MOLECULAR BIOLOGY OF SUGAR AND ANTHOCYANIN ACCUMULATION IN GRAPE BERRIES
3.2. The anthocyanin biosynthesis pathway
Mutations have been extensively studied in order to understand the pathway of flavonoid biosynthesis. Since the mutations affect an easily selectable phenotype - that of tissue pigmentation - and the fact that this pigmentation is not essential for the viability of the plants, there is much material available. Studies of pigmentation mutants have resulted in the characterisation of two main types of genes involved in anthocyanin synthesis. Some have been shown to only affect one structural gene of the biosynthetic pathway. Those that affect early stages in the biosynthesis of anthocyanins often result in colourless phe- notypes. However, if a mutation exists later in the pathway it may only affect the types of anthocyanins produced. Other mutations may affect the expression of one or more of the structural genes but do not map to these loci. These may be involved in the regula- tion of the anthocyanin pathway and are thus classified as regulatory genes. Mutations also exist that disrupt the transport of the colour compounds into the vacuoles or the tis- sue distribution of anthocyanin production.
3.2.2. The structural genes
The anthocyanin biosynthesis pathway has a branching nature, with the early steps re- sulting in products that act as precursors for many types of related compounds (Fig. 1.5).
Enzymes involved in the later biosynthetic steps act specifically for the production of anthocyanins. For extensive reviews on the structural genes of the anthocyanin pathway refer to articles by Martin and Gerats (l993a; 1993b), and Holton and Cornish (1995).
Phenylalanine ammonia lyase (PAL) is the first enzyme involved in anthocyanin pro- duction. It catalyses the production of cinnamic acid from phenylalanine (Hanson and Havir, 1981). A partial cDNA encoding pal has been isolated from grapevine using an An- tirrhinum pal clone as a probe (Sparvoli et al., 1994). This partial clone is in fact one member ofa large gene family in grapevine believed to consist of 15-20 pal genes (Spar- voli et aI., 1994). The cinnamic acid produced by the action of PAL is then converted to
Phenylalanine __ _ p-Coumaroyl-CoA PAL C4H 4CL CHS r 3 x Malonyl-CoA I Chalcones I ... --... ~ Aurones F3'H CHI I F3'5'H -- ... -Flavonones .... ~ Flavones, Isoflavonoids F3H~ ~ F3H~ ~ ~F3H Dihydroflavonols ... ~ Flavonols DFR ~ ~ DFR I Leucocyanidin J ... -... ~ Proanthocyanidins ... . I Leucodelphinidin I ~ LDOX LDOX ~ i , y (DEHYDRA rASE) t I Cyanidin I UFGT ~ ~ O.. MT "'-~.A. h'" "' . ... ~ ~:-H H OH OGIue otI ~Iuc Cyanidin-3- Peomdin-3-glucoside glucoside
Tannins - ~
OH HO OM .... Petunidin-3- glucoside MT ...-
: (DEHYDRATASE) ... I Delphlnldln I ~ U:GT ~ ... MT _ He) OH~ CH ~". "'. ~ .A. ()' OM • OH '()J:' Delphinidln-3-OM glucoside Malvldinã3- glucoside Figure 1.5. The anthocyanin biosynthesis pathway. This diagram of the anthocyanin biosynthesis pathway has been modified to account for the major products found in grapes, Further modifications and transport of the anthocyanin-3-glucosides into the vacuole also occur but these are not included in this figure as the exact order of these processes is unknown. The enzymes catalysing each reaction are indicated besides the solid arrows. The dehydratase is putative and is thus written in brackets and the exact points of action ofF3'H and F3'5'H have not been determined for grapes. The fine dotted lines indicate that some of the intermediates are precursors for other biosynthesis pathways with the final products indicated. Key: PAL phenylalanine ammonia lyase; C4H cinnamate 4-hydroxylase; 4CL 4-coumarate CoA ligase; CHS chalcone synthase; CHI chalcone isomerase; F3'H flavonoid 3' -hydroxylase; F3'5'H flavonoid 3' ,5' -hydroxylase; F3H flavonone 3-hydroxylase; DFR dihydroflavonol4- reductase; LDOX leucoanthocyanidin dioxygenase; UFGT UDP-glucose flavonoid 3-0-glucosyl transferase; MT methyltransferase.
V1 § ~ V1 ~ ~ n i V1 Z ~ tT1 t:C ~ V1 ... Ul
16 P.K. BOSS and C. DAVIES
p-coumaric acid by cinnamate 4-hydroxylase (C4H) (Nair and Vining, 1965). The en- zyme 4-coumarate CoA ligase (4CL) then ligates CoA to p-coumaric acid to produce a p-coumaroyl-CoA ester (Heller and Forkmann, 1988). Neither c4h nor 4cl have been cloned from grapevine. The first flavonoid produced is a chalcone, and the enzyme in- volved is chalcone synthase (CHS). The chalcone is produced by the condensation of p- coumaroyl-CoA with three molecules of malonyl CoA (Kreuzaler and Hahlbrock, 1972).
A maize chs clone was used by Sparvoli et al. (1994) to isolate a homologue from grape seedlings and it belongs to a small gene family with approximately 3-4 members. Chal- cones are then converted to flavanones by chalcone isomerase (CHI), which catalyses a stereo-specific ring closure (Moustafa and Wong, 1967). This can also occur spontane- ously, although at a slower rate (Kuhn et al., 1978). Because of the ability of chalconon- aringenin (the substrate for CHI) to isomerise non-enzymatically, mutations in chi are leaky. An Antirrhinum chi clone was isolated using homology to previously isolated chi clones (Martin et aI., 1991) and this clone was used to isolate a homologue from grape (Sparvoli et aI., 1994). It is believed that there is only one chi locus in the grape genome (Sparvoli et al., 1994). Flavanone 3-hydroxylase (F3H) then hydroxylates flavanones to form dihydroflavonols (Forkmann et aI., 1980). The j3h gene from Antirrhinum was cloned using differential screening and genetic mapping (Martin et al., 1991) and Spar- voli et al. (1994) used this snapdragonj3h clone to isolate a grape homologue. Southern analysis suggested that there are no more than two j3h genes in grapes (Sparvoli et aI., 1994). Enzymes, which catalyse the hydroxylation of the B rings appear to act on fla- vanones or dihydroflavonols, the substrates and products of F3H (Stolz et al., 1985;
Menting et al., 1994). These enzymes are important in determining the species ofantho- cyanins produced in a plant and are called flavonoid 3' -hydroxylases (F3 'H) and flavon- oid 3'5'-hydroxylases (F3'5'H). F3'H activity results in plants accumulating cyanidin- like anthocyanins rather than pelargonidin-like species. F3'5'H activity produces del- phinidin species, which result in blue colours. Thus, the lack of this enzyme is thought to be the reason for the absence of blue shades in tulips or roses, to name two well-known examples. Genes encoding F3'H and F3' 5'H have not been cloned from grapevine.
Dihydroflavonol 4-reductase (DFR) catalyses the first step in the conversion of dihy- droflavonols to anthocyanins. It causes a reduction at the 4 position of the C ring to give leucoanthocyanidin (Stafford and Lester, 1982). Sparvoli et al. (1994) cloned a grape dfr homologue by screening a seedling cDNA library with an Antirrhinum dfr clone and it is thought to be present as a single copy in the grape genome. The next steps in the produc- tion of anthocyanins from leucoanthocyanidin are not well characterised. They are be- lieved to involve a hydroxylase and a dehydratase (Heller and Forkmann, 1988). The candica (candi) locus in Antirrhinum majus (Martin et aI., 1991) and the A2 locus in maize (Menssen et aI., 1990) have been putatively identified as coding for leucoantho- cyanidin dioxygenase (ldox). An Idox cDNA has been cloned from grape using the can- dica cDNA as a probe (Sparvoli et aI., 1994). The incolorata I locus in Antirrhinum may encode the other enzyme predicted to be required for the synthesis of anthocyanidins, a dehydratase, but the gene has yet to be cloned (Martin and Gerats, 1993a).
Anthocyanidins can be stabilised through the addition of a glucose residue at the 3
SUGARS AND ANTHOCY ANINS IN GRAPE BERRIES 17 position of the C ring. This reaction is catalysed by UDP-glucose: flavonoid 3-0- glucosyltransferase (UFGT; Larson and Coe, 1977), and the product is believed to be able to be transported though the vacuolar membrane. A ufgt homologue from Antir- rhinum was used by Sparvoli et al. (1994) to isolate a partial ufgt cDNA from grape seedlings and it is thought to be present at a low copy number in the grape genome. This partial clone was later used to isolate a full-length ufgt gene from a ripe berry cDNA library made from the grapevine cultivar Shiraz (Boss, 1998; Ford et al., 1998).
Other genes are involved in the transport of anthocyanins into the vacuole and further modification of the anthocyanin species, but few have been cloned. The transport of an- thocyanins into the vacuole is thought to be mediated via glutathione conjugation by glutathione S-transferase (GST) and the subsequent transport of these conjugates into the vacuole by a glutathione pump (Marrs, 1996). This transport system is similar to that used by plants to recognise, transport and metabolise herbicides and xenobiotics, and seems to be the method that the plant uses to protect itself from anthocyanins which are in fact toxic to plant cells (Marrs, 1996). Other modifications of the anthocyanins in- clude further glycosylation, acylation and methylation, although the exact order or cellu- lar location of these modifications is unknown.
3.2.3. Genes involved in pathway regulation
Some pigmentation mutants are caused by the change in expression in more than one structural gene, or cannot be mapped to specific structural genes. It is thought that such mutants have lesions in genes that regulate the expression of the structural genes. These regulatory genes have been studied extensively in maize (reviewed by Dooner et al., 1991; Mol et al., 1996). Two families of genes called the R and the CI family control anthocyanin production in many maize tissues. Studies of the regulatory gene products revealed that R-type genes are homologous to myc protooncogenes from animals and are basic helix-loop-helix transcription factors (Ludwig et aI., 1989; Radicella et aI., 1991;
Consonni et al., 1993). The CI gene family encodes myb homologues, another group of transcription factors (Paz-Ares et al., 1987; Cone et al., 1993). The production ofantho- cyanins in maize requires both myc-like and myb-like proteins and these transcription factors regulate the expression of all the structural genes from chs down the anthocyanin pathway (reviewed by Martin and Gerats, I 993a). Similar regulatory genes have been isolated from other plant species. In snapdragon, the Delila gene encodes a myc-like transcription factor (Goodrich et al., 1992) and the delila (del) mutants have a loss of pigment in the floral tube. There is little effect on the expression of early genes (chs and chi) in these mutants, but there is inhibition ofthe expression of j3h, dfr, Idox and ufgt (Martin et al., 1991). So it seems that the mutant only affects the later genes of the path- way. A myb-like partner for Delila has not been identified, although Jackson et al.
(1991) isolated six genes with homology to myb-like genes that are expressed in snap- dragon flowers. The petunia mutants anI, an2, an4 and anll have been shown to be mutations in anthocyanin regulatory genes (Beld et aI., 1989; Quattrocchio et al., 1993).
The An2 gene has been cloned and shown to encode a myb-like transcription factor (Quattrocchio et aI., 1999). The petunia mutants lack dfr mRNA, and also have reduced
18 P.K. BOSS and C. DAVIES
ujgt activity but the expression of chs, chi andj3h is not altered (Beld et aI., 1989; Quat- trocchio et aI., 1993).
Control of anthocyanin biosynthesis appears to be exerted at the level of transcription (Martin and Gerats, 1993a), but the point at which the pathway is controlled differs in the three plant species mentioned above. In maize, it appears that the first major control point is chs, whereas in snapdragon and petunia the major control points are further on in the pathway, atj3h and dfr respectively (for reviews, see Martin and Gerats, 1993a; Mar- tin and Gerats, 1993b; Holton and Cornish, 1995).
The maize regulatory genes have been shown to be functional and thus have the ability to induce anthocyanin synthesis in other plant species, suggesting that anthocyanin synthe- sis is controlled by similar mechanisms in different plant species (Lloyd et aI., 1992;
Goldsbrough et aI., 1996; Bradley et al., 1998). Delila is able to enhance pigmentation of tobacco flowers and vegetative tissues of tomato (Mooney et aI., 1995) and the same re- sults were reported when two alleles of a myc-like gene from Perillafrutescens were over- expressed in tobacco and tomato (Gong et aI., 1999). Thus, it seems that anthocyanin bio- synthesis is controlled by similar regulatory factors in different plant species which show homology to myc-like and myb-like transcription factors. The manner in which the path- way is controlled in different plant species with regards to the tissue specificity of antho- cyanin synthesis and how much of the pathway is upregulated upon anthocyanin synthesis seems to be influenced by both the promoters of the anthocyanin structural genes and the properties ofthe endogenous and/or exogenous regulatory genes involved.