, Volume 256, Issue 1, pp 25–38 | Cite as

Histochemical and immunohistochemical analysis of enzymes involved in phenolic metabolism during berry development in Vitis vinifera L.

  • María Eugenia Molero de Ávila
  • María Victoria Alarcón
  • David Uriarte
  • Luis Alberto Mancha
  • Daniel Moreno
  • Javier Francisco-MorcilloEmail author
Original Article


Phenolics are involved in many of plants’ biological functions. In particular, they play important roles in determining the quality of grape berries and the wine made from them, and can also act as antioxidants with beneficial effects for human health. Several enzymes are involved in the synthesis of phenolic compounds. Among them, stilbene synthase (STS) is a key to the biosynthesis of stilbenes, which are considered to be important secondary metabolites in plants. Other enzymes, such as polyphenol oxidase (PPO) and peroxidase (POD), are involved in the degradation of phenolics, and become activated during late stages of berry ripening. In the present study, Vitis vinifera L. berries were sampled at eight stages of development, from 10 days after anthesis to late harvest. The PPO and POD enzymatic activities were determined at each stage. The presence of STS, PPO, and POD proteins in the grape exocarp and mesocarp was detected immunohistochemically and histochemically. The amount and intensity of the immunohistochemical and histochemical signals correlate with the variations in enzyme activities throughout fruit development. Strong STS immunoreactivity was detected until the onset of ripening. Labeled tissue increased gradually from mesocarp to exocarp, showing an intense signal in epidermis. At subcellular level, STS was mainly detected in cytoplasm grains and cell walls. The amount of PPO immunoreactivity increased progressively until the end of ripening. The PPO signal was detected in hypodermal layers and, to a lesser extent, in mesocarp parenchyma cells, especially in cytoplasm grains and cell walls. Finally, POD activity was stronger at the onset of ripening, and the POD histochemical signal was mainly detected in the cell walls of both exocarp and mesocarp tissue.


Berry development Histochemistry Immunohistochemistry Peroxidase Polyphenol oxidase Stilbene synthase Vitis vinifera 



During this work, M.E.M.C was a recipient of a PhD studentship from the Junta de Extremadura (PD12106). We also greatly thank Dr. Roque Bru (Departamento de Agroquímica y Bioquímica, University of Alicante, Spain) for the generous gifts of rabbit anti-PPO polyclonal antibody.


This work was supported by grants from Junta de Extremadura (GR15158 and AGA001), Fondo Social Europeo and Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-FEDER RTA-2012-00029-C01).


  1. Achnine L, Blancaflor EB, Rasmussen S, Dixon RA (2004) Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16:3098–3109CrossRefGoogle Scholar
  2. Alarcón MV, Lloret PG, Martín-Partido G, Salguero J (2016) The initiation of lateral roots in the primary roots of maize (Zea mays L.) implies a reactivation of cell proliferation in a group of founder pericycle cells. J Plant Physiol 192:105–110CrossRefGoogle Scholar
  3. Amrani-Joutei K, Glories Y, Mercier M (1994) Localization of tannins in grape berry skins. Vitis 33:133–138Google Scholar
  4. Andjelkovic M, Radovanovié B, Radovanovi A, Andjelkovic AM (2013) Changes in polyphenolic content and antioxidant activity of grapes cv vranac during ripening. S Afr J Enol Vitic 34(2):147–155Google Scholar
  5. Ayuso T, Moreno-Alías I, Valdés E, Uriarte D, Moreno D, Giraldo E, Prieto MH, Alarcón MV (2012) Estudio histológico de la distribución de los compuestos fenólicos en la piel de Vitis vinifera cv Tempranillo. Evolución durante la maduración. Acta Hortic 60:603–607Google Scholar
  6. Bejarano-Escobar R, Holguín-Arévalo MS, Montero JA, Francisco-Morcillo J, Martín-Partido G (2011) Macrophage and microglia ontogeny in the mouse visual system can be traced by the expression of Cathepsins B and D. Dev Dyn 240(7):1841–1855CrossRefGoogle Scholar
  7. Bejarano-Escobar R, Blasco M, Durán AC, Martín-Partido G, Francisco-Morcillo J (2013) Chronotopographical distribution patterns of cell death and of lectin-positive macrophages/microglial cells during the visual system ontogeny of the small-spotted catshark Scyliorhinus canicula. J Anat 223(2):171–184CrossRefGoogle Scholar
  8. Bejarano-Escobar R, Álvarez-Hernán G, Morona R, González A, Martín-Partido G, Francisco-Morcillo J (2015) Expression and function of the LIM-homeodomain transcription factor Islet-1 in the developing and mature vertebrate retina. Exp Eye Res 138:22–31CrossRefGoogle Scholar
  9. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  10. Bustamante CA, Budde CO, Borsani J, Lombardo VA, Lauxmann MA, Andreo CS, Lara MV, Drincovich MF (2012) Heat treatment of peach fruit: modifications in the extracellular compartment and identification of novel extracellular proteins. Plant Physiol Biochem 60:35–45CrossRefGoogle Scholar
  11. Cadot Y, Miñana-Castelló MT, Chevalier M (2006) Anatomical, histological, and histochemical changes in grape seeds from Vitis vinifera L. cv Cabernet franc during fruit development. J Agric Food Chem 54(24):9206–9215CrossRefGoogle Scholar
  12. Cadot Y, Chevalier M, Barbeau G (2011) Evolution of the localisation and composition of phenolics in grape skin between veraison and maturity in relation to water availability and some climatic conditions. J Sci Food Agric 91:1963–1976CrossRefGoogle Scholar
  13. Calderón AA, García-Florenciano E, Pedreño MA, Muñoz R, Ros Barceló A (1992) The vacuolar localization of grapevine peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes. Zeitschrift Naturforschung 47:215–221CrossRefGoogle Scholar
  14. Calderón AA, Zapata JM, Muñoz R, Ros Barceló A (1993) Localization of peroxidase in grapes using nitrocellulose blotting of freezing/thawing fruits. Hortscience 28:38–40Google Scholar
  15. Carbonell-Bejerano P, Rodríguez V, Hernáiz S, Grimplet J, Royo C, Martínez-Zapater JM (2012) Análisis transcriptómico de la maduración en uvas de ‘Tempranillo’ y ‘Albariño’ (Vitis vinífera L.) clasificadas según su densidad. Acta Hortic 60:554–557Google Scholar
  16. Casado-Vela J, Sellés S, Bru R (2005) Purification and kinetic characterization of polyphenol oxidase from tomato fruits (Lycopersicon esculentum Cv. Muchamiel). J Food Biochem 29:381–401CrossRefGoogle Scholar
  17. Chamkha M, Cathala B, Cheynier V, Douillard R (2003) Phenolic composition of champagnes from Chardonnay and Pinot Noir vintages. J Agric Food Chem 51:3179–3184CrossRefGoogle Scholar
  18. Dehon L, Mondolot L, Durand M, Chalies C, Andary C, Macheix JJ (2001) Differential compartmentation of o-diphenols and peroxidase activity in the inner sapwood of the Juglans nigra tree. Plant Physiol Biochem 39:473–477CrossRefGoogle Scholar
  19. Fang F, Tang K, Huang WD (2013) Changes of flavonol synthase and flavonol contents during grape berry development. Eur Food Res Technol 237:529–540CrossRefGoogle Scholar
  20. Fontes N, Gerós H, Delrot S (2011) Grape berry vacuole: a complex and heterogeneous membrane system specialized in the accumulation of solutes. Am J Enol Vitic 62:270–278CrossRefGoogle Scholar
  21. Fornara V, Onelli E, Sparvoli F, Rossoni M, Aina R, Marino G, Citterio S (2008) Localization of stilbene synthase in Vitis vinifera L. during berry development. Protoplasma 233:83–93CrossRefGoogle Scholar
  22. Fraignier MP, Michaux-Ferrière N, Kobrehel K (2000) Distribution of peroxidases in durum wheat (Triticum durum). Cereal Chem 77(1):11–17CrossRefGoogle Scholar
  23. García-Florenciano E, Calderón AA, Pedreño MA, Muñoz R, Ros Barceló A (1991) The vacuolar localization of basic isoperoxidases in grapevine suspension cell cultures and its significance in índole-3-acetic acid catabolism. Plant Growth Regul 10:125–138CrossRefGoogle Scholar
  24. García-Lara S, Arnason JT, Díaz-Pontones D, González E, Bergvinson DJ (2007) Soluble peroxidase activity in maize endosperm associated with maize weevil resistance. Crop Sci 47:1125–1130CrossRefGoogle Scholar
  25. Garrido I, Llerena JL, Valdés ME, Mancha LA, Uriarte D, del Henar Prieto M, Espinosa F (2014) Effects of defoliation and water restriction on total phenols and antioxidant activities in grapes during ripening. OENO One 48(1):31–42CrossRefGoogle Scholar
  26. Garrido I, Uriarte D, Hernández M, Llerena JL, Valdés ME, Espinosa F (2016) The evolution of total phenolic compounds and antioxidant activities during ripening of grapes (Vitis vinifera L., cv. Tempranillo) grown in semiarid region: effects of cluster thinning and water deficit. Int J Mol Sci 17(11):1923CrossRefGoogle Scholar
  27. Gatto P, Vrhovsek U, Muth J, Segala C, Romualdi C, Fontana P, Pruefer D, Stefanini M, Moser C, Mattivi F, Velasco R (2008) Ripening and genotype control stilbene accumulation in healthy grapes. J Agric Food Chem 56(24):11773–11785CrossRefGoogle Scholar
  28. Gómez C, Terrier N, Torregrosa L, Vialet S, Fournier-Level A, Clotilde Verriès C, Souquet JM, Mazauric JP, Klein M, Cheynier V, Ageorges A (2009) Grapevine MATE-type proteins act as vacuolar H+−dependent acylated anthocyanin transporters. Plant Physiol 150:402–415CrossRefGoogle Scholar
  29. Grotewold E, Davies K (2008) Trafficking and sequestration of anthocyanins. Nat Prod Commun 3:1251–1258Google Scholar
  30. Grundhöfer P, Niemetz R, Schilling G, Gross GG (2001) Biosynthesis and sub-cellular distribution of hydrolyzable tannins. Phytochemistry 57:915–927CrossRefGoogle Scholar
  31. Hall D, De Luca V (2007) Mesocarp localization of a bi-functional resveratrol/hydroxycinnamic acid glucosyltransferase of Concord grape (Vitis lambrusca). Plant J 49:579–591CrossRefGoogle Scholar
  32. Hrazdina G, Jensen RA (1992) Spatial organization of enzymes in plant metabolic pathways. Annu Rev Plant Physiol Plant Mol Biol 43:241–267CrossRefGoogle Scholar
  33. Huang XM, Huang HB, Wang HC (2005) Cell walls of loosening skin in post-veraison grape berries lose structural polysaccharides and calcium while accumulate structural proteins. Sci Hortic 104:249–263CrossRefGoogle Scholar
  34. Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, Moon RC (1997) Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275(5297):218–220CrossRefGoogle Scholar
  35. Jeandet P, Bessis R, Gautheron B (1991) The production of resveratrol (3,5,4′-trihydroxystilbene) by grape berries in different developmental stages. Am J Enol Vitic 42:41–45Google Scholar
  36. Jeandet P, Douillet-Breuil A-C, Bessis R, Debord S, Sbaghi M, Adrian M (2002) Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism. J Agric Food Chem 50:2731–2741CrossRefGoogle Scholar
  37. Jorgensen K, Rasmussen AV, Morant M, Nielsen AH, Bjarnholt N, Zagrobelny M, Bak S, Møller BL (2005) Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol 8:280–291CrossRefGoogle Scholar
  38. Kitamura S, Shikazono N, Tanaka A (2004) TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J 37:104–114CrossRefGoogle Scholar
  39. Kochhar S, Kochhar VK, Khanduja SD (1979) Changes in the pattern of isoperoxidases during maturation of grape berries cv Gulabi as affected by ethephon (2-chloethyl) phosponic acid. Am J Enol Vitic 30:275–277Google Scholar
  40. Li XB (1991) Molecular structure and physiological function of enzymes in plant cell wall. Plant Physiol Commun 27(4):246–252Google Scholar
  41. Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215:239–247CrossRefGoogle Scholar
  42. López-Miranda S, Hernández-Sánchez P, Serrano-Martínez A, Hellín P, Fenoll J, Núñez-Delicado E (2011) Effect of ripening on protein content and enzymatic activity of Crimson Seedless table grape. Food Chem 127(2):481–486CrossRefGoogle Scholar
  43. Lucena MA, Romero-Aranda R, Mercado JA, Cuartero J, Valpuesta V, Quesada MA (2003) Structural and physiological changes in the roots of tomato plants over-expressing a basic peroxidase. Physiol Plant 118:422–429CrossRefGoogle Scholar
  44. Mayer AM (2006) Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 67:2318–2331CrossRefGoogle Scholar
  45. Mayer AM, Harel E (1979) Polyphenoloxidase in plants. Phytochemistry 18:193–215CrossRefGoogle Scholar
  46. Miesle TJ, Proctor A, Lagrimini LM (1991) Peroxidase activity, isoenzymes, and tissue localization in developing highbush blueberry fruit. J Am Soc Hortic Sci 116(5):827–830Google Scholar
  47. Murata M, Tsurutani M, Hagiwara S, Homma S (1997) Subcellular location of polyphenol oxidase in apples. Biosci Biotechnol Biochem 61(9):1495–1499CrossRefGoogle Scholar
  48. Olah AF, Mueller WC (1981) Ultrastructural localization of oxidative and peroxidative activities in a carrot suspension cell culture. Protoplama 106(3):231–248CrossRefGoogle Scholar
  49. Ortega-García F, Blanco S, Peinado MA, Peragón J (2007) Polyphenol oxidase and its relationship with oleuropein concentration in fruits and leaves of olive (Olea europaea) cv. ‘Picual’ trees during fruit ripening. Tree Physiol 28:45–54CrossRefGoogle Scholar
  50. Ortega-García F, Blanco S, Peinado MA, Peragón J (2008) Phenylalanine ammonia-lyase and phenolic compounds in leaves and fruits of Olea europaea L. cv. Picual during ripening. J Sci Food Agric 89:398–406CrossRefGoogle Scholar
  51. Pan QH, Wang L, Li JM (2009) Amounts and subcellular localization of stilbene synthase in response of grape berries to UV irradiation. Plant Sci 176:360–366CrossRefGoogle Scholar
  52. Parish RW (1972) The intracellular location of phenol oxidases, peroxidase and phosphatases in the leaves of spinach beet (Beta vulgaris L. subspecies vulgaris). FEBS J 31(3):446–455Google Scholar
  53. Poustka F, Irani NG, Feller A, Lu Y, Pourcel L, Frame K, Grotewold E (2007) A trafficking pathway for anthocyanins overlaps with the endoplasmic reticulum-to-vacuole protein-sorting route in Arabidopsis and contributes to the formation of vacuolar inclusions. Plant Physiol 145:1323–1335CrossRefGoogle Scholar
  54. Rivera AP, Restrepo P, Narváez CE (2004) Polifenoloxidasa y peroxidasa de pulpa de uva Caimarona (Pourouma cecropiifolia). Rev Colomb Quim 33(1):57–66Google Scholar
  55. Robbins RJ (2003) Phenolic acids in foods: an overview of analytical methodology. J Agric Food Chem 51:2866–2887CrossRefGoogle Scholar
  56. Robinson DS (1991) Peroxidase and catalase. Elsevier Press, New YorkGoogle Scholar
  57. Saslowsky D, Winkel-Shirley B (2001) Localization of flavonoid enzymes in Arabidopsis roots. Plant J 27:37–48CrossRefGoogle Scholar
  58. Saslowsky DE, Warek U, Winkel BSJ (2005) Nuclear localization of flavonoid enzymes in Arabidopsis. J Biol Chem 25:23735–23740CrossRefGoogle Scholar
  59. Sherman TD, Vaughn KC, Duke SO (1991) A limited survey of the phylogenetic distribution of polyphenol oxidase. Phytochemistry 30(8):2499–2506CrossRefGoogle Scholar
  60. Singleton VL, Trousdale EK (1992) Anthocyanin-tannin interactions explaining differences in polymeric phenols between white and red wines. Am J Enol Vitic 43(1):63–70Google Scholar
  61. Thipyapong P, Hunt MD, Steffens JC (2004a) Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility. Planta 220:105–117CrossRefGoogle Scholar
  62. Thipyapong PJ, Melkoninan J, Wolfe DW, Steffens JC (2004b) Suppression of polyphenol oxidases increases stress tolerance in tomato. Plant Sci 164:693–703CrossRefGoogle Scholar
  63. Treutter D (2005) Significance of flavonoids in plant resistance: a review. Plant Biol 7:581–591CrossRefGoogle Scholar
  64. Valero E, Sánchez-Ferrer A, Varon R, Garcia-Carmona F (1989) Evolution of grape polyphenol oxidase activity and phenolic content during maturation and vinification. Vitis 28(2)Google Scholar
  65. Vamos-Vigyazo L (1981) Polyphenol oxidase and peroxidase in fruits and vegetables. Crit Rev Food Sci Nutr 15:49–127CrossRefGoogle Scholar
  66. Vaughn KC, Duke SO (1981) Tissue localization of polyphenol oxidase in Sorghum. Protoplasma 108(3):319–327CrossRefGoogle Scholar
  67. Vaughn KC, Duke SO (1984) Function of polyphenol oxidase in higher plants. Physiol Plant 60:257–261CrossRefGoogle Scholar
  68. Vaughn KC, Lax AR, Duke SO (1988) Polyphenol oxidase: the chloroplast oxidase with no established function. Physiol Plant 72:659–796CrossRefGoogle Scholar
  69. Versari A, Parpinello GP, Tronielli GB, Ferrarini R, Giulivo C (2001) Stilbene compounds and stilbene synthase expression during ripening, wilting and UV treatment in grape cv. Corvina. J Agric Food Chem 49:5531–5536CrossRefGoogle Scholar
  70. Walker JRL, Ferrar PH (1998) Diphenol oxidases, enzyme-catalysed browning and plant disease resistance. Biotechnol Genet Eng Rev 15(1):457–498CrossRefGoogle Scholar
  71. Wang W, Tang K, Yang HR, Wen PF, Zhang P, Wang HL, Huang WD (2010) Distribution of resveratrol and stilbene synthase in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon) and the effect of UV-C on its accumulation. Plant Physiol Biochem 48:142–152CrossRefGoogle Scholar
  72. Winkel-Shirley B (1999) Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Plant Physiol 107:142–149CrossRefGoogle Scholar
  73. Zhao J, Dixon RA (2010) The ‘ins’ and ‘outs’ of flavonoid transport. Trends Plant Sci 15:72–80CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Área de Biología Celular, Departamento de Anatomía, Biología Celular y Zoología, Facultad de CienciasUniversidad de ExtremaduraBadajozSpain
  2. 2.Departamento de HortofruticulturaInstituto de Investigaciones Agrarias Finca “La Orden-Valdesequera”, CICYTEX, Junta de ExtremaduraBadajozSpain
  3. 3.Departamento de Enología, INTAEX, CICYTEXJunta de ExtremaduraBadajozSpain

Personalised recommendations