CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis  Zhu Hui-Fen , Fitzsimmons Karen , Khandelwal Abha , Kranz Robert G.   Molecular Plant  Volume 2, Issue 4, Pages 790-802 (July 2009) DOI: 10.1093/mp/ssp030 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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 wild-type. Measurements are shown as the percentage of the WT level. Wild-type is Wassilewskija. Error bars show standard deviations. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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 co-delivered 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. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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 α-helices of both R2 and R3 domains are indicated in boxes; the conserved MYB–bHLH interaction motif on the first two α-helices of R3 domain is underlined with a gray bar. ‘*’, identical residues; ‘:’ and ‘.’, similar residues. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

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. Molecular Plant 2009 2, 790-802DOI: (10.1093/mp/ssp030) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions