Abstract
Apple fruit acidity, which affects the fruit’s overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid (Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of ‘Golden Delicious’ taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03–WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01–WAF02 and WAF10–WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16–WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially (P FDR < 0.05) between the high and low malate groups (or between the early and late developmental stages) but also in significant (P < 0.05) correlation with malate concentrations. The 3,066 genes fell in 648 MapMan (sub-) bins or functional classes, and 19 of them were significantly (P FDR < 0.05) co-enriched or co-suppressed in a malate dependent manner. Network inferring using the 363 genes encompassed in the 19 (sub-) bins, identified a major co-expression network of 239 genes. Since the 239 genes were also differentially expressed between the early (WAF03–WAF08) and late (WAF16–WAF20) developmental stages, the major network was considered to be associated with developmental regulation of apple fruit acidity in ‘Golden Delicious’.
Similar content being viewed by others
References
Angeli A, Baetz U, Francisco R, Zhang J, Chaves M, Regalado A (2013) The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera. Planta 238:283–291
Aoki K, Ogata Y, Shibata D (2007) Approaches for extracting practical information from gene co-expression networks in plant biology. Plant Cell Physiol 48:381–390
Assenov Y, Ramírez F, Schelhorn S-E, Lengauer T, Albrecht M (2008) Computing topological parameters of biological networks. Bioinformatics 24:282–284
Baggerly KA, Deng L, Morris JS, Aldaz CM (2003) Differential expression in SAGE: accounting for normal between-library variation. Bioinformatics 19:1477–1483
Bai Y, Dougherty L, Li M, Fazio G, Cheng L, Xu K (2012) A natural mutation-led truncation in one of the two aluminum-activated malate transporter-like genes at the Ma locus is associated with low fruit acidity in apple. Mol Genet Genomics 287:663–678
Bai Y, Dougherty L, Xu K (2014) Towards an improved apple reference transcriptome using RNA-seq. Mol Genet Genomics 289:427–438
Barbier-Brygoo H, De Angeli A, Filleur S, Frachisse J-M, Gambale F, Thomine S, Wege S (2011) Anion channels/transporters in plants: from molecular bases to regulatory networks. Annu Rev Plant Biol 62:25–51
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate—a practical and powerful approach to multiple testing. J R Stat Soc Ser B-Methodol 57:289–300
Beruter J (2004) Carbohydrate metabolism in two apple genotypes that differ in malate accumulation. J Plant Physiol 161:1011–1029
Blanke MM, Lenz F (1989) Fruit photosynthesis. Plant Cell Environ 12:31–46
Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman C, Christmas R, Avila-Campilo I, Creech M, Gross B, Hanspers K, Isserlin R, Kelley R, Killcoyne S, Lotia S, Maere S, Morris J, Ono K, Pavlovic V, Pico AR, Vailaya A, Wang P-L, Adler A, Conklin BR, Hood L, Kuiper M, Sander C, Schmulevich I, Schwikowski B, Warner GJ, Ideker T, Bader GD (2007) Integration of biological networks and gene expression data using Cytoscape. Nat Protoc 2:2366–2382
Collins NC, Shirley NJ, Saeed M, Pallotta M, Gustafson JP (2008) An ALMT1 gene cluster controlling aluminum tolerance at the Alt4 locus of rye (Secale cereale L.). Genetics 179:669–682
Delhaize E, Ma JF, Ryan PR (2012) Transcriptional regulation of aluminium tolerance genes. Trends Plant Sci 17:341–348
Emmerlich V, Linka N, Reinhold T, Hurth MA, Traub M, Martinoia E, Neuhaus HE (2003) The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier. P Natl Acad Sci USA 100:11122–11126
Eticha D, Stass A, Horst WJ (2005) Cell–wall pectin and its degree of methylation in the maize root–apex: significance for genotypic differences in aluminium resistance. Plant Cell Environ 28:1410–1420
Etienne A, Génard M, Lobit P, Mbeguié-A-Mbéguié D, Bugaud C (2013) What controls fleshy fruit acidity? A review of malate and citrate accumulation in fruit cells. J Exp Bot 64:1451–1469
Falcon S, Gentleman R (2007) Using GOstats to test gene lists for GO term association. Bioinformatics 23:257–258
Fernie AR, Carrari F, Sweetlove LJ (2004) Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr Opin Plant Biol 7:254–261
Gasic K, Hernandez A, Korban SS (2004) RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol Biol Rep 22:437–438
Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talón M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420–3435
Gowik U, Bräutigam A, Weber KL, Weber APM, Westhoff P (2011) Evolution of C4 photosynthesis in the genus Flaveria: how many and which genes does it take to make C4? Plant Cell 23:2087–2105
Gutierres S, Sabar M, Lelandais C, Chetrit P, Diolez P, Degand H, Boutry M, Vedel F, de Kouchkovsky Y, De Paepe R (1997) Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants. P Natl Acad Sci USA 94:3436–3441
Hoekenga OA, Maron LG, Pineros MA, Cancado GMA, Shaff J, Kobayashi Y, Ryan PR, Dong B, Delhaize E, Sasaki T, Matsumoto H, Yamamoto Y, Koyama H, Kochian LV (2006) AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. P Natl Acad Sci USA 103:9738–9743
Hu L, Sun H, Li R, Zhang L, Wang S, Sui X, Zhang Z (2011) Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage. Plant Cell Environ 34:1835–1848
Hulme AC, Wooltorton LSC (1957) The organic acid metabolism of apple fruits: changes in individual acids during growth on the tree. J Sci Food Agr 8:117–122
Jung S, Ficklin SP, Lee T, Cheng C-H, Blenda A, Zheng P, Yu J, Bombarely A, Cho I, Ru S, Evans K, Peace C, Abbott AG, Mueller LA, Olmstead MA, Main D (2014) The Genome Database for Rosaceae (GDR): year 10 update. Nucleic Acids Res 42:D1237–D1244
Kang C, Darwish O, Geretz A, Shahan R, Alkharouf N, Liu Z (2013) Genome-scale transcriptomic insights into early-stage fruit development in woodland strawberry Fragaria vesca. Plant Cell 25:1960–1978
Keller M, Shrestha P (2014) Solute accumulation differs in the vacuoles and apoplast of ripening grape berries. Planta 239:633–642
Kenis K, Keulemans J, Davey M (2008) Identification and stability of QTLs for fruit quality traits in apple. Tree Genet Genomes 4:647–661
Khan S, Beekwilder J, Schaart J, Mumm R, Soriano J, Jacobsen E, Schouten H (2013) Differences in acidity of apples are probably mainly caused by a malic acid transporter gene on LG16. Tree Genet Genomes 9:475–487
Klie S, Nikoloski Z (2012) The choice between mapman and gene ontology for automated gene function prediction in plant science. Front Genet 3:1–14
Kovermann P, Meyer S, Hortensteiner S, Picco C, Scholz-Starke J, Ravera S, Lee Y, Martinoia E (2007) The Arabidopsis vacuolar malate channel is a member of the ALMT family. Plant J 52:1169–1180
Liebhard R, Kellerhals M, Pfammatter W, Jertmini M, Gessler C (2003) Mapping quantitative physiological traits in apple (Malus x domestica Borkh.). Plant Mol Biol 52:511–526
Lisec J, Schauer N, Kopka J, Willmitzer L, Fernie AR (2006) Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat Protoc 1:387–396
Lohse M, Nagel A, Herter T, May P, Schroda M, Zrenner R, Tohge T, Fernie AR, Stitt M, Usadel B (2014) Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ 37:1250–1258
Maliepaard C, Alston FH, van Arkel G, Brown LM, Chevreau E, Dunemann F, Evans KM, Gardiner S, Guilford P, van Heusden AW, Janse J, Laurens F, Lynn JR, Manganaris AG, den Nijs APM, Periam N, Rikkerink E, Roche P, Ryder C, Sansavini S, Schmidt H, Tartarini S, Verhaegh JJ, Vrielink-van Ginkel M, King GJ (1998) Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers. Theor Appl Genet 97:60–73
Meyer S, Scholz-Starke J, De Angeli A, Kovermann P, Burla B, Gambale F, Martinoia E (2011) Malate transport by the vacuolar AtALMT6 channel in guard cells is subject to multiple regulation. Plant J 67:247–257
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628
Nelson DL, Lehninger AL, Cox MM (2008) Pathways of Amino Acid Degradation. Lehninger principles of biochemistry, 5th edn. W.H. Freeman, New York, pp 687-706
Noctor G, Dutilleul C, De Paepe R, Foyer CH (2004) Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism. J Exp Bot 55:49–57
Osorio S, Vallarino JG, Szecowka M, Ufaz S, Tzin V, Angelovici R, Galili G, Fernie AR (2013) 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 161:628–643
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Ruan Y, Mate C, Patrick J, Brady C (1995) Non-destructive collection of apoplast fluid from developing tomato fruit using a pressure dehydration procedure. Funct Plant Biol 22:761–769
Saeed A, Bhagabati N, Braisted J, Liang W, Sharov V, Howe E, Li J, Thiagarajan M, White J, Quackenbush J (2006) TM4 microarray software suite. Methods Enzymol 411:134–193
Sasaki T, Yamamoto Y, Ezaki B, Katsuhara M, Ahn SJ, Ryan PR, Delhaize E, Matsumoto H (2004) A wheat gene encoding an aluminum-activated malate transporter. Plant J 37:645–653
Schumacher K, Krebs M (2010) The V-ATPase: small cargo, large effects. Curr Opin Plant Biol 13:724–730
Secco D, Jabnoune M, Walker H, Shou H, Wu P, Poirier Y, Whelan J (2013) Spatio-temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery. Plant Cell 25:4285–4304
Smith E, Naik D, Cumming JR (2011) Genotypic variation in aluminum resistance, cellular aluminum fractions, callose and pectin formation and organic acid accumulation in roots of Populus hybrids. Environ Exp Bot 72:182–193
Sweetman C, Deluc LG, Cramer GR, Ford CM, Soole KL (2009) Regulation of malate metabolism in grape berry and other developing fruits. Phytochemistry 70:1329–1344
Thimm O, Blasing O, Gibon Y, Nagel A, Meyer S, Kruger P, Selbig J, Muller LA, Rhee SY, Stitt M (2004) MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J 37:914–939
Ulrich R (1970) Organic acids. In: Hulme A (ed) The biochemistry of fruit and their products. Academic Press, London and New York, pp 89–118
Usadel B, Nagel A, Steinhauser D, Gibon Y, Blasing O, Redestig H, Sreenivasulu N, Krall L, Hannah M, Poree F, Fernie A, Stitt M (2006) PageMan: an interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments. BMC Bioinformatics 7:535
Usadel B, Poree F, Nagel A, Lohse M, Czedik-Eysenberg A, Stitt M (2009) A guide to using MapMan to visualize and compare Omics data in plants: a case study in the crop species, Maize. Plant Cell Environ 32:1211–1229
Wang A, Xu K (2012) Characterization of two orthologs of reversion-to-ethylene sensitivity1 in apple. J Mol Biol Res 2:24–41
Wehr JB, Menzies NW, Blamey FPC (2003) Model studies on the role of citrate, malate and pectin esterification on the enzymatic degradation of Al- and Ca-pectate gels: possible implications for Al-tolerance. Plant Physiol Bioch 41:1007–1010
Wheeler MCG, Tronconi MA, Drincovich MF, Andreo CS, Flügge U-I, Maurino VG (2005) A comprehensive analysis of the nadp-malic enzyme gene family of Arabidopsis. Plant Physiol 139:39–51
Wilcoxon F (1945) Individual comparisons by ranking methods. Biometrics 1:80–83
Wong D, Sweetman C, Drew D, Ford C (2013) VTCdb: a gene co-expression database for the crop species Vitis vinifera (grapevine). BMC Genom 14:882
Xu K, Wang A, Brown S (2012) Genetic characterization of the Ma locus with pH and titratable acidity in apple. Mol Breeding 30:899–912
Yamaji N, Huang CF, Nagao S, Yano M, Sato Y, Nagamura Y, Ma JF (2009) A zinc finger transcription factor art1 regulates multiple genes implicated in aluminum tolerance in rice. Plant Cell 21:3339–3349
Yang XY, Zeng ZH, Yan JY, Fan W, Bian HW, Zhu MY, Yang JL, Zheng SJ (2013) Association of specific pectin methylesterases with Al-induced root elongation inhibition in rice. Physiol Plantarum 148:502–511
Yao YX, Li M, Liu Z, You CX, Wang DM, Zhai H, Hao YJ (2009) Molecular cloning of three malic acid related genes MdPEPC, MdVHA-A, MdcyME and their expression analysis in apple fruits. Sci Hortic 122:404–408
Yao Y-X, Li M, Zhai H, You C-X, Hao Y-J (2011) Isolation and characterization of an apple cytosolic malate dehydrogenase gene reveal its function in malate synthesis. J Plant Physiol 168:474–480
Zhang LY, Peng YB, Pelleschi-Travier S, Fan Y, Lu YF, Lu YM, Gao XP, Shen YY, Delrot S, Zhang DP (2004) Evidence for apoplasmic phloem unloading in developing apple fruit. Plant Physiol 135:574–586
Zhang XY, Wang XL, Wang XF, Xia GH, Pan QH, Fan RC, Wu FQ, Yu XC, Zhang DP (2006) A shift of phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry. Plant Physiol 142:220–232
Zhang YZ, Li PM, Cheng LL (2010) Developmental changes of carbohydrates, organic acids, amino acids, and phenolic compounds in ‘Honeycrisp’ apple flesh. Food Chem 123:1013–1018
Acknowledgments
We thank Dr. Zhangjun Fei and Dr. Yi Zheng for their helpful comments on the RNA seq data analysis. We also thank Drs Doreen Main and Stephen Ficklin for formatting the revised apple reference transcriptome and making it publically available through the Genome Database for Rosaceae (GDR). This work was supported in part by the Agriculture and Food Research Initiative competitive grant no. 2014-67013-21660 of the USDA National Institute of Food and Agriculture.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by S. Hohmann.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Bai, Y., Dougherty, L., Cheng, L. et al. A co-expression gene network associated with developmental regulation of apple fruit acidity. Mol Genet Genomics 290, 1247–1263 (2015). https://doi.org/10.1007/s00438-014-0986-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-014-0986-2