Skip to main content
SpringerLink
Log in

Expression and tissue and subcellular localization of anthocyanidin synthase (ANS) in grapevine

  • Original Article
  • Published:
Protoplasma Aims and scope Submit manuscript

Abstract

Anthocyanidin synthase (ANS) is one of the key enzymes in the biosynthesis of both anthocyanins and proanthocyanidins in grapevine. Although substantial researches have investigated ANS gene expression and regulation at the transcriptional level, little is yet known about protein expression and distribution in grapevine. Here, the expression and tissue and subcellular localization of ANS in different Cabernet sauvignon grapevine tissues were investigated by using the techniques of Western blotting, immunohistochemical localization, immuno-electron microscopy, and confocal microscopy. The results showed that the ANS was expressed in the grape berries, leaves, stems, petioles, and leaf buds. In grape berry skin and flesh, ANS expression is developmental dependent. Immunohistochemical analysis revealed that ANS is primarily distributed in the exocarp, mesocarp, and seed of the fruit; in palisade and spongy tissues of the leaves; in the primary phloem and pith ray in the stems; and in the growth point and leaf primordium of the leaf buds. Furthermore, at the subcellular level, the ANS was mainly localized in the cytoplasm regardless of cell types and some ANS were also found in the nucleus in the mesocarp vascular bundle and leaf bud cells. This research will give further insight for the biosynthesis and regulation of different flavonoid compounds in grapevine.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

ANR:

Anthocyanidin reductase

ANS:

Anthocyanidin synthase

BSA:

Bovine serum albumin

F3′5′H:

Flavonoid 3′, 5′-hydroxylase

IgG:

Immunoglobulin fractions

PAs:

Proanthocyanidins

PBS:

Phosphate-buffered saline

RSR:

Regular salt rinse solution

References

  • Abrahams S, Lee E, Walker AR, Tanner GJ, Larkin PJ, Ashton AR (2003) The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development. Plant J 35:624–636

    Article  PubMed  CAS  Google Scholar 

  • Adams DO (2006) Phenolics and ripening in grape berries. Am J Enol Vitic 57:249–256

    CAS  Google Scholar 

  • Berhow MA, Vandercook CE (1989) Biosynthesis of naringin and prunin in detached grapefruit. Phytochemistry 28:1627–1630

    Article  CAS  Google Scholar 

  • Bogs J, Downey MO, Harvey JS, Ashton AR, Tanner GJ, Robinson SP (2005) Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol 139:652–663

    Article  PubMed  CAS  Google Scholar 

  • Bogs J, Jaffe FW, Takos AM, Walker AR, Robinson SP (2007) The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol 143:1347–1361

    Article  PubMed  CAS  Google Scholar 

  • Bondada BR, Matthews MA, Shackel KA (2005) Functional xylem in the post-veraison grape berry. J Exp Bot 56:2949–2957

    Article  PubMed  CAS  Google Scholar 

  • Boss PK, Davies C, Robinson SP (1996a) Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape berries and the implications for pathway regulation. Plant Physiol 111:1059–1066

    PubMed  CAS  Google Scholar 

  • Boss PK, Davies C, Robinson SP (1996b) Expression of anthocyanin biosynthesis pathway genes in red and white grapes. Plant Mol Biol 32:565–569

    Article  PubMed  CAS  Google Scholar 

  • Braidot E, Zancani M, Petrussa E, Peresson C, Bertolini A, Patui S, Macrì F, Vianello A (2008a) Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.). Plant Signal Behav 3:626–632

    Article  PubMed  Google Scholar 

  • Braidot E, Petrussa E, Bertolini A, Peresson C, Ermacora P, Loi N, Passamonti S, Terdoslavich M, Macri F, Vianello A (2008b) Evidence for a putative flavonoid translocator similar to mammalian bilitranslocase in grape berries (Vitis vinifera. L.) during ripening. Planta 228:203–213

    Article  PubMed  CAS  Google Scholar 

  • Buer CS, Muday GK, Djordjevic MA (2007) Flavonoids are differentially taken up and transported long distances in Arabidopsis. Plant Physiol 145:478–490

    Article  PubMed  CAS  Google Scholar 

  • Cadot Y, Minana-castelloa 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 54:9206–9215

    Article  PubMed  CAS  Google Scholar 

  • Castellarin SD, PfeiVer A, Sivilotti P, Degan M, Peterlunger E, Di Gaspero G (2007) Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit. Plant Cell Environ 30:1381–1399

    Article  PubMed  CAS  Google Scholar 

  • Chamkha M, Cathala B, Cheynier V, Douillard R (2003) Phenolic composition of champagnes from Chardonnay and Pinot Noir vintages. J Agric Food Chem 51:3179–3184

    Article  PubMed  CAS  Google Scholar 

  • Charrier B, Trinh H, Poirier S, Kondorosi A, Ratet P (1998) Flavanone 3-hydroxylase (F3H) expression and flavonoid localization in nodules of three legume plants reveal distinct tissue specificities. Mol Plant Microbe In 11:924–932

    Article  CAS  Google Scholar 

  • Chen JY, Wen PF, Kong WF, Pan QH, Wan SB, Huang WD (2006) Changes and subcellular localizations of the enzymes that involved in phenylpropanoid metabolism during grape berry development. J Plant Physiol 163:115–127

    Article  PubMed  CAS  Google Scholar 

  • Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33–38

    Article  PubMed  CAS  Google Scholar 

  • Deytieux C, Geny L, Lapaillerie D, Claverol S, Bonneu M, Donèche B (2007) Proteome analysis of grape skins during ripening. J Exp Bot 58:1851–1862

    Article  PubMed  CAS  Google Scholar 

  • Famiani F, Walker RP, Tecsi L, Chen ZH, Proietti P, Leegood RC (2000) An immunohistochemical study of the compartmentation of metabolism during the development of grape berries. J Exp Bot 51:675–683

    Article  PubMed  CAS  Google Scholar 

  • Feucht W, Dithmar H, Polster J (2004) Nuclei of tea flowers as targets for flavanols. Plant Biol 6:696–701

    Article  PubMed  CAS  Google Scholar 

  • Fischer JFA (1968) Procedure for obtaining radioactive naringin from grapefruit leaves fed L-phenylalanine-14C. Phytochemistry 7:769–771

    Article  Google Scholar 

  • Gholami M (2004) Biosynthesis of anthocyanins in Shiraz grape berries. Acta Hortic (ISHS) 640:353–359

    CAS  Google Scholar 

  • Gollop R, Farhi S, Perl A (2001) Regulation of the leucoanthocyanidin dioxygenase gene expression in Vitis vinifera. Plant Sci 161:579–588

    Article  CAS  Google Scholar 

  • Gomez 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 150:402–415

    Article  PubMed  CAS  Google Scholar 

  • Gould KS, Markham KR, Smith RH, Goris JJ (2000) Functional role of anthocyanins in the leaves of Quintinia serrata A Cunn. J Exp Bot 51:1107–1115

    Article  PubMed  CAS  Google Scholar 

  • Grotewold E, Davies K (2008) Trafficking and sequestration of anthocyanins. Nat Prod Commun 3:1251–1258

    CAS  Google Scholar 

  • Halliwell B, Rafter J, Jenner A (2005) Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not? Am J Clin Nutr 81:268s–276s

    PubMed  CAS  Google Scholar 

  • Harborne JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504

    Article  PubMed  CAS  Google Scholar 

  • Hou ZX, Huang WD (2005) Immunohistochemical localization of IAA and ABP1 in strawberry shoot apexes during floral induction. Planta 222:678–687

    Article  PubMed  CAS  Google Scholar 

  • Hrazdina G, Jensen RA (1992) Spatial organization of enzymes in plant metabolic pathways. Ann Rev Plant Physiol Plant Mol Biol 43:241–267

    Article  CAS  Google Scholar 

  • Isla MI, Vattuone MA, Sampietro AR (1998) Essential group at the active site of Frapaeolum invertase. Phytochemistry 47:1189–1193

    Article  CAS  Google Scholar 

  • 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 8:280–291

    Article  PubMed  CAS  Google Scholar 

  • Kaltenbach M, Schroder G, Schmelzer E, Lutzl V, Schroder J (1999) Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants. Plant J 19:183–193

    Article  PubMed  CAS  Google Scholar 

  • Kennedy JA, Troup GJ, Pilbrow JR, Hutton DR, Hewitt D, Hunter CR, Ristic R, Iland PG, Jones GP (2000) Development of seed polyphenols in berries from Vitis vinifera L.cv. Shiraz. Aust J Grape Wine Res 6:244–254

    Article  CAS  Google Scholar 

  • Kitamura S, Shikazono N, Tanaka A (2004) TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J 37:104–114

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Nakajima J, Tanaka Y, Yamazaki M, Saito K (2001) Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis. J Biol Chem 276:25797–25803

    Article  PubMed  CAS  Google Scholar 

  • Naoumkina M, Farag MA, Sumner LW, Tang Y, Liu CJ, Dixon RA (2007) Different mechanisms for phytoalexin induction by pathogen and wound signals in Medicago truncatula. Proc Natl Acad Sci U S A 104:17909–17915

    Article  PubMed  CAS  Google Scholar 

  • Oparka KJ, Turgeon R (1999) Sieve elements and companion cells—traffic control centers of the phloem. Plant Cell 11:739–750

    Article  PubMed  CAS  Google Scholar 

  • Orak HH (2007) Total antioxidant activities, phenolics, anthocyanins, polyphenoloxidase activities of selected red grape cultivars and their correlations. Sci Hortic 111:235–241

    Article  CAS  Google Scholar 

  • Pang YZ, Peel GJ, Wright E, Wang ZY, Dixon RA (2007) Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol 145:601–615

    Article  PubMed  CAS  Google Scholar 

  • Pinelo M, Arnous A, Meyer AS (2006) Upgrading of grape skins: significance of plant cell-wall structural components and extraction techniques for phenol release. Trends Food Sci Tech 17:579–590

    Article  CAS  Google Scholar 

  • Polster J, Dithmar H, Burgemeister R, Friedemann G, Feucht W (2006) Flavonoids in plant nuclei: detection by laser microdissection and pressure catapulting (LMPC), in vivo staining, and uv–visible spectroscopic titration. Physiol Plantarum 128:163–174

    Article  CAS  Google Scholar 

  • 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 145:1323–1335

    Article  PubMed  CAS  Google Scholar 

  • Reddy AM, Reddy VS, Scheffler BE, Wienand U, Reddy AR (2007) Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential. Metab Eng 9:95–111

    Article  PubMed  CAS  Google Scholar 

  • Ruelland E, Campalans A, Selman-Housein G, Puigdomenech P, Rigau J (2003) Cellular and subcellular localization of the lignin biosynthetic enzymes caffeic acid-O-methyltransferase, cinnamyl alcohol dehydrogenase and cinnamoyl-coenzyme A reductase in two monocots, sugarcane and maize. Plant Physiol 117:93–98

    Article  CAS  Google Scholar 

  • Saito K, Yamazaki M (2002) Biochemistry and molecular biology of the late-stage of biosynthesis of anthocyanin: lessons from Perilla frutescens as a model plant. New Phytol 155:9–23

    Article  CAS  Google Scholar 

  • Saito K, Kobayshi M, Gong ZZ, Tanaka Y, Yamazaki M (1999) Direct evidence for anthocyanidin synthase as a 2-Oxoglutarate-dependent oxygenase: molecular cloning and functional expression of cDNA from a red forma of perilla frutescens. Plant J 17:181–189

    Article  PubMed  Google Scholar 

  • Saslowsky D, Winkel-Shirley B (2001) Localization of flavonoid enzymes in Arabidopsis roots. Plant J 27:37–48

    Article  PubMed  CAS  Google Scholar 

  • Saslowsky DE, Warek U, Winkel BS (2005) Nuclear localization of flavonoid enzymes in Arabidopsis. J Biol Chem 280:23735–23740

    Article  PubMed  CAS  Google Scholar 

  • Sheoran IS, Dumonceaux T, Datla R, Vipen K, Sawhney VK (2006) Anthocyanin accumulation in the hypocotyl of an ABA-over producing male-sterile tomato (Lycopersicon esculentum) mutant. Physiol Plantarum 127:681–689

    Article  CAS  Google Scholar 

  • Shimada S, Inoue YT, Sakuta M (2005) Anthocyanidin synthase in non-anthocyanin-producing Caryophyllales species. Plant J 44:950–959

    Article  PubMed  CAS  Google Scholar 

  • Tian L, Wan SB, Pan QH, Zheng JC, Huang WD (2008) A novel plastid localization of chalcone synthase in developing grape berry. Plant Sci 175:431–436

    Article  CAS  Google Scholar 

  • Turnbull JJ, Nakajima J, Welford RW, Yamazaki M, Saito K, Schofield CJ (2004) Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3′-hydroxylase. J Biol Chem 279:1206–1216

    Article  PubMed  CAS  Google Scholar 

  • Ueda T, Yamaguchi M, Uchimiya H, Nakano A (2001) Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. J Exp Bot 20:4730–4741

    CAS  Google Scholar 

  • Wang HL, Wang W, Zhang P, Pan QH, Zhan JC, Huang WD (2010) Gene transcript accumulation, tissue and subcellular localization of anthocyanidin synthase (ANS) in developing grape berries. Plant Sci. 10.1016/j.plantsci.2010.04.002

  • Wen PF, Chen JY, Kong WF, Pan QH, Wan SB, Huang WD (2005) Salicylic acid induced the expression of phenylalanine ammonia-lyase gene in grape berry. Plant Sci 169:928–934

    Article  CAS  Google Scholar 

  • Winkel-Shirley B (1999) Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Plant Physiol 107:142–149

    Article  CAS  Google Scholar 

  • Yu XH, Chen MH, Liu CJ (2008) Nucleocytoplasmic-localized acyltransferases catalyze the malonylation of 7-O-glycosidic (iso)flavones in Medicago truncatula. Plant J 55:382–396

    Article  PubMed  CAS  Google Scholar 

  • Zhang W, Curtin C, Kikuchi M, Franco C (2002) Integration of jasmonic acid and light irradiation for enhancement of anthocyanin biosynthesis in Vitis vinifera suspension cultures. Plant Sci 162:459–468

    Article  Google Scholar 

  • Zhao J, Dixon RA (2010) The ‘ins’ and ‘outs’ of flavonoid transport. Trends Plant Sci 15:72–80

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the National Natural Science Foundation of China (grant numbers 30871747) and major program of Beijing Municipal Science and Technology Commission (no. D07060500160701).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China

    Huiling Wang, Wei Wang, Hui Li, Ping Zhang, Jicheng Zhan & Weidong Huang

  2. Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Science, Beijing, 100093, China

    Wei Wang

Authors
  1. Huiling Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ping Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jicheng Zhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Weidong Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Weidong Huang.

Additional information

Handling Editor: Hanns H. Kassemeyer

Huiling Wang and Wei Wang contribute equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

Scheme of the grapevine seedling to show the experimental sampling sites, including: leaf bud, stem tip, leaf, root, stem (stem phloem and stem xylem). (GIF 345 kb)

High resolution image (TIFF 233 kb)

Supplementary Fig. 2

Subcellular localization of ANS–GFP fusion protein in the Arabidopsis protoplasts. ANS–GFP fusion protein was transiently expressed from its chimeric gene, and the localization of the fusion proteins was detected by confocal laser scanning microscopical imaging of GFP fluorescence. a–c Transient expression of the green fluorescent protein (GFP)–ANS fusion protein showing that the fusion protein was localized to the cytoplasm. d–f GFP of PEZS-NL empty vector expressed in the Arabidopsis protoplast, and no fluorescences were observed in the control protoplasts. The laser-scanning confocal microscopy images are the ANS–GFP fluorescence (a, d), merged images (b, e), and bright-field image (c, f) (GIF 128 kb)

High resolution image (TIFF 2281 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, H., Wang, W., Li, H. et al. Expression and tissue and subcellular localization of anthocyanidin synthase (ANS) in grapevine. Protoplasma 248, 267–279 (2011). https://doi.org/10.1007/s00709-010-0160-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-010-0160-6

Keywords

Search

Navigation