The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of. Shiraz grapevines. All of the tissues contained ...
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Plant Molecular Biology 32: 565-569, 1996. (~) 1996 Kluwer Academic Publishers. Printed in Belgium.
Short communication
Expression of anthocyanin biosynthesis pathway genes in red and white grapes P a u l K. B o s s 1'2'*, C h r i s t o p h e r D a v i e s 1 a n d S i m o n P. R o b i n s o n 1
ICooperative Research Centre for Viticulture, PO Box 145, Glen Osmond, SA 5064, Australia, and CSIRO, Division of Horticulture, GPO Box 350, Adelaide, SA 5001, Australia (*authorfor correspondence); 2Department of Horticulture, Viticulture and Oenology, University of Adelaide, SA 5064, Australia Received 5 March 1996; accepted in revised form 15 July 1996
Key words: anthocyanin, flavonoid, gene expression, grape, proanthocyanidin, Vitis vinifera
Abstract
The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes. The flavonoids are a diverse group of phenolic compounds found in higher plants, which consist of two aromatic rings joined by a C3 unit. They have many biological functions which include a role in plantmicrobe interactions and plant-pathogen interactions, pollen-tube growth, UV protection and tissue pigmentation [ 10]. One group of flavonoids, the anthocyanins, is a significant class of plant pigments and the metabolic pathway leading to their production has been well characterised in maize, petunia and snapdragon [5, 6, 9, 12]. This has been possible because mutations of genes in this pathway are often not lethal and the resulting phenotype is readily observed. The pathway leading to the production of anthocyanins can be divided into two parts (Fig. 1). Phenylalanine is first converted to 4-coumaroyl-CoA in the general phenylpropanoid metabolic pathway, and this compound may be used in the production of lignins, coumarins and stilbenes. Conversion of 4-coumaroyl-CoA in the flavonoid pathway results in the production of a range of flavonoid compounds, the most common being aurones, flavones, flavonols, isoflavonoids, proanthocyanidins and anthocyanins. Recently,
partial cDNAs for genes from the flavonoid pathway have been isolated from grape [17]. Here we describe studies using these cDNAs to investigate the expression of the first gene in phenylpropanoid metabolism (phenylalanine ammonia lyase, PAL) and six genes of the flavonoid pathway (chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT)), in a number of grapevine tissues including berry skins of both red and white cultivars. Total RNA was extracted from different tissues of the red grape cultivar Shiraz (Vitis vinifera L. cv. Shiraz), by a method described previously [4], in order to investigate the expression of the seven genes described above. These tissues included: young leaf tips; leaves expanded to ca. 5 cm (mid leaf); fully expanded leaves (old leaD; young tendrils; green cane tissue; root tips; seeds collected four weeks after flowering; flowers; berry skin tissue, 14 weeks after flowering; and berry flesh tissue, 14 weeks after flowering. Only the berry skin contained visible anthocyanin
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Phenylatanine 1
i ...................
¢4H,4CL
3~o_~a_~_o~L-C9
malonyl-CoA- ~ Aurones ~
_A. . . . . . . . . .
~
GeneralPhenylpropanoid ~?jhy?y_ ....
Flavonoidpathway
Chalcones
Flavones, ~ lsoflavonoids
Flavanones 1 ~-~
Flavonols ~
Dihydroflavonols
Proanthocyaaidins~ (tannins)
Leucoanthocyanidins 1
l (DEHYDRA TASE) Anthocyanidins Anthocyanir~s Figure 1. A schematic diagram of the general phenylpropanoid and flavonoidbiosynthesispathways. The dotted line indicates the start of the flavonoid pathway. The genes examined in this study are boxed and the dehydratase is putative. Arrows indicatethat the intermediates are precursors of other biosynthesis pathways with final products indicated. C4H, cinnamate4-hydroxylase; 4CL, 4coumarate CoA ligase.
pigments. The pattern of expression of each gene tested was similar in each of the tissues with the exception of U F G T (Fig. 2). Seeds and mid leaves showed the highest levels of expression of PAL, CHS, CHI, F3H, DFR and LDOX, but significant expression of these six genes was also detected in the young leaf, tendril, green cane, root and flower RNA samples. Lower levels of expression were detected for these six genes in fully expanded leaves and berry skin tissue, and in the berry flesh tissue only DFR expression was detected. U F G T was detected only in the berry skin sample, and this was also the only sample in which anthocyanins were detected (Table 1). These results suggest that the main point of control of anthocyanin production in grapevine is after LDOX, which is in contrast to other plants previously studied. In maize, expression of all the genes from
Figure 2. Northernanalysisof total RNA (4#g per lane) extracted from different Shiraz grapevine tissues probed with various clones as indicatedon the left of the figure. RNA was denatured and fractionated on 1.2% agarose gels containing8% formaldehyde, then transferred to ZetaProbe (BioRad) membranein 0.05 M NaOH for at least 15 h. Membranes were prehybridised for 2 h at 65 °C, in 0.25 M sodium phosphate pH 7.0, 1 mM EDTA, 7% SDS, and then hybridisedfor 15 h at 65 °C in the same buffer with the addition of denatured 32P-labelledprobes of the flavonoidgenes. The membranes were then washed for successive 15 rain periods in 2× SSC, 0.1% SDS (65 °C, twice) and 1x SSC, 0.1% SDS (65 °C ). The membraneswere exposed to KodakXAR film with intensifying screens, at -80 oC.
CHS to U F G T is induced when tissues become pigmented, whereas in snapdragon and petunia flowers, the control start points are further on in the pathway, being F3H and DFR respectively [for review, see 12]. We detected expression of all the flavonoid pathway genes, except UFGT, in all the unpigmented tissues except berry flesh. As depicted in Fig. 1, most of the intermediates of the pathway are precursors of other biosynthesis pathways, and this may explain why the expression of these genes occurs in the absence of anthocyanin synthesis in unpigmented tissues. For example, expression of the flavonoid genes up to and including DFR would be necessary for production of proanthocyanidins, which are precursors to tannin syn-
567 Table 2. The concentration of proanthocyanidins and anthocyanins in the berry skins of white and red grape cultivars. Soluble solids (measured as °Brix) were also measured to give an indication of the ripeness of the berries. Cultivar
°Brix
Proanthocyanidins
Anthocyanins
(mg/g fresh weight)
(A520/gfresh weight) n/d2
Riesling
14.8
2.1
Muscat Gordo
13.5
6.2
rdd
Semillon Chardonnay
17.0 21.3
8.7 5.1
n/d n/d
Sultana Cabernet Sauvignon
22.7 15.5
2.0 n/a I
n/d 84.4
Pinot Noir
21.6
n/a
137.8
Shiraz
20.8
ro'a
212.4
t rda = assay not applied as anthocyanins interfere with the proanthocyanidin assay. 2n/d = anthocyanins not detected (
