apple genotypes that differ in malate accumulation. J Plant Physiol. 2004; 161: 1011-29.

[5] Mignard P, Beguería S, Giménez R et al. Effect of genetics and climate on apple sugars and organic acids profiles. Agronomy. 2022; 12: 827.

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

[7] Zhang Y, Li P, Cheng L . Developmental changes of carbohydrates, organic acids, amino acids, and phenolic compounds in ‘Honeycrisp’ apple flesh. Food Chem. 2010; 123: 1013-8.

[8] Cuevas IC, Podestá FE . Purification and physical and kinetic characterization of an NAD⁺-dependent malate dehydrogenase from leaves of pineapple (Ananas comosus) . Physiol Plant. 2000; 108: 240-8.

[9] Miller SS, Driscoll BT, Gregerson RG et al. Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule-enhanced MDH. Plant J. 1998; 15: 173-84.

[10] Ding Y, Ma QH . Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat. Biochimie. 2004; 86: 509-18.

[11] KEJ T, Podestá FE . Purification and characterization of an NAD-dependent malate dehydrogenase from leaves of the crassulacean acid metabolism plant Aptenia cordifolia. Plant Physiol Biochem. 2003; 41: 97-105.

[12] Yao YX, Li M, Zhai H et al. Isolation and characterization of an apple cytosolic malate dehydrogenase gene reveal its function in malate synthesis. J Plant Physiol. 2011; 168: 474-80.

[13] Gibson N, McAlister-Henn L . Physical and genetic interactions of cytosolic malate dehydrogenase with other gluconeogenic enzymes. J Biol Chem. 2003; 278: 25628-36.

[14] Echeverria E, Burns JK . Vacuolar acid hydrolysis as a physiological mechanism for sucrose breakdown. Plant Physiol. 1989; 90: 530-3.

[15] 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.

[16] Wei L, Mao W, Jia M et al. FaMYB44.2, a transcriptional repressor, negatively regulates sucrose accumulation in strawberry receptacles through interplay with FaMYB10. J Exp Bot. 2018; 69: 4805-20.

[17] Yu J, Gu KD, Sun CH et al. The apple bHLH transcription factor MdbHLH3 functions in determining the fruit carbohydrates and malate. Plant Biotechnol J. 2021; 19: 285-99.

[18] Sun J, Zhang J, Larue CT et al. Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1: Roles of SPS isoforms in photosynthesis. Plant Cell Environ. 2011; 34: 592-604.

[19] Zhang L, Ma B, Wang C et al. MdWRKY126 modulates malate accumulation in apple fruit by regulating cytosolic malate dehydrogenase (MdMDH5). Plant Physiol. 2022; 188: 2059-72.

[20] Selinski J, König N, Wellmeyer B et al. The plastid-localized NAD-dependent malate dehydrogenase is crucial for energy homeostasis in developing Arabidopsis thaliana Seeds. Mol Plant. 2014; 7: 170-86.

[21] Schreier TB, Cléry A, Schläfli M et al. Plastidial NAD-dependent malate dehydrogenase: a moonlighting protein involved in early chloroplast development through its interaction with an FtsH12-FtsHi protease complex. Plant Cell. 2018; 30: 1745-69.

[22] Beeler S, Liu HC, Stadler M et al. Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis. Plant Physiol. 2014; 164: 1175-90.

[23] Teng X, Zhong M, Zhu X et al. FLOURY ENDOSPERM16 encoding a NAD-dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice. Plant Biotechnol J. 2019; 17: 1914-27.

[24] Wang Q, Sun H, Dong QL et al. The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants. Plant Biotechnol J. 2016; 14: 1986-97.

[25] Nan N, Wang J, Shi Y et al. Rice plastidial NAD-dependent malate dehydrogenase 1 negatively regulates salt stress response by reducing the vitamin B6 content. Plant Biotechnol J. 2020;18:172-84.

[26] Ollat N, Gaudillère J . Carbon balance in developing grapevine berries. Acta Hortic. 2000; 526: 345-50.

[27] 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.

[28] Li M, Feng F, Cheng L . Expression patterns of genes involved in sugar metabolism and accumulation during apple fruit development. PLoS One. 2012; 7: e33055.

[29] Li M, Li P, Ma F et al. Sugar metabolism and accumulation in the fruit of transgenic apple trees with decreased sorbitol synthesis. Hortic. Res. 2018; 5: 60.

[30] Stitt M . Limitation of photosynthesis by carbon metabolism: I. evidence for excess electron transport capacity in leaves carrying out photosynthesis in saturating light and CO₂ . Plant Physiol. 1986; 81: 1115-22.

[31] do Nascimento JRO, Cordenunsi BR, Lajolo FM et al. Banana sucrose-phosphate synthase gene expression during fruit ripening. Planta. 1997; 203: 283-8.

[32] Yu X, Wang X, Fan J et al. Cloning and characterization of a sucrose phosphate synthase-encoding gene from muskmelon. J Am Soc Hortic Sci. 2007; 132: 557-62.

[33] Komatsu A, Moriguchi T, Koyama K et al. Analysis of sucrose synthase genes in citrus suggests different roles and phylogenetic relationships. J Exp Bot. 2002; 53: 61-71.

[34] Verma AK, Upadhyay SK, Verma PC et al. Functional analysis of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in sugarcane (Saccharum) cultivars: Sugarcane functional sucrose enzyme analysis. Plant Biol. 2011; 13: 325-32.

[35] Ruan YL . Sucrose metabolism: Gateway to diverse carbon use and sugar signaling. Annu Rev Plant Biol. 2014; 65: 33-67.

[36] Baxter CJ, Foyer CH, Rolfe SA et al. A comparison of the carbohydrate composition and kinetic properties of sucrose phosphate synthase (SPS) in transgenic tobacco (Nicotiana tabacum) leaves expressing maize SPS protein with untransformed controls. Ann Appl Biol. 2001; 138: 47-55.

[37] Zhu YJ, Komor E, Moore PH . Sucrose accumulation in the sugarcane stem is regulated by the difference between the activities of soluble acid invertase and sucrose phosphate synthase. Plant Physiol. 1997; 115: 609-16.

[38] Worrell AC, Bruneau J-M, Summerfelt K et al. Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning. Plant Cell. 1991; 3: 1121.

[39] Ruffner HP, Kliewer WM . Phosphoenolpyruvate carboxykinase activity in grape berries. Plant Physiol. 1975; 56: 67-71.

[40] Famiani F, Farinelli D, Palliotti A et al. Is stored malate the quantitatively most important substrate utilised by respiration and ethanolic fermentation in grape berry pericarp during ripening? Plant Physiol Biochem. 2014; 76: 52-7.

[41] Famiani F, Farinelli D, Moscatello S et al. The contribution of stored malate and citrate to the substrate requirements of metabolism of ripening peach (Prunus persica L. Batsch) flesh is negligible. Implications for the occurrence of phosphoenolpyruvate carboxykinase and gluconeogenesis. Plant Physiol Biochem. 2016; 101: 33-42.

[42] Famiani F, Farinelli D, Frioni T et al. Malate as substrate for catabolism and gluconeogenesis during ripening in the pericarp of different grape cultivars. Biol Plant. 2016; 60: 155-62.

[43] Chen Q, Qi W, Reiter RJ et al. Exogenously applied melatonin stimulates root growth and raises endogenous indoleacetic acid in roots of etiolated seedlings of Brassica juncea. J Plant Physiol. 2009; 166: 324-8.

[44] Zhu L, 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. Proc Natl Acad Sci. 2021; 118: e2022788118.

[45] Sun HJ, Uchii S, Watanabe S et al. A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol. 2006; 47: 426-31.

[46] Zhang L, Sun S, Liang Y et al. Nitrogen levels regulate sugar metabolism and transport in the shoot tips of crabapple plants. Front Plant Sci. 2021; 12: 626149.