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The Grapevine R2R3-MYB Transcription Factor VvMYBF1 Regulates Flavonol Synt

Plant Physiology Preview. Published on September 9, 2009, as DOI:10.1104/pp.109.142059 Running title: Transcriptional Regulation of Flavonol Synthesis in Grapevine

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Corresponding author: Dr. Jochen Bogs, Heidelberger Institut für Pflanzenwissenschaften

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(HIP), Im Neuenheimer Feld 360, 69120 Heidelberg, Germany

phone: +49 6221 545537

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email: jbogs@hip.uni-heidelberg.de

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Research Area: System Biology, Molecular Biology and Gene Regulation

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The Grapevine R2R3-MYB Transcription Factor VvMYBF1 Regulates Flavonol

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Synthesis in Developing Grape Berries.

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Stefan Czemmel1, Ralf Stracke2, Bernd Weisshaar2, Nicole Cordon3,4,5, Nilangani N.

Harris3, Amanda R. Walker3,5, Simon P. Robinson3,5 and Jochen Bogs1*

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1Heidelberger Institut für Pflanzenwissenschaften, Im Neuenheimer Feld 360, 69120

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Heidelberg, Germany (S.C, J.B); 2Bielefeld University, Department of Biology, Genome 8

Research, 33594 Bielefeld, Germany (R.S,B.W); 3CSIRO Plant Industry, PO Box 350,

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Glen Osmond, 5064, Australia (N.C, N.N.H, A.R.W, S.P.R); 4Department of Horticulture,

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Viticulture and Oenology, The University of Adelaide, PMB 1, Glen Osmond, SA 5064 Australia (N.C.) 5CRC for Viticulture, PO Box 154, Glen Osmond, SA 5064, Australia

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(N.C., A.R.W., S.P.R.)

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Footnotes

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This research was financial supported by the Bundesministerium für Bildung und

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Forschung (BMBF) and its initiative Genomanalyse im biologischen System Pflanze 5

(GABI).

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CSIRO Plant Industry is a partner in the Wine Innovation Cluster and this research was 7

supported by the Grape and Wine Research and Development Corporation.

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*Corresponding author: Dr. Jochen Bogs

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phone: +49 6221 545537

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email: jbogs@hip.uni-heidelberg.de

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ABSTRACT

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Flavonols are important UV-protectants in many plants and contribute substantially 3

to the quality and health promoting effects of fruits and derived plant products. To study

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regulation of flavonol synthesis in fruit we isolated and characterized the grapevine (Vitis 5

vinifera L. cv ′Shiraz′) R2R3-MYB transcription factor VvMYBF1. Transient reporter

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assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and 7

several other promoters of grapevine and Arabidopsis thaliana genes involved in flavonol 8

synthesis. Expression of VvMYBF1 in Arabidopsis mutant myb12 resulted in

complementation of its flavonol-deficient phenotype and confirmed the function of

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VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of

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VvMYBF1 throughout grape berry development revealed its expression during flowering 12

and in skins of ripening berries, which correlates with the accumulation of flavonols and 13

expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression 14

was light-inducible, implicating VvMYBF1 in control of VvFLS1 transcription. Sequence 15

analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in

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promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid 17

sequence we identified the previously described SG7 domain and an additional sequence 18

motif conserved in several plant MYB factors. The described motifs have been used to

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identify MYB transcription factors from other plant species putatively involved in

regulation of flavonol biosynthesis. This is the first functional characterization of a light-

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inducible MYB transcription factor controlling flavonol synthesis in fruit.

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INTRODUCTION

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In plants, flavonoids represent a highly diverse class of low molecular weight

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polyphenolic compounds, synthesized via the general phenylpropanoid pathway, giving rise 4

to three major classes: anthocyanins, proanthocyanidins (PAs) and flavonols (Fig. 1).

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Flavonols play pivotal roles in fresh fruit and fruit products, including grape berries and

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wine, and contribute substantially to their taste, quality and nutrition. They exhibit anti-

inflammatory, antioxidant, and anti-proliferative capacities and thereby account for health 7

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promoting effects of the consumption of grapes and many other fruits (Havsteen et al.,

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2002; Butelli et al., 2008). Grapevine (Vitis vinifera) synthesizes derivatives of the

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flavonols quercetin, kaempferol, isorhamnetin and myricetin mainly found as glucosides 11

and glucuronides in grape berries (Cheynier and Rigaud, 1986; Price et al., 1995). These 12

flavonol glycosides have been shown to affect anthocyanin colour in wine and aqueous

solution by copigmentation (Dimitri?-Markovi? et al., 2005). Flavonols also protect plants

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against UV light. In this regard, sunlight and UV-B were shown to increase concentration 15

of quercetin glycosides in grapevine berries (Price et al., 1995; Downey et al., 2007),

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petunia (Ryan et al., 2002), mustard (Beggs et al., 1987) and soybean (Middleton et al.,

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1993) while a flavonol synthase (FLS)mutant was shown to be incapable of flavonol

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production in response to UV radiation in Arabidopsis thaliana (Wisman et al., 1998).

Interestingly, flavonol synthesis can also be induced by exposure to sunlight at stages

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during grape berry development when flavonols are normally not accumulating (Downey et al., 2004; Matus et al., 2009). Besides their important function as UV protection shields,

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flavonols are thought to be involved in regulation of auxin transport (Peer et al., 2004; Buer 23

and Muday, 2004; Besseau et al., 2007; Santelia et al., 2008). In plant sexual reproduction, 24

flavonols were identified as active compounds necessary and sufficient to allow formation 25

of functional pollen tubes thus complementing chalcone synthase (CHS) deficient petunia 26

(Mo et al., 1992), and promote Nicotiana tabacum pollen germination and pollen tube

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growth in vitro (Ylstra et al., 1992).

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The biosynthetic pathway forming the basis of accumulation of flavonoids has been 29

elucidated using genetic and biochemical information from many plant species, initially in 30

parsley (Kreuzaler et al., 1983), subsequently also in maize, petunia, Antirrhinum, (Holton 31

and Cornish, 1995), Arabidopsis (Shirley et al., 1995) and more recently in fruit crops

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including apple (Takos et al., 2006b; Espley et al., 2007), bilberry (Jaakola et al., 2002) and

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grapevine (Boss et al., 1996; Bogs et al., 2005; Bogs et al., 2006). It is notable that

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flavonoid composition among plant species can be remarkably different. In Arabidopsis for 3

example 5′-hydroxylated flavonoids and catechin-based PAs are not present because the

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Arabidopsis genome does not contain genes encoding the enzymes flavonoid 3′,5′-

hydroxylase (F3′5′H) and leucoanthocyanidin reductase (LAR), which have been

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characterized from grapevine (Bogs et al., 2005; Bogs et al., 2006) and other plant species. 7

The synthesis of flavonol aglycones is catalyzed by flavonol synthase 1 (FLS1) which

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employs dihydroflavonols as substrates (Fig. 1). Among other enzymes that use the same

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substrate are flavonoid 3′-hydroxylase (F3′H) and F3′5′H, which mediate addition of

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hydroxyl groups to the B-ring of flavanones, flavones and dihydroflavonols (Hagmann et al., 1983; Kaltenbach et al., 1999) and dihydroflavonol 4-reductase (DFR), which directly

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competes with FLS1 for the same substrate dihydrokaempferol (Martens et al., 2002).

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VvFLS1 is expressed in leaves, tendrils, buds, inflorescences and developing grape berries.

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In the skin of Shiraz berries, VvFLS1 expression is highest between flowering and fruit set, 15

then declines to veraison, which is the onset of ripening, and increases again during

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ripening coincident with accumulation of flavonols per berry (Downey et al., 2003b).

Consistent with their role as important UV screens, flavonols accumulate mainly in flowers

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and skins of grape berries, but they have also been reported in leaves and stems. No

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detectable amounts of flavonols have been identified in pulp and seed of Chardonnay and 20

Shiraz berries and expression of VvFLS1 was also not detected in these tissues (Souquet et 21

al., 2000; Downey et al., 2003b).

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The flavonoid biosynthetic pathway genes are predominantly regulated at the level of transcription, both developmentally and/or in response to various biotic and abiotic stress

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factors. Transcriptional regulators for anthocyanin and PA synthetic pathways are mainly 25

members of protein families containing R2R3-MYB domains, basic helix–loop–helix

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(bHLH) domains (also referred to as MYC proteins) and conserved WD repeats (WDR)

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(Weisshaar and Jenkins, 1998; Stracke et al., 2001; Marles et al., 2003). In various plant

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species, the combination and interaction of R2R3-MYB, bHLH and WDR factors

determine the set of genes expressed (Quattrocchio et al., 1998; Walker et al., 1999; Baudry

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et al., 2004). Two of them, the MYB and bHLH proteins are conserved in regulation of

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anthocyanin and PA pathways in all species analyzed to date (Koes et al., 2005). In

vertebrates, the MYB gene family includes C-MYB, A-MYB, and B-MYB, whereas in

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plants, the MYB family is much more extensive encompassing 126 R2R3-MYB genes in 3

the Arabidopsis genome which are involved in a wide range of developmental and defence 4

processes. All R2R3-MYB family members share two highly conserved imperfect repeats 5

(R2, R3) at the N-terminus, while the C-terminus is highly diverse (Stracke et al., 2001).

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The bHLH proteins may have overlapping regulatory targets (Zimmermann et al., 2004), but MYB proteins are believed to be key components by allocation of specific gene

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expression patterns (Stracke et al., 2001). In grapes, the MYB proteins MYBA1 & 2

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(Kobayashi et al., 2002; Walker et al., 2007); MYB5a(Deluc et al., 2006), MYB5b (Deluc 10

et al., 2008), and MYBPA1 (Bogs et al., 2007) act together with bHLH proteins and

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probably WDR proteins to control anthocyanin and/or PA synthesis in grape berries. Most 12

R2R3-MYB factors regulating flavonoid biosynthesis (except flavonol synthesis) depend on cofactors, including WDR proteins but, most importantly a small subgroup of bHLH

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proteins that share a common motif in their N termini that interacts with a signature motif 15

in the R3 repeat of the N terminal R2R3 domain of MYB factors (Grotewold et al., 2000).

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The first MYB factor that was shown to be active without binding a bHLH protein was

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ZmP from Zea mays, activating a subset of genes for 3-deoxyflavonoids and phlobaphene 18

biosynthesis (Grotewold et al., 1994). Recently, the first R2R3-MYB factors

(MYB12/PFG1, MYB11/PFG2 and MYB111/PFG3) regulating flavonol synthesis have

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been described in Arabidopsis (Mehrtens et al., 2005; Stracke et al., 2007). They are also cofactor-independent and individually regulate flavonol accumulation in different organs of

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developing seedlings (Stracke et al., 2007). MYB12 mainly controls flavonol biosynthesis 23

in roots, while MYB111 predominantly regulates biosynthesis in cotyledons. Flavonol

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accumulation requires these three factors because triple mutant myb11-12-111 seedlings

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can not produce flavonol glycosides, while synthesis of anthocyanins is not affected.

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Targets of these MYB factors are CHS, chalcone isomerase (CHI), flavanone 3-

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hydroxylase (F3H), FLS and UDP glycosyltransferases (UGT) family 1 genes (Mehrtens et 28

al., 2005; Stracke et al., 2007).

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Luo et al. (2008) showed that AtMYB12 can be used to produce transgenic

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tomatoes containing high amounts of flavonols in fruits. However, to improve flavonol

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content and composition of crops via agricultural management strategies or marker-assisted 32

breeding the endogenous genes and regulatory mechanisms of flavonol synthesis have to be

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examined. Recently, the genome sequence of V. vinifera has become available (Jaillon et

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al., 2007) and has provided a genomic platform for Matus et al. (2008) to classify 108

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members of the grape R2R3-MYB gene subfamily in terms of their genomic gene structures

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and similarity to their putative Arabidopsis orthologues. A partial cDNA clone (accession

no. FJ418175) of an AtMYB12-like gene was identified (Matus et al., 2008 and 2009),

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which is identical to the 3′ coding region of the flavonol regulator gene VvMYBF1.

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Grapevine is one of studied crop plant in terms of regulation of flavonoid synthesis

by transcription factors in fruit and regulators of flavonoid biosynthesis have also been

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found in other fruit-producing plants including strawberry (Aharoni et al., 2001) and apple

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(Takos et al., 2006a; Espley et al., 2007). However, a transcription factor which regulates

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flavonol synthesis in fruit has not been functionally characterized so far in grapevine or any

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other plant species. To elucidate regulation of flavonol synthesis in fruit, the flavonol-

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specific MYB transcription factor VvMYBF1 (accession no.FJ948477) from V. vinifera L.

cv ′Shiraz′ was characterized. The expression pattern of VvMYBF1 directly correlated with

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that of VvFLS1 and subsequent flavonol accumulation during grape berry development.

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Further, expression of both VvFLS1 and VvMYBF1 was strongly induced in grapevine cell

culture after exposure to light. VvMYBF1 specifically induced promoters of VvFLS1 and

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VvCHI, which are involved in flavonol synthesis, whereas promoters of genes involved in

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anthocyanin or PA synthesis were not controlled by VvMYBF1. Functionality of

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VvMYBF1 was shown by complementation of the flavonol-deficient phenotype of the

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Arabidopsis mutant myb12. Using amino acid motifs found to be conserved in flavonol

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regulators from Arabidopsis and grapevine, several other putative flavonol regulators have

been identified, providing a tool to screen plant databases for putative flavonol regulators 23

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from other fruit-producing crops.

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RESULTS

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Isolation and Sequence Analysis of the MYB Transcription Factor VvMYBF1

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Using the conserved R2R3 repeat region and the SG7 motif encoded by the

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AtMYB12 gene (CAB09172, Stracke et al., 2001), a similar sequence was detected in the

Vitis vinifera genomic sequence (highly homozygous genotype PN40024, Jaillon et al.,

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2007) provided by the French National Sequencing Center Genoscope. Primers were

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designed to the 5′ and 3′ untranslated regions (UTR) of the predicted gene to amplify a

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fragment of 1125 bp including the open reading frame (ORF), which was named

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VvMYBF1. The VvMYBF1 ORF was amplified from the cultivars V. vinifera L. cv ′Shiraz′

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and ′Chardonnay′ cDNA and sequence analysis showed a difference in two amino acids at

position 213 and 359 of the encoded protein sequences. The VvMYBF1 ORF isolated from 11

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Chardonnay cDNA was submitted to GenBank (accession no. GQ423422). The

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transcriptional start of the VvMYBF1 gene from Shiraz was determined by 5′ RACE and the 14

complete 5′ UTR and the ORF of VvMYBF1 were supplied to GenBank with the accession

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no. FJ948477. The ORF encodes a predicted protein sequence of 367 amino acid residues

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with a molecular mass of 40.9 kD and a pI of 4.98. Analysis of the deduced amino acid

sequence revealed that VvMYBF1 contains an N-terminal R2R3 repeat that corresponds to

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the DNA binding domain of plant MYB-type proteins (Fig. 2A). Although sequence

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similarity between MYB proteins is generally restricted to the N-terminus, the SG7 motif

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(GRTxRSxMK, Stracke et al., 2001) characteristic of flavonol regulators of Arabidopsis

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was also found to be present in the C-terminus of VvMYBF1 with one amino acid

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substitution (GRTSRWAMK). Additionally, one previously unidentified motif designated

SG7-2 ([W/x][L/x]LS) was detected at the C-terminal ends of AtMYB12, AtMYB11,

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AtMYB111 and VvMYBF1. The conserved R2R3 repeat and the two SG7 motifs have

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been used as a tool to identify further putative flavonol-specific transcriptional regulators of

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other plant species including Sorghum bicolor, Lotus japonicus, Oryza sativa and Gerbera

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hybrida (Fig. 2A). We could also detect another R2R3-MYB transcription factor from V.

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vinifera on chromosome 5 (accession no. GSVIVT00033103001) that we named

VvMYBF2 with both, the SG7 and SG7-2 motifs differing significantly from the other

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flavonol MYB-type transcription factors. The homology within the SG7 domain in the

other putative flavonol regulatory proteins allowed us to expand and redefine the motif

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introduced by Stracke et al. (2001) to ([K/R][R/x][R/K]xGRT[S/x][R/G]xx[M/x]K). Except 1

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in VvMYBF2, the SG7 motif is not conserved in SlMYB12 from Solanum lycopersicum

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while the SG7-2 domain is less conserved in ZmP and Malus x domestica MdMYB22 (Fig. 4

2A). Phylogenetic analysis revealed the similarity of VvMYBF1 to the flavonol regulators 5

from Arabidopsis, AtMYB12, AtMYB11 and AtMYB111 and other plant MYB proteins, 6

(Fig. 2B). The predicted protein sequence of VvMYBF1 was most closely related to

MdMYB22 with 92 % identity in the R2R3 domain, while complete protein sequence

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showed only 39 % identity. The similarity between the R2R3 domains of VvMYBF1 and 9

AtMYB12, AtMYB11 and AtMYB111 (Fig. 2A), which have been shown to regulate

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flavonol synthesis in young seedlings of Arabidopsis (Stracke et al., 2007), was 83, 81 and 11

85 % amino acid identity, respectively. The complete protein sequence of VvMYBF1

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showed 41 % amino acid identity to AtMYB12, 45 % identity to AtMYB11 and 38 %

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identity to AtMYB111. Additionally, the R2R3 repeat region of VvMYBF1 does not

contain the motif [D/E]Lx2[R/K]x3Lx6Lx3R for interaction with bHLH proteins

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(Zimmermann et al., 2004).

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Expression of VvMYBF1 Correlates with Flavonol Accumulation during Fruit

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Development

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To determine if VvMYBF1 expression during grape berry development correlates with

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flavonol accumulation we analyzed Shiraz grape berries collected during the season 2000-21

2001 by quantitative PCR (qPCR). The identical Shiraz developmental series was

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previously used by Downey et al. (2003b) and (2004) to analyze VvFLS1 expression and flavonol accumulation. The pattern of VvMYBF1 expression during early grape

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development, in skins and seeds is shown in Figure 3A-C. VvMYBF1 expression was

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highest around flowering from nine to seven weeks prior to veraison (Fig. 3A). This

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expression of VvMYBF1 in developing flowers and grape berries with highest expression 27

throughout flowering correlates with the expression of VvFLS1 and flavonol accumulation 28

in developing Shiraz berries (Downey et al., 2003b and 2004). In berry skins beginning

with four weeks before veraison, VvMYBF1 was expressed in skins of developing berries

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with small peaks at four weeks prior to veraison, at veraison and six weeks after veraison 31

(Fig. 3B). Concomitantly, VvFLS1 expression and flavonol accumulation (measured as

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mg/berry) also peaked six weeks after veraison in Shiraz berry skins (Downey et al.,

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2003b). In seeds, VvMYBF1 was expressed only at very low levels during the entire grape

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berry development (Fig. 3C) coinciding with no detectable flavonol accumulation and no

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detectable expression of VvFLS1 in seeds of grape berries (Downey et al., 2003b).

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Induction of VvMYBF1 Expression by Light in Cell Cultures Correlates with

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Flavonol Accumulation

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Previous studies by Downey et al. (2004) demonstrated light induction of flavonol

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biosynthesis in Shiraz berries. To determine if light-induced synthesis of flavonols could be

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the result of an increase in VvMYBF1 gene expression, dark-grown Chardonnay petiole cell

cultures were exposed to light, and VvMYBF1 expression monitored over an eight day time 11

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period, and correlated with flavonol accumulation and VvFLS1 expression data. After

exposure to light, the transcript levels of VvMYBF1 increased by ~twenty-fourfold over the

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first twelve hours compared to expression in the dark after twelve hours. Transcript levels

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of VvMYBF1 continued to increase with light exposure leading to ~one hundred thirtyfold

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change in its expression by 24 hours (Fig. 4A). Concomitantly, the transcript level of

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VvFLS1 was also steadily increasing upon light exposure during the first 24 hours. VvFLS1

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transcript levels were highest after 24 hours of light induction, with a ~twelve thousand

three hundredfold increase compared to expression of VvFLS1 after 24 hours in the dark

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(Fig. 4B). Both transcripts rapidly decreased with 36 hours of light induction with almost

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no detectable expression of VvMYBF1 and low expression of VvFLS1 after two days of

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light induction (Fig. 4A, B). Corresponding with the increase in VvMYBF1 and VvFLS1

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transcript levels, flavonols were detected after two days of light exposure, with the highest

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levels of flavonols observed at day six of the light induction experiment (Fig. 4C,

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supplementary Fig. S2 A, B). The early induction of VvMYBF1 gene expression upon light

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exposure in Chardonnay cells correlates well with a role for this gene in regulating the

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expression pattern of VvFLS1. The subsequent increase in flavonol levels in Chardonnay

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cells confirms the inducibility of flavonol biosynthesis providing evidence that this

response is likely to be mediated by VvMYBF1 in response to light.

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After demonstrating that VvMYBF1 and VvFLS1 are light-inducible genes, the

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promoter sequences of VvMYBF1 and VvFLS1 from the grapevine cultivars Shiraz, Pinot

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Noir and Chardonnay were determined and bioinformatic analysis performed (for accession 2

no. see Material and Methods). Sequence analysis revealed that the VvMYBF1 and VvFLS1 3

promoter sequences of these three grapevine cultivars are very similar and contain several 4

putative light regulatory elements conserved between the cultivars and respective promoters (Fig. 4D, supplementary Fig. S1). Hartmann et al. (2005) presented evidence that light

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responsiveness of the Arabidopsis CHS promoter is mediated by a light regulatory unit

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(LRU) necessarily containing MYB recognition- (MRE) and bZIP recognition (ACE)

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sequence elements. This LRU is also present in the promoter of the VvMYBF1 and VvFLS1 9

genes with elements similar to MRE and ACE, which were designated MRS and ACS (Fig.

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4D, supplementary Fig. S1). By comparing the promoter sequences with the PLACE

database (Higo et al., 1999), additional putative light regulatory elements were identified in

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the VvFLS1 promoter (Fig. 4D, supplementary Fig. S1). These elements are present

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upstream of the LRU and include an IBOXCORE element involved in binding of MYB

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factors of light regulated genes in tomato (Rose et al., 1999) and a LREBOXIPCCHS1

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consensus sequence conferring light responsiveness in the parsley CHS1 promoter

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(Schulze-Lefert et al., 1989). In addition, two MYBCORE elements, which are similar to binding sites for MYB factors in Arabidopsis (Urao et al., 1993) and petunia PhMYB3

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(Solano et al., 1995) were identified in the VvFLS1 promoter while the VvMYBF1 promoter 19

shows one additional MRS located ~350 bp upstream to the transcriptional start codon (Fig.

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4D, supplementary Fig. S1 B). Except for the LRU none of the light regulatory elements 21

are present in the VvMYBF1 promoter. The VvMYBF1 promoter also contains elements

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similar to a R response element (RRS) that include the bHLH factor consensus binding site (Hartmann et al., 2005) indicating another level of regulation independent of light and

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which is absent in the first 1000 bp proximal to the transcriptional start of the VvFLS1 gene 25

(Fig. 4D, supplementary Fig. S1). The similarity of the described promoter sequences for 26

Shiraz, Pinot Noir and Chardonnay and the conservation of putative light-regulatory

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elements indicates that light-regulation of flavonol synthesis in red and white grapevine

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cultivars is very similar on the molecular level.

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VvMYBF1 Activates Promoters of the Flavonoid Pathway Genes Required for

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Flavonol Synthesis in Fruits

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To identify the structural genes of the flavonoid pathway activated by VvMYBF1, transient 2

expression experiments using Chardonnay grape suspension culture cells and the dual-

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luciferase assay system were conducted (Horstmann et al., 2004; Bogs et al., 2007).

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VvMYBF1 was introduced into pART7, a vector allowing expression of genes under control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter. The VvCHI promoter 5

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was chosen as an example for a general flavonoid pathway gene involved in synthesis of

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flavonols, anthocyanins and proanthocyanidins (PAs) (Fig. 1). In contrast, VvUFGT and

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VvANR are specifically required for anthocyanin and PA synthesis, respectively, whereas 9

VvFLS1catalyzes the final step leading to synthesis of flavonols (Fig. 1). The grapevine 10

promoters of the genes encoding these proteins were then tested as potential targets of

VvMYBF1. Figure 5A summarizes the results of the transient expression experiments

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carried out to compare the transactivation potential of VvMYBF1, AtMYB12, VvMYBA2 13

and VvMYBPA1 on the VvFLS1 promoter. VvMYBF1 activated the promoter of the gene 14

VvFLS1~tenfold and AtMYB12 activated the promoter ~threefold compared to the

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corresponding control, consisting of promoter without MYB factor, both showing their

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ability to induce the flavonol-specific branch point gene of grapevine. As negative controls, VvMYBPA1 and VvMYBA2 did not activate the VvFLS1 promoter confirming their

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specificity for PA- and anthocyanin synthesis, respectively. The addition of AtEGL3, a

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bHLH factor needed as a cofactor during transcription initiation by the transcription factors 20

VvMYBPA1 and VvMYBA2 in this assay, decreased the activation potential of

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VvMYBF1 on the VvFLS1 promoter (Fig. 5A). VvMYBF1 also induced the promoters of 22

the general flavonoid pathway gene VvCHI (~twelvefold, Fig. 5B) suggesting it can

activate the general pathway providing substrates for flavonol synthesis. The Arabidopsis

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homologue, AtMYB12 activated the VvCHI promoter approximately half as strong as the 25

grapevine factor indicating AtMYB12 binding and activation of the VvFLS1 promoter in a 26

heterologous system. As already published, VvMYBPA1 activates the VvCHI promoter

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(Bogs et al., 2007) to the same extent as VvMYBF1, showing the necessity of the general 28

pathway gene VvCHI for both PA and flavonol synthesis (Fig. 5B). VvMYBA2 does not activate the VvCHI promoter confirming previous results indicating that VvMYBA2

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specifically controls VvUFGT (Fig. 5E, Walker et al., 2007, Deluc et al., 2008). VvMYBF1 31

also displayed a potential to activate the leucoanthocyanidin dioxygenase (LDOX) promoter

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with a ~ninefold induction compared to the negative control (Fig. 5C). VvMYBPA1

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activates the VvANR promoter ~two hundred eightyfold, showing its specific

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transactivation potential for the PA branch of the pathway, whereas VvMYBA2 activates 4

the VvANR promoter only 0.6 fold, VvMYBF1 ~ninefold and AtMYB12 ~twofold (Fig.

5D). The anthocyanin-specific promoter of VvUFGT was strongly induced by VvMYBA2 5

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(~one hundred thirty-threefold), whereas VvMYBF1, VvMYBPA1 and AtMYB12 did not 7

activate the VvUFGT promoter substantially, therefore confirming the specificity of

8

VvMYBA2 to the VvUFGT promoter and of VvMYBF1 to the VvFLS1 promoter (Fig. 5E). 9

Finally basal levels of the promoters without effectors (referred to as controls in Fig. 5)

10

indicate that VvFLS1 and VvCHI promoters possess higher background activity compared to the promoters of the VvUFGT, VvANR and VvLDOX genes suggesting endogenous

11

12

transcription factors and/or environmental stress conditions that might induce these

13

promoters during the transient assay (Fig. 5F). To confirm that VvMYBF1 can also induce 14

pathway genes in the heterologous system of Arabidopsis, At7 cells were cotransfected

15

with VvMYBF1 and different Arabidopsis flavonoid pathway gene promoter constructs. 16

Figure 5G summarizes the results and shows that VvMYBF1 is able to induce AtCHS, the 17

initial enzyme in the flavonoid pathway and to a lower but still significant extent AtFLS but 18

not AtDFR whose gene product directly competes with FLS for the same substrate

19

(dihydroflavonols). In summary, these results suggest VvMYBF1 to be a specific regulator 20

of flavonol synthesis, potentially regulating the general flavonoid pathway (CHS, CHI) and the FLS1 gene specifically directing flavonoid precursors to flavonol formation in

21

22

grapevine.

23

24

Complementation of the Flavonol-Deficient Phenotype of the Arabidopsis Mutant

25

myb12 by Heterologous Expression of VvMYBF1

26

The MYB transcription factor AtMYB12 has been shown to control flavonol synthesis in the root of young Arabidopsis seedlings, while AtMYB111 controls flavonol biosynthesis

27

28

primarily in the cotyledons (Stracke et al., 2007). To confirm the function of VvMYBF1 as

a regulator of flavonol synthesis, VvMYBF1 was expressed under control of the root-

29

30

specific AtMYB12 promoter in Arabidopsis ecotype Columbia (Col-0) wild type and myb12 31

mutant plants. Whereas the myb12 mutant showed a flavonol-deficient phenotype in roots,

all BASTA-resistant independent transgenic Col-0 lines (Col-0/proAtMYB12::VvMYBF1 1

2

Col/VvMYBF1 01-10, ten lines) and transgenic myb12 lines

3

(myb12/proAtMYB12::VvMYBF1 010,-012,-013,-017,-018, five lines) transformed with

4

VvMYBF1 showed flavonol accumulation and therefore normal wild type phenotype in

5

roots. The accumulation patterns of flavonols in Col-0, myb12 and complemented mutants

6

have been visualized with in situ diphenylboric acid-2-aminoethylester(DPBA)staining of

flavonols. This is a powerful method for detection of flavonols in plant tissues that can be 7

8

used to differentiate in vivo between quercetin (orange fluorescence) and kaempferol

9

(greenish fluorescence) (Peer et al., 2001). In line with previous results of Stracke et al.

10

(2007), the Col-0 seedlings showed intense orange fluorescence five days after germination

11

(5 dag) indicating massive accumulation of quercetin (Fig. 6A). Root-specific expression of

12

VvMYBF1 in Col-0 (Col-0/proAtMYB12::VvMYBF1) did not alter flavonol-specific

staining in roots and cotyledons as well as hypocotyl root transition zone compared with

13

14

untransformed Col-0 (Fig. 6B). The myb12 mutant seedlings display drastically reduced

15

flavonol levels in the root and the hypocotyl root transition zone while flavonol

16

accumulation in cotyledons and the distal elongation zone in the root was unaffected (Fig.

17

6C). Complementation of the myb12 phenotype was obtained in roots of the transgenic

18

lines myb12/proAtMYB12::VvMYBF1 010, -012, -013, -017 and -018, which all express

VvMYBF1 under a root-specific promoter in myb12Arabidopsis seedlings and showed

19

20

orange staining indicative for quercetin accumulation in the root (Fig. 6D). To confirm in

vivo staining results, the total flavonol accumulation profiles of the complemented lines

21

22

were analyzed by high-performance liquid chromatography (HPLC), and compared with

23

flavonol profiles of Col-0, myb12 (supplementary Fig. S2 C) and myb11-12-111 mutant

24

lines. The triple mutant consistently displayed a dramatic reduction in flavonol content: no

25

flavonols were detected in this mutant compared to Col-0 (Stracke et al., 2007; Fig. 6E)

26

while the single mutant myb12 shows a fivefold decrease in accumulation of flavonols

27

relative to Col-0 (Fig.6E). Complemented lines always show significantly elevated levels of

28

flavonols compared to the myb12 mutant with lines m yb12/proAtMYB12::VvMYBF1 010,

-012 and -013 showing seven, nine and sixfold increased values compared to myb12,

29

30

respectively. These lines even show augmented levels of total flavonol content compared to

31

Col-0. Lines myb12/proAtMYB12::VvMYBF1 017 and -018 display lower levels of

32

flavonol accumulation (Fig. 6E) showing four- and threefold increased values compared to

myb12, although these lines exhibited the strongest staining phenotypes (Fig. 6D). These 1

2

results clearly support the in vivo staining data and confirm that VvMYBF1 is able to

3

complement the Arabidopis myb12 mutant deficient in flavonol synthesis. Therefore,

VvMYBF1 from V. vinifera is a functional R2R3-MYB transcription factor involved in 4

5

regulation of flavonol biosynthesis.

1

DISCUSSION

2

Amino-Acid Sequence Features of VvMYBF1 and the Flavonol Clade of MYB

3

Transcription Factors

4

Analysis of the amino-acid sequence of VvMYBF1 revealed the presence of the

highly conserved N-terminal R2R3 domain characteristic for MYB-related proteins. Similar 5

6

to the MYB12 factor from Arabidopsis and the MYB protein P from Zea mays, which

7

function independently of a bHLH cofactor (Mehrtens et al., 2005; Grotewold et al., 2000),

8

the R2R3 domain of VvMYBF1 does not contain the bHLH binding site for interaction

9

with R/B-like bHLH proteins (Zimmermann et al., 2004). This cofactor independency

10

seems to be specific for the MYB factors of the flavonol/phlobaphene clade, whereas other

MYB transcription factors require the interaction with the bHLH proteins to control gene

11

12

expression (Fig. 2B). Similar to the other 126 members of the R2R3-MYB protein family

13

in Arabidopsis, the R2R3 repeat region of VvMYBF1 is highly conserved, whereas the C-

14

terminal region is highly diverse (Stracke et al., 2001; Fig. 2A). Albeit this C-terminal

15

diversity the MYB factor families in Arabidopsis and rice have been categorized into

16

subgroups on the basis of conserved amino-acid sequence motifs detected in the C-terminus

of the MYB proteins (Kranz et al., 1998; Jiang et al., 2004). The C-terminal SG7 motif

17

18

described in Stracke et al. (2001) was also found in VvMYBF1 suggesting similarities in

19

function to the flavonol regulators of Arabidopsis. An additional previously unidentified

20

motif, SG7-2, was detected by examining the protein alignment of VvMYBF1, AtMYB12,

21

AtMYB11 and AtMYB111 at the very C-terminal end of these proteins (Fig. 2A). Due to

22

the high sequence similarity of the R2R3 domain of VvMYBF1 to the members of the

MYB subgroup 7 and the presence of the SG7 motif, VvMYBF1 was classified as a

23

24

putative flavonol regulator of the MYB subgroup 7. The R2R3 domain and the putative

25

flavonol regulator-specific SG7- and SG7-2 motifs were used to screen EST databases for

26

other putative flavonol regulators elucidating that both motifs are also present in MYB

27

factors of other plant species (Fig. 2A). Consensus sequences for both motifs have been re-

28

defined expanding previous results obtained by Stracke et al. (2001) for the SG7 motif (Fig.

2A). Both motifs were detected in the maize transcription factor ZmP, in MYB-type

29

30

transcription factors from Sorghum bicolor, Lotus japonicus, Oryza sativa, and in MYB1

31

from Gerbera hybrida (Fig. 2A). Single motifs have been detected in SlMYB12 from

Solanum lycopersicum and MdMYB22 from Malus x domestica which has been shown to 1

2

regulate CHS promoter of different plant species (Hellens et al., 2005), while neither motifs 3

could be detected in VvMYBF2, another R2R3-MYB transcription factor from grapevine 4

(Fig. 2A).Therefore, the combination of the SG7 and SG7-2 motifs with the R2R3 domain 5

will be a useful tool for the initial identification of R2R3-MYB proteins that might regulate 6

flavonol synthesis. Both motifs may be part of a specific functional domain outside the

DNA binding region of flavonol regulators. Evidence for the functional importance of C-

7

8

terminal motifs as activation domains of MYB proteins has been found for the MYB

9

factors MYB2 and WEREWOLF of Arabidopsis (Urao et al., 1996; Lee and Schiefelbein, 10

2001).

11

Additionally, sequence analysis revealed a highly conserved sequence N-terminal to 12

the R2R3 domain (MGR[A/T]PCC[E/D]K[V/I]G) in the presumptive MYB type flavonol regulators analyzed during this study (Fig. 2A). This motif is also present in VvMYBF2

13

14

and in the proanthocyanidin (PA)-specific regulator MYBPA1 but not in the anthocyanin 15

regulator MYBA2 (data not shown), indicating that, based on sequence similarities flavonol regulators seem to be more closely related to PA- than to anthocyanin regulators (Fig. 2B).

16

17

This hypothesis is intriguing, because the flavonoid biosynthetic pathway leading to

18

flavonols and PAs first concomitantly appeared in the group Filicopsida (ferns) while

19

anthocyanins finally made their appearance in the gymnosperms later during development 20

(Markham et al., 1988). It is worth considering if this motif did belong to the R2R3 domain 21

in the common ancestors of flavonoid regulators and was lost during the speciation process 22

leading to the formation of anthocyanin regulators but only further investigations regarding 23

evolutionary relationships between MYB-type transcription factors in plants can strengthen 24

this hypothesis.

25

In summary, our analyses clearly support the categorization of VvMYBF1 and the 26

other putative flavonol MYB factors from different plant species as members of the

27

flavonol/phlobaphene clade of R2R3-MYB transcription factors (Fig. 2A, B).

28

29

Spatiotemporal- and Light-Dependent Expression of VvMYBF1

During grape berry development the synthesis of flavonols, anthocyanins, PAs and

1

2

expression of the respective flavonoid pathway genes are temporally separated presumably 3

to avoid competition for common substrates during synthesis of the different flavonoid

4

compounds (Robinson and Davies, 2000; Downey et al., 2003a, b and 2004; Bogs et al., 5

2005). Flavonols accumulate before anthesis and increase during flowering, probably to

6

protect the inflorescence and pollen against UV light. After bloom, accumulation of

flavonols and expression of flavonol synthase1 (FLS1) decreases and PA synthesis is

7

8

induced (Downey et al., 2003a, b and 2004; Bogs et al., 2005). VvMYBF1 is highly

9

expressed in buds and flowers early in berry development with a decrease of expression 10

two weeks after flowering (Fig. 3). This early expression of VvMYBF1 correlates with the 11

accumulation of flavonols and expression of VvFLS1 during flowering, although

12

VvMYBF1 expression does not precede the highest expression of VvFLS1 and

accumulation of flavonols. It was shown that expression of transcription factors normally

13

14

precedes the expression of the corresponding regulated genes (Bogs et al., 2005; Bogs et 15

al., 2007). This consecutive induction of VvMYBF1 and VvFLS1 expression, might be

16

present in flower buds between nine and ten weeks before veraison, which have not been 17

analyzed. Additionally, the decrease of VvMYBF1 expression after flowering seems to be 18

delayed when compared with VvFLS1 expression, which may be due to an additional

function of VvMYBF1 or the repression of VvFLS1 by another factor. In developing grape

19

20

berries, flavonols seem to be present only in skins with an increase of synthesis and

VvFLS1 expression with ripening of the berries (Downey et al., 2003b). VvMYBF1 is

21

22

expressed in skins of berries and could regulate flavonol synthesis; however expression is 23

not increasing when berry ripening proceeds (Fig. 3). This increase of flavonol synthesis 24

before harvest of the berries could be regulated by VvMYBF2, a MYB factor also

25

belonging to the flavonol MYB factor clade (Fig. 2B). It will be interesting to analyze the 26

expression pattern of VvMYBF2 and its function in regulation of flavonol synthesis. The 27

transcription factor VvMYB5b does not belong to the SG7 flavonol subgroup of R2R3-

28

MYB factors (Fig. 2), but it is also expressed early and late in berry development when 29

flavonols are synthesized (Deluc et al., 2008). Ectopic VvMYB5b expression in tobacco

30

increased expression of a number of structural genes in the phenylpropanoid pathway and 31

modified the levels of various flavonoid compounds in flowers including flavonols. The 32

authors suggest that VvMYB5b is involved in a general upregulation of the flavonoid

pathway and acts together with the specific factors VvMYBA and VvMYBPA1 to control 1

2

anthocyanin and PA synthesis, respectively (Deluc et al., 2008). In this respect, VvMYB5b

3

could also play a role in regulation of flavonol synthesis during grape berry development.

4

In seeds, VvMYBF1 is expressed only at very low levels during grape berry development

5

(Fig. 3C) coinciding with the absence of detectable flavonol accumulation and low

6

expression of VvFLS1 in seeds (Downey et al., 2003b). Comparing expression data of

VvMYBF1 with VvFLS1 expression and accumulation of flavonols in developing Shiraz

7

8

berries (Downey et al., 2003b and 2004), we postulate that VvMYBF1 is responsible for

9

induction of flavonol synthesis in buds, flowers and in skins of young developing berries

10

(Fig 3).

11

Flavonol biosynthesis can be induced by light in Shiraz (Downey et al., 2004) and

12

Cabernet Sauvignon (Matus et al., 2009) berries even during developmental stages when

flavonols are normally not synthesized. In the latter study, Matus et al. (2009) were able to 13

14

induce the expression of VvFLS1 (termed VvFLS4) and a partial cDNA (accession no.

15

FJ418175) corresponding to the 3′ end of VvMYBF1 by exposure to sunlight in post-

16

veraison grape berry skins. Our expression analysis in grape suspension culture cells

17

confirmed these results: expression levels of VvMYBF1 and VvFLS1 were significantly

18

increased after six hours exposure of Chardonnay cell culture to light leading to a

concomitant increase in total flavonol content (Fig. 4A-C, supplementary Fig. S2 A). The

19

20

early induction of VvMYBF1 gene expression upon light exposure in the Chardonnay cells

after six hours (Fig. 4A) correlates well with a role for this gene in regulating the

21

22

expression of VvFLS1 (Fig. 4B). The co-induction of VvFLS1 and VvMYBF1 implies that

23

VvFLS1 light induction is not exclusively mediated via VvMYBF1, because induction of

24

VvMYBF1 should then precede the expression of the VvFLS1 gene. However, as we have

25

not measured gene expression before six hours after light exposure, early expression of

26

VvMYBF1, which precedes induction of VvFLS1 can not be excluded. The delay of about

27

12 hours for the increase of flavonol content of the Chardonnay cells compared with the

28

onset of VvFLS1 and VvMYBF1 expression (Fig. 4) might reflect the period of time, which

29

is needed to subsequently translate VvFLS1 and synthesize flavonols. This increase in

30

flavonol levels of the Chardonnay cells eventually confirms the light induction of flavonol

31

biosynthesis and providing evidence that VvMYBF1 is involved in mediating this light

32

induction response in Chardonnay (Fig. 4C, supplementary Fig. S2 A).

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