SUPERMAN regulates floral whorl boundaries through control of auxin biosynthesis
2018; Springer Nature; Volume: 37; Issue: 11 Linguagem: Inglês
10.15252/embj.201797499
ISSN1460-2075
AutoresYifeng Xu, Nathanaël Prunet, Eng‐Seng Gan, Yanbin Wang, Darragh Stewart, Frank Wellmer, Jiangbo Huang, Nobutoshi Yamaguchi, Yoshitaka Tatsumi, Mikiko Kojima, Takatoshi Kiba, Hitoshi Sakakibara, Thomas Jack, Elliot M. Meyerowitz, Toshiro Ito,
Tópico(s)Plant Gene Expression Analysis
ResumoArticle15 May 2018free access Source DataTransparent process SUPERMAN regulates floral whorl boundaries through control of auxin biosynthesis Yifeng Xu Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Nathanaël Prunet Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Department of Biological Sciences, Dartmouth College, Hanover, NH, USA Search for more papers by this author Eng-Seng Gan Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Search for more papers by this author Yanbin Wang Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Search for more papers by this author Darragh Stewart Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland Search for more papers by this author Frank Wellmer Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland Search for more papers by this author Jiangbo Huang Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Nobutoshi Yamaguchi Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan Search for more papers by this author Yoshitaka Tatsumi Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Mikiko Kojima Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Takatoshi Kiba Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Hitoshi Sakakibara Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Thomas P Jack Department of Biological Sciences, Dartmouth College, Hanover, NH, USA Search for more papers by this author Elliot M Meyerowitz Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Search for more papers by this author Toshiro Ito Corresponding Author [email protected] orcid.org/0000-0002-8206-2787 Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Yifeng Xu Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Nathanaël Prunet Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Department of Biological Sciences, Dartmouth College, Hanover, NH, USA Search for more papers by this author Eng-Seng Gan Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Search for more papers by this author Yanbin Wang Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Search for more papers by this author Darragh Stewart Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland Search for more papers by this author Frank Wellmer Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland Search for more papers by this author Jiangbo Huang Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Nobutoshi Yamaguchi Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan Search for more papers by this author Yoshitaka Tatsumi Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Mikiko Kojima Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Takatoshi Kiba Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Hitoshi Sakakibara Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Thomas P Jack Department of Biological Sciences, Dartmouth College, Hanover, NH, USA Search for more papers by this author Elliot M Meyerowitz Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA Search for more papers by this author Toshiro Ito Corresponding Author [email protected] orcid.org/0000-0002-8206-2787 Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Author Information Yifeng Xu1,2, Nathanaël Prunet3,4,5, Eng-Seng Gan1, Yanbin Wang1, Darragh Stewart6, Frank Wellmer3,6, Jiangbo Huang1,7, Nobutoshi Yamaguchi2,8, Yoshitaka Tatsumi2, Mikiko Kojima8,9, Takatoshi Kiba8,9, Hitoshi Sakakibara8,9, Thomas P Jack5, Elliot M Meyerowitz3,4 and Toshiro Ito *,1,2,3,7 1Temasek Life Sciences Laboratory (TLL), National University of Singapore, Singapore, Singapore 2Plant Stem Cell Regulation and Floral Patterning Laboratory, Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan 3Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA 4Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA 5Department of Biological Sciences, Dartmouth College, Hanover, NH, USA 6Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland 7Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore 8Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama, Japan 9RIKEN Center for Sustainable Resource Science, Yokohama, Japan *Corresponding author. Tel: +81 0743 72 5500; Fax: +81 0743 72 5500; E-mail: [email protected] EMBO J (2018)37:e97499https://doi.org/10.15252/embj.201797499 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Proper floral patterning, including the number and position of floral organs in most plant species, is tightly controlled by the precise regulation of the persistence and size of floral meristems (FMs). In Arabidopsis, two known feedback pathways, one composed of WUSCHEL (WUS) and CLAVATA3 (CLV3) and the other composed of AGAMOUS (AG) and WUS, spatially and temporally control floral stem cells, respectively. However, mounting evidence suggests that other factors, including phytohormones, are also involved in floral meristem regulation. Here, we show that the boundary gene SUPERMAN (SUP) bridges floral organogenesis and floral meristem determinacy in another pathway that involves auxin signaling. SUP interacts with components of polycomb repressive complex 2 (PRC2) and fine-tunes local auxin signaling by negatively regulating the expression of the auxin biosynthesis genes YUCCA1/4 (YUC1/4). In sup mutants, derepressed local YUC1/4 activity elevates auxin levels at the boundary between whorls 3 and 4, which leads to an increase in the number and the prolonged maintenance of floral stem cells, and consequently an increase in the number of reproductive organs. Our work presents a new floral meristem regulatory mechanism, in which SUP, a boundary gene, coordinates floral organogenesis and floral meristem size through fine-tuning auxin biosynthesis. Synopsis SUPERMAN (SUP) controls the specification and maintenance of the boundary between stamens in whorl 3 and carpels in whorl 4. This boundary gene functions as an active repressor of auxin biosynthesis that regulates flower development, the absence of which leads to flowers with supernumerary stamens. SUP recruits the Polycomb Group (PcG) component CURLY LEAF (CLF) to directly repress auxin biosynthesis genes YUCCA1 and 4 (YUC1/4) locally. The derepression of YUC1/4 in sup mutants causes ectopic auxin activity at the whorl 3/4 boundary region. Perturbed auxin activity in sup flowers results in delayed termination of stem cell activity and increase in FM size, which then leads to increased stamen number. Introduction In many angiosperms, floral patterning is tightly controlled by the precise coordination of stem cell proliferation in the floral meristem (FM), commitment of stem cell descendants to specific floral organs, and establishment of meristem-to-organ and organ-to-organ boundaries. By such mechanisms, the number and position of floral organs for a given species are well defined. Wild-type (WT) Arabidopsis flowers consist of four types of organs arranged in a series of concentric whorls: four sepals in the outermost whorl 1, followed by four petals in whorl 2, six stamens in whorl 3, and two fused carpels in the innermost whorl 4. While three classes of homeotic genes, classes A, B, and C, function alone or in combination to determine the cell identities of floral organs (Bowman et al, 1991; Coen & Meyerowitz, 1991), the precise developmental regulations of the FM that determine the family- and/or species-specific numbers of floral organs and whorls remain unknown. In Arabidopsis, a negative feedback loop between the WUSCHEL (WUS)-expressing organizing center and CLAVATA3 (CLV3)-expressing stem cells maintains the appropriate size of both FMs and shoot apical meristems (SAMs; Brand et al, 2000). FM activity is associated with the number of floral organs. Mutation in WUS causes plants to lose the ability to maintain stem cells and prematurely stops organ formation (Laux et al, 1996). In contrast, stem cells accumulate in clv3 mutants due to unrestricted WUS expression, leading to the formation of more organs (Clark et al, 1995; Fletcher et al, 1999). Unlike the indeterminate SAM, the FM is determinate and ceases to maintain stem cells after the initiation of carpels. Another negative feedback between WUS and the class C gene AGAMOUS (AG) plays a central role in this termination process (Lenhard et al, 2001; Lohmann et al, 2001; Sun et al, 2009, 2014). AG is induced at floral stage 3 by WUS and the FM regulator LEAFY (LFY) in whorls 3 and 4 of floral primordia where stamens and carpels will develop in later stages (Lohmann et al, 2001). AG in turn represses WUS, both directly by affecting the recruitment of polycomb group (PcG) proteins to the WUS locus and indirectly through the C2H2 zinc finger protein KNUCKLES (KNU), to terminate stem cell maintenance at floral stage 6, approximately 2 days after AG induction (Sun et al, 2009; Liu et al, 2011). In ag and knu loss-of-function mutants, WUS expression remains active beyond stage 6, which is sufficient to induce FM indeterminacy, leading to the production of extra whorls of reproductive organs (Lenhard et al, 2001; Sun et al, 2009). AG also activates the YABBY family transcription factor CRABS CLAW to regulate carpel organogenesis and FM determinacy through the establishment of auxin maxima in the fourth whorl (Yamaguchi et al, 2017). The number and position of floral organs are also controlled by boundary genes, which function through various mechanisms, including the crosstalk with the phytohormone auxin (Zadnikova & Simon, 2014). The NAC family transcription factors CUP-SHAPED COTYLEDON1-3 (CUC1-3), which participate in the formation of boundaries between organs and between organs and meristems, are negatively regulated by auxin-dependent signaling pathways (Takada et al, 2001; Daimon et al, 2003). In the Arabidopsis SAM, new floral primordia are initiated in the peripheral zone, at the region where auxin concentration is highest. As the primordium forms, auxin is depleted from the boundary separating the emerging primordium from the meristem and flows toward the incipient position of the next primordium (Heisler et al, 2005). Thus, CUC genes are restricted in the boundary regions of low auxin activity. Auxin also controls the size of the root meristem non-cell autonomously; this auxin signaling is antagonistic to cytokinin signaling, and cytokinin negatively controls the root meristem size (Dello Ioio et al, 2007). In contrast to root meristems, cytokinin signaling and WUS activity in the SAM could reinforce each other in a positive feedback (Leibfried et al, 2005; Gordon et al, 2009; Zhao et al, 2010). Although auxin and cytokinin show opposite functions in the regulation of shoot and root meristems, the function of auxin in FMs is not well understood (Werner et al, 2003; Schaller et al, 2015). The SUPERMAN (SUP) gene encodes a transcription factor with a C2H2-type zinc finger motif and is proposed to function as a boundary gene to separate the stamen-producing whorl 3 from the carpel-producing whorl 4 (Sakai et al, 1995). Loss of function of SUP leads to an increased number of stamens, suggesting that SUP is involved in both floral patterning and FM determinacy (Bowman et al, 1992; Gaiser et al, 1995). AG is a positive regulator of SUP transcription, and SUP mRNA level is greatly reduced in ag mutants (Bowman et al, 1992). Notably, the transient and weak expression of SUP in ag mutants is sufficient for some level of function, since ag sup double mutants show strong synergistic effects on FM size, causing enlarged and fasciated FMs (Bowman et al, 1992). Although sup mutants were identified and well characterized decades ago, how SUP functions to bridge floral organogenesis and FM determinacy is still unclear. A recent study showed that SUP cell autonomously prevents the ectopic expression of class B/stamen identity genes in whorl 4, and non-cell autonomously promotes stem cell termination in developing flowers (Prunet et al, 2017). The ectopic expression of SUP in different plant species leads to dwarf plants with organs of reduced size, which could be associated with both auxin and cytokinin signaling defects (Hiratsu et al, 2002; Nibau et al, 2011). However, it is difficult to distinguish the causal factors of the sup phenotypes from the consequence of altered morphology. Here, we elucidate how SUP functions to control floral organogenesis and FM size non-cell autonomously. SUP interacts with PcG proteins to exert its function as an active repressor and negatively regulates auxin biosynthesis in the stamen-to-carpel boundary region. In the sup mutant, the derepression of YUCCA (YUC) genes YUC1 and YUC4 leads to increased auxin accumulation and the formation of extra primordia of reproductive organs. Consistently, treatment with an anti-auxin (p-chlorophenoxyisobutyric acid, PCIB) can rescue the stamen number and carpel defects of sup mutants. Increased local auxin biosynthesis in the SUP-expressing region leads to sup-like floral phenotypes. Our work presents a new mechanism on how the boundary gene SUP coordinates floral organogenesis and FM size through fine-tuning of auxin biosynthesis. Results SUP regulates floral stem cells non-cell autonomously We first tested whether the formation of supernumerary stamens in sup mutants is associated with WUS function in FMs. A loss of WUS activity leads to the premature termination of FMs so that both wus-1 single-mutant and wus-1 sup-5 double-mutant flowers typically form only a single stamen and no carpels (Laux et al, 1996; Fig 1A and B). Thus, wus-1 is fully epistatic to sup, suggesting that the sup phenotype of supernumerary stamens is dependent on WUS function. In contrast, flowers of ag-1 sup-5 double mutants show enhanced meristem indeterminacy (Bowman et al, 1991; Uemura et al, 2017). Taken together, these results suggest that SUP may regulate WUS in FMs and that this regulation might be at least in part independent from the known AG-WUS feedback pathways. Figure 1. SUP spatially controls the FM size in a non-cell-autonomous manner A, B. wus-1 (A) and sup-5 wus-1 (B) mutant flowers with one stamen and without carpels. Scale bars, 1 mm. C. The comparison of the number of cells with the stem cell marker pCLV3::GFP-ER signals in WT and sup-5. The numbers of cells with the signals were counted based on the z-stack images. From stage 4 (s4) onwards, the sup-5 floral buds showed increased numbers of CLV3-expressing stem cells compared with those of WT. Error bars indicate s.d. of 12–15 samples; two-tailed Student's t-test, *P < 0.05. D, E. The pCLV3::GFP-ER (green) in WT (D) and sup-5 (E) floral buds at different floral stages. Scale bars, 20 μm. Download figure Download PowerPoint To address whether SUP regulates floral stem cell activities, we monitored the expression of the stem cell marker CLV3 in sup-5 mutant flowers (Fig 1C–E). Using a pCLV3::GFP-ER reporter (Reddy & Meyerowitz, 2005), we determined that there is no obvious difference of fluorescence intensity between WT and sup; however, the CLV3 expression region appeared slightly broader in sup flowers from stage 4 onward (Fig 1D and E). To further test this, we counted the number of cells expressing pCLV3::GFP-ER in sup and WT flowers at different stages and found that while the number of cells expressing pCLV3::GFP-ER was comparable between WT and sup at stage 3, from stage 4 onward, it was significantly higher in sup floral buds (Fig 1C–E). This result suggests that there are an increased number of floral stem cells in sup mutants. To confirm this observation, we employed a floral induction system (denoted: ap1 cal p35S::AP1-GR), which is based on the activation of a fusion protein between the APETALA1 (AP1) transcription factor and the steroid-binding domain of the rat glucocorticoid receptor (GR) in the inflorescence-like meristems of ap1 cauliflower (cal) double mutants by dexamethasone (DEX) treatment and allows the collection of a large number of synchronized floral buds for analysis (Wellmer et al, 2006). Using real-time quantitative reverse transcription PCR (qRT–PCR), we detected increased transcription levels for both CLV3 and WUS in stage 6 flowers of ap1 cal p35S::AP1-GR sup-5 plants relative to those of ap1 cal p35S::AP1-GR plants (Fig EV1A). We also detected pCLV3::GFP-ER expression at later floral stages in sup-5 than in the wild type (Fig EV1B), confirming previous reports that floral stem cell termination is delayed in sup (Prunet et al, 2017). Altogether, our data show that SUP influences floral stem cells both spatially and temporally. Click here to expand this figure. Figure EV1. WUS and CLV3 are increased at stage 6 floral buds in sup-5 A. Expression of WUS and CLV3 (in floral buds of approximately stage 6) 5 days after treatment of ap1 cal p35S::AP1-GR sup-5 inflorescences with 1 μM DEX. The relative values to equally treated ap1 cal p35S::AP1-GR plants are shown. Error bars indicate standard errors from qRT–PCR experiments of four biological repeats. P = 0.028 and 0.031 for WUS and CLV3 based on a Student's t-test, respectively. B, C. The pCLV3::GFP-ER (green) can be detected in a sup-5 floral bud (C) but not in WT (B) at a floral stage later than 6. Scale bars, 20 μm. Download figure Download PowerPoint We also analyzed the expression of the meristem marker SHOOT MERISTEMLESS (STM) by using a translational reporter pSTM::STM-VENUS and a transcriptional reporter pSTM::CFP-N7 (Fig EV2; Heisler et al, 2005; Landrein et al, 2015). Up to stage 4, STM expression appears identical in sup-5 and WT flowers: STM is initially expressed throughout stage 1–2 flower buds, before fading from developing sepals at stage 3 (Fig EV2A and B). STM expression domain appears larger in sup than in the wild type at late stage 5 (Fig EV2A and B), which is associated with an enlarged FM in sup. By stage 6, STM expression ceases in whorls 2 and 3 in both wild-type and sup-5 flowers (Fig EV2A and B). STM then becomes restricted to emerging carpel primordia in the fourth whorl of wild-type flowers, whereas in sup-5 flowers, its expression domain in the center becomes enlarged. Later on, STM only remains expressed at the carpel margins/placenta region in the WT (Fig EV2C and D). Conversely, in sup-5, STM is expressed in a larger domain, which encompasses the FM that keeps proliferating; STM is also transiently expressed in the emerging extra stamen primordia that form in the center of sup-5 flowers (Fig EV2C and D). The expression domain of SUP forms a ring at the boundary between whorls 3 and 4 (Appendix Fig S1A and B) that is mostly non-overlapping with that of CLV3 or WUS throughout flower development, indicating that SUP affects floral stem cells non-cell autonomously (Prunet et al, 2017). Click here to expand this figure. Figure EV2. STM expression is expanded in sup floral buds A, B. The expression of pSTM::CFP-N7 (light blue) in WT and sup-5 inflorescence. In both WT and sup-5, the STM-VENUS fusion protein was highly expressed in the center of floral buds younger than stage 5 as well as in the boundary regions of sepals. C, D. A stem cell marker pSTM::STM-VENUS (red) in WT (C) and sup-5 (D) floral buds at the stages 5 and 6. From late stage 5 (s5), STM was reduced in the regions with developing stamens. The STM expression domain in FM centers is relatively larger in sup-5 (1,680 ± 167 μm2, n = 15) than that in WT (1,150 ± 160 μm2, n = 16). P < 0.05 based on a Student's t-test. Dashed lines mark the FM regions and whorl 3/4 boundary regions with STM-VENUS. E, F. The expression of pSTM::CFP-N7 (light blue) only remains expressed at the carpel margins/placenta region in the WT at stage 7 (s7) and greatly diminished at stage 9 (s9) (E). Conversely, in sup-5, STM is expressed in a larger domain, which encompasses the FM that keeps proliferating as well as in emerging extra stamen primordia up to stage later than 10 (s10+) (F). Data information: Scale bars, 20 μm. Download figure Download PowerPoint Auxin signaling is disrupted in sup mutants To investigate how SUP regulates organ boundaries, FM size, and differentiation, we compared the expression of a CUC2 reporter in wild-type and sup flowers. CUC genes encode closely related members of the NAC family of transcription factors, which participate in shoot meristem and boundary formation (Takada et al, 2001; Daimon et al, 2003). In situ hybridization analysis showed CUC2 mRNA accumulation in the center of FMs in sup-1 (Breuil-Broyer et al, 2004). As ectopic expression of CUC2 is associated with an increased number of petals (Huang et al, 2012), we monitored CUC2 expression using pCUC2::CUC2-3xVENUS-N7 (Heisler et al, 2005) in the sup mutant (Fig EV3A–D). CUC2 is widely expressed in stage 3 floral buds in both WT and sup (Fig EV3A and B). From stage 4 onward, clear differences in CUC2 expression were observed between WT and sup flowers. In the WT, high CUC2 expression was observed in cells at the boundary regions between the sepal primordia, and in the inner part of the whorl 3/4 boundary regions, while the central region of the FM showed no CUC2 expression (Fig EV3A and C). In sup, CUC2 expression was also observed in the FM region (Fig EV3B and D), in a domain where SUP is not normally expressed, suggesting that CUC2 is not a direct target of SUP. Since it has been shown that CUC2 is induced by low levels of auxin but repressed by high levels of auxin (Heisler et al, 2005), we hypothesized that auxin signaling or accumulation could be disturbed in sup mutant flowers. To test this hypothesis, we monitored the activity of the auxin response reporters pDR5rev::2xGFP-N7 and pDR5rev::GFP-ER in sup and WT flowers (Xu et al, 2006a; Liao et al, 2015). In WT stage 4 flower buds, DR5 expression occurs only at the sites of petal primordia initiation and at the tips of sepals (Fig 2A and Appendix Fig S2A). In contrast, in sup mutant stage 4 flower buds, DR5 is also expressed at the whorl 3/4 boundary, indicating an increase in auxin response in that region (Fig 2B and Appendix Fig S2B). The DII-VENUS auxin sensor, expressed under the control of the ubiquitous RPS5A promoter, is degraded in presence of auxin (Liao et al, 2015). In WT flower buds at stage 4, DII-VENUS is detected at the boundary between whorls 3 and 4 but not in the center of the flower, indicating that auxin is depleted at the boundary, but not in the FM region (Fig 2C). In contrast, in sup flower buds at stage 4, DII-VENUS is observed in the center of the FM but not at the boundary between whorls 3 and 4, showing that auxin is depleted in the FM region rather than at the boundary (Fig 2D). These data imply that the loss of SUP function leads to auxin accumulation, rather than depletion, at the boundary between whorl 3 and 4, and to a reduction in auxin in the center of the FM. The increase in auxin at the whorl 3/4 boundary in sup could be due either to an increase in auxin biosynthesis or to a perturbation of auxin transport. To test whether the sup phenotype is due to the cell-autonomous effect of an increase in auxin levels or due to perturbed auxin transport, we treated sup mutant inflorescences with p-chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action (Oono et al, 2003). PCIB treatment strongly rescued both the stamen number and carpel defects in sup-5 (Fig 2E–G). Next, we tested the stage-specific rescue effect of PCIB by measuring time to anthesis. Generally, stage 4–5 floral buds were rescued better than stage 6 floral buds in terms of carpel morphology and stamen numbers (Fig 2G). We also tested the effect of PCIB treatment on CUC2 and DR5 expressions in sup (Fig EV4). CUC2 expression was not reversed to a WT-like pattern following treatment with PCIB. Instead, CUC2 was ectopically expressed through most of the flower bud (Fig EV4A and B), which may be due to CUC2 activation by low auxin levels. In contrast, DR5 expression at the whorl 3/4 boundary is almost completely absent in sup flowers 5 h after PCIB treatment (Fig EV4C and D), indicating that PCIB restores a wild-type pattern of auxin response in sup flowers, which is consistent with the fact that PCIB treatments rescue the sup phenotype (Fig 2E–G). Click here to expand this figure. Figure EV3. pCUC2::3xVENUS-N7 expression is expanded in the epidermal cells of sup mutants pCUC2::3xVENUS-N7 in WT floral buds at stages 3-6. CUC2 was highly expressed in the floral buds of stage 3, and its expression started to be constrained to the boundary regions between the sepals after stage 3. At stage 5 and stage 6 floral buds, CUC2 was only detected in the small boundary regions at the bottom of the sepals. pCUC2::3xVENUS-N7 in sup-5 floral buds at stages 3–6. At stage 3, the CUC2 expression pattern was similar to that in WT. From stage 4, the CUC2 expression pattern in sup-5 started to show differences from that of WT. CUC2 expression was detected in FM region of at stage 4. At stages 5 and 6, the CUC2 expression was still relatively high
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