, Volume 245, Issue 5, pp 1021–1035 | Cite as

Fine-tuning of the flavonoid and monolignol pathways during apple early fruit development

  • Paolo BaldiEmail author
  • Mirko Moser
  • Matteo Brilli
  • Urska Vrhovsek
  • Massimo Pindo
  • Azeddine Si-Ammour
Original Article


Main conclusion

A coordinated regulation of different branches of the flavonoid pathway was highlighted that may contribute to elucidate the role of this important class of compounds during the early stages of apple fruit development.

Apple (Malus × domestica Borkh.) is an economically important fruit appreciated for its organoleptic characteristics and its benefits for human health. The first stages after fruit set represent a very important and still poorly characterized developmental process. To enable the profiling of genes involved in apple early fruit development, we combined the suppression subtractive hybridization (SSH) protocol to next-generation sequencing. We identified and characterized genes induced and repressed during fruit development in the apple cultivar ‘Golden Delicious’. Our results showed an opposite regulation of genes coding for enzymes belonging to flavonoid and monolignol pathways, with a strong induction of the former and a simultaneous repression of the latter. Two isoforms of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, key enzymes located at the branching point between flavonoid and monolignol pathways, showed opposite expression patterns during the period in analysis, suggesting a possible regulation mechanism. A targeted metabolomic analysis supported the SSH results and revealed an accumulation of the monomers catechin and epicatechin as well as several forms of procyanidin oligomers in apple fruitlets starting early after anthesis, together with a decreased production of other classes of flavonoids such as some flavonols and the dihydrochalcone phlorizin. Moreover, gene expression and metabolites accumulation of ‘Golden Delicious’ were compared to a wild apple genotype of Manchurian crabapple (Malus mandshurica (Maxim.) Kom.). Significant differences in both gene expression and metabolites accumulation were found between the two genotypes.


Apple Catechin Epicatechin Flavonoids Monolignols Next-generation sequencing SSH 



We are grateful to Pierluigi Magnago for the maintenance of the orchard. This work was supported by the Autonomous Province of Trento “TranscrApple” grandi progetti 2012 to ASA.

Supplementary material

425_2017_2660_MOESM1_ESM.pptx (65 kb)
Supplementary material 1 (PPTX 64 kb)
425_2017_2660_MOESM2_ESM.pptx (66 kb)
Supplementary material 2 (PPTX 65 kb)
425_2017_2660_MOESM3_ESM.pptx (35 kb)
Supplementary material 3 (PPTX 35 kb)
425_2017_2660_MOESM4_ESM.pptx (824 kb)
Supplementary material 4 (PPTX 824 kb)
425_2017_2660_MOESM5_ESM.pptx (126 kb)
Supplementary material 5 (PPTX 126 kb)
425_2017_2660_MOESM6_ESM.pptx (63 kb)
Supplementary material 6 (PPTX 63 kb)
425_2017_2660_MOESM7_ESM.docx (15 kb)
Supplementary material 7 (DOCX 15 kb)
425_2017_2660_MOESM8_ESM.xlsx (1.8 mb)
Supplementary material 8 (XLSX 1893 kb)
425_2017_2660_MOESM9_ESM.xlsx (14 kb)
Supplementary material 9 (XLSX 14 kb)
425_2017_2660_MOESM10_ESM.xlsx (101 kb)
Supplementary material 10 (XLSX 101 kb)
425_2017_2660_MOESM11_ESM.xlsx (15 kb)
Supplementary material 11 (XLSX 14 kb)
425_2017_2660_MOESM12_ESM.xlsx (11 kb)
Supplementary material 12 (XLSX 11 kb)
425_2017_2660_MOESM13_ESM.xlsx (19 kb)
Supplementary material 13 (XLSX 19 kb)
425_2017_2660_MOESM14_ESM.xlsx (13 kb)
Supplementary material 14 (XLSX 13 kb)
425_2017_2660_MOESM15_ESM.xlsx (14 kb)
Supplementary material 15 (XLSX 14 kb)


  1. Allan AC, Hellens RP, Laing WA (2008) MYB transcription factors that colour our fruit. Trends Plant Sci 13:99–102CrossRefPubMedGoogle Scholar
  2. Bain JM, Robertson RN (1951) The physiology of growth in apple fruits. I. Cell size, cell number, and fruit development. Aust J Sci Res B 4:75–107CrossRefPubMedGoogle Scholar
  3. 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 57:289–300Google Scholar
  4. Besseau S, Hoffmann L, Geoffroy P, Lapierre C, Pollet B, Legrand M (2007) Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell 19:148–162CrossRefPubMedPubMedCentralGoogle Scholar
  5. Botton A, Rasori A, Ziliotto F, Moing A, Maucourt M, Bernillon S, Deborde C, Petterle A, Varotto S, Bonghi C (2016) The peach HECATE3-like gene FLESHY plays a double role during fruit development. Plant Mol Biol 91:97–114CrossRefPubMedGoogle Scholar
  6. Buer CS, Kordbacheh F, Truong TT, Hocart CH, Djordjevic MA (2013) Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture. Planta 238:171–189CrossRefPubMedGoogle Scholar
  7. Carpenter JL, Caruso FL, Tata A, Vorsa N, Neto CC (2014) Variation in proanthocyanidin content and composition among commonly grown North American cranberry cultivars (Vaccinium macrocarpon). J Sci Food Agric 94:2738–2745CrossRefPubMedGoogle Scholar
  8. Dardick CD, Callahan AM, Chiozzotto R, Schaffer RJ, Piagnani MC, Scorza R (2010) Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence. BMC Biol 8:13CrossRefPubMedPubMedCentralGoogle Scholar
  9. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085–1097CrossRefPubMedPubMedCentralGoogle Scholar
  11. Doughty J, Aljabri M, Scott RJ (2014) Flavonoids and the regulation of seed size in Arabidopsis. Biochem Soc Trans 42:364–369CrossRefPubMedGoogle Scholar
  12. Ehlting J, Buttner D, Wang Q, Douglas CJ, Somssich IE, Kombrink E (1999) Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms. Plant J 19:9–20CrossRefPubMedGoogle Scholar
  13. Feng F, Li M, Ma F, Cheng L (2013) Phenylpropanoid metabolites and expression of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to sun exposure. Plant Physiol Biochem 69:54–61CrossRefPubMedGoogle Scholar
  14. Flachowsky H, Peil A, Sopanen T, Elo A, Hanke V (2007) Overexpression of BpMADS4 from silver birch (Betula pendula Roth.) induces early-flowering in apple (Malus × domestica Borkh.). Plant Breed 126:137–145CrossRefGoogle Scholar
  15. Gillaspy G, Ben-David H, Gruissem W (1993) Fruits: a developmental perspective. Plant Cell 5:1439–1451CrossRefPubMedPubMedCentralGoogle Scholar
  16. Gottardini E, Cristofori A, Pellegrini E, La Porta N, Nali C, Baldi P, Sablok G (2016) Suppression substractive hybridization and NGS reveal differential transcriptome expression profiles in wayfaring tree (Viburnum lantana L.) treated with ozone. Front Plant Sci 7:713CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gui J, Shen J, Li L (2011) Functional characterization of evolutionarily divergent 4-coumarate:coenzyme a ligases in rice. Plant Physiol 157:574–586CrossRefPubMedPubMedCentralGoogle Scholar
  18. Guo WL, Chen RG, Gong ZH, Yin YX, Li DW (2013) Suppression subtractive hybridization analysis of genes regulated by application of exogenous abscisic acid in pepper plant (L.) leaves under chilling stress. PLoS One 8:e66667CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hectors K, Van Oevelen S, Geuns J, Guisez Y, Jansen MA, Prinsen E (2014) Dynamic changes in plant secondary metabolites during UV acclimation in Arabidopsis thaliana. Physiol Plant 152:219–230CrossRefPubMedGoogle Scholar
  20. Henry-Kirk RA, McGhie TK, Andre CM, Hellens RP, Allan AC (2012) Transcriptional analysis of apple fruit proanthocyanidin biosynthesis. J Exp Bot 63:5437–5450CrossRefPubMedPubMedCentralGoogle Scholar
  21. Honda C, Kotoda N, Wada M, Kondo S, Kobayashi S, Soejima J, Zhang ZL, Tsuda T, Moriguchi T (2002) Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin. Plant Physiol Biochem 40:955–962CrossRefGoogle Scholar
  22. Hu WJ, Kawaoka A, Tsai CJ, Lung J, Osakabe K, Ebinuma H, Chiang VL (1998) Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides). Proc Natl Acad Sci USA 95:5407–5412CrossRefPubMedPubMedCentralGoogle Scholar
  23. Huang J, Gu M, Lai Z, Fan B, Shi K, Zhou YH, Yu JQ, Chen Z (2010) Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress. Plant Physiol 153:1526–1538CrossRefPubMedPubMedCentralGoogle Scholar
  24. Jackson JE (2003) The biology of apples and pears. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  25. Jacobs M, Rubery PH (1988) Naturally occurring auxin transport regulators. Science 241:346–349CrossRefPubMedGoogle Scholar
  26. Janssen BJ, Thodey K, Schaffer RJ, Alba R, Balakrishnan L, Bishop R, Bowen JH, Crowhurst RN, Gleave AP, Ledger S, McArtney S, Pichler FB, Snowden KC, Ward S (2008) Global gene expression analysis of apple fruit development from the floral bud to ripe fruit. BMC Plant Biol 8:16CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protocols 10:845–858CrossRefPubMedGoogle Scholar
  28. Kim DS, Hwang BK (2014) An important role of the pepper phenylalanine ammonia-lyase gene (PAL1) in salicylic acid-dependent signalling of the defence response to microbial pathogens. J Exp Bot 65:2295–2306CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lee KW, Kim YJ, Kim DO, Lee HJ, Lee CY (2003) Major phenolics in apple and their contribution to the total antioxidant capacity. J Agric Food Chem 51:6516–6520CrossRefPubMedGoogle Scholar
  30. Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659CrossRefPubMedGoogle Scholar
  31. Li J, Yuan R (2008) NAA and ethylene regulate expression of genes related to ethylene biosynthesis, perception, and cell wall degradation during fruit abscission and ripening in ‘Delicious’ apples. J Plant Growth Regul 27:283–295CrossRefGoogle Scholar
  32. Li S, Zachgo S (2013) TCP3 interacts with R2R3-MYB proteins, promotes flavonoid biosynthesis and negatively regulates the auxin response in Arabidopsis thaliana. Plant J 76:901–913CrossRefPubMedGoogle Scholar
  33. Li W, Jaroszewski L, Godzik A (2001) Clustering of highly homologous sequences to reduce the size of large protein databases. Bioinformatics 17:282–283CrossRefPubMedGoogle Scholar
  34. Lindermayr C, Mollers B, Fliegmann J, Uhlmann A, Lottspeich F, Meimberg H, Ebel J (2002) Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family. Eur J Biochem 269:1304–1315CrossRefPubMedGoogle Scholar
  35. Lin-Wang K, Micheletti D, Palmer J, Volz R, Lozano L, Espley R, Hellens RP, Chagne D, Rowan DD, Troggio M, Iglesias I, Allan AC (2011) High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. Plant Cell Environ 34:1176–1190CrossRefPubMedGoogle Scholar
  36. Lukyanov SA, Gurskaya NG, Lukyanov KA, Tarabykin VS, Sverdlov ED (1994) Highly efficient subtractive hybridization of cDNA. Bioorg Khim 20:701–704Google Scholar
  37. Montero L, Herrero M, Ibanez E, Cifuentes A (2013) Profiling of phenolic compounds from different apple varieties using comprehensive two-dimensional liquid chromatography. J Chromatogr A 1313:275–283CrossRefPubMedGoogle Scholar
  38. Naoumkina MA, Zhao Q, Gallego-Giraldo L, Dai X, Zhao PX, Dixon RA (2010) Genome-wide analysis of phenylpropanoid defence pathways. Mol Plant Pathol 11:829–846PubMedGoogle Scholar
  39. Ogah O, Watkins CS, Ubj BE, Oraguzie NC (2014) Phenolic compounds in rosaceae fruit and nut crops. J Agric Food Chem 62:9369–9386CrossRefPubMedGoogle Scholar
  40. Onkokesung N, Reichelt M, van Doorn A, Schuurink RC, van Loon JJ, Dicke M (2014) Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae. J Exp Bot 65:2203–2217CrossRefPubMedPubMedCentralGoogle Scholar
  41. Payyavula RS, Navarre DA, Kuhl JC, Pantoja A, Pillai SS (2012) Differential effects of environment on potato phenylpropanoid and carotenoid expression. BMC Plant Biol 12:39CrossRefPubMedPubMedCentralGoogle Scholar
  42. Peer WA, Bandyopadhyay A, Blakeslee JJ, Makam SN, Chen RJ, Masson PH, Murphy AS (2004) Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana. Plant Cell 16:1898–1911CrossRefPubMedPubMedCentralGoogle Scholar
  43. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefPubMedPubMedCentralGoogle Scholar
  44. Pratt C (1988) Apple flower and fruit: morphology and anatomy. Horticultural reviews. Wiley, New York, pp 273–308Google Scholar
  45. Raes J, Rohde A, Christensen JH, Van de Peer Y, Boerjan W (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol 133:1051–1071CrossRefPubMedPubMedCentralGoogle Scholar
  46. Renard CMGC, Dupont N, Guillermin P (2007) Concentrations and characteristics of procyanidins and other phenolics in apples during fruit growth. Phytochemistry 68:1128–1138CrossRefPubMedGoogle Scholar
  47. Rohde A, Morreel K, Ralph J, Goeminne G, Hostyn V, De Rycke R, Kushnir S, Van Doorsselaere J, Joseleau JP, Vuylsteke M, Van Driessche G, Van Beeumen J, Messens E, Boerjan W (2004) Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino Acid, and carbohydrate metabolism. Plant Cell 16:2749–2771CrossRefPubMedPubMedCentralGoogle Scholar
  48. Szankowski I, Flachowsky H, Li H, Halbwirth H, Treutter D, Regos I, Hanke M-V, Stich K, Fischer TC (2009) Shift in polyphenol profile and sublethal phenotype caused by silencing of anthocyanidin synthase in apple (Malus sp.). Planta 229:681–692CrossRefPubMedGoogle Scholar
  49. Treutter D (2001) Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regul 34:71–89CrossRefGoogle Scholar
  50. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74CrossRefPubMedPubMedCentralGoogle Scholar
  51. Vieira PM, Coelho AS, Steindorff AS, de Siqueira SJ, Silva Rdo N, Ulhoa CJ (2013) Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application. BMC Genom 14:177CrossRefGoogle Scholar
  52. Vinson JA, Su XH, Zubik L, Bose P (2001) Phenol antioxidant quantity and quality in foods: fruits. J Agric Food Chem 49:5315–5321CrossRefPubMedGoogle Scholar
  53. Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20CrossRefPubMedGoogle Scholar
  54. Vrhovsek U, Rigo A, Tonon D, Mattivi F (2004) Quantitation of polyphenols in different apple varieties. J Agric Food Chem 52:6532–6538CrossRefPubMedGoogle Scholar
  55. Vrhovsek U, Masuero D, Gasperotti M, Franceschi P, Caputi L, Viola R, Mattivi F (2012) A versatile targeted metabolomics method for the rapid quantification of multiple classes of phenolics in fruits and beverages. J Agric Food Chem 60:8831–8840CrossRefPubMedGoogle Scholar
  56. Yin R, Han K, Heller W, Albert A, Dobrev PI, Zazimalova E, Schaffner AR (2013) Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots. New Phytol 201:466–475CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zhong R, Ye Z-H (2009) Transcriptional regulation of lignin biosynthesis. Plant Signal Behav 4:1028–1034CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Paolo Baldi
    • 1
    Email author
  • Mirko Moser
    • 1
  • Matteo Brilli
    • 1
  • Urska Vrhovsek
    • 1
  • Massimo Pindo
    • 1
  • Azeddine Si-Ammour
    • 1
  1. 1.Fondazione Edmund Mach (FEM)San Michele all’ AdigeItaly

Personalised recommendations