SUMMARY. Keywords: anthocyanin, MYB, bhlh, transcription factor, petunia. INTRODUCTION

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1 The Plant Journal (2011) 65, doi: /j X x Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning Nick W. Albert 1,2,*, David H. Lewis 1,*, Huaibi Zhang 1, Kathy E. Schwinn 1, Paula E. Jameson 3 and Kevin M. Davies 1 1 New Zealand Institute for Plant and Food Research Limited, Private Bag , Palmerston North, New Zealand, 2 Institute of Molecular BioSciences, Massey University, Private Bag , Palmerston North, New Zealand, and 3 School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand Received 13 August 2010; revised 16 November 2010; accepted 13 December 2010; published online 14 January * For correspondence (fax ; nicholas.albert@plantandfood.co.nz; david.lewis@plantandfood.co.nz). SUMMARY We present an investigation of anthocyanin regulation over the entire petunia plant, determining the mechanisms governing complex floral pigmentation patterning and environmentally induced vegetative anthocyanin synthesis. DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) encode members of the R2R3-MYB transcription factor family that regulate anthocyanin synthesis in petunia, and control anthocyanin production in vegetative tissues and contribute to floral pigmentation. In addition to these two MYB factors, the basic helix loop helix (bhlh) factor ANTHOCYANIN1 (AN1) and WD-repeat protein AN11, are also essential for vegetative pigmentation. The induction of anthocyanins in vegetative tissues by high light was tightly correlated to the induction of transcripts for PHZ and AN1. Interestingly, transcripts for PhMYB27, a putative R2R3-MYB active repressor, were highly expressed during non-inductive shade conditions and repressed during high light. The competitive inhibitor PhMYBx (R3-MYB) was expressed under high light, which may provide feedback repression. In floral tissues DPL regulates vein-associated anthocyanin pigmentation in the flower tube, while PHZ determines light-induced anthocyanin accumulation on exposed petal surfaces (bud-blush). A model is presented suggesting how complex floral and vegetative pigmentation patterns are derived in petunia in terms of MYB, bhlh and WDR co-regulators. Keywords: anthocyanin, MYB, bhlh, transcription factor, petunia. INTRODUCTION Anthocyanins are coloured products of the flavonoid biosynthetic pathway, produced in response to a range of developmental and environmental signals. In flowers and fruits, they provide visual cues to pollinators and seed distributors (Grotewold, 2006). In vegetative tissues, they are frequently produced in response to stress. Anthocyanins are now generally accepted to be important photoprotectants, although their modes of action in vegetative tissues are still debated as they both absorb light and are also powerful antioxidants (Steyn et al., 2002; Gould, 2004). It may be that anthocyanins have multiple physiological roles in vegetative tissues, or that their roles vary between species. Some plants lack anthocyanins, and presumably achieve photoprotection through alternative mechanisms, for example through non-flavonoid phenolic compounds (such as hydroxy cinnamic acid derivatives). Given both their importance as stress-responsive compounds in vegetative tissues and their ability to absorb photosynthetic light, the production of anthocyanins needs to be tightly regulated to achieve a balance between photoprotection and light harvesting. The flavonoid biosynthetic pathway is the best characterised secondary metabolic pathway in plants (Figure 1), and much is now understood of its regulation. The primary point of regulation for anthocyanin biosynthesis occurs at the transcriptional level, and several regulators of anthocyanin metabolism have been identified (Cone et al., 1986; Paz-Ares et al., 1986, 1987; Ludwig et al., 1989; Goodrich et al., 1992; de Vetten et al., 1997; Quattrocchio et al., 1998, 1999; Walker et al., 1999; Borevitz et al., 2000; Spelt et al., 2000, 2002; Carey et al., 2004; Schwinn et al., 2006). In all species studied to date, anthocyanin and proanthocyanidin regulation is con- 771 The Plant Journal ª 2011 Blackwell Publishing Ltd

2 772 Nick W. Albert et al. Figure 1. The flavonoid biosynthetic pathway. The flavonoid biosynthetic pathway branches off the greater phenylpropanoid biosynthetic pathway leading to the production of flavonoids such as flavonols and anthocyanins. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase, CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3 H, flavonoid 3 -hydroxylase; F3 5 H, flavonoid 3 5 -hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; 3GT, 3-glucosyl transferase; 3RT, 3-rhamnosyl transferase; 5GT, 5-glucosyl transferase; AT, anthocyanin acyl transferase; MT, anthocyanin methyl transferase; GST, glutathione-s-transferase; DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM, dihydromyricetin. trolled by a transcriptional activation complex consisting of R2R3-MYB and basic helix loop helix (bhlh) transcription factors (TFs) and a WD-repeat (WDR) protein (MBW complex), which activates transcription of flavonoid biosynthetic genes (Baudry et al., 2004; Koes et al., 2005; Lepiniec et al., 2006). The R2R3-MYB genes are thought to be key in determining the spatial and temporal patterning of anthocyanins in plants (Schwinn et al., 2006). In addition to the proteins involved in activating the anthocyanin biosynthetic genes, distinct R2R3 and R3-MYB factors have been identified as repressors of anthocyanin synthesis. R2R3-MYB repressors actively repress target genes, mediated by motifs in their C-terminal domains (Tamagnone et al., 1998; Jin et al., 2000). FaMYB1, an R2R3- MYB from strawberry, was shown to interact with the petunia bhlh factors JAF13 and AN1, and ectopic expression of FaMYB1 in tobacco repressed the levels of ANS transcripts (an anthocyanin biosynthetic gene) and anthocyanin synthesis in flowers (Aharoni et al., 2001). Recently an R3 MYB factor (containing a truncated R2 domain), AtMYBL2, from Arabidopsis was shown to actively repress anthocyanin synthesis (Dubos et al., 2008; Matsui et al., 2008). Another class of MYB factors involved in anthocyanin regulation are the small R3 repressors, which are proposed to act as competitive inhibitors of R2R3-MYB activators by binding and titrating bhlh cofactors (Kroon, 2004; Koes et al., 2005; Zhang et al., 2009). The R3-MYB CAPRICE (CPC) from Arabidopsis has been proposed to control anthocyanin production in addition to its primary role in determining epidermal cell fates (Zhang et al., 2009), and ectopic expression of CPC in tobacco repressed anthocyanin synthesis (Zhu et al., 2009). Together, the MBW activators and MYBrepressor factors involved in anthocyanin regulation offer one of the best current systems for elucidating mechanisms of transcriptional regulation in plants. Petunia is one of the classic model systems for investigating anthocyanin biosynthesis and regulation, particularly for floral pigmentation. It has complex floral pigmentation patterns, including petal limb, petal tube, anthers and veinassociated patterning (Wiering, 1974; Cornu and Maizonnier, 1983). Known regulators of some of these patterns are the R2R3-MYB factors, AN2 (petal limb) and AN4 (petal tube and anthers) (Quattrocchio et al., 1998, 1999; Kroon, 2004), a bhlh factor, AN1 (Spelt et al., 2000, 2002), and a WDR protein, AN11 (de Vetten et al., 1997). An additional MYB clone, MYBb, has been isolated from petunia petals and shares high sequence identity with AN4 (Kroon, 2004) but remains to be fully characterised. AN11 is expressed throughout all major plant organs (de Vetten et al., 1997), and low levels of AN1 transcripts have been detected in sepals, stems and leaves (Spelt et al., 2000; Quattrocchio et al., 2006). A second bhlh factor, JAF13, has been identified in petunia and may contribute to floral pigmentation, although it is not functionally redundant with AN1 (Quattrocchio et al., 1998; Tornielli et al., 2008). R2R3- and R3-MYB repressors have also been identified in petunia, but have yet to be functionally characterised. The R2R3-MYB, PhMYB27, has high amino acid similarity with strawberry FaMYB1, including a putative ERF-repression motif (EAR) (Murr, 1995; Aharoni et al., 2001), and a petunia homologue of CPC expressed in developing flowers, PhMYBx, represses anthocyanin synthesis when over-expressed (Kroon, 2004; Koes et al., 2005; Tornielli et al., 2008). Petunia also produces anthocyanin in vegetative tissues (stems, leaves and sepals) in response to stressors such as high light (Albert et al., 2009). However, the regulators of vegetative anthocyanin synthesis have yet to be identified. In this study, we identified the MYB transcriptional activators that regulate pigmentation patterns in vegetative tissues of

3 Anthocyanin pigmentation patterning in petunia 773 petunia. The previous identification of R2R3-MYB activators, R2R3-MYB repressors, R3-MYB repressors and bhlh and WDR factors involved in floral patterning in petunia provides an opportunity to investigate whether distinct floral and vegetative regulators exist, which factors are the key determinants for pigmentation patterns, and what interactions may occur between the floral and vegetative regulatory pathways. We propose a model of the regulation of anthocyanin production in floral and vegetative tissues that integrates the data on the TFs identified with the activity of known anthocyanin regulators in petunia. RESULTS Two MYB TFs identified, DEEP PURPLE and PURPLE HAZE Previous research suggested that R2R3-MYB factors, distinct from AN2 and AN4, are required for light-induction of anthocyanin biosynthesis in vegetative tissues of petunia (Albert et al., 2009). Thus, a candidate-gene approach was taken to identify MYB TFs that regulate the accumulation of vegetative anthocyanin in petunia. Two R2R3-MYB TFs were identified by a combination of PCR, using degenerate oligonucleotides to the region encoding the MYB DNA-binding domain, and 3 and 5 rapid amplification of cdna ends (RACE) to obtain the full-length cdna sequences. The genes were named DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) based on their gain-of-function phenotypes. DPL was cloned from shade-grown Mitchell petunia (MP) leaves (no anthocyanin), while PHZ was cloned from high-light grown leaves (producing anthocyanins). DPL and PHZ encode R2R3-MYB TFs that share 70 and 79% nucleotide identity, respectively, to AN2 across their coding sequences. A phylogenetic tree, based on the alignment of the deduced amino acid sequence of DPL and PHZ to other R2R3-MYB genes, is shown in Figure 2a. DPL and PHZ form a clade with other known R2R3- MYB anthocyanin regulators, particularly with those from the Solanaceae. DPL groups very closely to the petunia MYBs, AN4 and MYBb. Both DPL and PHZ contain the amino acid signature motif ([DE]Lx 2 [RK]x 3 Lx 6 Lx 3 R) that has been identified as important for interaction with bhlh type co-regulators (Zimmermann et al., 2004) and is a feature of other known anthocyanin regulators. DPL and MYBb are allelic As the nucleotide sequence identity between AN4, MYBb and DPL is very high, we investigated the possibility that they are alleles rather than separate genes. AN4 and MYBb were originally isolated from Petunia hybrida (line R27 for MYBb) and DPL from MP [Petunia axillaris (P. axillaris P. hybrida); double haploid]. A PCR amplification of all three genes was performed on genomic DNA from a P. hybrida background (V30) and MP. With both V30 and MP DNA, an AN4 PCR product was obtained distinct from the DPL/MYBb products, demonstrating it is a separate gene. Figure 2. DPL and PHZ phylogenetically group with anthocyanin regulators. (a) Phylogenetic tree comparing the deduced amino acid sequence of DEEP PURPLE and PURPLE HAZE to other R2R3-MYB transcription factors. EOBII, ODORANT1, PhMYB1, PhMYB2, PhMYB3 from Petunia and MIXTA from Antirrhinum were included as outgroups. The scale bar represents amino acid substitutions. Bootstrap values from 1000 replicates are indicated. Am, Antirrhinum majus; At, Arabidopsis thaliana; Ca, Capsicum annuum; Gt, Gentiana triflora; Md, Malus domestica; Ph, Petunia hybrida; Sl, Solanum lycopersicum. (b) Gene structures of DPL and PHZ: boxes indicate exons, dashed lines indicate introns. The triangle indicates the position of a putative long terminal repeat (LTR)-retrotransposon element, present within the W59 petunia allele of DPL. However, amplification of DPL/MYBb resulted in a single product in both V30 and MP, and these were very similar to each other, including introns, suggesting that MYBb and DPL are alleles rather than separate genes. The DPL/MYBb alleles from V26 and W59 petunia (both P. hybrida) were also amplified and were very similar to the V30 sequence (MYBb-like), suggesting that the small sequence differences

4 774 Nick W. Albert et al. are simply polymorphisms between MP and P. hybrida cultivars. (GenBank accessions are given in the Experimental procedures.) The allelism of DPL and MYBb was confirmed by segregation analysis of the cross W59 (MP W59) (a cross that was performed to investigate the segregation of DPL and venation patterning, as explained later). W59 contains a MYBb-like (P. hybrida) allele of DPL (dpl::ltr) that segregated with the MP allele of DPL as a single gene in the backcross population. Using PCR amplification and sequence analysis, DPL and MYBb were demonstrated to be inherited as alleles of the same gene. DPL and PHZ control anthocyanin production To confirm the function of DPL and PHZ as anthocyanin regulators, complementation of Antirrhinum rosea dorsea (ros dor ) petals was performed. The petals of ros dor plants are only weakly pigmented in the abaxial epidermis due to the loss of function of the anthocyanin-related R2R3-MYB factors Rosea1 and Rosea2 (Schwinn et al., 2006). Transient expression of DPL or PHZ driven by the CaMV35S promoter resulted in the production of red foci in the acyanic adaxial epidermis (Figure 3a). Petunia lines expressing DPL and PHZ under the control of the CaMV35S promoter were generated in the MP background. During transformation, the leaf discs became pigmented along the cut surfaces within 48 h of incubation with Agrobacterium tumefaciens containing the MYB over-expression (OE) constructs. The coloured leaf discs gave rise to highly pigmented shoots and, subsequently, highly pigmented plants were regenerated. Of the >20 independent lines generated for each construct, four representative lines for each were chosen for further analyses. The PHZ OE plants consistently had a dusky bronze appearance, while DPL OE plants were always more intensely pigmented (Figure 3b). One PHZ OE line (MP/14/5) was non-pigmented and did not express the transgene, but was included in analyses as a negative control. In addition to generating over-expression lines in the MP petunia background, transgenic V26 petunia (V26 is wild-type for all known anthocyanin regulators, except AN4) were also generated. These had similar vegetative pigmentation phenotypes as the MP OE lines (Figure S1 in Supporting Information). Mitchell petunia is an2, an4, resulting in acyanic petals and anthers, respectively (Figure 3b). Both DPL and PHZ expressed from a CaMV35S promoter were able to comple- Figure 3. DPL and PHZ regulate anthocyanin production. (a) Biolistic transformation of Antirrhinum rosea dorsea petal tissue with 35S pro :DPL or 35S pro :PHZ (+35S pro :GFP internal control). Anthocyanin-producing cells were viewed with white light, GFP was viewed with blue light. (b) Pigmentation phenotypes of leaves, petals and mature flower buds from untransformed Mitchell petunia, 35S pro :DPL, 35S pro :PHZ petunia lines. Selected lines shown are representative of the vegetative and floral pigmentation observed in other independent lines.

5 Anthocyanin pigmentation patterning in petunia 775 ment these mutations, restoring anthocyanin synthesis to the corolla and anthers: DPL OE resulted in purple flower buds, which faded upon flower opening; PHZ OE resulted in very pale mauve buds, which appeared white upon flower opening. This fading of flower colour is due to a dominant trait that affects anthocyanin stability or synthesis in the petal limbs of MP, which may be due to a dominant allele of the FADING locus (de Vlaming and van Eekeres, 1982). DPL or PHZ OE resulted in anthocyanin accumulation in immature anthers, but pollen was always yellow. Anthocyanins accumulated to high levels in the leaves of the DPL and PHZ OE lines, correlating to their pigmentation phenotypes (Figure S2). MP/12/9 was the most darkly coloured line and had the highest concentration of anthocyanins, while the untransformed controls and line MP/14/5 that had green leaves had no detectable anthocyanins. Flavonol levels in leaves were severely reduced in all lines that accumulated anthocyanins, compared with control lines (Figure S2), indicating that the common dihydroflavonol precursors were being redirected towards anthocyanin synthesis (Figure 1). Anthocyanin accumulation was enhanced in the flower tube in both the DPL and PHZ OE lines. In the flower limb, DPL lines had strongly increased anthocyanin levels, while PHZ lines had a much smaller increase in anthocyanins, which correlated with the off-white/pale mauve phenotype of PHZ lines. Flavonols were produced at high levels in the flower limb and tube of control lines, and their levels were not altered in the MYB OE lines. The major anthocyanin peaks detected in the flowers and leaves of the MYB OE lines by HPLC (Figure S2) corresponded to various delphinidin, petunidin and malvidin glycosides previously identified from the leaves and flowers of petunia (Bloor et al., 1998; Albert et al., 2009). Over-expression of DPL resulted in enhanced transcript levels for the general phenylpropanoid genes PAL, C4H and several flavonoid genes (CHS, CHI, F3H, F3 H, F3 5 H, DFR, ANS, 3RT, 5GT, MT and GST but not FLS, 3GT or AT ) (Figure 4a). The same set of genes was activated in PHZ OE lines, although at lower levels to those of the DPL lines (Figure 4a). DPL OE lines accumulated transgene transcripts at approximately 10-fold higher levels than the PHZ OE lines (Figure 4b). Transgene expression was determined by qrt- PCR (Figure 4b) and showed that the DPL OE lines accumulated transcripts at approximately 10-fold higher levels than the PHZ OE lines. The MP/14/5 line, which failed to express the PHZ transgene and performed as the untransformed controls, showed low levels of transcripts for PAL, CHI, FLS and 3GT in leaves. In the flower, only a subset of the genes activated in leaves was activated by DPL or PHZ OE (F3 H, F3 5 H, DFR, ANS, 3RT, 5GT, MT and GST) (Figure S3), as the phenylpropanoid (PAL) and flavonol biosynthetic genes (CHS, CHI, F3H, FLS, 3GT and AT) were already highly expressed. Figure 4. Phenylpropanoid and flavonoid gene induction in DPL and PHZ over-expression OE petunias. (a) RNA gel blots of phenylpropanoid and flavonoid biosynthetic genes expressed in the leaves of untransformed Mitchell petunia controls (UC), 35S pro :DPL (MP/12/ ) and 35S pro :PHZ (MP/14/ ) petunias, with each sample representing an independent line. 25/26S rrna is shown as a loading control. (b) Transgene expression was determined by quantitative RT-PCR. Line MP/ 14/5 does not express the transgene and was included as a control. Light-induced vegetative pigmentation involves both activator and repressor TFs High-light treatment induces anthocyanin accumulation in vegetative tissues of wild-type MP plants, while

6 776 Nick W. Albert et al. shade-grown plants remain acyanic (Albert et al., 2009). In order to determine why anthocyanin synthesis is absent in shade conditions and induced by high-light conditions, transcript abundance in the leaves of MP was determined for both activator and putative repressor TFs involved in anthocyanin regulation (Figure 5). High-light-induced anthocyanin production positively correlated with an increase in transcripts for the two R2R3-MYB factors, PHZ (>60-fold induction) and DPL (5-fold induction). Transcripts for AN2, which contains a frame-shift mutation in MP, were not detected in leaves (Figure 5) but were found at the expected high-levels in flowers (data not shown). Transcript levels for AN1 (bhlh) were not detectable in shade-grown leaves, but increased with high light. Transcripts for both AN11 (WDR) and JAF13 (bhlh) were present at significant levels in both shade and high-light plants, and their levels were not affected by the light environments. PhMYB27 (R2R3-MYB repressor) had an inverse expression profile to that of the MYB activators, with transcript levels high in shade-grown leaves and reduced 10-fold with high-light treatment. Transcript levels for the putative R3-MYB repressor, PhMYBx, increased with high-light. AN1 and AN11 are described as essential regulators of anthocyanin synthesis throughout the plant (Quattrocchio et al., 1993), although these studies focused on floral patterning. The vegetative phenotypes for stable an1 ) (W225) and an11 ) (W134) petunia lines were assessed, and confirmed to be completely free of anthocyanins throughout vegetative tissues (Figure S4a). DPL and PHZ have distinct roles regulating floral pigmentation patterns The possible roles of DPL or PHZ in floral tissues in addition to vegetative tissues were examined using gene expression and genetic mutant analysis. The full petal colour phenotype in petunia is controlled by the R2R3-MYB factor AN2, while flower tube and anther colour is regulated by AN4 (Quattrocchio et al., 1993, 1999). In petunia species or lines with AN2 and AN4 active, the full colour masks more subtle floral pigmentation patterns such as vein-associated anthocyanin pigmentation in the flower tube (venation) and light-regulated anthocyanin accumulation on the abaxial petal surface of flower buds (bud-blush). Mitchell petunia (an2 an4) displays anthocyanin venation patterning of the flower tube (Figure 6a), and if grown under high-light environments the external bud surface is weakly pigmented, which is particularly noticeable in petunia over-expressing the maize Lc bhlh factor where the existing pigmentation patterns are enhanced (Figure S4b; Bradley et al., 1998). This blush is not easily observed in wild-type MP due to a dominant trait reducing anthocyanin pigmentation in flowers, which may be due to the activity of FADING. The petunia line W59 (an2 an4) completely lacks flower tube venation but the petals are blushed with anthocyanin on the abaxial light-exposed bud surfaces resulting in a tie-dyed appearance in open flowers (Figure 6b). Both MP and W59 produce anthocyanins in vegetative tissues. DPL and PHZ transcripts were expressed early during flower bud development in MP, overlapping with AN1 expression, and coinciding with the appearance of veinassociated anthocyanin in the flower tube (stage 2 3) and bud-blush (stage 2 3) (Figure 6c). In W59 petunia, PHZ transcripts were expressed early and overlapped with AN1 during stages 1 3, which coincided with the bud-blush phenotype. DPL transcript levels were severely reduced in W59 compared with MP. The reduced transcript levels for DPL in W59 suggested that W59 may contain a nonfunctional allele of DPL, which may account for the lack of flower tube venation. Alleles for DPL and PHZ were isolated from W59 and found to be highly similar to the P. hybrida V30 alleles. However, the W59 DPL allele contained a large (1.6 kb) insertion within intron two (Figure 2b), displacing the TA-rich sequences from the adjacent splice donor site. The insertion contains 226 bp inverted terminal repeats Figure 5. High light induces PHZ and AN1 to determine vegetative pigmentation. Transcript abundance of anthocyanin regulators in leaves of shade or high-light grown Mitchell petunia plants. Relative transcript abundance was determined by quantitative RT-PCR. Means SE, n = 4 are reported. WDR, WD-repeat; bhlh, bacis helix loop helix.

7 Anthocyanin pigmentation patterning in petunia 777 Figure 6. DPL and PHZ activity correlates to flower tube venation and bud-blush anthocyanin patterning in flowers. Floral phenotypes of (a) Mitchell petunia (MP) and (b) W59 petunia. (c) Relative transcript abundance for DPL, PHZ and AN1 in developing flower buds of MP and W59 petunia, determined by quantitative (q)rt-pcr. Means SE, n = 3 are reported. (d) AN1 and PHZ expression in dark- or light-grown W59 flower buds, determined by qrt-pcr. Means SE, n = 3 are reported, expressed as fold-differences relative to dark treatment. Floral phenotypes of representative progeny from the MP W59 (F 1 ) (e) and backcross (MP W59) W59 populations (f). Inset shows the flower tube, individual plant ID numbers are indicated. (g) Genotyping of MP, W59, F 1 and backcross population for the presence of the dpl::ltr allele from W59 petunia, or DPL allele from MP. The venation phenotype of the individuals is indicated (). resembling long terminal repeat (LTR) retrotransposons, but lacks any identifiable open reading frames (ORFs). The expression of AN1 and PHZ was determined in W59 flower buds grown with or without light, and showed that PHZ transcript levels were significantly higher in light-grown buds (Figure 6d). To determine whether the insertion within DPL in W59 petunia (dpl::ltr) was responsible for the lack of flower tube venation, W59 was crossed with MP, and subsequently backcrossed to W59 to allow segregation analysis. The W59 MP F 1 plants had blushed flower buds and flower tube venation (Figure 6e). The backcross progeny all had blushed buds, but segregated for flower tube venation (Figure 6f) and the presence of the functional DPL allele from MP co-segregated with flower tube venation (Figure 6g). Localisation of DPL and PHZ promoter activity To further link the activities of DPL and PHZ to specific pigmentation patterns throughout the petunia plant, the promoter region for these genes was isolated and used for promoter:intron GUS (igus) localisation studies. Stable MP plants containing DPL pro :igus, PHZ pro :igus, 2 35S pro : igus or part27 (empty vector) constructs were generated. Regenerated shoots were stained for GUS activity. Double- 35S pro lines exhibited intense blue staining, DPL pro lines had a vein-associated pattern, and PHZ pro weak staining to leaf margins and apical tissues (data not shown). The plants were grown in a greenhouse and stained for GUS activity. However, material from mature greenhouse-grown plants browned excessively and failed to stain. The browning, presumably due to the oxidation of phenolic compounds, inhibited GUS activity, preventing the histochemical localisation of GUS activity with 5-bromo-chloro-3-indoyl-b- D-glucuronic acid (X-gluc). Difficulties in staining certain tissues for GUS activity, erratic staining patterns and browning have previously been reported in petunia and cranberry (Koes et al., 1990; van der Meer et al., 1992; Serres et al., 1997). The inclusion of oxidation catalysts severely exacerbated this effect. Extensive optimisation with the 2 35S:iGUS and part27 empty vector lines demonstrated that leaf, stem, sepals and flower tubes could not be reliably stained. Flower limb tissues were able to be stained after treatment to allow substrate penetration. We repeated the analysis using T 1 seedlings, germinated and grown in tissue culture (young tissues, low light). Seedlings grown under these conditions proved reliable for GUS staining. The DPL promoter directed expression predominantly to the vasculature (Figure 7a). Due to the long incubation times required and the lack of oxidation catalysts, the primary X-gluc cleavage products diffused from the vasculature, resulting in a diffuse localisation pattern particularly in cotyledons and leaves. Inclusion of 1 mm K 3 Fe(CN) 6 /K 4 Fe(CN) 6 reduced the GUS signal, but showed that the DPL pro -directed GUS activity was restricted to the

8 778 Nick W. Albert et al. vasculature (Figure 7b). The PHZ promoter directed expression in vegetative tissues that become pigmented with anthocyanin in response to light, particularly to the leaf margins and exposed apical tissues (Figure 7a). Lightinduction experiments for the PHZ promoter were not possible, as these treatments resulted in tissue browning and unreliable staining in all lines (including 2 35S:iGUS). The petal limb was able to be stained for GUS activity. PHZ pro directed GUS activity to the abaxial surface of the petal (Figure 7c), matching the observed petal blush phenotype in W59 petunia. DPL pro directed expression to the petal limb, with localisation in the reticulated network of fine veins, and some diffusion to surrounding cells (Figure 7c). The flower tube was not able to be stained because of GUS inhibition. This inhibition is apparent in the white tissue surrounding the major veins entering the flower limb from the tube region in the DPL pro lines (Figure 7c). DISCUSSION Our previous investigation into the regulation of lightinduced vegetative anthocyanin accumulation in petunia led us to hypothesise that the expression of R2R3-MYB TFs was induced during high-light conditions to regulate anthocyanin synthesis, and that these factors differed from AN2 and AN4, which regulate anthocyanin synthesis in petunia flowers (Albert et al., 2009). In this study we have isolated and characterised two R2R3-MYB TFs from petunia, DPL and PHZ. We have confirmed both DPL and PHZ as positive regulators of anthocyanin synthesis through transient complementation of the Antirrhinum ros dor R2R3-MYB mutant phenotype, complementation of the an2 and an4 R2R3-MYB mutations in stably transformed MP petunia, and ectopic synthesis of anthocyanin pigments in vegetative and floral tissues of petunia stably over-expressing DPL or PHZ. Target gene selectivity of DPL and PHZ in the OE lines was similar, in that the same genes were activated by both factors. The level of activation, however, was consistently lower in the PHZ lines, matching the weaker pigmentation phenotypes and reflecting the level of transgene expression. It is interesting that only weak expressors of PHZ were able to be regenerated, which could be due to high PHZ levels having negative effects upon growth and development. However, it may also be due to post-transcriptional mechanisms that limit PHZ transcript levels. MYB activators and repressors control vegetative anthocyanin synthesis The induction of PHZ in leaves grown under high light, correlating strongly with the induction of anthocyanin synthesis, in conjunction with the W59 (an2, an4, dpl) genetic data, suggests that PHZ is the predominant regulator of vegetative anthocyanin pigmentation in petunia. DPL was only modestly activated by high light, and is expressed at a basal level under non-inductive conditions, suggesting it makes a smaller contribution to pigmentation patterning in vegetative tissues. Transcripts for AN1 (bhlh) were not detected in leaves of shade-grown petunias, but high-light induced the expression of AN1. While the total level of AN1 transcripts is relatively low, this correlates well with the weak pigmentation phenotype, which is limited to subepidermal cells (Bradley et al., 1998; Albert et al., 2009). Furthermore, the mutant analysis confirms AN1, and the WDR AN11, as essential regulators of anthocyanin synthesis in the vegetative tissues (Figure S4), petals and seed coat (Spelt et al., 2000; 2002). The role of JAF13 (bhlh) in regulating anthocyanin synthesis is unclear. JAF13 activates the Figure 7. The promoter activities of DPL and PHZ determine venation and blushed pigmentation patterns. (a c) Histochemical localisation of GUS (b-glucuronidase) activity in DPL pro :igus and PHZ pro :i- GUS petunia lines: (a) seedlings, (b) seedlings in the presence of 1 mm K 3 Fe(CN) 6 /K 4 Fe(CN) 6 ; (c) petal limb tissues in the presence of 1 mm K 3 Fe(CN) 6 /K 4 Fe(CN) 6. The staining patterns are representative of the 15 independent lines generated for each construct (based on petal limb staining in T 0 ). Three representative lines were selected for each construct and self-crossed (T 1 seedlings). The GUS localisation for two independent lines per construct is shown.

9 Anthocyanin pigmentation patterning in petunia 779 promoters of anthocyanin biosynthetic genes in transient assays (Quattrocchio et al., 1998; Spelt et al., 2000), yet cannot replace AN1 activity in flowers (Spelt et al., 2000) or in vegetative tissues, where it is constitutively expressed at higher levels than AN1. In shade-grown leaves, DPL (MYB), JAF13 (bhlh) and AN11 (WDR) are all expressed, yet anthocyanins fail to accumulate. This raises the possibilities that: (i) these three factors are expressed in different cell types within the vegetative tissues, (ii) DPL cannot activate anthocyanin synthesis with JAF13, or (iii) active repressors are preventing anthocyanin synthesis during shade conditions. The an1 phenotype together with the induction of AN1 by high light suggests that AN1 plays a central role in regulating light-induced anthocyanin pigmentation in conjunction with PHZ, DPL and AN11, but that the role of JAF13 in anthocyanin synthesis requires further investigation. Our results support previous observations suggesting that anthocyanin synthesis is actively repressed during shade conditions. In Arabidopsis, the R3-MYB active repressor AtMYBL2 is highly expressed in shade conditions, and repressed with high-light (Dubos et al., 2008). High-light also up-regulates the Arabidopsis R2R3-MYB activators PAP1 and PAP2 and their bhlh partner TT8 (AN1- like) (Dubos et al., 2008). AtMYBL2 binds bhlh TFs and requires a C-terminal motif to assert its repressive function upon anthocyanin biosynthetic genes (Matsui et al., 2008). Although PhMYB27 is an R2R3-MYB rather than an R3-MYB, like AtMYBL2 it contains residues for bhlh interaction and a repression motif (an EAR-type motif), and is similar to FaMYB1 from strawberry, which can bind the petunia bhlh factors PhJAF13 and PhAN1 and repress anthocyanin synthesis (Aharoni et al., 2001). The expression patterns of the TFs are also similar to Arabidopsis, as PhMYB27 is highly expressed in shade-grown leaves and down-regulated by high light, while transcript levels for PHZ, DPL and AN1 are elevated in response to high light and, interestingly, PhMYB27 is a direct target of AN1 in petals (Spelt et al., 2000). PhMYB27 warrants further investigation for its role, if any, in repressing anthocyanin synthesis, as the similarities between the Arabidopsis and petunia data suggest that there is a conserved mechanism for regulation of anthocyanin biosynthesis in vegetative tissues involving activators and multiple types of repressor (R3-MYB and R2R3-MYB). The small R3-MYB, PhMYBx, was expressed in response to high-light treatment in MP leaves. Transcript levels were low relative to the other TF genes assayed. However, such levels may be expected when considering that only low levels of anthocyanins are produced in leaves, and they are restricted to a few cell layers within the leaf. The transcript levels were similar to those for AN1, which is known to be active in vegetative pigmentation. PhMYBx is proposed to act as a competitive inhibitor in anthocyanin biosynthesis in petunia, as it binds the bhlh factors JAF13 and AN1, and OE suppresses floral anthocyanin production (Kroon, 2004; Koes et al., 2005). In addition, PhMYBx may act as part of a feedback mechanism, as PhMYBx transcripts are reduced in anthocyanin-lacking flowers of an1 and an11 lines (Kroon, 2004; Koes et al., 2005). This proposed role of PhMYBx is supported by data for the small R3-MYB of Arabidopsis, CAPRICE (CPC), which has known roles in regulating epidermal cell fate (Zhu et al., 2009). Over-expression of CPC represses anthocyanin biosynthetic gene expression, and cpc mutants produce higher levels of anthocyanins than wild-type plants during stress conditions (Zhu et al., 2009). The upregulation of PhMYBx when tissues begin to accumulate anthocyanin pigments (Figure 5) could provide feedback inhibition to provide fine control and limits on anthocyanin levels. Thus, anthocyanin regulation in vegetative tissues may be by a combination of R2R3-MYB activators (DPL, PHZ), with their bhlh (AN1) and WDR (AN11) partners, and MYB repressors (PhMYBx and, potentially, PhMYB27) (Figure 8), with suppression in shade conditions as well as activation and de-repression under high light. R2R3-MYB family members control specific floral pigmentation patterns Based on gene expression patterns, segregation analysis and vascular-associated promoter activity, DPL controls flower tube venation in petunia, and W59 is identified as carrying a non-functional DPL allele. The VENATION1 (VE1) locus is a positive regulator of venation to the flower tube and limb. The descriptions of VE1 + individuals as having close, reticulate venation to the flower tube and ve1 ) individuals completely lacking venation or having only weak longitudinal pigmentation over the veins (Wiering, 1974) accurately matches our observations in MP and W59 petunia, raising the possibility that DPL may reside at this locus. Interestingly, while VE1 + is essential for vein-patterning of the tube and limb, two other loci control vein patterning in the flower limb. VENATION2 prevents vein-patterning in the flower limb (dominant negative), while VENATION3 enhances the degree of veining in the limb (in VE1 + ve2 ) genotypes) (Wiering, 1974). Vein-associated pigmentation in Antirrhinum flowers is controlled by the R2R3-MYB factor Venosa (Ven) (Schwinn et al., 2006). Ven transcripts are expressed radiating from vascular tissues to the adaxial epidermis, where they intersect the expression domain of the epidermal-specific bhlh factors, resulting in vein-associated pigmentation to the overlying epidermal cells (Shang et al., 2010). That similar R2R3-MYB genes define venation in petunia (Solanaceae, order Solanales) and Antirrhinum (Plantaginaceae, order Lamiales), suggests that a common mechanism may determine venation pigmentation in a wide range of plant species. PHZ determines the petal bud-blush phenotype in petunia. PHZ transcript levels were shown to be activated in vegetative tissues and flower buds in response to light,

10 780 Nick W. Albert et al. Figure 8. Proposed models for the regulation of complex floral and vegetative pigmentation patterns. (a) Existing model for floral patterning in petunia. AN2 (MYB) determines strong petal limb pigmentation, while AN4 (MYB) controls flower tube and anther pigmentation, with common basic helix loop helix (bhlh) (AN1) and WD-repeat (WDR) (AN11) partners. (b) Extended floral pigmentation model to include bud-blushing, determined by PHZ, and flower tube venation determined by DPL. (c) Model for vegetative anthocyanin pigmentation. supporting its function in regulating light-induced floral pigmentation. W59 petunia lacks functional alleles for all known R2R3-MYB anthocyanin regulators (an2, an4, dpl) except for PHZ, and has wild-type light-regulated vegetative pigmentation and blushed flowers. PHZ transcript accumulation in developing flower buds overlaps with AN1 in stages 1 3, preceding the accumulation of anthocyanin on the exposed bud surfaces, and decreases at later stages of development (Figure 6c). This results in a tie-dye appearance because mature flower buds (stage 5) do not express PHZ and, therefore, newly exposed petal tissue in opening flowers remains non-pigmented, which is supported by PHZ pro -directed GUS localisation. Only that petal tissue exposed to light during early development, when PHZ is expressed, becomes pigmented. The link between environmentally regulated vegetative pigmentation and bud-blush is seen in a range of species, suggesting that stress-induced vegetative pigmentation and floral bud-blush may be determined by PHZ-like genes in genera other than Petunia. For example, gentian lines lacking functional alleles of GtMYB3 (the predominant floral MYB regulator) have white flowers, except under stress conditions (high light, cold) when vegetative tissues and exposed flower bud surfaces accumulate anthocyanins (Nakatsuka et al., 2008). Apple also has blushed flowers, producing anthocyanins early during flower development in response to light (Dong et al., 1998). The light-responsive anthocyanin regulator MdMYB1 that controls fruit colour is expressed in flower buds (Takos et al., 2006), suggesting that it may also control bud-blush. In both petunia and apple, light-responsive bud-blush pigmentation only occurs during the early stages of bud development, while mature flower buds apparently lose competency to respond to environmental signals. This contrasts with the full petal colour phenotypes controlled by AN2 in petunia and Rosea1 in Antirrhinum. The expression of these genes is upregulated in maturing buds, resulting in peak anthocyanin accumulation prior to flower opening (Quattrocchio et al., 1998; Spelt et al., 2000; Schwinn et al., 2006). The current model for regulation of anthocyanin pigmentation in petunia flowers is that full petal limb colour is controlled by the R2R3-MYB factor AN2, while flower tube and anther pigmentation are regulated by AN4, sharing common bhlh (AN1) and WDR (AN11) factors (Figure 8a) (de Vetten et al., 1997; Quattrocchio et al., 1998, 1999; Spelt et al., 2000). This model can now be extended (Figure 7b). In addition to the activities of AN2 and AN4, PHZ determines light-regulated anthocyanin synthesis in the exposed petal surface in developing flower buds, resulting in a tie-dyed appearance (Figure 8b), as well as vegetative pigmentation (Figure 8c). DPL controls vein-associated anthocyanin pigmentation in the flower tube (Figure 8b). Although expressed in leaves under high light, DPL may make only a weak contribution to vegetative pigmentation patterns, and AN2 and AN4 probably do not contribute significantly, as W59 (an2, an4, dpl) has wild-type levels of anthocyanin in vegetative tissues. The petunia model is consistent with the findings for Antirrhinum (Schwinn et al., 2006) and maize

11 Anthocyanin pigmentation patterning in petunia 781 (Piazza et al., 2002) in suggesting that R2R3-MYB gene family members, having diverse expression patterns, are key to determining the spatial distribution of anthocyanins throughout the plant. The findings presented here demonstrate that in petunia, vegetative-specific anthocyanin regulators do not exist, rather, environmentally regulated patterns are controlled by a common MYB factor active throughout the plant. The R2R3-MYB factors are key determinants for controlling distinct pigmentation patterns throughout the plant, while bhlh and WDR factors are shared between the regulatory mechanisms that govern floral and vegetative pigmentation. EXPERIMENTAL PROCEDURES Identifying DEEP PURPLE and PURPLE HAZE Degenerate primers NA1/NA2 (Table S1) were designed based upon MYB DNA-binding domains of known anthocyanin-regulating MYB genes from dicot species. First-strand cdna was made with RNAs extracted from shade- and high-light-grown MP leaves using Superscript III Reverse Transcriptase and oligo dt (Invitrogen, Reverse transcription PCR was performed from 2 ll first-strand cdna using the primers NA1/NA2 with Taq polymerase (Qiagen, The PCR was performed at 94 C for 2 min (94 C 30 sec, 50 C 30 sec, 72 C 1 min) 35 cycles, 72 C for 5 min. The PCR products were sequenced, and the sequences were used to design gene-specific primers for 3 -RACE and 5 -RACE. NA8 primer was used for generating tagged cdna. 3 -RACE was performed using the primers NA9 and NA1, followed by a nested round using NA9 and the genespecific primers NA7 and NA6 for DPL and PHZ, respectively. 5 -RACE was performed using the 5 -RACE System version 2.0 (Invitrogen) following the manufacturer s instructions with genespecific primers NA18 and NA19 for DPL and NA16 and NA17 for PHZ. The RACE PCRs were performed under the same conditions as described above, except that 52 C annealing was used for 5 -RACE. Full cdnas for DPL and PHZ were amplified with gene-specific primers NA27/NA12 and NA26/NA15. Genomic sequences for AN4, MYBb, DPL and PHZ were amplified from petunia V30, V26, W59 and MP DNA by PCR with the primers NA158/NA159, NA156/NA157, NA34/NA35 and NA24/NA25, respectively, with Faststart High Fidelity Polymerase (Roche, DPL/dpl::LTR genotyping was performed by PCR upon genomic DNA with the primers NA34/NA35 (DPL +, 1.5 kb) and the dpl::ltr-specific primers NA134/NA209 (dpl::ltr, 350 bp). GenBank accessions: AN4, MP HQ428104, V30 HQ428105; PHZ, MP HQ116170, HQ428100, V30 HQ428102, V26 HQ428101, W59 HQ428103; DPL, MP HQ116169, HQ428106, V30 HQ428108, V26 HQ428107, W59 HQ Phylogenetic analysis of DEEP PURPLE and PURPLE HAZE The translated coding sequences for DPL and PHZ were aligned to other MYB sequences using ClustalX (v. 2.0) and a neighbour-joining phylogenetic tree was generated with 1000 bootstrap replicates (Larkin et al., 2007). The tree was rendered in Dendroscope (v ) (Huson et al., 2007). GenBank accession numbers are AN2 AF (Quattrocchio et al., 1999); AN4 EB175066, MYBb EB (Kroon, 2004); MdMYB1 DQ (Takos et al., 2006); MdMYB10 EU (Espley et al., 2006); GtMYB3 AB (Nakatsuka et al., 2008); EOBII EU (Spitzer-Rimon et al., 2010); ANT1 AY (Mathews et al., 2003); A AJ (Borovsky et al., 2004); Rosea1 DQ275529, Rosea2 DQ275530, Venosa DQ (Schwinn et al., 2006), PAP1/AtMYB75 AF and PAP2/AtMYB90 AF (Borevitz et al., 2000); ODORANT1 AY (Verdonk et al., 2005), PhMYB1 Z13996, PhMYB2 Z13997, PhMYB3 Z13998 (Avila et al., 1993); Mixta X79108 (Noda et al., 1994); DPL HQ116169, PHZ HQ this study. Quantitative reverse-transcription PCR (qrt-pcr) RNA was extracted from pooled leaves of MP petunia grown under shade ( lmol m )2 sec )1 ) or high-light conditions (750 lmol m )2 sec )1 ), with four biological replicates per treatment, as described in Albert et al. (2009). Petal tissue (tube and limb combined) from bud stages 1 5 (Figure 6c) was collected from three biological replicates from MP and W59 petunia and RNA extracted (Chomczynski and Sacchi, 1987); each sample consisted of three to six pooled buds from an individual plant. Petals from dark- or lightgrown stage 1 2 W59 buds were collected (three biological replicates). Total RNA (1 lg) was treated with RNAse-free DNAse 1 (Invitrogen) and used for first-strand cdna synthesis, prepared with Transcriptor reverse transcriptase (Roche) and oligo dt The cdna was diluted 20-fold for qrt-pcr. Transgene expression in the MYB OE lines was determined in pooled leaf samples using qrt- PCR. The primer pairs used were AN11, NA82/NA83; AN1, NA84/ NA85; JAF13, NA86/NA87; DPL (MP allele) NA90/NA91, DPL (P. hybrida allele) NA90/NA136; PHZ, NA88/NA89; MYBx, NA103/ NA104 and AN2, NA105/NA106. The primers for the reference genes ACT2 (PhACT2F/PhACT2R) and EF1a (PhEF1aF/PhEF1aR) were designed by Snowden et al. (2005). Quantitative RT-PCR was performed using LightCycler Ò 480 SYBR Green I Master (Roche) reagents and the Rotor Gene 3000 real-time PCR machine and Rotor Gene 6000 series software 1.7 (Corbett Research, corbettlifescience.com), with three replicates per cdna sample. The PCR was performed as follows: 95 C for 10 min (95 C 10 sec, 60 C 15 sec, 72 C 20 sec) 40 cycles, C melt. Fluorescence measurements were performed at 72 C for each cycle and during final melting. Relative transcript abundance was determined by comparative quantification (Pfaffl, 2001) to the geometric mean of ACT2 and EF1a determined using BestKeeper (Pfaffl et al., 2004). Over-expression constructs and transformation The ORFs of DPL and PHZ were amplified using the primers NA34/ NA35 and NA24/NA25, respectively, and cloned into the part7 expression vector (Gleave, 1992) generating pnwa1 (35S pro :DPL) and pnwa2 (35S pro :PHZ). The NotI fragment of pnwa1 and pnwa2 were cloned into the part27 binary vector (Gleave, 1992), generating pnwa12 (DPL) and pnwa14 (PHZ). Biolistic transformation of Antirrhinum petals was performed as described in Schwinn et al. (2006) using a particle inflow gun and conditions as described by Shang et al. (2007). DNA (10 lg) of pnwa1 or pnwa2 was transformed together with 2 lg ppn93 (35S pro :GFP-m-ER) into the adaxial epidermis of A. rosea dorsea petals. Negative controls (GFP only) were performed. Images were collected as described in Albert et al. (2010). Stable transgenic petunia lines were generated by leaf disc transformation with A. tumefaciens LBA4404 (Invitrogen) harbouring pnwa12 or pnwa14, as described by Conner et al. (2008). More than 20 independent lines were established for each construct, with at least 10 independent lines each with three clones being assessed in a greenhouse. Plant material and growing conditions Mitchell petunia (an2 an4 DPL PHZ AN1 AN11), V26 (AN2 an4 DPL PHZ AN1 AN11), V30 (AN2 AN4 DPL PHZ AN1 AN11), W59 (an2 an4 dpl PHZ AN1 AN11), W134 (AN2 an4 AN1 an11) and W225 (AN2 an4 an1 AN11) petunias, and A. rosea dorsea plants were grown in a

12 782 Nick W. Albert et al. greenhouse that was heated at 15 C and vented at 25 C with ambient lighting. Light/dark expression studies in W59 petunia flowers used very small buds (pre-stage 1) enclosed by sepals. Buds were either covered in aluminium foil (dark) or grown under ambient greenhouse conditions (light). When the buds reached stage 2, petals were collected (three biological replicates), RNA extracted and qrt-pcr performed for AN1 and PHZ, ACT2 and EF1a as described earlier. RNA gel blots Total RNA was extracted from pooled leaves, from the MYB OE petunia lines (Chomczynski and Sacchi, 1987). Tissue was sampled at the same time of day, to standardize possible diurnal effects upon flavonoid gene expression. RNA gel blots were prepared, probed and washed to high stringency as described in Albert et al. (2009). Radiolabelled probes ([a- 32 P]dATP) were generated by random priming (Feinberg and Vogelstein, 1983). Probe templates have been described previously: PAL, C4H, CHS-A, CHI-A, F3H, F3 H, F3 5 H, FLS, ANS, 3GT, 25/26S rrna (Bradley et al., 1998), and GST/ AN9 (Alfenito et al., 1998). Complementary DNA clones of 3RT, 5GT, MT, AT and DFR-A were amplified for probe templates by RT-PCR from V26 petunia mixed stage flower bud cdna. Primers were designed based on publicly available sequence data from GenBank: 3RT (X71059) NA54/NA55; 5GT (AB027455) NA50/NA51; AT (AR944737) NA52/NA53; MT (DD178352) NA56/NA57; DFR-A (X79723) NA58/NA59. Anthocyanin and flavonoid analysis Flavonoids including anthocyanins were extracted from 50 mg dry weight (DW) of leaf, petal tube and limb tissue from the transgenic petunia lines as described previously (Albert et al., 2009) and quantified by HPLC as described in Bradley et al. (1998), using a Phenomenex Prodigy (5 lm, mm) RP-18 end-capped column. Anthocyanidins were extracted from 50 mg DW tissue in 6N HCl, partitioned with isoamyl alcohol (Mitchell et al., 1998) and separated by HPLC. Promoter:GUS petunia lines The promoters for DPL and PHZ were isolated using the Universal GenomeWalker Ò kit (Clontech, with the gene-specific primers NA60, NA61 (DPL) and NA99, NA98 (PHZ). The promoter and 5 -untranslated region (UTR) for DPL (1.7 kb) and PHZ (1 kb) was amplified from MP genomic DNA with the primers NA78/NA79 and NA81/NA102, respectively, and directionally cloned into pdah3 (NheI/NcoI), placing the promoter in front of an intron GUS (igus) reporter gene (uida) encoding b-glucuronidase. The DPL pro :igus and PHZ pro :igus cassettes were sub-cloned (NotI) into part27, generating pnwa35 and pnwa36, respectively. The control constructs were 2 35S pro :igus/part27 and part27 empty vector. Stable transgenic petunia lines were generated by leaf disc transformation with A. tumefaciens GV3101 harbouring the binary vector constructs. Fifteen independent lines were established for each construct, each with four clones being assessed in a greenhouse. The primary transgenic lines were self-crossed, and the seed was collected. Seed was sterilised and germinated on half-strength MS medium (25 C, 30 lmol m )2 sec )1 light, 16-h photoperiod). Seedlings were stained for GUS activity 4 days after germination. Histochemical GUS staining Petal limb tissue was pre-treated with diethyl ether for 30 sec, rinsed for 1 min in 90% (v/v) ice-cold acetone and rinsed twice in 50 mm phosphate buffer (ph 7) prior to staining. Germinated seedlings were washed in ice-cold 90% (v/v) acetone for 5 min, and rinsed twice in 50 mm phosphate buffer (ph 7) prior to staining. Seedlings or petal limb tissues were covered in X-gluc staining solution [0.63 mm X-gluc; 10 mm EDTA; 0.1% (v/v) TritonX-100; 50 mm phosphate buffer (ph 7):methanol 80:20 (v/v), with or without 1 mm K 3 Fe(CN) 6 /K 4 Fe(CN) 6 as indicated in figure legends], vacuum infiltrated for 10 min and incubated overnight at 37 C. Seedlings were destained with ethanol:acetic acid (95:5 v/v) at 55 C for 30 min. Stained tissues were stored in 70% (v/v) ethanol. ACKNOWLEDGEMENTS We thank Professor Michael McManus for providing academic supervision to NWA during his PhD; Steve Arathoon for technical assistance; Ian King and Julie Ryan for care of plants; Murray Boase and Dr Simon Deroles for plant transformation advice; Tony Corbett for photography; Dr Donald Hunter for helpful discussions; and Professor Cathie Martin for helpful discussions and critique. We thank International Flower Development Pty Ltd for use of the petunia plasmid clones and Professor Virginia Walbot for the AN9 clone. We thank Professor Ronald Koes for the petunia cultivars W134, W225, W59 and V30. NWA was supported by a Bright Futures Top Achiever Doctoral Scholarship awarded by the Tertiary Education Commission of New Zealand. This work was funded by the New Zealand Foundation for Research Science and Technology (From Tools to Traits: Functional Genomics for Metabolite Accumulation, contract C0ZX0805). SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Vegetative pigmentation phenotypes of 35S pro :DPL and 35S pro :PHZ V26 petunia transformants. Figure S2. Anthocyanin and flavonoid content of 35S pro :DPL and 35S pro :PHZ over-expression lines. Figure S3. Phenylpropanoid and flavonoid gene expression in floral tissues of DPL and PHZ over-expression lines. Figure S4. Vegetative pigmentation phenotype of W134 (an11) and W225 (an1) petunia, and blushed bud phenotype in Lc petunia flowers. Table S1. Primers sequences used in this study. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. REFERENCES Aharoni, A., De Vos, C.H.R., Wein, M., Sun, Z.K., Greco, R., Kroon, A., Mol, J.N.M. and O Connell, A.P. (2001) The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28, Albert, N.W., Lewis, D.H., Zhang, H., Irving, L.J., Jameson, P.E. and Davies, K.M. (2009) Light-induced vegetative anthocyanin pigmentation in Petunia. J. Exp. Bot. 60, Albert, N.W., Arathoon, S., Collette, V.E., Schwinn, K.E., Jameson, P.E., Lewis, D.H., Zhang, H. and Davies, K.M. 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