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Temporal Control of Trichome Distribution by MicroRNA156-Targeted SPL Genes in W OA

Nan Yu,a,b,1 Wen-Juan Cai,a,b,1 Shucai Wang,c Chun-Min Shan,a,b Ling-Jian Wang,a and Xiao-Ya Chena,2 a National Key Laboratory of Plant Molecular Genetics, Institute of and Ecology, Shanghai Institutes for Biological Sciences, 200032 Shanghai, P.R. China b Graduate School of Chinese Academy of Sciences, 200032 Shanghai, P.R. China c Department of , University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

The production and distribution of plant trichomes is temporally and spatially regulated. After entering into the flowering stage, Arabidopsis thaliana have progressively reduced numbers of trichomes on the inflorescence stem, and the floral organs are nearly glabrous. We show here that SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, which define an endogenous flowering pathway and are targeted by microRNA 156 (miR156), temporally control the trichome distribution during flowering. Plants overexpressing miR156 developed ectopic trichomes on the stem and floral organs. By contrast, plants with elevated levels of SPLs produced fewer trichomes. During plant development, the increase in SPL transcript levels is coordinated with the gradual loss of trichome cells on the stem. The MYB transcription factor genes TRICHOMELESS1 (TCL1) and TRIPTYCHON (TRY) are negative regulators of trichome development. We show that SPL9 directly activates TCL1 and TRY expression through binding to their promoters and that this activation is independent of GLABROUS1 (GL1). The phytohormones cytokinin and gibberellin were reported to induce trichome formation on the stem and inflorescence via the C2H2 transcription factors GIS, GIS2, and ZFP8, which promote GL1 expression. We show that the GIS-dependent pathway does not affect the regulation of TCL1 and TRY by miR156-targeted SPLs, represented by SPL9. These results demonstrate that the miR156-regulated SPLs establish a direct link between developmental programming and trichome distribution.

INTRODUCTION tified, including TRANSPARENT TESTA GLABRA1 (TTG1) (Galway et al., 1994; Walker et al., 1999; Bouyer et al., 2008), Trichomes are specialized epidermal cells that act as barriers to GLABRA1 (GL1) (Oppenheimer et al., 1991), GL3, and EN- protect plants from , UV irradiation, and excessive HANCER OF GLABRA3 (EGL3) (Payne et al., 2000; Szymanski (Johnson, 1975; Mauricio and Rausher, 1997). In et al., 2000; Zhang et al., 2003). TTG1, GL1, and GL3+EGL3 Arabidopsis thaliana, the distribution of trichomes is spatially and encode a WD40 protein, an R2R3 MYB transcription factor, and temporally regulated. During the early vegetative phase, tri- two basic helix-loop-helix–type transcription factors, respec- chomes are evenly distributed on the adaxial side of juvenile tively, and they form a ternary complex that initiates trichome cell rosette . As trichome production initiates on the abaxial development by activating GL2, which encodes a homeodo- side of leaves, the plant prepares for the transition from the main/leucine zipper t ranscription factor (Rerie et al., 1994; vegetative to the reproductive stage (Telfer et al., 1997). After Masucci et al., 1996; Schiefelbein, 2003; Pesch and Hu¨ lskamp, entering into the reproductive stage, the number of trichomes 2004; Ramsay and Glover, 2005). Another group of genes produced on the main inflorescence stem is gradually reduced. encode the single-repeat R3 MYB factors TRIPTYCHON (TRY), Floral organs are nearly glabrous except for a few trichomes CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1), ETC2, present on the abaxial surface of . ETC3, and TRICHOMELESS1 (TCL1) (Wada et al., 1997; Esch Previous studies of Arabidopsis trichome development have et al., 2004; Kirik et al., 2004a, 2004b; Schellmann et al., 2002; focused on rosette leaves (Marks, 1997; Hu¨ lskamp et al., 1999; Simon et al., 2007; Wang et al., 2007). They suppress trichome Larkin et al., 2003; Ishida et al., 2008). A series of genes positively initiation in a redundant manner, although TRY, TCL1, and CPC regulating trichome initiation and development have been iden- exert a major influence on rosette leaves, inflorescences, and ( ), respectively (Wada et al., 1997; Schnittger et al., 1998; 1999; Schellmann et al., 2002; Wang et al., 2007). It 1 These authors contributed equally to this work. 2 Address correspondence to [email protected]. was suggested that expression of some of the negative regulator The author responsible for distribution of materials integral to the genes, including TRY, CPC, ETC1, and ETC3, was completely or findings presented in this article in accordance with the policy described partially dependent on the GL1-GL3-TTG1 protein complex in the Instructions for Authors (www.plantcell.org) is: Xiao-Ya Chen (Morohashi et al., 2007), whereas TCL1 and ETC2 were regulated ([email protected]). by yet unidentified mechanisms (Wang et al., 2008b). The single- W Online version contains Web-only data. OA Open Access articles can be viewed online without a subscription. repeat MYB proteins can move from the trichome into neigh- www.plantcell.org/cgi/doi/10.1105/tpc.109.072579 boring cells where they compete with GL1 for the binding site of

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GL3 to prevent the formation of the active protein complex, density on successive stem internodes, thus linking the devel- resulting in blockage of trichome initiation. This lateral inhibition opmental timing to trichome development. mechanism explains how the trichome spacing pattern is gen- erated in rosette leaves (Schellmann et al., 2002; Digiuni et al., 2008). RESULTS Although trichome production is gradually repressed in Arabi- dopsis plants after bolting, the phytohormones of gibberellins SPLs Levels Are Inversely Correlated to Trichome Numbers (GAs) and cytokinins can stimulate trichome initiation on the stem and inflorescence (Chien and Sussex, 1996; Telfer et al., 1997; In the juvenile vegetative stage, trichomes were evenly distributed Perazza et al., 1998; Gan et al., 2006, 2007). A group of tran- on the adaxial side of rosette leaves of Arabidopsis. After entering scription factor genes involved in phytohormone-activated into the reproductive stage, when the plant was bolting, the trichome formation has been identified: GLABROUS INFLORES- numbers of emerging trichomes on the successive internodes of CENCE STEMS (GIS), GIS2, and ZINC FINGER PROTEIN8 the main stem were gradually reduced. The third (counting from (ZFP8) (Gan et al., 2006, 2007). GIS was first reported to act in the bottom) and upper internodes became nearly glabrous (Figure a GA-responsive pathway; loss-of-function mutation of GIS 1A). In flowers, all other floral organs, including , were decreases trichome initiation on the stem, whereas overexpres- glabrous or nearly so (Figure 1B), with the exception that un- sion of GIS leads to excessive trichome production on most of branched trichomes were found sparsely on the abaxial side of the the floral organs (Gan et al., 2006). Later, it was shown that both . The spatial distribution of trichomes indicates a coordina- GIS2 and ZFP8 play important roles in mediating GA and tion of developmental timing and trichome patterning. cytokinin responses in initiating trichome development (Gan Recent reports demonstrated that miR156-regulated SPLs et al., 2007). define an endogenous flowering pathway and promote phase For developmentally regulated trichome patterning in the transition (Wang et al., 2009; Wu et al., 2009; Yamaguchi et al., reproductive stage, TCL1 plays an important role. Loss-of-func- 2009). To explore the relation between floral transition and tion mutants of TCL1 induce ectopic trichome development on trichome repression, we first examined trichome phenotypes of the main inflorescence stem and pedicels (Wang et al., 2007). transgenic Pro35S:MIR156f plants, which overexpress MIR156f, TCL1 has a high sequence identity to other single-repeat MYB an RNA encoded by one of the eight loci of MIR156 in Arabi- proteins, such as TRY, CPC, ETC1, 2, and 3, and it also dopsis. Pro35S:MIR156f plants produced ectopic trichomes on negatively regulates GL1 transcription (Wang et al., 2007). How- both inflorescence stems and pedicels, a phenotype also ob- ever, among the genes that encode single-repeat R3 MYB served with the tcl1-1 mutant (Figures 1B and 1C). We then proteins, TCL1 is unique in that it is activated after bolting and analyzed several mutants of floral induction pathways. It has its expression does not require the GL1-GL3-TTG1 complex been reported that LFY and the MADS box genes of SOC1 and (Wang et al., 2008b). Thus, dissection of TCL1 regulation is key to FUL are direct targets of SPL transcription factors (Wang et al., discovering the signal that links the developmental timing and 2009; Yamaguchi et al., 2009); FLOWERING LOCUS T (FT) trichome patterning at the reproductive stage. encodes a small protein that is a movable flowering signal Flowering time is precisely controlled by both endogenous and (Kardailsky et al., 1999; Kobayashi et al., 1999), and TERMINAL environmental cues (Baurle and Dean, 2006). Previous studies of FLOWER1 (TFL1) encodes an FT homolog with an opposite Arabidopsis revealed four major pathways that are integrated function (Bradley et al., 1997; Ohshima et al., 1997). These results into the flowering control network: the photoperiod, vernali- suggest that miR156 plays an important role in determining the zation, autonomous, and the phytohormone GA pathways production of trichomes on stems and inflorescences. (Komeda, 2004; He and Amasino, 2005). Two types of positive In the Arabidopsis genome, 11 of the 17 SPLs are targeted by regulators, the plant-specific transcription factor LEAFY (LFY) miR156 (Rhoades et al., 2002; Gandikota et al., 2007; Wang and members of the APETALA1 (AP1)/FRUITFULL (FUL) clade of et al., 2008a; Wu et al., 2009). These SPL genes can be further MADS box transcription factors, are activated during phase classified into four clades, represented by SPL3, SPL10, SPL13, transition and promote flowering (Litt and Irish, 2003; Benlloch and SPL9, respectively (Guo et al., 2008; Wu et al., 2009). To et al., 2007). More recently, miR156 and its target genes, SQUA- check if those miR156-targeted SPL genes were responsible for MOSA BINDING PROMOTER BINDING PROTEIN-LIKE (SPL), the trichome phenotype observed in the Pro35S:MIR156f plants, have been identified to define an endogenous flowering timing we analyzed transgenic plants expressing a mimicry target of pathway. SPLs promote flowering by directly activating LFY and miR156 (Pro35S:MIM156), which resulted in elevated levels of a group of MADS box genes, including SUPPRESSOR OF SPLs (Franco-Zorrilla et al., 2007; Wang et al., 2008a). We found OVEREXPRESSION OF CONSTANS1 (SOC1), FUL, and AP1 that the Pro35S:MIM156 plants indeed had a dramatic decrease (Wang et al., 2009; Yamaguchi et al., 2009). of trichome densities on the stem (Figure 1D). Here, we show that the miR156-regulated SPL genes also To provide more direct evidence that increased levels of SPLs control trichome patterning on the inflorescence stems and floral were the cause of reduced trichome production, we examined organs, a phenotype that was also described by a recent report transgenic plants expressing the miR156-resistant form (rSPL)of (Shikata et al., 2009). We demonstrate that SPL transcription four SPL genes, Pro35S:rSPL3, ProSPL10:rSPL10, ProSPL13: factors suppress trichome formation by directly activating TCL1 rSPL13,andProSPL9:rSPL9, which contained synonymous sub- and TRY gene expression and show that an increase in SPL stitutions that changed the miR156f-pairing sequence(Wang et al., transcript levels was coordinated with the reduced trichome 2009). Compared with the wild type, trichome formation on the miR156 Influences Trichome Distribution 3 of 14

Figure 1. Regulation of Inflorescence Trichomes by miR156-Targeted SPL Genes.

(A) Spatial distribution of trichomes on aerial parts of Arabidopsis plants. Int. represents the three main stem internodes numbered from bottom to top. Red rectangles show locations of inset photographs. Bars = 1 cm in main photograph and 1 mm in insets. (B) Trichomes on inflorescences of wild-type (left), Pro35S:MIR156f (middle), and tcl1-1 mutant (right) plants. Arrows indicate the ectopic trichomes on the main inflorescence stem and pedicels. The bottom panels represent the area marked by the red square in the top panels. Bars in top panels = 2 mm, and bars in bottom panels = 500 mm. (C) Trichome density of wild-type, Pro35S:MIR156f,andtcl1-1 plants. Top: the main inflorescence stem internodes below (numbered 0) and above (numbered 1 to 7) the site of the first pedicel (flower); bottom: the first seven main inflorescence pedicels (numbered 1 to 7 from bottom to top). Data are given as mean 6 SD (n >15). (D) Trichome density of the main stem of wild-type and Pro35S:MIM156 plants. 1st to 3rd represent the main stem internodes from bottom to top. Data are given as mean 6 SD (n > 15) and analyzed by t test. **P # 0.01, very significant difference.

main stem of these transgenic plants was suppressed to vary- TCL1 and TRY Act Downstream of SPLs ing degrees (Figure 2A). Together, these data suggest that the miR156-targeted SPLs are involved in trichome regulation. Previous investigation showed that the single-repeat MYB tran- To further examine if SPLs were directly involved in suppress- scription factors TCL1, TRY, and CPC are involved in repressing ing trichome formation on the inflorescence stem and floral trichome development on the inflorescence stems and pedicels organs, we used an ethanol-inducible cassette of miR156 (Wang et al., 2007). In comparison with the tcl1-1 mutant, the (ProAlcA:MIR156f) and a hormone-inducible cassette of SPL9, Pro35S:MIR156f plants showed a similar, or even severe, phe- (ProSPL9:rSPL9-GR), in which the genomic fragment of SPL9 notypic change regarding to the ectopic trichomes produced on was translationally fused to the hormone binding domain of the the inflorescence (Figure 1B, Table 1). To understand the genetic glucocorticoid receptor. When ethanol was applied to ProAlcA: interaction between SPLs and TCL1, we generated a double MIR156f plants, an increased number of stem trichomes was mutant of tcl1-1 Pro35S:MIM156. We found that tcl1-1 was observed (Figure 2B). By contrast, treatment of ProSPL9:rSPL9- epistatic to Pro35S:MIM156 as all the tcl1-1 Pro35S:MIM156 GR plants with the GR ligand of dexamethasone (DEX) resulted in plants showed a similar trichome phenotype to tcl1-1 single reduced trichome formation on the stem (Figure 2C). mutant in terms of ectopic production of trichomes on the 4 of 14 The Plant Cell

Figure 2. SPLs Mediate Temporal Control of Trichome Patterning.

(A) Trichome density of the main stems of wild-type, Pro35S:rSPL3, ProSPL10:rSPL10, ProSPL13:rSPL13,andProSPL9:rSPL9 plants. 1st to 3rd represent the main stem internodes from bottom to top. Data are given as mean 6 SD (n > 15). **P < 0.01 compared with the wild type. (B) Trichome density of the main inflorescence stem, pedicels, and sepals of ProAlcA:MIR156f plants treated with 1% ethanol. Water was used as mock control. Inf. Stem1 represents the main inflorescence stem internode between the first two pedicels. Pedicel and sepal were from the first lower inflorescence. Data are given as mean 6 SD (n > 15). **P < 0.01 compared with mock control. (C) Trichome density of the main stem of ProSPL9:rSPL9-GR plants treated with 10 mM DEX. DMSO was used as mock control. 1st to 3rd represent the main stem internodes from bottom to top. Data are given as mean 6 SD (n > 15). **P < 0.01 compared with mock control. inflorescence stems and pedicels, while the Pro35S:MIM156 cences, however, both TCL1 and TRY were highly upregulated inflorescence was glabrous (Figure 3A, Table 1). These data and the transcript levels were comparable between the wild type suggest that SPLs act upstream of TCL1. and the gl1 mutant; by contrast, the CPC expression level We then examined the expression of TCL1, TRY, and CPC decreased in the wild-type inflorescences compared with - genes in wild-type and gl1 mutant plants. Quantitative real-time ling rosette leaves (see Supplemental Figure 1A online). These RT-PCR (qRT-PCR) analyses showed that in rosette leaves, TRY data suggest that TRY and TCL1 expression in inflorescences is and CPC were clearly expressed in the wild type, but their activated and maintained by a pathway that is independent expression levels were drastically reduced in the gl1 mutant, of GL1. whereas the TCL1 transcript level was low in either the wild-type Since the trichome density was inversely correlated to the or the gl1 mutant seedling rosette leaves (Figure 3B). In inflores- progress of floral transition, we compared the TCL1, TRY, CPC,

Table 1. Trichome Production in Wild-Type, Mutant, and Transgenic Lines

No. of Trichomes

Internode 1st Rosette Genotypes Cotyledon Leaves 1st 2nd 3rd 1st Pedicel 1st Sepal

Wild type 0.0 6 0.0 31.0 6 1.7 234.0 6 10.1b1 41.8 6 20.4b1 1.2 6 1.2b1 0.0 6 0.0 6.3 6 1.6b2 tcl1-1 0.0 6 0.0 33.1 6 2.7 298.2 6 16.6a1,b1 149.7 6 20.1a1,b1 62.5 6 15.1a1,b1 34.8 6 5.9a1,b1 14.3 6 3.0a1,b1 Pro35S:MIM156 0.0 6 0.0 27.1 6 5.0 94.8 6 8.2a1 2.4 6 1.3a1 0.0 6 0.0a1 0.0 6 0.0 3.7 6 2.1a2 Pro35S:MIM156 tcl1-1 0.0 6 0.0 28.3 6 4.3 230.9 6 15.0b1 86.3 6 18.6a2,b1 35.1 6 10.3a1,b1 15.6 6 7.2a1,b1 7.2 6 3.3b2

At least 10 plants for each line were analyzed. Values indicate mean 6 SD. aRelative to the corresponding wild-type line. a1P < 0.01, very significant difference; a2P < 0.05, significant difference. bRelative to the corresponding Pro35S:MIM156 line. b1P < 0.01, very significant difference; b2P < 0.05, significant difference. miR156 Influences Trichome Distribution 5 of 14

Figure 3. SPLs Positively Regulate TCL1 and TRY Expression. (A) Trichomes on Pro35S:MIM156 (left) and tcl1-1 Pro35S:MIM156 (right) inflorescence. The bottom panels represent the area marked by the red square in the top panels. Bars in top panels = 2 mm, and bars in bottom panels = 1 mm. (B) Expression of TCL1 (left) and TRY (right) in seedling rosette leaves and inflorescences. Seedling (10-d-old) rosette leaves and inflorescences (Inf.) of the wild type and gl1 mutant were collected and analyzed by qRT-PCR. b-TUBULIN-2 was used as the internal standard. Error bars indicate SD of three technical replicates, and the results were consistent in three biological replicates. (C) Expression of TCL1, TRY,andSPL9 in three successive internodes of the main stem and the inflorescence of wild-type and gl1 plants. Error bars indicate SD of three technical replicates, and the results were consistent in three biological replicates. (D) Expression levels of SPL9, TCL1,andTRY and in the main stem (top) and developing inflorescence (bottom) of wild-type and Pro35S:MIR156f plants. Error bars indicate SD of three technical replicates, and the results were consistent in three biological replicates. (E) Expression levels of SPL9, TCL1,andTRY in rosette leaves of 10-d-old wild-type and Pro35S:MIM156 plants. Error bars indicate SD of three technical replicates, and the results were consistent in three biological replicates. and SPL9 expression levels in the main stem internodes 1 to 3 manifested by the first internode, and this dependence was then (bottom to top) and inflorescence (Figures 1A and B). SPL9 was lost along with plant development. chosen here as a representative of miR156-targeted SPL genes. Because of the similar tendency of SPL9, TCL1, and TRY Transcript levels of both TCL1 and TRY were gradually elevated expression in the stem and inflorescence, we wondered whether from internode 1 to internode 3, and the highest level was SPL9, and possibly other SPLs too, played a role in elevating the detected from the inflorescence (Figure 3C). SPL9 exhibited a expression level of TCL1 and TRY during floral transition. As similar change of spatial expression as did TCL1 and TRY (Figure described above, stem trichome numbers were inversely corre- 3C). CPC, on the contrary, showed an opposite expression lated to expression levels of SPL genes in transgenic plants. pattern in the stem as its transcript level was decreasing from qRT-PCR analyses revealed that expression of both TRY and internode 1 to 3, although the expression in inflorescence was TCL1 genes was indeed downregulated in Pro35S:MIR156f evident (see Supplemental Figure 1B online). The gl1 mutation plants (Figure 3D) but upregulated in ProSPL13:rSPL13, did not affect TCL1 expression in the main stem and the inflo- ProSPL10:rSPL10, ProSPL9:rSPL9, Pro35S:rSPL3 (see Supple- rescence, but it did cause an obvious reduction of TRY transcript mental Figure 2 online), and Pro35S:MIM156 plants (Figure 3E). level in the first internode of the main stem (Figure 3C). Therefore, However, accumulation of CPC transcripts was slightly en- while TCL1 expression was unrelated to GL1, TRY expression hanced rather than downregulated in Pro35S:MIR156f plants, was still dependent on GL1 at the early reproductive stage, as and there was little change of its expression in Pro35S:MIM156 6 of 14 The Plant Cell

plants (see Supplemental Figure 1C online). Taken together, both mutations of the TRY promoter dramatically decrease GUS genetic and gene expression data support that TCL1 and TRY, activity in the stem and flowers (Figures 4Q to 4S), indicating but not CPC, act downstream of SPLs in repressing trichome that the SPL binding sites are also important for TRY expression production in the stem and inflorescence. in these organs. Because TCL1 expression level in rosette leaves were rather SPL9 Directly Targets TCL1 and TRY Promoters low (Figure 3B), GUS activities of ProTCL1:GUS were then detected in trichome cells of cauline leaves of high-expression To know whether TCL1 and TRY were directly regulated by SPL lines (see Supplemental Figure 3A online). Mutation of single transcription factors, we tested the activity of SPL9 in activating GTAC motifs in the TCL1 promoter did not severely affect its TCL1 and TRY gene expression. One-week-old ProSPL9:rSPL9- activity and trichome specificity; mutation of five GTAC cis- GR seedling rosette leaves were sprayed with 10 mM DEX to elements, however, decreased the promoter activity (see Sup- allow of the translocation of the rSPL9-GR fusion protein into the plemental Figures 3B to 3F online). For TRY promoter, mutation nucleus. A significant increase of TRY and TCL1 transcript of single GTAC motifs did not decrease its activity or trichome abundance was observed after 5 h (Figure 4A), suggesting that specificity either (see Supplemental Figures 3G to 3J online). SPL9 rapidly activated the transcription of TRY and TCL1.By These data demonstrate that the cis-elements recognized by contrast, downregulation of SPLs by a 6-h ethanol treatment of SPL transcription factors are less important for TCL1 and TRY the 1-month-old ProAlcA:MIR156 plants almost blocked the TRY expression in leaves than in inflorescences. and TCL1 expression in inflorescences (Figure 4B). To further demonstrate that SPL9 could bind directly to the To view the spatial expression pattern of TCL1, we generated a promoters of TCL1 and TRY, we performed chromatin immuno- reporter line by fusing the 2-kb upstream regulatory fragment of precipitation (ChIP) assay. We used transgenic lines in which a TCL1 to the coding sequence of b-glucuronidase (GUS) miR156-resistant form of SPL9 was translationally fused to the (ProTCL1:GUS; Figure 4C). Most of the ProTCL1:GUS trans- green fluorescent protein (GFP) coding region (ProSPL9:GFP- genic plants (17/20) exhibited a strong GUS expression in the rSPL9). The ProSPL9:GFP-rSPL9 plants had a similar phenotype inflorescence (Figure 4D). The GUS staining was increasingly as Pro35S:MIM156 with fewer trichomes on the stem internodes intensive in the main inflorescence stem from bottom to top, (Figure 2A) compared with the wild type. We chose four frag- which was consistent with the expression pattern of TCL1 ments spanning the GTAC motifs from TCL1 promoter (Figure revealed by qRT-PCR (Figure 3C). GUS staining became paler 3C). Fragments I, III, and IV were enriched after ChIP with GFP in Pro35S:MIR156f and stronger in Pro35S:MIM156 plants, re- antibody in the transgenic ProSPL9:GFP-rSPL9 compared with spectively (Figures 3E and 3F). In addition, we observed tri- the wild-type plants (Figure 5A). We also detected the enrich- chome-specific distribution of GUS activity in young rosette ment of the TRY promoter fragments (Figures 4C and 5B). These leaves (Figure 4G) and in cauline leaves of the high-expression data further support that TCL1 and TRY are direct targets of lines (Figure 4H), a pattern different from the universal expression SPL9. of TCL1 in the inflorescence stem , possibly due to the higher level of SPLs in the latter. Expression of SPL9, TCL1,andTRY Is Not Dependent A consensus sequence, GTAC, has been identified as the on GISs core binding motif of SPLs (Klein et al., 1996; Birkenbihl et al., 2005; Kropat et al., 2005; Liang et al., 2008). There are seven The putative C2H2 transcription factors GIS, GIS2, and ZFP8 GTAC motifs within the 2-kb upstream fragment of the TCL1 mediate another a phytohormone-dependent pathway in which gene (Figure 4C). To determine the role of these GTAC motifs, GAs and cytokinins induce trichome formation on the stem and we constructed four mutant versions of the TCL1 promoter inflorescence. It has been proposed that these proteins are (ProTCL1mu1:GUS, ProTCL1mu2:GUS, ProTCL1mu3:GUS, involved in positive regulation of GL1 (Gan et al., 2006, 2007). We and ProTCL1mu4:GUS), in which the GTAC motif was changed tested whether there is a close relation between GIS-dependent to AAGA, respectively (Figure 4C). In addition, we made a trichome production and SPL-dependent regulation of the neg- reporter line (ProTCL1mu5:GUS) with five motifs simultaneously ative regulators of trichome initiation. First, we treated the wild- mutated. ProTCL1mu1:GUS, ProTCL1mu2:GUS, ProTCL1mu3: type plants with cytokinin 6-benzylaminopurine (6-BA), which GUS, and ProTCL1mu4:GUS plants showed a reduction of GUS resulted in a clear increase in the expression levels of GIS, GIS2, activity in the inflorescences to a different extend in comparison and ZFP8, although the induction magnitudes varied (Figure 6A). with ProTCL1:GUS (Figures 4I to 4L), whereas GUS staining was The GL1 expression level was also elevated in the 6-BA–treated not observed in ProTCL1mu5:GUS plants (Figure 4M), indicating plants (Figure 6A), consistent with the previous report (Gan et al., that these GTAC motifs are indispensable for the TCL1 promoter 2007). If SPL9, TCL1, and TRY were involved in this hormone- activity in the inflorescences. induced trichome pathway, we would expect a repression of We also scanned the TRY promoter for SPL binding site these genes in the 6-BA–treated plants. However, none of them (GTAC) and constructed three single mutant versions (Pro- were downregulated after the hormone treatment (Figure 6A). We TRYmu1:GUS, ProTRYmu2:GUS, and ProTRYmu3:GUS), using also treated the plants with GA3 and a similar induction pattern the same method as for TCL1 promoter (Figure 4C). Most of the was observed, although the induction effect was not as great as ProTRY:GUS transgenic plants showed a clear GUS staining in that by 6-BA in our experimental conditions. the inflorescence stem, floral organs (Figure 4N), and trichomes Because overexpression of GIS or GIS2 produced a similar of rosette and cauline leaves (Figures 4O and 4P), whereas single phenotype as did the hormone treatments with regard to ectopic miR156 Influences Trichome Distribution 7 of 14

Figure 4. TCL1 and TRY as Direct Targets of SPL9. (A) Expression of TCL1 and TRY in rosette leaves of 10-d-old ProSPL9:rSPL9-GR seedlings after a 5-h DEX treatment or a mock treatment with DMSO. Error bars indicate SD of three technical replicates, and the results were consistent in three biological replicates. (B) Expression of SPL9, TCL1,andTRY in ProAlcA:MIR156f inflorescences after a 6-h ethanol treatment or a mock treatment with water. Error bars indicate SD of three technical replicates, and the results were consistent in three biological replicates. (C) Schematic diagram of ProTCL1:GUS (top) and ProTRY:GUS (bottom) constructs. Top diagram: A 1941-bp 59-upstream fragment of TCL1 was fused to GUS. Bottom diagram: A 2138-bp 59-upstream fragment of TRY was fused to GUS. In both diagrams, triangles indicate GTAC boxes in the promoter, mu1 to mu4 represent the GTAC motifs mutated, and I to IV represent the DNA fragments used for ChIP analyses. 8 of 14 The Plant Cell

internode of the gis Pro35S:MIM156 plants were decreased significantly, and overexpression of MIR156f in gis mutant led to ectopic trichomes on inflorescences (see Supplemental Figure 4 and Supplemental Table 1 online). Thus, the regulation of TCL1 and TRY by SPLs was not impaired by the gis mutation. These data suggest that the GIS-dependent pathway does not play a major role in regulating SPL and the trichome negative regulator genes of TCL1 and TRY. We then examined the effects of altered levels of SPLs on GIS expression. In plants with increased levels of SPLs due to Pro35S:MIM156 expression, expression levels of the GIS genes were largely unchanged in comparison with those in the wild- type plants. On the other hand, in Pro35S:MIR156f plants in which SPLs were repressed and trichomes were formed on the stem and inflorescence, expression of the GIS genes was either unchanged or even slightly downregulated (Figure 7A). Finally, we applied 6-BA to the Pro35S:MIM156 and Pro35S:MIR156f plants. We found that GIS, GIS2, and ZFP8 genes were clearly upregulated in both Pro35S:MIR156f and Pro35S:MIM156 plants after treatments (Figures 7B and 7C), as observed with the wild- type plants (Figure 6A). GL1 gene was also upregulated by 6-BA treatment of the Pro35S:MIM156 plants. In Pro35S:MIR156f Figure 5. Binding of SPL9 to TCL1 and TRY Promoters. plants, GL1 expression was induced only slightly (Figure 7B), ChIP enrichment of TCL1 (A) and TRY (B) promoter regions bound by possibly due to a high basal level. Thus, altered levels of SPLs did GFP-SPL9. Rosette leaves of the 3-week-old ProSPL9:GFP-rSPL9 and not interfere with 6-BA induction of GISs genes, and GL1 gene wild-type plants were used. The DNA fragments were quantitatively induction was not affected, at least not in plants with an elevated analyzed by PCR, and the b-TUBULIN-2 promoter was used as a level of SPLs. reference. Error bars indicate SD of three quantitative PCR replicates, Together, these data demonstrate that while the GIS-depen- and the results were consistent in three precipitations. dent pathway promotes trichome initiation through the positive regulator gene GL1, the miR156-targeted SPLs repress trichome formation by upregulating the negative regulator genes TCL1 and trichome production in the stem and inflorescence, we analyzed TRY. It is likely that the two pathways act in parallel in determining the changes of gene expression in Pro35S:GIS2 plants. We trichome formation in the stem and inflorescences. found that, while the GL1 transcript level highly correlated with that of GIS2, as previously reported (Gan et al., 2007), the transcript levels of SPL9, TCL1, and TRY were not affected by DISCUSSION GIS2 overexpression (Figure 6B). Furthermore, the SPL9, TCL1, and TRY transcript levels were similar in the wild type, gis, and The epidermis is the outermost layer covering plant aerial organs. the gis zfp8-R1 gis2 (Gan et al., 2006, 2007) triple mutant plants During colonization of land, plants evolved many typical epider- (Figure 6C). To further examine whether the regulation of TCL1 mal features. One of the specialized cell types of aerial epidermis and TRY by SPL transcription factors requires GIS, we intro- is the trichome, which generally forms a barrier to protect the duced Pro35S:MIM156 and Pro35S:MIR156f into the gis mutant, plant. However, the excess of trichomes on floral organs may respectively. Following the changed levels of SPL9, the TCL1 offer a few disadvantages to cleistogamous plants, in which self- and TRY transcript levels were elevated in the former and occurs before flower opening. It was reported that the decreased in the latter (Figure 6D). Consistent with the change rate of natural self-pollination was negatively correlated with the of transcript levels, trichome numbers on the first mainstem distance from the to anther (Shimizu and Ichimura, 2002).

Figure 4. (continued). (D) to (F) GUS staining of inflorescences showing ProTCL1:GUS expression in wild-type (D), Pro35S:MIR156f (E),andPro35S:MIM156 (F) backgrounds. (G) and (H) GUS staining showing ProTCL1:GUS expression in trichomes of rosette (G) and cauline (H) leaves in the wild-type background. (I) to (M) GUS staining of inflorescences expressing mutated versions of ProTCL1:GUS, mu1 to mu5, respectively. Note that GUS activities were greatly reduced in mu1 to mu4 and to an undetectable level in mu5 inflorescences. (N) GUS staining of ProTRY:GUS inflorescences. (O) and (P) GUS staining of ProTRY:GUS activities in trichomes of rosette (O) and cauline (P) leaves in the wild-type background. (Q) to (S) GUS staining of inflorescences expressing mutated versions of ProTRY:GUS, mu1 to mu3, respectively. Bars = 2 mm. miR156 Influences Trichome Distribution 9 of 14

Figure 6. Expression of SPL9, TCL1,andTRY in Plants with Altered Levels of GISs.

(A) qRT-PCR analysis of the expression of GISs, SPL9, and trichome regulatory genes (GL1, TCL1,andTRY) in inflorescences of wild-type plantsafter6-BAtreatment.ErrorbarsindicateSD of three technical replicates, and the results were consistent in two biological replicates. Cyt, 6-benzylaminopurine. (B) Expression of SPL9, TCL1,andTRY in inflorescences of Pro35S:GIS2 plants. Error bars indicate SD of three biological replicates. (C) Expression of SPL9, TCL1,andTRY in inflorescences of gis single and gis ZFP8-R1 gis2 triple mutant plants. Error bars indicate SD of three biological replicates. (D) Expression of SPL9, TCL1,andTRY in the first pair of rosette leaves of Pro35S:MIM156 and Pro35S:MIR156f in the gis mutant background. Error bars indicate SD of three biological replicates.

This could be an explanation why the carpel and of such the stem internodes and floral organs of Arabidopsis. First, cleistogamous plants as Arabidopsis and Pisum sativum do not expression of miR156/SPLs is age regulated and tightly coupled bear branched trichomes. The conical-papillate cells form another to the transition from the vegetative to the reproductive stage. type of epidermal structures, which are found only on in the The miR156 levels decrease as the plant ages. In short days, majority of entomophilous (insect-pollinated) flowers, and they miR156 levels were the highest in young seedling rosette leaves allow the to grip flowers (Martin and Glover, 2007). and subsequently declined over several weeks. On the contrary, From these points of view, it is reasonable to assume that plants the expression levels of miR156 target genes, including SPL9, couple epidermal cell differentiation to floral transition. were inversely related to miR156 expression with a gradual There could be alternate ways of transducing phase transition increase during reproductive stage (Schmid et al., 2005; Wang signals to trichome repression in the stem and inflorescence at the et al., 2009). This temporal expression pattern fits the timing of gene expression level: developmental factors could downregulate trichome repression during plant development. Secondly, the GL1 or other positive regulator genes of trichome initiation, or the miR156-regulated SPL transcription factors are master regula- developmental factors could upregulate negative regulators of the tors of Arabidopsis phase transition and one of the endogenous trichome pathway. Previous identification of the TCL1 gene and flowering pathways. They directly activate expression of tran- phenotypic analysis of the tcl1-1 try double mutant plants sup- scription factor genes of LFY, FUL, AP1, and SOC1, which ported the latter possibility: both negative regulators of TCL1 and control the timing of flowering (Wang et al., 2009; Yamaguchi TRY are required for progressive repression of trichome formation et al., 2009). Our transgenic plant and ChIP analyses data in the reproductive stage (Wang et al., 2007, 2008b). revealed that SPL9, and likely other SPLs, positively regulates Recently, Shikata et al. (2009) reported reduced trichome TCL1 and TRY gene expression through binding to and activating initiation on cauline leaves and floral organs in plants expressing their promoters. Thus, the key factors of promoting flowering chimeric repressors of SPL10, SPL11, and SPL12. In this inves- also directly specify certain epidermal features of inflorescence tigation, we demonstrated that it is the miR156-regulated SPLs stems and floral organs. Nevertheless, the results presented here that activate the TCL1 and TRY expression. Our results establish do not exclude the possibility that other flowering factors also a direct link between transition to flowering stage and trichome participate in controlling trichome formation in specific floral patterning and explain the gradual loss of trichome formation on organs. 10 of 14 The Plant Cell

epidermal cells, resulting in increasingly higher ratios of negative (such as TCL1 and TRY) to positive (mainly GL1) regulators and the gradual lose of trichome formation. Interestingly, overexpression of SPL genes promoted, rather than inhibited, trichome formation on abaxial surface of leaves, likely due to an accelerated transition to adult phase (Cardon et al., 1997; Wang et al., 2009; Wu et al., 2009). Thus, suppression of trichome formation by SPLs seems not to be universal, and the factors controlling trichome initiation on abxial surfaces re- main to be identified. In addition to TRY and TCL1, Arabidopsis has at least four more single-repeat R3 MYB factors that function as negative regulators of trichome development. CPC, for exam- ple, is involved in suppression of trichome formation and flavonoid biosynthesis, in addition to its role in specifying root hairs (Wada et al., 1997; Wang et al., 2008b; Zhu et al., 2009; Broun, 2005). Our data presented herein revealed that CPC is progressively down- regulated in stems during plant development, and its expression is not regulated by SPLs. Therefore, these negative regulator genes must be differentially regulated in different organs during devel- opment, emphasizing that the regulatory network controlling trichome patterning requires further investigation. Previous investigations demonstrated that promotion of tri- chome formation by phytohomones of GAs and cytokinins is mediated by GIS and related transcription factors of GIS2 and ZFP8. Unlike SPLs that activate the trichome negative regulator Figure 7. Expression of GIS, GIS2,andZFP8 in Pro35S:MIM156 and genes, the GIS-dependent pathway upregulates the positive Pro35S:MIR156f Plants. regulator gene GL1 (Perazza et al., 1998; Gan et al., 2006, 2007). Our analysis of gis mutants and the transgenic plants with altered (A) Expression of GIS, GIS2,andZFP8 in inflorescences of the Pro35S: SPL levels suggested that GIS factors were not involved in the MIM156 and Pro35S:MIR156f plants. Error bars indicate SD of three biological replicates. SPL-dependent regulation of trichome negative regulator genes (B) and (C) Expression of GIS, GIS2,andZFP8 in inflorescences of of TCL1 and TRY, neither did the SPLs play a major role in Pro35S:MIR156f (B) and Pro35S:MIM156 (C) plants after 6-BA treat- regulating GIS and related genes. Collectively, data of previous ment. Error bars indicate SD of three technical replicates, and the results reports and data presented herein suggest that the miR156/ were consistent in two biological replicates. Cyt, 6-benzylaminopurine. SPL- and the GIS-dependent pathways function in parallel in regulating trichome regulator complex in the main stem and inflorescences. However, at present, a possible interaction of the In the lateral inhibition model proposed for rosette leaf tri- two pathways at an early step cannot be excluded, since both chome pattering, the negative regulator genes, such as TRY, are are involved regulating phase transition, maturation, and activated in trichome cells by the GL1-GL3-TTG1 protein com- flowering, besides trichome initiation. Further genetic and bio- plex and moved to neighboring cells to repress trichome initiation chemical investigations are needed to answer how the two (Schellmann et al., 2002; Digiuni et al., 2008). Consistent with pathways are integrated into plant development programming. GL1, the TRY expression in rosette leaves is also trichome In addition to the endogenous hormone-mediated flowering specific (Schellmann et al., 2002; Esch et al., 2003). However, signals, environmental conditions also influence trichome devel- this model requires the presence of trichomes as a precondition opment. It was reported that the trichome density is closely to prevent trichome initiation and does not explain the glabrous related to temperature, light, soil moisture, wounding, and phenotype of such aerial organs as , pedicel, and upper other factors in various plant (Wellso and Hoxie, 1982; internodes of inflorescence stem. Activation of TRY and TCL1 by Gianfagna et al., 1992; Pe´ rez-Estrada et al., 2000; Yoshida et al., age-related SPLs provides a solution to this paradox. At the 2009). It will be interesting to examine the regulation of trichome basal part of stem, TRY expression is still influenced by GL1, but production by these exogenous and endogenous cues and their this effect becomes negligible along with the increasing levels of crosstalk with the miR156/SPLs pathway. SPLs during shoot growth. In agreement with the expression patterns of SPL genes (Ito et al., 2004), promoter activities of TRY METHODS and TCL1 are widely distributed in at least certain regions of epidermal cells of the inflorescence stems and floral organs, with Plant Materials and Primers higher activities in the upper internodes despite the absence of Plants of Arabidopsis thaliana, ecotype Columbia (Columbia-0) were trichomes. We propose that, after plants entering into the repro- grown at 228C in long days (16 h light/8 h dark). The trichome numbers ductive stage, the progressive elevation of SPL levels causes a were counted, and data were given as mean 6 SD and were analyzed by t general activation of TRY and TCL1 genes in stem and floral test. miR156 Influences Trichome Distribution 11 of 14

Primers used in PCR are listed in Supplemental Table 2 online. ChIP

ChIP experiments were performed according to published protocols Constructs and Plant Transformation (Wang et al., 2002, 2009). About 3 g tissues of the 3-week-old ProSPL9: To make ProTCL1:GUS and ProTRY:GUS constructs, the promoters of GFP-rSPL9 plants were harvested. After fixation, the material was TCL1 and TRY (;2 kb) were PCR amplified using Pyrobest DNA poly- washed and ground under liquid nitrogen, the resultant powder was merase (TaKaRa) and individually fused into the GUS coding region. resuspended in extraction buffer (0.4 M sucrose, 10 mM Tris-HCl, pH 8.0,

Promoter mutations were created by two-round PCR, using primers as 10 mM MgCl2, 5 mM mercaptoethanol, 0.1 mM PMSF, and 13 protease shown in Supplemental Table 2 online. inhibitor [Roche]) and lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, For ProAlcA:MIR156f, a genomic fragment of MIR156f was cloned 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate sodium, and 0.1% between AlcA promoter and OCS terminator. The resulting fragment was SDS), successively, followed by sonification (Output 3, 6 3 10 s). One- inserted into pMLBart vector, which harbors a Pro35S:AlcR cassette third of the solution was saved as input total DNA without precipitation. An (Roslan et al., 2001). anti-GFP antibody (Abmart) was added to another one-third and the Constructs were delivered into Agrobacterium tumefaciens strain remaining one-third was precipitated in parallel with anti-HA (Sigma- GV3101 (pMP90). Arabidopsis plants were transformed using a flower- Aldrich) as a negative control. The resulting DNA samples were purified dip method (Clough and Bent, 1998). Transgenic seedlings were selected using a PCR purification kit (Qiagen). The relative concentrations of the with 40 mg/mL kanamycin (for pCAMBIA2300) on plates or 0.1% DNA fragments were analyzed by quantitative PCR, using the b-TUBU- glufosinate (BASTA, for pCAMBIA3300) in soil. For each construct at LIN2 gene promoter as the reference. Relative enrichment fold was least 25 T1 seedlings were analyzed. calculated first by normalizing the amount of a target DNA fragment against the TUBULIN2 promoter fragment (1153 to 1309 bp upstream of Plant Treatments the translation start codon) and then by normalizing the value for anti-GFP against the value for anti-HA. For DEX induction, 1-week-old ProSPL9:rSPL9-GR seedling rosette leaves were sprayed twice a week with 10 mM DEX (Sigma-Aldrich) or Accession Numbers DMSO (mock control) solutions until the plants started bolting. For ethanol treatment, ProAlcA:MIR156f plants were grown in soil until Arabidopsis Genome Initiative gene identifiers are as follows: SPL9 flowered. The plants were then sprayed with 1% ethanol or water (At2g42200), SPL3 (At2g33810), SPL10 (AT1G27370), SPL13 (At5g50670), (mock control) twice a week. MIR156f (AT5G26147), TFL1 (At5g03840), FUL (At5g60910), SOC1 For measuring the effect of transient downregulation of miR156-reg- (At2g45660), LFY (At5g61850), FT (At1g65480), GL1 (AT3G27920), TCL1 ulated SPLs on TCL1 and TRY expression, ProAlcA:MIR156f plants (n > (At2g30432), TRY (At5G53200), GIS (At3g58070), GIS2 (At5g06650), ZFP8 18) were grown in soil until the inflorescence stem had reached a length of (At2g41940), and b-TUBULIN-2 (At5g62690). Mutants and transgenic 2 to 3 cm. The plants were then sprayed with 1% ethanol or water (mock plants of gl1 (SALK_039478), try (SALK_029760), tcl1-1 (SALK_055460), control), and the were harvested after 6 h. For transient upregu- Pro35S:MIR156f, Pro35S:MIM156, soc1-6 (SALK_138131), ful-7 (SALK_ lation of SPL9, 1-week-old ProSPL9:rSPL9-GR seedling rosette leaves 033647), lfy-12, ft-10, tfl1-1, ProSPL13:rSPL13, ProSPL10:rSPL10, m m were sprayed with either 10 M DEX or 10 M DMSO (control). The plants Pro35S:rSPL3, ProSPL9:rSPL9, ProSPL9:rSPL9-GR, ProSPL9:GFP- were harvested 5 h after treatments, followed by RNA extraction. rSPL9, gis (GABI _423G08), gis2 (NASC_N119489), and gis zfp-R1 gis2 Hormone treatments were performed as described (Gan et al., 2007). were as described previously (Wang et al., 2004, 2007, 2008a, 2009; Gan Briefly, the cytokinin 6-BA (Sigma-Aldrich) and the gibberellin GA3 et al., 2006, 2007). (Sigma-Aldrich) were used, and the plants were grown in pots at 228C in long-day conditions (16 h light/8 h dark). Until the first five to six leaves Supplemental Data had emerged, the pots were soaked in 6-BA (50 mM) or GA3 (100 mM) for 4 h, respectively, once every 2 d. After shoots had reached 6 to 8 cm The following materials are available in the online version of this article. in length, the plants were treated for another 4 h with the same hormone, Supplemental Figure 1. Expression Pattern of CPC in Wild-Type, gl1, and the inflorescences were harvested for RNA extraction. and Transgenic Plants.

Expression Analyses Supplemental Figure 2. Overexpression of SPLs Upregulates TCL1 and TRY Expression. Total RNAs were extracted with Trizol reagent (Invitrogen), from which Supplemental Figure 3. GUS Staining of Leaves of the ProTCL1: 1 mg was used for reverse transcription in a 20-mL reaction system with GUS and ProTRY:GUS Plants. the RNA PCR (AMV) kit (Promega). Quantitative real-time RT-PCR was performed with SYBR-Green PCR Mastermix (Takara), and amplification Supplemental Figure 4. Trichome Production on Inflorescences of was real-time monitored on a Mastercycler ep RealPlex2 (Eppendorf). The gis, gis Pro35S:MIR156f,andgis Pro35S:MIM156 Plants. level of transcript abundance relative to reference gene (termed DC )was T Supplemental Table 1. Trichome Numbers of the Main Stem of gis determined according to the function DC =C (test gene) 2 C (reference T T T and gis Pro35S:MIM156 Plants. gene). To compare untreated and treated expression levels, the function Supplemental Table 2. Oligonucleotide Primers. DDCT was first determined using the equation DD CT = D CT (treatment) 2

D CT (control) where control represented mock-treated plants. The induction ratio of treatment/control was then calculated by the equation 2DD C 2 T. The primer sequences are listed in Supplemental Table 2 online. ACKNOWLEDGMENTS GUS activity was detected by staining plant tissues at 378C in 0.1 M sodium phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.5 We thank the ABRC for Arabidopsis T-DNA insertion lines, Jia-Wei mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 0.5 mg/ Wang, Detlef Weigel, and Yinbo Gan for sharing materials and for mL X-glucuronide. Subsequent washes with 70% ethanol were per- their helpful discussions. This research was supported by grants from formed to remove and enhance contrast. At least six inde- the National Natural Science Foundation of China (30630008 pendent transformants for each constructs were selected. and 30721061), the State Key Basic Research Program of China 12 of 14 The Plant Cell

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Yamaguchi, A., Wu, M.F., Yang, L., Wu, G., Poethig, R.S., and Zhang, F., Gonzalez, A., Zhao, M., Payne, C.T., and Lloyd, A. Wagner, D. (2009). The microRNA-regulated SBP-Box transcription (2003). A network of redundant bHLH proteins functions in all factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL,and TTG1-dependent pathways of Arabidopsis. Development 130: APETALA1. Dev. Cell 17: 268–278. 4859–4869. Yoshida, Y., Sano, R., Wada, T., Takabayashi, J., and Okada, K. Zhu, H.F., Fitzsimmons, K., Khandelwal, A., and Kranz, R.G. (2009). Jasmonic acid control of GLABRA3 links inducible defense (2009). CPC, a single-repeat R3 MYB, is a negative regulator and trichome patterning in Arabidopsis. Development 136: 1039– of anthocyanin biosynthesis in Arabidopsis. Mol. Plant 2: 1048. 790–802. Temporal Control of Trichome Distribution by MicroRNA156-Targeted SPL Genes in Arabidopsis thaliana Nan Yu, Wen-Juan Cai, Shucai Wang, Chun-Min Shan, Ling-Jian Wang and Xiao-Ya Chen PLANT CELL published online Jul 9, 2010; DOI: 10.1105/tpc.109.072579

This information is current as of July 9, 2010

Supplemental Data http://www.plantcell.org/cgi/content/full/tpc.109.072579/DC1

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