CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis

Molecular Plant

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Volume 2

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Number 4

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Pages 790–802

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July 2009

RESEARCH ARTICLE

CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis Hui-Fen Zhu, Karen Fitzsimmons, Abha Khandelwal and Robert G. Kranz1 Department of Biology, Washington University, St Louis, MO 63130, USA

ABSTRACT Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3–MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs (e.g. root hairs, trichomes), but can influence anthocyanin pigment synthesis. Key words:

CPC (CAPRICE); anthocyanin biosynthesis; negative regulator; Arabidopsis.

INTRODUCTION Research on anthocyanin pigments has begun to elucidate their biological functions, including attracting insects for pollination and as a protective response to environmental stress (for reviews, see Gould, 2004). The biosynthesis of flavonoid pigments is one of the best understood pathways in nature, and its regulation has been recently studied (for reviews, see Koes et al., 2005; Lepiniec et al., 2006). Most of the genes encoding structural enzymes for anthocyanin biosynthesis are well conserved among plant species, but the factors involved in regulation differ (Quattrocchio et al., 1993; Albert et al., 1997; Quattrocchio et al., 1998; Devic et al., 1999; Serna, 2004; Ramsay and Glover, 2005; Bogs et al., 2005; Quattrocchio et al., 2006; Serna and Martin, 2006; Bogs et al., 2007). Typically, regulation occurs at the level of transcription with a hierarchy of transcription factors (Devic et al., 1999; Lepiniec et al., 2006). This has been shown for several anthocyanin biosynthesis regulators in different species, including maize (Zea mays) C1 and R proteins, petunia (Petunia hybrida) AN11, AN1, AN2, and AN4, and Arabidopsis (Arabidopsis thaliana) TTG1, TT8, (E)GL3, and PAP1/2 factors (Mol et al., 1999; Zhang et al., 2003; Hernandez et al., 2004; Gonzalez et al., 2008). In A. thaliana, anthocyanin biosynthesis

is controlled through a TTG1–bHLH–MYB regulatory complex (Quattrocchio et al., 1998; Larkin et al., 1999; Walker et al., 1999; Zhang et al., 2003; Carey et al., 2004; Schwinn et al., 2006; Gonzalez et al., 2008). TRANSPARENT TESTA GLABRA1 (TTG1) possesses WD-repeats involved in protein–protein interaction (Walker et al., 1999); GL3 and EGL3 are bHLH proteins shown to enhance anthocyanin biosynthesis together with the R2R3–MYB proteins, PAP1 and PAP2 (Zhang et al., 2003). Yeast two-hybrid studies also have demonstrated that GL3/EGL3 interact with both TTG1 and PAP1/2, thus the formation of a ternary complex (Zhang et al., 2003; Zimmermann et al., 2004). According to a recent review by Wang et al. (2008), the Arabidopsis genome has a total of six single-repeat R3 MYB factors: CPC (At2g46401), TRY (At5g53200), TCL (At2g30432), ETC1 (At1g01380), ETC2 (At2g30420), and ETC3 (At4g01060).

1 To whom correspondence should be addressed. E-mail kranz@biology. wustl.edu, fax 314-935-4432, tel. 314-935-4278.

ª The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssp030, Advance Access publication 2 June 2009 Received 1 December 2008; accepted 19 April 2009

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These single-repeat R3 MYBs play important roles in regulating root hair differentiation and trichome initiation (Wada et al., 1997; Schellmann et al., 2002; Wada et al., 2002; Koshino-Kimura et al., 2005). ETC3, also known as CAPRICELIKE MYB3 (CPL3), was shown to control endoreduplication and flowering development in addition to trichome and root hair formation (Tominaga et al., 2008). Besides these small R3 MYB members, Arabidopsis MYB-LIKE2 (MYBL2), a factor with a full R3 MYB domain and a part of R2 domain, was recently reported as a new regulator of flavonoid biosynthesis (Dubos et al., 2008; Matsui et al., 2008). However, the six Arabidopsis single-repeat R3 MYBs have not, to our knowledge, been reported to regulate anthocyanin biosynthesis CPC was first reported to be involved in both trichome and root hair development (Wada et al., 1997). It is suggested that CPC reduces the formation of the TTG1–(E)GL3–GL1 complex by competing with the R2R3–MYB GL1 for bHLH binding in trichome initiation (Schiefelbein, 2000; Schellmann et al., 2002; Larkin et al., 2003; Schiefelbein, 2003; Zhang et al., 2003; Kirik et al., 2004a, 2004b; Serna and Martin, 2006; Zhao et al., 2008). For root hair differentiation, the R2R3–MYB WER and bHLH interaction is responsible for transcription regulation of a negative regulator, GLABRA2 (GL2). CPC acts competitively with WER to regulate this epidermal cell fate (Schiefelbein, 2000; Schellmann et al., 2002; Wada et al., 2002). CPC was recently found to be a positive regulator of stomatal formation (Serna, 2008). Here, for the first time, we show that CPC is involved in the regulation of anthocyanin biosynthesis in Arabidopsis. Plants overexpressing CPC in leaves have reduced or no trichomes; plants overexpressing CPC in roots have more root hairs (Wada et al., 1997). Interestingly, we found that accumulation of anthocyanin in CPC overexpression plants is inversely related to the levels of CPC. The results with 35S::CPC plants, microarrays, Q–PCR analysis, and promoter activity assays lead us to propose that CPC competes with the binding of PAP1(2) to (E)GL3, thus preventing activation of the structural genes for anthocyanin biosynthesis. Thus, CPC is a negative regulator of anthocyanin biosynthesis.

RESULTS Characterization of Selected GAL-4-Mediated CPC Overexpression Transgenic Lines GAL4/UAS–CPC overexpression transgenic plants were generated by transforming 5xUAS–CPC into the previously developed driver lines (see details in Methods). T2 plants of selected independent transgenic lines were shown with LUC, GUS, GFP expression, and reduced trichomes (Figure 1). Tissue-specific expression patterns (leaf-only, leaf-and-root) were identified; in leaf-only expression lines, the majority (95–96%) of transgenic plants showed no or reduced trichome numbers, but no increase in root hairs (Table 1); in leaf-and-root expression lines (Figure 1B), 96–100% plants showed no or reduced trichome numbers (Table 1); 76% of the tested plants have more root hairs (Table 1 and Figure 1B). Based on the plants tested for

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trichome changes and/or root hair growth, correlations between reporter genes and CPC traits were confirmed.

CPC Overexpression Represses Anthocyanin Biosynthesis in Response to Nitrogen Stress During our studies on plant growth using low nitrogen media, we observed that CPC overexpression lines produced dramatically less anthocyanins (Figure 2A and 2B). Analyzed by whole seedling LUC spray assays (see Methods), LUC activities in three tested GAL4/UAS–CPC lines were shown with a range of luciferase expression levels as #354 . #225 . #268 (Figure 2B (I, H, G) and 2C); real-time Q–PCR results indicated that relative CPC overexpression levels in these lines share the same pattern as #354 . #225 . #268 (Figure 2D); and this inversely correlated with anthocyanin accumulation, #354 , #225 , #268 (Figure 2B (C, B, A) and 2E). CPC levels therefore control anthocyanin accumulation. One explanation for the CPC-mediated control of anthocyanin could be that an increase in root hairs from CPC overexpression confers more nutrient absorption from the growth media; thus, plants are less stressed. However, note that GAL4/UAS–CPC plants with different CPC expression patterns exclude this possibility. For example, no increased root hairs were observed in the leaf-only lines #268 and #225 (Table 1), but only 20–30% of anthocyanin biosynthesis was detected in these lines compared with the control lines (Figure 2E). This suggests that CPC itself negatively controls anthocyanin biosynthesis, not through increased root hair growth.

Microarray Analysis Shows that the Late Anthocyanin Biosynthesis Genes Are Repressed by GAL4/UAS–CPC Overexpression The results with different driver lines of CPC overexpression suggested that CPC regulates anthocyanin biosynthesis. To determine what genes are changed due to CPC overexpression, a microarray analysis was carried out using Agilent Arabidopsis 3 chips to test the GAL4/UAS–CPC (line #225–11, leaf-only) and driver line control plants. Two independent biological replicates were included, and the same amounts of total RNA were applied to microarray analysis with a dye swap. Of approximately 37 000 features tested, 85 genes were down-regulated by more than three-fold in GAL4/UAS–CPC plants, and 26 genes (besides CPC) were up-regulated (Supplemental Table 1). Analysis of the 85 down-regulated genes revealed that seven are structural genes for anthocyanin biosynthesis, and nine are regulatory genes (Table 2). RT–PCR was also carried out to validate the microarray data. RNA was isolated from independent biological samples grown under the same conditions used for microarray analysis. As a result, 16 out of 20 genes that were down-regulated by more than three-fold in the microarray analysis were confirmed by RT–PCR; part of the data are shown in Figure 3A. These results indicate that late genes in anthocyanin biosynthesis pathway (see ** in Figure 4) are controlled by CPC, possibly through the transcription factor regulatory complex.

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Figure 1. Reporter Expression and CPC Overexpression Traits in GAL4/UAS–CPC Lines. (A) CPC overexpression in leaf-only plant line (#268) and wild-type (Col). A, C, E, and G show wild-type plants (Col) with an image of a whole plant (A), a hairy leaf (C), with no LUC (E) and no GUS (G) expression. B, D, F, and H show GAL4/UAS–CPC line #268, a whole plant image with glabrous leaf surface (B), a close-up of a leaf with no trichomes (D), LUC expression in leaves (F) (LUC expression is depicted in red) and GUS expression (H). (B) CPC overexpression in leaf-and-root plant line #354. I, wild-type (Col); J, increased root hair growth (line #354); K, LUC expression in whole seedling (merged image with red-yellow representing LUC expression); L, GUS expression in both leaves and roots; M, GFP expression in roots. Scale bars: 1 mm in I and J; 0.5 mm in M.

Table 1. Several GAL4/UAS–CPC Lines with Reduced Trichome and/or Enhanced Root Hair Growth. Plant line #a 99a

Expression pattern

Total plants analyzedb,c

Plants with no or reduced trichome

Plants with increased root hair (.24 mm 1)d

Leaf

24b

96%

Not tested

268

Leaf

40b + 29c

95%

0

225

Leaf

93b + 36c

95%

0

354

Leaf + root

160b + 34c

96%

169a

Leaf + root

22b

100%

Not tested

316a

Leaf + root

5b

100%

Not tested

76%

a T2 plants were used except where noted with an ‘a’, in which case, T1 plants were studied. b Plants observed for trichome change. c Plants counted for root hair numbers. d Primary roots were counted for 2 mm starting from 1 mm behind the root tip; 18 mm 1 is the control root hair number counted from wild-type plants.

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Figure 2. CPC Overexpression Leads to Reduced Anthocyanin Accumulation. (A) Plants were grown on MS-N media (1 mM) under long-day light conditions for 15 d. Plant image showing that anthocyanin accumulation in GAL4/UAS–CPC line #225–11 (left) is less than in control plants (driver line #225) (right). (B) CPC tightly controls the anthocyanin accumulation. A–F: 5-day-old seedlings grown on MS-N media. Anthocyanin accumulation in GAL4/ UAS–CPC plant line #268 (A) is more than line #225 (B), and more than line #354 (C); D, E and F are driver control plants, respectively. Seedlings were pulled out, put on the surface of media, and sprayed with 1 mM luciferin for LUC expression detection. G–I: merged images showing LUC expression (red-yellow) in GAL4/UAS–CPC plants. Line #268 (G) and line #225 (H) are leaf-only lines; no signal was detected in roots; line #354 (I) is a leaf-and-root line, showing LUC expression detected in both leaves and roots. LUC images indicated that CPC overexpression level was higher through lines #268, #225, and #354. Scale bars: 1 mm in A–F. (C) LUC image quantitation in lines #268, #225, and #354. LUC image was taken and quantification was carried out using ImageGauge software. Error bars show standard deviations. (D) Relative expression levels of CPC in lines #268, #225, and #354 detected by real-time Q–PCR. The CPC expression levels in the corresponding driver line control were set as 1; the numbers shown in the figure are increased CPC expression fold in those three lines. Error bars show standard deviations. (E) Relative anthocyanin content in lines #268, #225, and #354. Anthocyanin contents of each CPC overexpression line and its driver line control were measured, the content of each driver line control was taken as 100%, and the numbers shown in the figure are anthocyanin content as of the percentage level of the control line. Error bars show standard deviations.

Anthocyanin Biosynthesis Pathway Is Inhibited by CPC In order to further confirm that CPC regulates anthocyanin biosynthesis gene expressions, WS, cpc-1, and 35S::CPC transgenic plants (both in WS background) (Wada et al., 1997) were used for real-time Q–PCR analysis. In 35S::CPC plants, significant decreases in mRNA levels of late genes as DFR and LDOX (see ** in Figure 4) were observed, and early genes as CHS, CHI, F3#H, and F3H (see * in Figure 4) were detected with less decreases in mRNA levels (Figure 3B). In cpc-1 mutant, four

of these six structure genes showed higher expression levels (Figure 3B). These results suggested that CPC inhibits anthocyanin biosynthesis pathway.

Anthocyanin Repression by CPC Is Common in Response to Many Stress Conditions To verify that CPC does play a role in anthocyanin accumulation under nitrogen stress, we analyzed the cpc-1 mutant the 35S::CPC overexpression line and the WS wild-type. Cpc-1, WS, and 35S::CPC seedlings were grown side by side on

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Table 2. A List of Selected Genes that Were Down-Regulated in CPC Overexpression Plants. Gene ID

Description of gene products

Fold change

Gene name or category

Structural genes for anthocyanin synthesis At1g02940

glutathione S-transferase

–7.74

GSTF5

At1g25460

oxidoreductase family protein similar to DFR

–5.57

Similar to DFR

At2g26480

UDP-glucoronosyl/UDP-glucosyl transferase family protein

–4.72

UGT

At5g42800

dihydroflavonol 4-reductase

–3.98

DFR

At5g17220

glutathione S-transferase

–3.51

GST12

At4g22870

leucoanthocyanidin dioxygenase,

–3.31

LDOX

At4g22880

leucoanthocyanidin dioxygenase

–3.19

LDOX

At1g79840

homeobox protein (GLABRA2)

–12.50

GL2

At5g40330

myb family transcription factor (MYB23)

–11.81

MYB23

At5g53980

homeobox-leucine zipper family protein

–8.69

HB52

At1g71692

MADS-box protein (AGL12)

–7.70

AGL12

At3g13540

myb family transcription factor (MYB5)

–7.32

MYB5

At1g56650

myb family transcription factor (MYB75)

–4.33

PAP1

At2g37260

WRKY family transcription factor (TTG2)

–4.30

TTG2

At2g25820

transcription factor

–3.67

/

At4g09820

basic helix-loop-helix (bHLH) family protein

–3.28

TT8

Regulatory genes

Figure 3. Anthocyanin Biosynthesis Genes Are Regulated by CPC. (A) RT–PCR results validated down-regulated gene expressions in GAL4/UAS–CPC overexpression plants (line #225) in microarray study. (B) Real-time Q–PCR analysis of the expression levels for the flavonoid biosynthesis genes in cpc-1, 35S::CPC plants compared with wildtype. Measurements are shown as the percentage of the WTlevel. Wild-type is Wassilewskija. Error bars show standard deviations.

nitrogen stress media (N–) with no ammonium or nitrate. Compared with wild type, cpc-1 mutant exhibited fewer numbers of root hairs and 35S::CPC plants exhibited more (Figure 5A). Aerial tissues were collected and extracted for anthocyanin and chlorophyll measurements. We observed no significant differences in chlorophyll levels (data not shown). Consistently, less anthocyanin accumulation was observed in 35S::CPC overexpression plants, while more anthocyanin was observed in cpc-1 mutant (Figure 5A and 5B). No obvious difference in anthocyanin contents was measured under replete nitrogen conditions (N+). In addition to nitrogen stress, we tested osmotic, salt, and cold stresses. Cpc-1, WS, and 35S::CPC plants were tested side

by side on different stress media. As shown in Figure 6, under all three tested stress conditions, 35S::CPC plants show less anthocyanin accumulation (Figure 6A (F, I, L)). These results were also confirmed by anthocyanin measurements (Figure 6B). Therefore, one of the functions of CPC is to repress anthocyanin biosynthesis.

The Mechanism of CPC Control on Anthocyanin Genes Involves Competition with PAP1/2 Previous studies have shown anthocyanin biosynthesis is regulated through a TTG1–bHLH–MYB regulatory complex. In vitro GST-tagged protein pull-down experiments showed that GL3/EGL3 and PAP1/2 form a bHLH–MYB complex

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Figure 4. Anthocyanin Biosynthesis Pathway in Arabidopsis. Enzymes catalyze respective steps. Enzymes marked with ** were repressed due to CPC overexpression in both microarray study and Q–PCR analysis. Enzymes marked with * were tested with decreased expressions in Q–PCR experiments. Putative steps are shown as dotted arrows. PAL, phenylalanine ammonia-lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3#H, flavonoid 3#-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; GST, glutathione S-transferase.

(Zimmermann et al., 2004); yeast two-hybrid and transient expression experiments verified the bHLH–MYB interaction and further indicated that the complex binds to the promoter of the anthocyanin synthesis gene, DFR (Zimmermann et al., 2004). This DFR is also repressed in both our microarray and Q–PCR analysis of CPC overexpression (Figures 3 and 4 and Table 2). Since a comprehensive study of Arabidopsis MYB transcription factors revealed that CPC interacts with both GL3 and EGL3 by yeast two-hybrid (Zimmermann et al., 2004), we suggest that CPC competes with PAP1/2 for binding to (E)GL3, thereby reducing activation of the DFR gene by PAP1/2. To test this hypothesis, transient expression experiments were carried out using a pDFR:GUS plasmid as reporter and a 35S:LUC plasmid as an internal control. A combination of EGL3 with PAP1 was assayed using Arabidopsis roots from both wild-type and 35S::CPC overexpression plants. Significantly lower reporter activation was observed in assays using 35S::CPC roots compared with assays using wild-type roots (Figure 7A). In addition, using the same transient expression system, different combinations of effectors including PAP1 only, EGL3 only, PAP1 + EGL3, PAP1 + EGL3 + CPC were delivered into wild-type (Col) roots. Similarly, a reporter activity repression by CPC was observed (Figure 7B). Both results suggest that CPC negatively influences the activity of the EGL3/PAP1 complex, thus negatively controlling anthocyanin biosynthesis.

Figure 5. CPC Controls Anthocyanin Accumulation under N Stress. (A) Root hair of cpc-1, Ws, and 35S::CPC showed that 35S::CPC has more root hair while cpc-1 has less (upper panel). Plant images showed that, under nitrogen stress, 35S::CPC plant has lower anthocyanin accumulation than wild-type, and cpc-1 has more (lower panel). (B) Anthocyanin contents in cpc-1, WS, and 35S::CPC plants under N+ ( ) and N ( ) conditions. Error bars show standard deviations.

DISCUSSION CPC Is Involved in Regulating Anthocyanin Biosynthesis In this study, we report that CPC is involved in anthocyanin biosynthesis. We observed an up to seven-fold repression of anthocyanin in GAL4/UAS–CPC overexpression plants under nitrogen stress conditions (Figure 2A, 2B, and 2E). By testing cpc-1, WS, and 35S::CPC overexpression plants, we also found 35S::CPC plants showed less anthocyanin accumulation than the wild-type (Figure 5A and 5B), indicating that CPC regulates

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Figure 6. CPC Overexpression Reduces Less Anthocyanin Accumulation under Different Stress Conditions. (A) Seeds were germinated on ½ MS media for 7 d, and transferred to different stress media for another 7 d before photos were taken. Images are arranged in order from left to right as cpc-1, WS, 35S::CPC. A, B, and C: plants were grown on control media; D, E and F: on osmotic stress media; G, H and I: on salt stress media; J, K and L: cold stress. (B) Anthocyanin quantitations of plants grown under salt, osmotic, and cold stress conditions. Error bars show standard deviations.

anthocyanin synthesis. Similar repression of anthocyanin accumulation in 35S::CPC plants is consistently found when tested on different stress conditions, including osmotic, salt, and cold stress (Figure 6). Microarray analysis comparing gene expression profilings between CPC overexpression plants (GAL4/UAS–CPC) and control plants showed that 85 genes had been down-regulated more than three-fold upon CPC overexpression (Supplemental Table 1), seven of which are structural genes for anthocyanin biosynthesis (Table 2). RT–PCR using GAL4/UAS–CPC overexpression plants and real-time Q–PCR analysis using cpc-1, 35S::CPC plants confirmed that anthocyanin biosynthesis gene expressions are inhibited by CPC (Figure 3A and 3B). Additionally, microarray experiments showed that nine regulatory genes, including GL2, TTG2, PAP1, etc., were negatively regulated (Table 2), suggesting that CPC is involved in regulatory feedback to TTG/bHLH/MYB transcriptional complexes.

The Repressor Role of CPC on Anthocyanin Biosynthesis Is to Prevent PAP1/2 from Binding to Its bHLH Partners One argument might be that CPC controls PAP1/2 transcription (Table 2), thus affecting indirectly the anthocyanin regulation. However, our results with transient expression of PAP1 and EGL3 suggest that CPC competes with PAP1/2 for (E)GL3 directly (Figure 7). It is known that full-length R2R3–MYB factors contain two DNA-binding domains, R2 and R3. Each domain has three a-helices, and the third helices from both R2 and R3 domains are critical for contacting with DNA (Gabrielsen et al., 1991; Jia et al., 2004). The first two a-helices of the R3 domain contain a conserved motif DLX2RX3LX6LX3R involved in MYB–bHLH interactions (Figures 8 and 9) (Gabrielsen et al., 1991; Ogata et al., 1994; Jia et al., 2004; Zimmermann et al., 2004; Uhrig, 2006; Tominaga et al., 2007). CPC, a single-repeat R3–MYB, does not contact DNA directly, because it only has a single R

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domain, and the third a-helix of the R3 domain is significantly diverged from R2R3–MYBs (Figure 8), supporting the contention that single-repeat MYB factors do not behave as direct transcription activators. The PAP1/2, R2R3–MYB transcription factors directly activate structural genes for anthocyanin biosynthesis (Borevitz et al., 2000; Dare et al., 2008) and studies have shown that they interact with bHLH proteins (E)GL3 (Bernhardt et al., 2003; Zhang et al., 2003; Zimmermann et al., 2004; Bernhardt et al., 2005). Since CPC contains the conserved MYB–bHLH interaction motif (helices 1 and 2 of the R3 domain) (see Figures 8 and 9) (Wada et al., 1997; Zimmermann et al., 2004; Chen et al., 2007; Tominaga et al., 2007), it is capable of binding to bHLH proteins. Indeed, previous studies confirmed that CPC physically interacts with GL3/EGL3 in yeast cells (Bernhardt et al., 2003; Zimmermann et al., 2004). Promoter activity assays in our transient expression study have shown that CPC reduces the PAP1/EGL3 activation of DFR expression (Figure 7). The results suggest that CPC competes with PAP1/2 to contact with GL3/EGL3, thus negatively controlling anthocyanin biosynthesis (see Figure 9). Similar working models have been proposed that R3–MYBs (single repeat MYBs) act as inhibitors by sequestration of the bHLH protein into an inactive complex involved in trichome development (Koes et al., 2005; Serna and Martin, 2006; Wang et al., 2007). Figure 7. CPC Inhibits EGL3–PAP1 Transcriptional Activity. Transient promoter activity assays were carried out using a DFR-promoter driving GUS as a reporter, effectors (EGL3, PAP1, CPC or combination(s)), and 35S:LUC was always included as an internal control. Results shown represent the mean value of seven independent assays, and error bars show standard deviations. (a) A combination of 35S:EGL3, 35S:PAP1, pDFR:GUS, and 35S:LUC was codelivered into wild-type and 35S:CPC plant roots. (b) Different combinations of effectors (PAP1 only, EGL3 only, PAP1 + EGL3, PAP1 + EGL3 + CPC) were delivered into wild-type roots.

CPC Is a Single-Repeat R3–MYB Involved in Anthocyanin Biosynthesis Regulation PhMYBx, an R3–MYB ortholog of CPC and TRY, was reported to be a suppressor of anthocyanin biosynthesis in petunia (Petunia hybrida) (Koes et al., 2005). In Arabidopsis, the genome contains six single-repeat R3–MYB members (Simon et al., 2007; Wang et al., 2008); most studies on this group of small

Figure 8. The Amino Acid Sequence Alignment of Selected R2R3 MYBs and R3 MYBs. Sequence alignment of Arabidopsis R2R3–MYB members including PAP1, PAP2, WER, MYB23, GL1, MYB113, MYB114, TT2, MYB4, MYB5, MYB57; and R3-MYB members including CPC, TRY, TCL1, ETC1, ETC2, and ETC3, using ClustalW program. R3 and/or R2 domains are marked with black bars under the corresponding residues. Three a-helices of both R2 and R3 domains are indicated in boxes; the conserved MYB– bHLH interaction motif on the first two a-helices of R3 domain is underlined with a gray bar. ‘*’, identical residues; ‘:’ and ‘.’, similar residues.

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Figure 9. Working Model Depicting the Role of CPC Together with TTG1/(E)GL3/PAP1(2) Complex in Transcription Activation of a Structural Anthocyanin Biosynthesis Gene (DFR) as Described in the Text. TTG1/(E)GL3/PAP1(2) form an active complex for transcription activation for structural genes. CPC, a R3–MYB, acts as an inhibitor by competing with R2R3–MYBs for the binding site of bHLH proteins, keeping R2R3–MYBs from forming the active complex with bHLHs for anthocyanin synthesis.

R3–MYBs are with trichome and root hair development. Our study is the first report showing that CPC also acts functionally in anthocyanin biosynthesis. Besides single-repeat R3–MYBs, MYBL2 encodes a MYB protein of 195 amino acids, which is longer than CPC (94 amino acids) and contains a full R3 domain and part of an R2 domain (see Figure 8); MYBL2 was very recently reported to be a new regulator of flavonoid biosynthesis (Dubos et al., 2008; Matsui et al., 2008), indicating that MYBL2 and small R3–MYB factors may function redundantly in regulating anthocyanin biosynthesis. Anthocyanin pigments are induced by environmental stresses; CPC’s regulatory involvement in anthocyanin biosynthesis indicates that other than developmental processes, these R3–MYBs might also be involved in stress responses. Still unresolved is the actual molecule(s) sensed under stress that feed into the PAP1, CPC, and MYBL2 circuitry to activate the pathway and the mechanisms on this feedback. In a recent review, Lepiniec et al. (2006) have wondered whether the small R3–MYBs might be involved in flavonoid and seed mucilage synthesis. We tested whether CPC is also involved in proanthocyanidin and seed mucilage biosynthesis using cpc-1, wild-type WS, and 35S::CPC seeds, and in GAL4/UAS– CPC driver lines, but no significant difference was found (data not shown). Further studies will be needed to confirm that CPC is not involved in these biosynthesis pathways.

METHODS Plant Materials and Growth Conditions The GAL4/UAS driver system plants used in this study are all based on Arabidopsis thaliana Columbia(Col-0) ecotype. The cpc-1 insertion mutant (CS6399), and 35S::CPC overexpression plant (CS6526) were described by Wada et al. (1997), seeds were obtained from

the Arabidopsis Resources Center (Columbus, Ohio), and both are in Arabidopsis Wassilewskija (WS) background. For all experiments mentioned in this article, seeds were sterilized followed by Engineer and Kranz (2007), and germinated on 0.6% Agar plates (Acumedia, Cat No. 7178A). After an overnight at 4
C, plates were transferred to a Percival growth chamber with a 16 h light/8 h dark light cycle at an intensity of 100 lE at 22
C/20
C. MS Modified Basal Salt Mixture without Nitrogen (PhytoTechnology Laboratories, KS) was used for the growth media. 10 mM of NH4NO3 was added for the +N media, and 1 mM for the –N media. 1x vitamin (PhytoTechnology Laboratories, KS) and 2% sucrose were always added and pH adjusted to 5.7 in all the media. All the +N and –N media in this article followed this formula unless indicated differently. For a harsher nitrogen-free media (N–), we followed Lam et al. (2003) with 1x MS basal salt without nitrogen (PhytoTechnology Laboratories, KS), 3% sucrose, 0.8% agar, 0.5 mM glycine and pH to 5.7 with KOH; 1x MS media were used as control media (N+).

Stress Conditions Seeds were germinated on ½x MS for 7 d, and seedlings were then transferred to different stress media. The basic media used in this experiment was ½x MS, with 1x vitamin and 3% sucrose, and pH adjusted to 5.7; and this was used as control media. An additional 200 mM mannitol was added to make osmotic media; and an additional 150 mM NaCl was added for salt media. Plants were grown under the same conditions as above. For cold stress, plants were transferred onto ½x MS media and put in a 4
C chamber with similar light conditions.

Generation of a GAL4/UAS–CPC Driver System of LUC, rsGFP, and GUS Co-Expression In a previous study, we developed an enhancer trap system (GAL4-based) with a GAL4/5XUAS and a minimal 35S promoter directing luciferase (LUC) as a preliminary reporter, and GFP/ GUS as secondary reporters in Arabidopsis, and a library of 10 000 events was produced (Engineer et al., 2005), which we used for the present study. To find driver lines from the GAL4-based transgenic library, a total of 3714 seedlings representing all 19 pools were checked for luminescence. Of those, 180 were selected with a range of LUC expression. Tissue-specific expression was also considered: leaf-only, root-only, and leaf-and-root expression. GFP and GUS expression were both analyzed, and 61 lines with correlated LUC, GFP, and GUS activities were further evaluated as T3 plants. No lines were ultimately isolated as root-only. Carrying on through generations and reporter assays, 20 driver lines were selected based on matched expression patterns among LUC, GFP, and GUS. The vector pRGK337 (Engineer et al., 2005) was used to create the responder vector of pRGK374 (see Supplemental Figure 1 for map) with kanamycin resistance selection in plant and a multiple cloning site (MCS) to insert the genes of interest.

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pRGK337 was digested with XmnI to remove 35S/Hygromycin resistance gene/3#UTR, ligated with the fragment containing the promoter and tetracycline resistance gene (Tet) released by SspI from pACYC (New England BioLabs). The resulting plasmid gained a HindIII site and tetracycline resistance so that Tet/ Kan double selection can be used to obtain positive clones. The plasmid was digested with HindIII to remove the tetracycline resistance gene, and linked with a fragment containing 35S promoter/kanamycin resistance gene/3#UTR, which was obtained by PCR amplification from vector pPZP212 (Hajdukiewicz et al., 1994) with primers 5#-TTGTGGAGCAAGCTTATTGACGCT-3’ (HindIII site was engineered in underlined region) and 5#-TCGTATGTTGTGTGGAATCGTG-3#. The resulting plasmid was confirmed by sequencing at Washington University Sequencing Center and named pRGK374 (GeneBank accession number FJ268773). CPC was chosen to test the tissue specificity of driver lines because it is a positive regulator for root hair growth and a negative regulator for trichome initiation; thus, CPC overexpression plants carry a phenotype of no (or reduced) trichomes and more root hairs when compared with the wild-type plant (Wada et al., 1997). A GAL4/UAS–CPC driver system was generated by transforming 5XUAS:CPC into 15 selected driver lines (including both leaf-only and leaf-and-root expression patterns). 1053 bp of CPC genomic DNA fragment was isolated from wild-type DNA (Col) by PCR amplification using the following primers: 5#-CTTTTCGTCGACCTCTCTCTCTCACTCTTTTCTTTTC-3’ (SalI site engineered in as underlined region) and 5#-CCAAACGGGTTACCTCATTTCCTAAAAAAGTCTCTTC-3’ (underlined region as BstEII site). The full-length cDNA fragment of CPC was PCR-amplified from Col cDNA sample using the same primer set as listed above. Both the genomic DNA and the cDNA fragments were separately inserted into responder vector pRGK374 (Supplemental Figure 1 for map) through SalI and BstEII sites; thus, the GFP:GUS reporter region was replaced. The clones were then verified by sequencing and resulting plasmids were named as pRGK374+CPC (gDNA) and pRGK374+CPC (cDNA), respectively. This 5XUAS was initially tested for GAL4-dependence by first cloning in GFP:GUS. GFP and GUS expression were completely dependent on GAL4 in these experiments (data not shown).

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in units of kAU. For GUS staining, seedlings were incubated in 5-bromo-4-chloro-3-indolyl-b-glucuronic acid staining buffer for 2–4 h, de-stained, and imaged by light microscope. GFP images were captured using a Zeiss Stemi SV11 microscope with a 500-nm filter and AxioCam camera and software.

Trichome and Root Hair Observation T2 seedlings were selected on MS media with 50 lg ml 1 kanamycin, grown for 12–15 d, and observed under light microscope for trichome changes. For root hair counting, to avoid the affect caused by resistance, both control seeds and GAL4/UAS–CPC seeds were germinated on ½x ATS with 0.6% Gelrite (Sigma G1910) in constant light (Okada and Shimura, 1990; Wada et al., 2002). After 5 d, seedlings were examined with images captured using the Zeiss Stemi SVII microscope, Axio Cam camera and software, and then transferred onto MS media with 50 lg ml 1 kanamycin and labeled corresponding to root hair image. Positive transgenic plants were then counted for root hairs on the captured images; 2 mm of primary root from 1 mm behind the root tip was counted for the number of root hairs.

Anthocyanin and Chlorophyll Measurement Anthocyanin and chlorophyll determinations were largely based on a combination of two previous protocols (Mita et al., 1997; Lichtenthaler, 1987) with modifications to ensure a single extraction for both anthocyanin and chlorophyll measurements. Aerial parts of seedlings were collected, weighed, and extracted in 1 ml of 100% methanol for an overnight at 4
C with continuous shaking. After centrifuging for 10 min at 13 000 rpm, 200 ll of supernatant were taken out for chlorophyll determination and read at A650 and A666. Another 200 ll of extracts were removed for anthocyanin measurement, concentrated HCl were added by a 1% volume, and thoroughly mixed; A530 and A657 were read, and (A530– 0.25*A657)/fresh weight was counted as an anthocyanin unit shown in the figures. At least three biological replicates were included, and each of these biological replicates contained at least 12 seedlings.

Microarray Arabidopsis Transformation Arabidopsis plants were transformed following the previously described protocol (Clough and Bent, 1998) via Agrobacterium tumefaciens-mediated transformation.

Imaging of Reporters Imaging of LUC, GFP, and GUS expression in plants were decided by Engineer et al. (2005). For LUC imaging, plants were sprayed with luciferin substrate at a concentration of 1 mM, and imaged using a CCD darkbox camera system (Fuji LAS1000) by software IR LAS-100 Pro. LUC image quantification was carried out using ImageGauge software; a uniform area covering each seedling was counted for signal density

GAL4/UAS–CPC plants (line #225) and driver line control plants were grown side by side on MS –N plates for 15 d. Only those showing reduced trichomes were picked up as CPC overexpression sample; the same number of control plants was collected. Two different sets (four plates each set) of plants were grown and collected at different times. The same amount of total RNA from each set were pooled together and used for array analysis. All the array experiments were carried out using Agilent Arabidopsis 3 chips (www.chem.agilent.com/Scripts/ PDS.asp?lPage = 50879) containing ;37 000 features. Two independent biological replicates of CPC overexpression samples were hybridized with controls and a dye swap was performed for each replicate. Hybridization and data analysis were

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carried out commercially by MOgene LC Company (Agilent Technologies’ Certified Microarray Service Provider). Microarray data were analyzed using the Agilent error model and replicates were combined using Luminator software that performs an ANOVA analysis to identify genes with significantly different expression than control (p-value < 0.01).

RT–PCR and Q–PCR Total RNA was isolated from leaf tissues using the Agilent Plant RNA Isolation Mini Kit (Agilent Technologies, Wilmington, DE, USA). 5 lg of total RNA was treated with DNaseI (Ambion, Austin, TX) to eliminate DNA contamination and reversetranscribed using the SuperScript III transcriptase kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The synthesized cDNA was used as a template for RT–PCR or Q–PCR. Primer sets used for RT–PCR validation of microarray results and Q–PCR analysis of 35S::CPC plants are listed in Supplemental Table 2. Q–PCR was carried out using the Cepheid SmartCycler (Sunnyvale, CA) following the previously described method (Engineer and Kranz, 2007); SYBR Green qPCR Master Mix was purchased from Fermentas (Glen Burnie, MD).

Constructs for Transient Promoter Activity Assays The 1040 bp of genomic fragment with promoter region of DFR (At5g42800) was PCR-amplified using wild-type Col DNA with primers pDFR–EcoRI–F: 5#- ACGAATTCTCTGACGTCTTACGATACAACA-3#, and pDFR–KpnI–R: 5#-AAGGTACCACGTTCTAGTAATCGCATC-3#. The primers have EcoRI and KpnI restriction sites engineered into them (underlined region). Purified PCR product and a binary vector (pBAR–GUS) (Ge et al., 2005) were digested with EcoRI/KpnI and ligated. The resulting plasmid pDFR:GUS was then confirmed by restriction digest and sequencing, and served as a reporter construct in the transient promoter activity assay experiment. For effector constructs, full coding regions of PAP1 (At1g56650) and EGL3 (At1g63650) were PCR-amplified using Col cDNA sample and the following primer sets: PAP1–Kpn–F (5#-TTGGTACCATGGAGGGTTCGTCCAA-3’) and PAP1–Xba–R (5#-AATCTAGAATCAAATTTCACAGTCTCT-3’) for PAP1 gene; EGL3–Kpn–F (5#-AAGGTACCATGGCAACCGGAGAAAAC-3’) and EGL3–Xba–R (5#-ATCTAGAACATATCCATGCAACCCTT-3’) for EGL3; and CPC-Kpn-F (5‘-TTGGTACCATGTTTCGTTCAGACAAG-3‘) and CPC-Xba-R (5‘-AATCTAGATTTCCTAAAAAAGTCTC-3‘) for CPC. All three primer sets have KpnI and XbaI restriction sites (underlined region); PCR products were digested with KpnI/ XbaI, and inserted into a 35S-promoter driving binary vector 35S–FAST (Ge et al., 2005) through KpnI and XbaI sites. Resulting plasmids 35S–PAP1, 35S-EGL3 and 35S-CPC were verified by restriction digests and sequencing, and served as effector constructs. The coding region of LUC gene was released from pSP–luc+ NF fusion vector (Promega, Madison WI) by digestion with KpnI/ XbaI, and inserted into KpnI/XbaI cut 35S–FAST vector. The resulting plasmid 35S:LUC was confirmed by sequencing and included as an internal control in all transient promoter assay experiments.

Transient Expression and Assays A transient expression assay was developed using particle bombardment delivery (BioRad Biolistic Particle Delivery PDS-1000) into Arabidopsis fresh roots based on two previous protocols (Lanahan et al., 1992; Bogs et al., 2007) with modifications. In brief, fresh roots were arranged in a small circle (2.5 cm in diameter) on the surface of ½ MS media plates, so that all would be in the direct path to capture the gold particles. For each shot, gold particles were coated with a mixture of reporter plasmid (pDFR:GUS), effector plasmids (35S:PAP, 35S:EGL3, 35S:CPC or combination(s)) and internal control plasmid of 35S:LUC; 300 ng of each plasmid was included per shot. After incubation in the dark at 22
C for 48 h, two shots were combined as one sample, and the harvested samples were extracted by 500 ll of extraction buffer (100 mM Na-PO4 (pH 7.2), 5 mM DTT) and assayed for LUC activities and GUS activities by following the protocol of Lanahan et al. (1992); final relative GUS activities were normalized based on the internal control LUC activities included in every sample to indicate the particle delivery efficiency. For every independent experiment, 14 shots were included to make seven biological replicates, and at least three independent experiments were carried out separately.

Accession Numbers Sequence data of the cloning vector RGK374 from this article can be found in the GenBank data libraries under accession number FJ268773.

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING This study was supported by the Monsanto/Washington University Collaboration Agreement to R.G.K.

ACKNOWLEDGMENTS We thank Dr Yiji Xia for pBAR–GUS and 35S–FAST vector plasmids. No conflict of interest declared.

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