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Flowering is delayed by mutations in homologous genes CAPRICE and TRYPTICHON in the early flowering Arabidopsis cpl3 mutant
Journal of Plant Physiology 170 (2013) 1466–1468
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short communication
Flowering is delayed by mutations in homologous genes CAPRICE and TRYPTICHON in the early flowering Arabidopsis cpl3 mutant Rumi Tominaga-Wada ∗ , Yuka Nukumizu, Takuji Wada Interdisciplinary Research Organization, University of Miyazaki, 1-1, Gakuen Kibanadai-Nishi, Miyazaki 889-2192, Japan
a r t i c l e
i n f o
Article history: Received 28 March 2013 Received in revised form 23 May 2013 Accepted 23 May 2013 Available online 21 June 2013 Keywords: Arabidopsis Flowering Root hair Trichome Transcription factor
a b s t r a c t CAPRICE (CPC) and CAPRICE-like (CPL) myeloblastosis (MYB) family members [including TRYPTICHON (TRY) and ENHANCER OF TRYPTICHON AND CAPRICE (ETC)] of Arabidopsis thaliana encode R3-type MYB transcription factors that promote root hair differentiation and inhibit trichome formation in a redundant manner. Previously, we reported that the CPL3 gene affects flowering. The cpl3 mutant plants flower earlier and with fewer leaves than the wild type. In this study, we show that mutations in CPC or TRY delay flowering of cpl3 plants. A mutation in ETC1 did not further delay flowering but reduced plant size. Our study provides insight into the regulation of flowering time by the CPC-like MYB gene family. © 2013 Elsevier GmbH. All rights reserved.
Introduction The CAPRICE (CPC) gene, which encodes an R3-type myeloblastosis (MYB) transcription factor, is a key regulator of epidermal cell development including trichome initiation and root-hair differentiation in Arabidopsis thaliana (Wada et al., 1997). Several additional CPC-like MYB genes have been identified in the Arabidopsis genome, including TRYPTICHON (TRY), ENHANCER OF TRY AND CPC1 and 2 (ETC1 and ETC2), ENHANCER OF TRY AND CPC3/CPCLIKE MYB3 (ETC3/CPL3), and TRICHOMELESS1 and 2/CPC-LIKE MYB4 (TCL1 and TCL2/CPL4) (Esch et al., 2004; Gan et al., 2011; Kirik et al., 2004a,b; Schellmann et al., 2002; Simon et al., 2007; Tominaga et al., 2008; Tominaga-Wada and Nukumizu, 2012a,b; Wang et al., 2007). These seven CPC-like MYB transcription factors are thought to act as positive regulators of root hair differentiation and as negative regulators of trichome differentiation. The regulation of cell differentiation is a crucial step in the development of multicellular organisms. Thus, epidermal cell differentiation in Arabidopsis has been used extensively as a model system for analyzing cell fate determination. Several additional regulatory factors of the CPClike MYB gene family are known to be involved in epidermal cell differentiation. Mutations in the GLABRA2 (GL2) and WEREWOLF (WER) genes cause an increase in root hairs (Lee and Schiefelbein,
Abbreviations: CPC, CAPRICE; CPL, CAPRICE-like; ETC, ENHANCER OF TRYPTICHON AND CAPRICE; MYB, myeloblastosis; TRY, TRYPTICHON. ∗ Corresponding author. Tel.: +81 985 58 7864; fax: +81 985 58 7864. E-mail address: [email protected] (R. Tominaga-Wada). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.05.013
1999; Masucci et al., 1996). The GL2 gene encodes a homeodomain leucine-zipper protein, and the WER gene encodes an R2R3-type MYB transcription factor and induces GL2 expression (Di Cristina et al., 1996; Lee and Schiefelbein, 1999; Masucci et al., 1996; Rerie et al., 1994). The GL1 and MYB23 genes encode R2R3-type MYB genes that are closely related to WER. The GL1 gene induces trichome formation and is also expressed in the trichomes (Larkin et al., 1993; Oppenheimer et al., 1991). Constitutive expression of the MYB23 gene induces ectopic trichome formation (Kirik et al., 2001). GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) encode basic helix-loop-helix (bHLH) transcription factors that inhibit root hair differentiation in a redundant manner (Bernhardt et al., 2003). The TRANSPARENT TESTA GLABRA1 (TTG1) gene encodes a WD40repeat protein that reduces root hair differentiation and promotes trichome formation (Galway et al., 1994; Walker et al., 1999). Using a yeast two-hybrid system, GL3 and EGL3 were shown to interact with CPC-like MYBs and WER (Bernhardt et al., 2003; Tominaga et al., 2008) and with a WD40 protein (TTG1) (Esch et al., 2003; Payne et al., 2000; Zhang et al., 2003). Previously, we identified the CPL3 gene independently of Simon et al. (2007) and examined the functions of the CPL3 gene in Arabidopsis (Tominaga et al., 2008). CPL3 redundantly regulates root hair and trichome development along with other CPC-like MYBs. Among the homologs, the CPL3 gene has a special effect on flowering. The cpl3 mutant plants flower earlier than the wild type and had fewer leaves (Tominaga et al., 2008). Here, we examine the effect of CPC-like MYB genes and CPL3 on flowering time. Notably, mutations in CPC or TRY can negate the cpl3 mutant early flowering phenotype.
R. Tominaga-Wada et al. / Journal of Plant Physiology 170 (2013) 1466–1468
1467
Materials and methods
Results and discussion
Plant material and growth conditions
cpl3 displays early flowering in vivo and in vitro
The Arabidopsis thaliana (L.) Heynh. Col-0 ecotype was used as wild-type. The cpc-2 mutant used in this study was described previously (Kurata et al., 2005). The cpl3-1 mutant was isolated from a Wisconsin T-DNA population as described previously (Tominaga et al., 2008). The try-29760, etc1-1 and etc2-2 mutants were from a SALK T-DNA population. All mutants carried the Col-0 background. Double and triple mutants of cpc, try, etc1, etc2 and cpl3 were screened from F2 progeny using PCR to identify homozygous cpc2, try-29760, etc1-1, etc2-2 and cpl3-1 plants. Selected double and triple mutants were checked and documented in the F3 generation as described previously (Tominaga et al., 2008). Seeds were surfacesterilized, sown on 1.5% agar plates as described previously (Okada and Shimura, 1990) and germinated to record the seedling phenotypes. Seeded plates were kept at 4 ◦ C for 2 d and then incubated at 22 ◦ C under constant white light (50–100 mol m−2 s−1 ). For each mutant line, at least ten individual 18-d-old seedlings were assayed for rosette leaf number.
Previously, we reported an intriguing feature of the CPL3 gene that other CPC-like MYB genes may not carry. The cpl3 mutant displayed early flowering when grown in soil under continuous light (Tominaga et al., 2008). As shown in Fig. 1, early flowering was also observed by the bolting of all 18-d-old seedlings of the cpl3 mutant grown on agar plates under continuous light (Fig. 1). This result demonstrates that early flowering of cpl3 is similarly expressed when growing in soil or on agar plates. The etc1 cpl3 and etc2 cpl3 double mutants also produced early flowering phenotypes as judged by the bolting of seedlings in a manner similar to that of the cpl3 mutant (Fig. 1). On the other hand, the cpc-2 cpl3 and the try cpl3 double mutant plants did not show early flowering compared to the cpl3 mutant or the wild type (Fig. 1) with try having a less pronounced effect compared to cpc-2. Furthermore, the cpc-2 cpl3 double mutant had longer petioles than the wild type or the other mutants (Fig. 1). The cpc-2 cpl3 try and etc1 cpl3 try triple mutants produced extremely small plants and flowered late (Fig. 1).
Fig. 1. Phenotypes associated with the CPC-like MYB family loss-of function mutants. Arabidopsis 18-d-old seedlings of the wild-type Col-0, the cpc-2, try, etc1, etc2 and cpl3 single mutants, the cpc-2 cpl3, try cpl3, etc1 cpl3 and etc2 cpl3 double mutants, and the cpc-2 cpl3 try and etc1 cpl3 try triple mutants were observed. The cpl3, etc1 cpl3 and etc2 cpl3 mutant lines bolted significantly earlier than the wild type or other mutant lines. Bar (black) = 1 cm.
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R. Tominaga-Wada et al. / Journal of Plant Physiology 170 (2013) 1466–1468
12
Number of leaves
10 8 6
*
*
*
4 2 0
Fig. 2. The rosette leaf number of the wild-type Col-0, the cpc-2, try, etc1, etc2 and cpl3 single mutant, the cpc-2 cpl3, try cpl3, etc1 cpl3 and etc2 cpl3 double mutant, and the cpc-2 cpl3 try and etc1 cpl3 try triple mutant lines in 18-d-old Arabidopsis seedlings. The number of true leaves was determined by counting a minimum of ten 18-d-old seedlings from each line. Error bars indicate the standard deviation. Bars marked with asterisks indicate a significant difference between the wild-type Col-0 and the mutant lines by Student’s t-test (P < 0.050).
cpc and try mutants suppress the early flowering phenotype of the cpl3 mutant The early flowering phenotype of the cpl3 mutant was concomitant with a fewer number of true leaves at bolting time when grown in soil (Tominaga et al., 2008). To verify this phenotype of the cpl3 mutant on agar, we counted the number of true leaves in 18-dold Arabidopsis seedlings as described in M&M. Although the total number of true leaves was less than for seedlings grown in soil (Tominaga et al., 2008), the cpl3 mutant had significantly fewer leaves compared with the wild type Col-0 (Fig. 2). By contrast, the leaf number of cpc-2, try, etc1 and etc2 mutant plants was not significantly different from Col-0 (Fig. 2). Thus, we confirmed that both the early flowering and reduced leaf number phenotype of the cpl3 mutant were observed together under agar plate growth conditions. The etc1 cpl3 and the etc2 cpl3 double mutant plants flowered earlier with significantly fewer leaves than wild type, similar to the cpl3 mutant (Figs. 1 and 2). On the other hand, the cpc-2 cpl3 and the try cpl3 double mutant plants did not show any significant difference in leaf number in comparison with Col-0 (Fig. 2). These results suggest that mutations in CPC or TRY suppress the early flowering phenotype of the cpl3 mutant. The cpc-2 cpl3 try and the etc1 cpl3 try triple mutant plants all displayed a dwarf phenotype, but produced true leaf numbers comparable with Col-0 (Fig. 2). Our observation reveals a unique function of the CPC-like MYB family that is involved in flowering time. WER, a regulator of root hair pattern formation, was reported to control flowering time (Seo et al., 2011). WER encodes an R2R3type MYB protein that interacts with GL3 or EGL3 to make a transcription factor complex that promotes non-hair cell differentiation (Tominaga-Wada et al., 2011). The CPC-like MYB family proteins, such as CPC or TRY, are known to competitively interrupt WER-GL3/EGL3 complex formation (Koshino-Kimura et al., 2005; Tominaga et al., 2007). Recently, we reported that at least two amino acid substitutions convert WER to a CPC-like function (Tominaga-Wada and Nukumizu, 2012a,b). Therefore, it is a reasonable function for the CPC-like MYB family to control flowering time. It seems peculiar that CPL3 and CPC/TRY have opposite functions. Wang et al. (2008) suggested that although CPC-like MYB genes have largely overlapping roles in controlling epidermal
development, the precise functions of the gene products are not the same, and the transcriptional regulation of these CPC-like MYB genes involve different and distinct mechanisms (Wang et al., 2008). For example, expression of ETC1 under the control of the TRY promoter could not perfectly rescue a try mutant (Esch et al., 2004). CPC, TRY and ETC1 expression was mainly detected in trichomes and non-hair cells; on the other hand, ETC2 and CPL3 expression was mainly detected in young leaf blades and guard cells (Tominaga et al., 2008). These different expression patterns may cause the opposite functions of CPC/TRY and CPL3 in flowering time. In fact, the cpc and try mutant plants produce an increased number of trichomes and more trichome branching; on the other hand, the cpl3 mutant plants produce a reduced number of trichomes and trichome branches (Hulskamp et al., 1994; Schellmann et al., 2002; Tominaga et al., 2008; Wada et al., 1997). Acknowledgements This work was financially supported by the program “Improvement of Research Environment for Young Researchers” from the Ministry of Education, Culture, Sports, Science and Technology (Monbusho), a grant for Scientific Research on Priority Areas from the University of Miyazaki, and Grants-in-Aid from the Japan Society for the Promotion of Science (No. 23570057) and Monbusho (No. 23012035). References Bernhardt C, Lee MM, Gonzalez A, Zhang F, Lloyd A, Schiefelbein J. Development 2003;130:6431–9. Di Cristina M, Sessa G, Dolan L, Linstead P, Baima S, Ruberti I, et al. Plant J 1996;10:393–402. Esch JJ, Chen M, Sanders M, Hillestad M, Ndkium S, Idelkope B, et al. Development 2003;130:5885–94. Esch JJ, Chen MA, Hillestad M, Marks MD. Plant J 2004;40:860–9. Galway ME, Masucci JD, Lloyd AM, Walbot V, Davis RW, Schiefelbein JW. Dev Biol 1994;166:740–54. Gan L, Xia K, Chen JG, Wang S. BMC Plant Biol 2011;11:176. Hulskamp M, Misra S, Jürgens G. Cell 1994;76:555–66. Kirik V, Schnittger A, Radchuk V, Adler K, Hulskamp M, Baumlein H. Dev Biol 2001;235:366–77. Kirik V, Simon M, Huelskamp M, Schiefelbein J. Dev Biol 2004a;268:506–13. Kirik V, Simon M, Wester K, Schiefelbein J, Hulskamp M. Plant Mol Biol 2004b;55:389–98. Koshino-Kimura Y, Wada T, Tachibana T, Tsugeki R, Ishiguro S, Okada K. Plant Cell Physiol 2005;46:817–26. Kurata T, Ishida T, Kawabata-Awai C, Noguchi M, Hattori S, Sano R, et al. Development 2005;132:5387–98. Larkin JC, Oppenheimer DG, Pollock S, Marks MD. Plant Cell 1993;5:1739–48. Lee MM, Schiefelbein J. Cell 1999;99:473–83. Masucci JD, Rerie WG, Foreman DR, Zhang M, Galway ME, Marks MD, et al. Development 1996;122:1253–60. Okada K, Shimura Y. Science 1990;250:274–6. Oppenheimer DG, Herman PL, Sivakumaran S, Esch J, Marks MD. Cell 1991;67:483–93. Payne CT, Zhang F, Lloyd AM. Genetics 2000;156:1349–62. Rerie WG, Feldmann KA, Marks MD. Genes Dev 1994;8:1388–99. Schellmann S, Schnittger A, Kirik V, Wada T, Okada K, Beermann A, et al. EMBO J 2002;21:5036–46. Seo E, Yu J, Ryu KH, Lee MM, Lee I. Plant Physiol 2011;156:1867–77. Simon M, Lee MM, Lin Y, Gish L, Schiefelbein J. Dev Biol 2007;311:566–78. Tominaga R, Iwata M, Okada K, Wada T. Plant Cell 2007;19:2264–77. Tominaga R, Iwata M, Sano R, Inoue K, Okada K, Wada T. Development 2008;135:1335–45. Tominaga-Wada R, Nukumizu Y. Int J Mol Sci 2012a;13:3478–91. Tominaga-Wada R, Ishida T, Wada T. Int Rev Cell Mol Biol 2011;286:67–106. Tominaga-Wada R, Nukumizu Y, Wada T. Plant Sci 2012b;183:37–42. Wada T, Tachibana T, Shimura Y, Okada K. Science 1997;277:1113–6. Walker AR, Davison PA, Bolognesi-Winfield AC, James CM, Srinivasan N, Blundell TL, et al. Plant Cell 1999;11:1337–50. Wang S, Kwak SH, Zeng Q, Ellis BE, Chen XY, Schiefelbein J, et al. Development 2007;134:3873–82. Wang S, Hubbard L, Chang Y, Guo J, Schiefelbein J, Chen JG. BMC Plant Biol 2008;8: 81. Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A. Development 2003;130:4859–69.
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short communication
Flowering is delayed by mutations in homologous genes CAPRICE and TRYPTICHON in the early flowering Arabidopsis cpl3 mutant Rumi Tominaga-Wada ∗ , Yuka Nukumizu, Takuji Wada Interdisciplinary Research Organization, University of Miyazaki, 1-1, Gakuen Kibanadai-Nishi, Miyazaki 889-2192, Japan
a r t i c l e
i n f o
Article history: Received 28 March 2013 Received in revised form 23 May 2013 Accepted 23 May 2013 Available online 21 June 2013 Keywords: Arabidopsis Flowering Root hair Trichome Transcription factor
a b s t r a c t CAPRICE (CPC) and CAPRICE-like (CPL) myeloblastosis (MYB) family members [including TRYPTICHON (TRY) and ENHANCER OF TRYPTICHON AND CAPRICE (ETC)] of Arabidopsis thaliana encode R3-type MYB transcription factors that promote root hair differentiation and inhibit trichome formation in a redundant manner. Previously, we reported that the CPL3 gene affects flowering. The cpl3 mutant plants flower earlier and with fewer leaves than the wild type. In this study, we show that mutations in CPC or TRY delay flowering of cpl3 plants. A mutation in ETC1 did not further delay flowering but reduced plant size. Our study provides insight into the regulation of flowering time by the CPC-like MYB gene family. © 2013 Elsevier GmbH. All rights reserved.
Introduction The CAPRICE (CPC) gene, which encodes an R3-type myeloblastosis (MYB) transcription factor, is a key regulator of epidermal cell development including trichome initiation and root-hair differentiation in Arabidopsis thaliana (Wada et al., 1997). Several additional CPC-like MYB genes have been identified in the Arabidopsis genome, including TRYPTICHON (TRY), ENHANCER OF TRY AND CPC1 and 2 (ETC1 and ETC2), ENHANCER OF TRY AND CPC3/CPCLIKE MYB3 (ETC3/CPL3), and TRICHOMELESS1 and 2/CPC-LIKE MYB4 (TCL1 and TCL2/CPL4) (Esch et al., 2004; Gan et al., 2011; Kirik et al., 2004a,b; Schellmann et al., 2002; Simon et al., 2007; Tominaga et al., 2008; Tominaga-Wada and Nukumizu, 2012a,b; Wang et al., 2007). These seven CPC-like MYB transcription factors are thought to act as positive regulators of root hair differentiation and as negative regulators of trichome differentiation. The regulation of cell differentiation is a crucial step in the development of multicellular organisms. Thus, epidermal cell differentiation in Arabidopsis has been used extensively as a model system for analyzing cell fate determination. Several additional regulatory factors of the CPClike MYB gene family are known to be involved in epidermal cell differentiation. Mutations in the GLABRA2 (GL2) and WEREWOLF (WER) genes cause an increase in root hairs (Lee and Schiefelbein,
Abbreviations: CPC, CAPRICE; CPL, CAPRICE-like; ETC, ENHANCER OF TRYPTICHON AND CAPRICE; MYB, myeloblastosis; TRY, TRYPTICHON. ∗ Corresponding author. Tel.: +81 985 58 7864; fax: +81 985 58 7864. E-mail address: [email protected] (R. Tominaga-Wada). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.05.013
1999; Masucci et al., 1996). The GL2 gene encodes a homeodomain leucine-zipper protein, and the WER gene encodes an R2R3-type MYB transcription factor and induces GL2 expression (Di Cristina et al., 1996; Lee and Schiefelbein, 1999; Masucci et al., 1996; Rerie et al., 1994). The GL1 and MYB23 genes encode R2R3-type MYB genes that are closely related to WER. The GL1 gene induces trichome formation and is also expressed in the trichomes (Larkin et al., 1993; Oppenheimer et al., 1991). Constitutive expression of the MYB23 gene induces ectopic trichome formation (Kirik et al., 2001). GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) encode basic helix-loop-helix (bHLH) transcription factors that inhibit root hair differentiation in a redundant manner (Bernhardt et al., 2003). The TRANSPARENT TESTA GLABRA1 (TTG1) gene encodes a WD40repeat protein that reduces root hair differentiation and promotes trichome formation (Galway et al., 1994; Walker et al., 1999). Using a yeast two-hybrid system, GL3 and EGL3 were shown to interact with CPC-like MYBs and WER (Bernhardt et al., 2003; Tominaga et al., 2008) and with a WD40 protein (TTG1) (Esch et al., 2003; Payne et al., 2000; Zhang et al., 2003). Previously, we identified the CPL3 gene independently of Simon et al. (2007) and examined the functions of the CPL3 gene in Arabidopsis (Tominaga et al., 2008). CPL3 redundantly regulates root hair and trichome development along with other CPC-like MYBs. Among the homologs, the CPL3 gene has a special effect on flowering. The cpl3 mutant plants flower earlier than the wild type and had fewer leaves (Tominaga et al., 2008). Here, we examine the effect of CPC-like MYB genes and CPL3 on flowering time. Notably, mutations in CPC or TRY can negate the cpl3 mutant early flowering phenotype.
R. Tominaga-Wada et al. / Journal of Plant Physiology 170 (2013) 1466–1468
1467
Materials and methods
Results and discussion
Plant material and growth conditions
cpl3 displays early flowering in vivo and in vitro
The Arabidopsis thaliana (L.) Heynh. Col-0 ecotype was used as wild-type. The cpc-2 mutant used in this study was described previously (Kurata et al., 2005). The cpl3-1 mutant was isolated from a Wisconsin T-DNA population as described previously (Tominaga et al., 2008). The try-29760, etc1-1 and etc2-2 mutants were from a SALK T-DNA population. All mutants carried the Col-0 background. Double and triple mutants of cpc, try, etc1, etc2 and cpl3 were screened from F2 progeny using PCR to identify homozygous cpc2, try-29760, etc1-1, etc2-2 and cpl3-1 plants. Selected double and triple mutants were checked and documented in the F3 generation as described previously (Tominaga et al., 2008). Seeds were surfacesterilized, sown on 1.5% agar plates as described previously (Okada and Shimura, 1990) and germinated to record the seedling phenotypes. Seeded plates were kept at 4 ◦ C for 2 d and then incubated at 22 ◦ C under constant white light (50–100 mol m−2 s−1 ). For each mutant line, at least ten individual 18-d-old seedlings were assayed for rosette leaf number.
Previously, we reported an intriguing feature of the CPL3 gene that other CPC-like MYB genes may not carry. The cpl3 mutant displayed early flowering when grown in soil under continuous light (Tominaga et al., 2008). As shown in Fig. 1, early flowering was also observed by the bolting of all 18-d-old seedlings of the cpl3 mutant grown on agar plates under continuous light (Fig. 1). This result demonstrates that early flowering of cpl3 is similarly expressed when growing in soil or on agar plates. The etc1 cpl3 and etc2 cpl3 double mutants also produced early flowering phenotypes as judged by the bolting of seedlings in a manner similar to that of the cpl3 mutant (Fig. 1). On the other hand, the cpc-2 cpl3 and the try cpl3 double mutant plants did not show early flowering compared to the cpl3 mutant or the wild type (Fig. 1) with try having a less pronounced effect compared to cpc-2. Furthermore, the cpc-2 cpl3 double mutant had longer petioles than the wild type or the other mutants (Fig. 1). The cpc-2 cpl3 try and etc1 cpl3 try triple mutants produced extremely small plants and flowered late (Fig. 1).
Fig. 1. Phenotypes associated with the CPC-like MYB family loss-of function mutants. Arabidopsis 18-d-old seedlings of the wild-type Col-0, the cpc-2, try, etc1, etc2 and cpl3 single mutants, the cpc-2 cpl3, try cpl3, etc1 cpl3 and etc2 cpl3 double mutants, and the cpc-2 cpl3 try and etc1 cpl3 try triple mutants were observed. The cpl3, etc1 cpl3 and etc2 cpl3 mutant lines bolted significantly earlier than the wild type or other mutant lines. Bar (black) = 1 cm.
1468
R. Tominaga-Wada et al. / Journal of Plant Physiology 170 (2013) 1466–1468
12
Number of leaves
10 8 6
*
*
*
4 2 0
Fig. 2. The rosette leaf number of the wild-type Col-0, the cpc-2, try, etc1, etc2 and cpl3 single mutant, the cpc-2 cpl3, try cpl3, etc1 cpl3 and etc2 cpl3 double mutant, and the cpc-2 cpl3 try and etc1 cpl3 try triple mutant lines in 18-d-old Arabidopsis seedlings. The number of true leaves was determined by counting a minimum of ten 18-d-old seedlings from each line. Error bars indicate the standard deviation. Bars marked with asterisks indicate a significant difference between the wild-type Col-0 and the mutant lines by Student’s t-test (P < 0.050).
cpc and try mutants suppress the early flowering phenotype of the cpl3 mutant The early flowering phenotype of the cpl3 mutant was concomitant with a fewer number of true leaves at bolting time when grown in soil (Tominaga et al., 2008). To verify this phenotype of the cpl3 mutant on agar, we counted the number of true leaves in 18-dold Arabidopsis seedlings as described in M&M. Although the total number of true leaves was less than for seedlings grown in soil (Tominaga et al., 2008), the cpl3 mutant had significantly fewer leaves compared with the wild type Col-0 (Fig. 2). By contrast, the leaf number of cpc-2, try, etc1 and etc2 mutant plants was not significantly different from Col-0 (Fig. 2). Thus, we confirmed that both the early flowering and reduced leaf number phenotype of the cpl3 mutant were observed together under agar plate growth conditions. The etc1 cpl3 and the etc2 cpl3 double mutant plants flowered earlier with significantly fewer leaves than wild type, similar to the cpl3 mutant (Figs. 1 and 2). On the other hand, the cpc-2 cpl3 and the try cpl3 double mutant plants did not show any significant difference in leaf number in comparison with Col-0 (Fig. 2). These results suggest that mutations in CPC or TRY suppress the early flowering phenotype of the cpl3 mutant. The cpc-2 cpl3 try and the etc1 cpl3 try triple mutant plants all displayed a dwarf phenotype, but produced true leaf numbers comparable with Col-0 (Fig. 2). Our observation reveals a unique function of the CPC-like MYB family that is involved in flowering time. WER, a regulator of root hair pattern formation, was reported to control flowering time (Seo et al., 2011). WER encodes an R2R3type MYB protein that interacts with GL3 or EGL3 to make a transcription factor complex that promotes non-hair cell differentiation (Tominaga-Wada et al., 2011). The CPC-like MYB family proteins, such as CPC or TRY, are known to competitively interrupt WER-GL3/EGL3 complex formation (Koshino-Kimura et al., 2005; Tominaga et al., 2007). Recently, we reported that at least two amino acid substitutions convert WER to a CPC-like function (Tominaga-Wada and Nukumizu, 2012a,b). Therefore, it is a reasonable function for the CPC-like MYB family to control flowering time. It seems peculiar that CPL3 and CPC/TRY have opposite functions. Wang et al. (2008) suggested that although CPC-like MYB genes have largely overlapping roles in controlling epidermal
development, the precise functions of the gene products are not the same, and the transcriptional regulation of these CPC-like MYB genes involve different and distinct mechanisms (Wang et al., 2008). For example, expression of ETC1 under the control of the TRY promoter could not perfectly rescue a try mutant (Esch et al., 2004). CPC, TRY and ETC1 expression was mainly detected in trichomes and non-hair cells; on the other hand, ETC2 and CPL3 expression was mainly detected in young leaf blades and guard cells (Tominaga et al., 2008). These different expression patterns may cause the opposite functions of CPC/TRY and CPL3 in flowering time. In fact, the cpc and try mutant plants produce an increased number of trichomes and more trichome branching; on the other hand, the cpl3 mutant plants produce a reduced number of trichomes and trichome branches (Hulskamp et al., 1994; Schellmann et al., 2002; Tominaga et al., 2008; Wada et al., 1997). Acknowledgements This work was financially supported by the program “Improvement of Research Environment for Young Researchers” from the Ministry of Education, Culture, Sports, Science and Technology (Monbusho), a grant for Scientific Research on Priority Areas from the University of Miyazaki, and Grants-in-Aid from the Japan Society for the Promotion of Science (No. 23570057) and Monbusho (No. 23012035). References Bernhardt C, Lee MM, Gonzalez A, Zhang F, Lloyd A, Schiefelbein J. Development 2003;130:6431–9. Di Cristina M, Sessa G, Dolan L, Linstead P, Baima S, Ruberti I, et al. Plant J 1996;10:393–402. Esch JJ, Chen M, Sanders M, Hillestad M, Ndkium S, Idelkope B, et al. Development 2003;130:5885–94. Esch JJ, Chen MA, Hillestad M, Marks MD. Plant J 2004;40:860–9. Galway ME, Masucci JD, Lloyd AM, Walbot V, Davis RW, Schiefelbein JW. Dev Biol 1994;166:740–54. Gan L, Xia K, Chen JG, Wang S. BMC Plant Biol 2011;11:176. Hulskamp M, Misra S, Jürgens G. Cell 1994;76:555–66. Kirik V, Schnittger A, Radchuk V, Adler K, Hulskamp M, Baumlein H. Dev Biol 2001;235:366–77. Kirik V, Simon M, Huelskamp M, Schiefelbein J. Dev Biol 2004a;268:506–13. Kirik V, Simon M, Wester K, Schiefelbein J, Hulskamp M. Plant Mol Biol 2004b;55:389–98. Koshino-Kimura Y, Wada T, Tachibana T, Tsugeki R, Ishiguro S, Okada K. Plant Cell Physiol 2005;46:817–26. Kurata T, Ishida T, Kawabata-Awai C, Noguchi M, Hattori S, Sano R, et al. Development 2005;132:5387–98. Larkin JC, Oppenheimer DG, Pollock S, Marks MD. Plant Cell 1993;5:1739–48. Lee MM, Schiefelbein J. Cell 1999;99:473–83. Masucci JD, Rerie WG, Foreman DR, Zhang M, Galway ME, Marks MD, et al. Development 1996;122:1253–60. Okada K, Shimura Y. Science 1990;250:274–6. Oppenheimer DG, Herman PL, Sivakumaran S, Esch J, Marks MD. Cell 1991;67:483–93. Payne CT, Zhang F, Lloyd AM. Genetics 2000;156:1349–62. Rerie WG, Feldmann KA, Marks MD. Genes Dev 1994;8:1388–99. Schellmann S, Schnittger A, Kirik V, Wada T, Okada K, Beermann A, et al. EMBO J 2002;21:5036–46. Seo E, Yu J, Ryu KH, Lee MM, Lee I. Plant Physiol 2011;156:1867–77. Simon M, Lee MM, Lin Y, Gish L, Schiefelbein J. Dev Biol 2007;311:566–78. Tominaga R, Iwata M, Okada K, Wada T. Plant Cell 2007;19:2264–77. Tominaga R, Iwata M, Sano R, Inoue K, Okada K, Wada T. Development 2008;135:1335–45. Tominaga-Wada R, Nukumizu Y. Int J Mol Sci 2012a;13:3478–91. Tominaga-Wada R, Ishida T, Wada T. Int Rev Cell Mol Biol 2011;286:67–106. Tominaga-Wada R, Nukumizu Y, Wada T. Plant Sci 2012b;183:37–42. Wada T, Tachibana T, Shimura Y, Okada K. Science 1997;277:1113–6. Walker AR, Davison PA, Bolognesi-Winfield AC, James CM, Srinivasan N, Blundell TL, et al. Plant Cell 1999;11:1337–50. Wang S, Kwak SH, Zeng Q, Ellis BE, Chen XY, Schiefelbein J, et al. Development 2007;134:3873–82. Wang S, Hubbard L, Chang Y, Guo J, Schiefelbein J, Chen JG. BMC Plant Biol 2008;8: 81. Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A. Development 2003;130:4859–69.