The Plant Journal (1995) 7(2), 333-339

SHORT C O M M U N I C A T I O N

Altered regulation of tomato and tobacco pigmentation genes caused by the delila gene of Antirrhinum

Mark Mooney 1, Thierry Desnos 1,t, Kate Harrison z, Jonathan Jones 2, Rosemary Carpenter 1 end Enrico Coen 1,* I Genetics Department, John Innes Institute, Colney Lane, Norwich NR4 7UH, UK, and 2Sainsbury Laboratory, The John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

Summary

Delila (de/), a regulatory gene of Antirrhinum, alters antho- cyanin pigmentation when introduced into two Solana- ceous species. In tomato, pigmentation in vegetative tissues is strongly increased while in tobacco, intensifica- tion of pigment is restricted to flowers. Although del transcripts are ubiquitous in the transgenic plants, tran- script levels of host anthocyanin biosynthetic genes are only increased in pigmented regions. Constructs carrying the maize trensposon Ac, inserted at the 3' end of the 35S promoter prior to the start of translation of the de/gene, give variegated leaves in tomato, suggesting that de/acts cell-autonomously end that it may be used as a phenotypic marker. In Arabidopsis, del has no strong phenotypic effects, suggesting that del may not be able to function effectively in ell plant hosts.

Introduction

Plant pigmentation provides an amenable system for study- ing the evolution of pattern. Anthocyanin pigments confer a wide variety of coloration patterns and have been subject to detailed genetic and molecular analysis in several species, including maize, Antirrhinum, Petunia, Arabi- dopsis, pea and tomato (Almeida et al., 1989; Dooner et al., 1991; Harker et al., 1990; Kubasek et al., 1992; Martin et al., 1991; van Tunen et al., 1988; von Wettstein-Knowles et al., 1968). Several structural genes, encoding enzymes of the anthocyanin biosynthetic pathway, have been identi- fied. In addition, genes that directly regulate the transcrip- tion of structural genes have been isolated from maize and Antirrhinum (Cone et al., 1986; Dellaporta et al., 1988;

Received 13 June 1994; revised 18 October 1994; accepted 24 October 1994. *For correspondence (fax + 44 603 56844). tPresent address: Laboratoire de Biologie Cellulaire, INRA, Route De St Cyr, 78026 Versailles, France.

Goodrich et aL, 1992; Grotewold et aL, 1994; Jackson et al., 1991; Paz-Ares et al., 1987; Sablowski et al., 1994). Changes in either the structural or regulatory genes could be involved in the evolution of pigmentation patterns. The role of structural genes may be limited because extending pigmentation requires altered expression of an entire biosynthetic pathway and hence mutations in each of the structural genes. In contrast, a single mutation in a regulatory gene could affect the anthocyanin pathway coordinately (Radicella eta/., 1992), suggesting that altered activity of such genes may more readily account for evolu- tion of new pigmentation patterns (Goodrich et al., 1992). According to this view, altered pigmentation phenotypes should result when regulatory genes are expressed in novel ways in various species. To test this hypothesis, we have examined the effects of the regulatory gene de/, from Antirrhinum, on both the pigmentation phenotypes and expression patterns of host structural genes of three species.

The de/gene encodes a protein with limited similarity to a myc-type transcription factor and is required for pigmentation of the corolla tube and a variety of other tissues (Goodrich et al., 1992). It regulates the transcription levels of several structural genes, including flavanone- 3-hydroxylase, dihydroflavonol-4-reductase (DFR), uridine flavonoid 3-O-glucosyl-transferase (UF3GT) and, to a lesser extent, chalcone synthase (CHS), an early step in the pathway (Almeida et al., 1989; Martin et al., 1991). Several functional homologues of de/have also been described in maize, including R, Lc, Sn and B, collectively termed the R gene family (Ludwig and Wessler, 1990). These genes affect anthocyanin pigmentation in various parts of the plant by regulating structural genes in a similar way to de/, although their effects on CHS expression are more marked (Dooner, 1983; Ludwig et al., 1989). In addition to myc-type factors, some pigment regulatory genes of the myb-type have also been described and include the C1, P/and Pgenes of maize and the myb305 gene of Antirrhinum (Dooner et al., 1991; and Sablowski et al., 1994). In maize, the myc and myb types of transcription factor are hypothesized to interact to form a functional heterodimer that activates structural gene transcription (Goff et al., 1992).

Representatives of both myc and myb regulatory genes from maize have been introduced into two dicot species, tobacco and Arabidopsis, under the control of the CaMV- 35S promoter (Lloyd et al., 1992). In both species,

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expression of Lc intensifies pigmentation. In Arabidopsis, Lc also confers excess trichomes and an absence of root hairs and complements the transparent testa and glabrous (ttg) mutant. Plants homozygous for ttg lack both anthocy- anin pigmentation and trichomes and have an excessive number of root hairs. C1 alone produces no phenotypic effects but Arabidopsis lines transgenic for both Lc and CI are more extensively pigmented than those carrying Lc alone. In all cases pigmentation is not constitutive but is restricted to certain tissues of the plants. Similar restrictions are also seen within maize: the starchy endosperm does not become pigmented when both Lc and Clare overexpressed there (Ludwig et al., 1990). These observations raise the question of how pigmentation pattern is limited and to what extent limitations reflect the taxonomic origin of the introduced regulatory genes.

Here we show that de/, a dicot representative of the myc family, can alter pigmentation in two Solanaceous species, tobacco and tomato, by increasing transcript levels of host structural genes. This occurs in particular regions of the plant, even though de/transcripts are widespread, showing that de/is not the only factor limiting pigmenta- tion. In Arabidopsis, de/does not have strong phenotypic effects, unlike the Lc gene of maize. Furthermore, we show that constructs carrying the transposon Ac give a variegated phenotype in tomato and that de~therefore acts cell-autonomously in a novel host and may be useful as a phenotypic marker.

Results

Two constructs were used in the transformation experi- ments. The first comprised the cauliflower mosaic virus 35S promoter fused to the start site of translation of a de/ cDNA. The second construct had the 35S promoter fused to the translation start site of a genomic clone of de/ (containing introns and the de/3' region). Both constructs carried a kanamycin-resistance gene and were introduced via Agrobacterium-mediated transformation into wild-type lines of tomato and Arabidopsis. Tobacco was transformed with only the de/cDNA construct.

Del enhances pigmentation in tomato and tobacco

Ten independent tomato transformants were obtained with the 35S-cDNA construct and seven of these showed a similar pattern of enhanced pigmentation. Comparable pigmentation phenotypes were also observed in five out of the eight tomato transformants carrying the 35S-genomic construct. DNA blots showed that all transformants carried DNA inserts hybridizing to the del cDNA. One of the 35S-cDNA transformants with enhanced pigmentation (TC69-2) was selected for detailed genetic and phenotypic

analysis. The primary transformant was shown from DNA blots to contain three, unlinked T-DNA insertions and was self-pollinated to analyse their segregation. Out of 70 progeny, three individuals had no enhanced pigmentation and were shown not to carry T-DNA insertions. These unpigmented plants bred true and were used as control populations in further comparisons. Individuals that carried single insertions were also identified and .two of these (TD34 and TD37) were self-pollinated for progeny testing. TD34 was homozygous for a single insertion (a single band of 9 kb in Hindlll digests) and bred true for the enhanced phenotype and kanamycin resistance (>200 progeny tested). TD37 proved to be heterozygous for a different insertion (4.5 kb in Hindlll digests) and segregated for enhanced and wild-type pigmentation in a 3:1 ration (>100 progeny tested). When adult plants were sprayed with kanamycin, resistance was always found to be linked to the enhanced pigmentation phenotype (all plants without enhanced pigmentation were found to be sensitive).

The characteristics of the phenotype in transgenic plants (TD34 homozygotes) were analysed throughout the plant growth cycle and compared with controls (Figure lb, c, d, f and g). In general, the regions that showed pigmentation in the transgenic plants were also pigmented in the controls but to a much lesser extent. Following seed germination, the first evidence of an altered phenotype was an increased purple pigmentation in the hypocotyl and cotyledons. As the first leaves emerged, the rachis and leaflets became intensely pigmented. The leaflets first accumulated pig- ment at their tips, but as they developed, pigmentation was observed throughout (Figure lb). Measurement of pigment in extracts showed that the transformants had a mean of 23 (standard deviation (SD) 4.5) times more anthocyanin in their mature leaflets than the controls. The most intensely pigmented cells of young leaflets were the subepidermal cells overlying vascular tissue but after leaflet expansion, intense pigmentation was also observed in the epidermal ceils (Figure lf). The subepidermal cells in the stem also accumulated pigment, most notably after internode elongation. The leaf and stem phenotypes were maintained as plants matured and were further enhanced when plants were grown under high light (see Experimental procedures). The roots were normally unpigmented, but after exposure to light they became strongly pigmented within 2 days. The pigment was seen only in the cells of the root cortex and was not observed in the root tip (Figure lg), By comparison, control roots only showed weak pig- mentation on exposure to light after a delay of 1 week. Upon flowering, enhanced pigmentation was observed in the sepals and in the subepidermal cells overlying the main vein of each petal (Figure ld). Fruits and the testa of the seed showed no detectable enhancement of normal pigmentation (Figure lc).

Seven independent tobacco transformants were

Altered pigmentation caused by the delila gene 335

Figure 1. Phenotypes resulting from del cDNA expression in tomato and tobacco. The wild-type is on the left and transgenic on the right. (a) Tobacco flowers showing enhanced pig- mentation of the petals and anther filaments in the transgenic. (b) Tomato leaflets. (c) Tomato stems and developing fruit showing increased pigmentation in the stems but no observable anthocyanin pigmentation in the young fruits. (d) Tomato flowers showing anthocyanin associated with the main vein of the petal of the transgenic (the stamens show no anthocyanin pigmentation). (e) Tomato leaf from 35S:dek.:Actransformant, showing cell autonomous sectors of antho- cyanin pigmentation. (f) Cross-section of a tomato leaflet from a transgenic plant, Anthocyanin can be seen in the upper and lower epidermis. Antho- cyanin is also seen as a dark green pig- mentation (resulting from the overlay of green from chlorophyll) in cells of the spongy meso- phyll and palisade layers. (g) Cross-section through a lateral root of a transgenic tomato plant, showing pig- mentation of the cortex (c). Pigment was never observed in the epidermis (e) or stele (s).

obtained with the 35S--cDNA construct. Five of these had excess pigmentation and were shown to contain the con- struct by DNA blots and progeny testing on kanamycin. One of the primary transformants (NB10-3), containing a single T-DNA insertion, was chosen for detailed analysis. From the progeny of this plant, one pigmented individual was selected and shown to breed true for kanamycin resistance and pigmentation. A line derived from this homozygote was used for further molecular analysis and compared with control plants that derived from the same original transformant but had lost the T-DNA insertion through segregation.

None of the transformants showed altered anthocyanin pigmentation in the vegetative parts of the plant: in all cases the leaves and stems were green, as seen in the controls. Roots also showed no evidence of pigmentation, even on prolonged exposure to light. Enhanced pigmenta- tion was seen in both the petals and the stamens of flowers (Figure la). Pigments extracted from the homozygous line

showed that the corolla lobes accumulated a mean of 40 (SD 5.7) times more anthocyanin than the controls and the stamen filaments in excess of 50 times the control level. The intensity of the pigmentation phenotype was enhanced by high light. Although the pigmentation was more intense its distribution was similar to that observed in the controls: pigmentation being strongest in the lobes of the corolla and the distal parts of the stamen filament. Anthocyanin was restricted to the epidermal cells of petals and was particularly intense in the cells overlying the veins; as a consequence of this the corolla had a reticulate appearance (Figure la). By comparison, pigmentation in the petals of control plants was restricted to the epidermis but was not noticeably enhanced in cells overlying the veins, giving the corolla a more uniform colour. In the stamen filaments, pigment was observed in both epidermal and subepidermal cells. Anthocyanin distribution in the control stamens could not be determined because of the very low levels. No anthocyanin pigmentation was seen in

336 Mark Mooney et al.

the sepals or carpels of the flower in either the transgenic plants or the controls.

Del produces no strong phenotype in Arabidopsis

Neither of the constructs produced a consistent phenotype in Arabidopsis (seven transgenic plants produced). One transgenic line (AH105), homozygous for the 35S-genomic construct and known to express de/on the basis of RNA blots, sometimes showed a slight increase in the pigmenta- tion of older leaves, young bolts and siliques. The weak and variable phenotype could be explained if the action of de/ila was obscured by endogenous myc-like regulators. To test this possibility, the transgene from this line was introduced into a ttg background. The ttg mutant has been shown to lack pigmentation and trichomes, possibly because of insufficient myc-like regulator activity. The F 2 from a cross between the transgenic line AH105 and ttg was grown on kanamycin and segregated for wild-type and ttg in a 3:1 ratio, showing that the construct did not restore any aspect of the glabrous or transparent testa phenotypes of the mutant. The transgenic line was also crossed to a transgenic line of Arabidopsis homozygous for the C1 gene of maize expressed under the control of the 35S promoter (Lloyd et al., 1992). No obvious pheno- type was observed in the FI. This suggests that DELILA acts inefficiently or not at all in Arabidopsis.

Del increases host gene transcript levels

Although the del constructs were able to enhance pig- mentation, their effects were not uniform throughout the plant. This could have several explanations: (i) the con- structs were not expressed in the unpigmented regions; (ii) de/was expressed throughout the plant but was unable to activate target genes in all regions; (iii) target genes were activated in unpigmented regions but other factors, such as the availability of precursors, limited pigmentation.

To test these possibilities, we analysed the expression pattern of de/ and its potential target genes encoding pigment biosynthetic enzymes, mRNA was extracted from tissues of tomato (leaves and young fruit), tobacco (leaves and petals) and Arabidopsis (leaves). The RNA blots showed that a de/transcript of the expected size (2 kb) was observed in all transgenic plants and tissues tested, even though some of these tissues showed no evidence of enhanced pigmentation (Figure 2). In tobacco, additional bands below 1.9 kb were also observed and presumably reflect other products of the introduced gene. No de/ transcript was detected in control plants.

Probing with biosynthetic genes, showed that DFR tran- scripts were increased in pigmented tissues, 10-fold in

Figure 2. Northern analysis of del, CHS and DFR expression in control and transgenic plants. In tobacco: de/is detected in both the leaves and petals of the transgenic plant. CHS is only detected in the petals of tobacco and is slightly increased in both the de/and Lc transformants. DFR is also restricted to the petals and expression is obviously increased in both the de/and Lc transgenics. In tomato: de/is expressed in both leaves and fruit of the transformed plant. CHS is expressed in the leaves of the control and the transgenic and also to a lesser extent in the transgenic fruit (fruit of control plant is not tested). In the leaves of the deltrensgenic plant the level of CHS expression is slightly enhanced and DFR is clearly increased. There is a very low level of DFR expression in fruit of transgenic plants (visible on long exposure). As a control, blots were reprobed with ubiquitin (Ubi).

tomato and fourfold in tobacco (Figure 2). Increased levels of another biosynthetic gene, CHS were also observed to a small but reproducibleextent (twofold in tobacco and threefold in tomato; Figure 2b). Ethidium staining of RNA and reprobing blots with ubiquitin confirmed that the loading was the same for control and transgenic plants. In unpigmented tissue, no CHS or DFR transcripts were detected except for tomato fruit which showed some expression of CHS and very low levels of DFR. Presumably, the low levels observed in the fruit were not sufficient to confer visible pigmentation.

In the case of tobacco it was possible to compare directly the effects of de/ with those of Lc gene of maize (Lloyd et al., 1992). Transgenic plants containing Lc under the control of the 35S promoter showed similar patterns of CHS and DFR expression to the transgenic de/plants (Figure 2). The effect of Lc on DFR expression was more pronounced (an increase of 10-fold) than that of de/, con- sistent with the more intense pigmentation of the flowers.

The effects of del are cell autonomous

To test whether expression of de/was able to activate pigment production autonomously within individual cells,

we made DNA constructs that carried the maize transposon Ac, inserted within the leader sequence of the 35SdelcDNA construct. Ac is known to transpose in tomato (Yoder et al., 1988) and so expression of de/should be restored upon excision of Ac. Eight primary transformants were produced and three of these showed a variegated leaf phenotype. The phenotype of one of the primary trans- formants was investigated in detail (Figure le). The leaves showed clonal sectors of anthocyanin pigmentation of various sizes. On examination under the light microscope, cells at the edge of the sectors could be clearly distin- guished from adjacent unpigmented cells, suggesting that de/was acting cell-autonomously.

Discussion

Expression of de/alters anthocyanin pigmentation in two So/anaceous species, tobacco and tomato. The transgenic plants contain higher transcript levels of DFR, showing that the effects on pigmentation result from regulation of endogenous anthocyanin biosynthetic genes. Transcript levels of CHS are also slighly elevated, consistent with the action of de/in Antirrhinum (Almeida et al., 1989). The variegated phenotype of de/constructs carrying the trans- poson Ac suggests that de/acts cell-autonomously in the new hosts, again similar to its behaviour in Antirrhinum (Goodrich et al., 1992).

These results indicate that the interaction between de/ and its target genes has been conserved since the diver- gence of the Scrophu/ariaceae (the family to which Antirrhinum belongs) and the Solanaceae. Furthermore, they suggest that the levels of myc-type transcription factors may normally limit structural gene expression, and therefore provide one important means of evolutionary change. This raises the question as to why mutations in de/or members of the maize R gene family are recessive, because heterozygous plants should have half the dose of gene activity and hence (assuming no dosage compensa- tion) be less pigmented than homozygous wild-types. One explanation is that heterozygosity only alters gene expres- sion by a factor of 2 whereas the levels in transgenic plants are more greatly affected and therefore easier to discern. Another possibility is that artificial selection for intensely pigmented varieties of maize and Antirrhinum may have exploited natural variation in regulatory genes, resulting in pigmentation that is less strictly limited by myc-type factors.

The phenotypic effects of de/on tomato and tobacco appear to be quite different. In tomato, de/ strongly increases pigmentation in vegetative tissues while in tobacco, effects are only seen in the flowers. The differ- ences in phenotype are not the result of the expression pattern of the introduced construct because all tissues

Altered pigmentation caused by the delila gene 337

analysed, even those that lack pigment, contain similar high levels of de/transcript. The pigmentation phenotypes are mirrored, however, by the pattern of anthocyanin biosynthetic gene expression: in tobacco flowers, tran- scripts of DFR are clearly increased whereas in leaves both DFR and CHS transcripts can not be detected. In all cases, tissues showing strong pigmentation in transgenic plants also exhibit a lesser degree of pigmentation in control plants. Therefore, although de/may affect the levels of target gene transcription and hence the intensity of pig- mentation, other factors act to restrict the overall patterns of expression.

The effects of de/in tobacco are qualitatively similar to those of Lc, both in terms of pigmentation pattern and host gene expression. This suggests that the same factors limit the action of de/ and Lc on tobacco host gene expression. However, the Lctransgenic lines analysed have more intensely pigmented flowers than those of de/. This correlates with the greater effects of Lc on DFR transcript levels. Similar to the effects of de/, the level of CHS transcripts is only slightly elevated by Lc, even though the R gene family strongly regulates CHS in maize (Dooner, 1983). This shows that the degree to which CHS expression is dependent upon myc-type transcription factors depends to a large extent on the host background rather than the origin of the introduced gene. Similar results have also been obtained using transient assays to study the effect of Lc on CHS expression in Petunia (Quattrocchio et al., 1993).

Introduction of the de/constructs into Arabidopsis has no strong phenotypic effects, even though de/transcripts are detected in these transgenic plants. It is possible that none of the seven Arabidopsis transformants express de/ optimally. However, comparable numbers of tomato and tobacco transformants give a majority of plants showing phenotypic effects. This indicates that relative to its action in Solanaceae, de/may not function well in Arabidopsis and that the interaction between de/and its targets has therefore diverged since the separation of the Scrophularia- ceae and the Brassicaceae. The ability of de/to confer a phenotype may reflect taxonomic distance: the Scrophular- iacea are thought to be much more closely related to the Solanaceae than to the Brassicaceae. Accordingly, de/ homologues from other species might be expected to show similar phylogenetic restrictions. However, this is contrary to results obtained with Lc. Expression of Lc under the control of the 35S promoter enhances pigmentation in both tobacco and Arabidopsis. The ability of Lc to function more broadly than de/in some dicot plants suggests that the DEL protein may be more discriminating than that of Lc. This could be because DEL has different requirements for interacting with promoter sequences or other host factors. In support of this, introduction of the 35S-de/cDNA construct into the aleurone layer of maize kernels by particle bombardment does not complement an r mutant

338 Mark Mooney et al.

(our unpublished results). Another possibility is that DEL

may interact more readily with negative regulatory host

factors of dicots, whereas the greater divergence of Lc allows it to evade such control.

The ability of del and Lc to change the transcription of anthocyanin biosynthetic genes in various species suggests that alterations in the expression of myc-type regulatory genes provide one possible mechanism for evolutionary change in pigmentation. Pigmentation pattern does not simply fol low the expression pattern of these

regulatory genes but it is restricted to certain domains by other host regulatory and environmental factors. Each of these factors in turn wil l have its domain of action limited by the myc-type gene expression pattern.

Experimental procedures

conditions, either in high light (without shading), or in low light (with shading). As conditions were ambient, fluence varied.

Anthocyanin extractions

Anthocyanin was extracted from tissues using a solution of acidic methanol (99% methanol, 1% HCI) according to the method of Rabino and Mancinelli (1986), and the absorbance of the resulting extract determined in a spectrophotometer (SP8-200 UV/VIS Pye/ Unicam). Absorbance was measured between wavelenghts 450 nm and 600 nm; a peak was observed at 530 nm.

Sectioning and light microscopy

Tissue was embedded in low melting point agarose and then cut into thick sections (50 p~m) using a vibratome (BioRad micro-cut H1200). Sections were then placed on slides and viewed under a light microscope (Zeiss, Stemi SV8) and photographed.

Constructs

Both the delcDNA, p JAM121 and genomic coding sequence, ~,G4 (Goodrich et al., 1992) were cloned into vector pSLJ4D4 (Jones et al., 1992) replacing the GUS gene in this plasmid. The genomic coding sequence carried introns and a I kb 3' region incorporating the de/ polyadenylation sequences. To facilitate cloning, site- directed mutagenesis (Amersham Sculptor TM in vitro mutagenesis system RPN1526) on both the cDNA and genomic clones was used to introduce Ncol sites at the start of translation and BamHI sites after the 3' end of the ORF. This resulted in constructs p JAM173 (cDNA) and p JAM174 (genomic sequence). Fragments were then released from this plasmid by cutting with Bgltl and Hindlll, and inserted in to the binary vector pSLJ456 (Jones et aL, 1992) to give p JAM175 (cDNA) and p JAM176 (genomic). These binary vectors were then transferred to appropriate strains of Agrobacterium tumefaciens, C58C1/pGV2260 (Deblaere et al., 1985) for use in Arabidopsis transformation and LBA4404 (Hoek- ema et al., 1983) for transformation of both tomato and tobacco. The 35S:del:Ac construct was produced by excising the de/cDNA from p JAM173 using Xhol and BamHI, and ligating this into the similarly cut binary vector pSLJ6562 (Jones et al., 1992). Ac sequences, derived from pSJ7C3 by digestion with Sstt and Salt (Jones et al., 1992), were inserted at Ss~ and Xhol sites in the 35S-CaMV promoter, resulting in the plasmid p JAM177.

Plant manipulations

Tomato (Lycopersicon esculentum var. Moneymaker) transforma- tions were performed as in Fillati et aL (1987). Tobacco (Nicotiana tabacum var. Samsun) as in Horsch et eL (1985) and Arabidopsis thaliana ecotype Landsberg erecta, as in Valvekans et aL (1988). Kanamycin selection of seedlings was performed by sterilizing the seed using a solution of 10% hypochlorite (bleach) and then plating on the appropriate germination media for each species, supplemented with kanamycin (kan), at concentrations of 50 lig m1-1 for Arabidopsis, 10011g m1-1 for tobacco and 300 lig m1-1 for tomato. Kanamycin selection was also carried out on adult plants (Weide et aL, 1989), by spraying with a solution of 300 lig m1-1 kanamycin. This allowed resistant and sensitive individuals to be distinguished without the loss of sensitive plants. All species were grown in the glasshouse under ambient

Analysis of nucleic acids

The methods for DNA and RNA extraction and Southern and Northern analysis were as described previously (Coen et aL, 1986). Poly(A) ÷ RNA extraction was performed using the promega polyATtrect kit (Z5310) and 5 tig loaded per lane of the agarose gels, which were then blotted on to nitrocellulose filters. Extrac- tions were repeated a minimum of two times, RNA being pooled from a number of individual plants. Probes were isolated from the cDNA clones of tomato CHS I and DFR (provided by Dr J. Yoder, UCSF), tobacco CHS (provided by Dr T. Beals, UCLA) and ubiquitin (provided by Dr C. Martin, J.l.I.). The level of tobacco DFR RNA was assayed by probing with the tomato DFR probe and then washing at medium stringency (60°C, lxSSC, 0.5% SDS). Probes were labelled using random hexamer primers (Fein- berg and Volgelstein, 1983). RNA levels were quantified using a Joyce Loebl chromoscan 3 densitometer, slit width 0.3 mm.

Acknowledgements

We thank Alan Lloyd for the kind provision of both transgenic plant lines and information relating to the Lc transformants; John Yoder and T. Beals for providing probes for anthocyanin biosynthetic genes; Cathie Martin for the ubiquitin probe and stimulating discussions; Justin Goodrich for providing clones of the De/gene and helpful guidance and David Jones for care and analysis of the 35S:deh:Ac plants. We also thank Des Bradley, Sylvie Pouteau, Yongbiao Xue, Sandra Doyle, Pilar Cubas and Elisabeth Schultz for comments on the manuscript. T.D. gratefully acknowledges the support of a grant from Diatech UK.

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