This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online reduces the time to publication by several weeks. Temporal Control of Trichome Distribution by MicroRNA156-Targeted SPL Genes in Arabidopsis thaliana 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 Plant Physiology 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 Botany, 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 plants 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 herbivores, UV irradiation, and excessive HANCER OF GLABRA3 (EGL3) (Payne et al., 2000; Szymanski transpiration (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 leaves. 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 sepals. 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 roots (root hairs), 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 The Plant Cell Preview, www.aspb.org ã 2010 American Society of Plant Biologists 1 of 14 2 of 14 The Plant Cell 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 pedicel, 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 sepal. 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