Access the full text.
Sign up today, get DeepDyve free for 14 days.
Downloaded from https://academic.oup.com/jxb/article/70/21/e6499/4962458 by DeepDyve user on 19 July 2022 Journal of Experimental Botany, Vol. 70, No. 21 pp. e6499–e6501, 2019 doi:10.1093/jxb/ery098 Advance Access publication 6 April 2018 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) COMMENTARY My favourite flowering image: an Arabidopsis inflorescence expressing fluorescent reporters for the APETALA3 and SUPERMAN genes Nathanaël Prunet Howard Hughes Medical Institute, California Institute of Technology, Division of Biology and Biological Engineering, Pasadena, CA 90028, USA firstname.lastname@example.org Editor: Frank Wellmer, Trinity College Dublin, Ireland When asked to provide a picture for the cover of the was easy: it was an important breakthrough in my research Flowering Newsletter, I picked this image of an Arabidopsis on the role of SUP in the separation of stamens in whorl thaliana inflorescence expressing fluorescent reporters for two 3 and carpels in whorl 4; and among the images of flowers key regulators of flower development: APETALA3 (AP3), I have taken with a confocal microscope, it is also one my which promotes petal and stamen identity, and SUPERMAN favourites aesthetically. The image won awards at the 2015 (SUP), which encodes a transcriptional repressor that defines Nikon Small World and FASEB BioArt competitions and is the boundary between stamens and pistil (Fig. 1). The choice published in Prunet et al. (2017). The molecular mechanisms underlying the determination of or fl al organ identity have been extensively studied over the last three decades, from the description of mutants with floral organ homeosis (Bowman et al., 1989, 1991; Irish and Sussex, 1990) to the characterization of the corresponding genes, most of which encode transcription factors of the MADS- box family (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994), and the identifi- cation of their targets (Kaufmann et al., 2009, 2010; Wuest et al., 2012; Ó’Maoiléidigh et al., 2013). Floral organ iden- tity is determined by the combinatorial action of four classes of MADS-box transcription factors [class A, AP1; class B, AP3 and PISTILLATA (PI); class C, AGAMOUS (AG); and class E, SEPALLATAs (SEPs)], which form different protein quartets in each whorl (reviewed in Prunet and Jack, 2014). For instance, quartets composed of class B, C, and E tran- scription factors orchestrate stamen development in whorl 3, while quartets composed of class C and E transcription fac- tors alone determine carpel identity in whorl 4. These quar- tets recruit different transcription co-regulators and histone modification factors to regulate the transcription of their targets (Smaczniak et al., 2012). While the genetic networks downstream of these quartets have been partially deciphered (reviewed in Stewart et al., 2016), questions remain about Fig. 1. AP3 and SUP expression in young Arabidopsis flower buds. Arabidopsis inflorescence expressing gAP3-GFP (green) and gSUP- how boundaries between floral whorls are established. 3xVenusN7 (red) fluorescent reporters. Cell walls were stained with Mutations in SUP disrupt the boundary between whorls propidium iodide (grey). Siliques and older flower buds were removed, 3 and 4, with the formation of numerous extra stamens, usu- and the inflorescence was prepared and imaged on a Zeiss LSM780 with ally at the expense of carpels, which are reduced or missing in a 20× water-dipping lens as described in Prunet (2017) and Prunet et al. most alleles (Schultz et al., 1991; Bowman et al., 1992). This (2016). Background noise was digitally removed for aesthetic reasons. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/70/21/e6499/4962458 by DeepDyve user on 19 July 2022 e6500 | Prunet phenotype is associated with the expansion of the expression WUSCHEL in sup flowers compared to the wild type, sug- of AP3 and PI towards the center of the flower (Bowman gesting that the increase in floral organ number resulted et al., 1992; Goto and Meyerowitz, 1994), but does not result from delayed termination of the floral stem cells rather than from a simple homeotic conversion of carpels into stamens: from an over-proliferation of cells in whorl 3 (Prunet et al., the overall number of floral organs is increased in sup com- 2017). Time-lapse experiments also demonstrated that a ring pared to the wild type, indicating an excess of cell prolifer- of cells in whorl 4, adjacent to the boundary with whorl 3, ation in sup o fl wers. Two models have been proposed for the starts expressing AP3 ectopically at the transition between developmental origin of the extra stamens in sup flowers. It whorl 4 and 5 in sup mutant flowers, thus confirming that was first suggested that these extra stamens form in whorl 4, extra stamens form in the fourth whorl in sup (Prunet et al., due to the ectopic expression of class B genes, and that the 2017). Our data also seemed to point at a mostly non cell- increase in floral organ number comes from delayed termin- autonomous effect of SUP, which, based on hard-to-inter- ation of the or fl al stem cells (Schultz et al., 1991; Bowman pret in situ hybridizations, was believed to be expressed in et al., 1992). However, when the SUP gene was identified, in whorl 3, and not in whorl 4 (Sakai et al., 1995). We generated situ hybridization experiments suggested that SUP was co- a translational fluorescent reporter for SUP to have a closer expressed with AP3 and PI in the inner part of whorl 3, but look at the SUP expression pattern. It turned out to be a not expressed in whorl 4, casting doubts on the fact that SUP slow and painful process—it took 4 years and some pretty might function to prevent ectopic expression of class B genes acrobatic cloning by Tom—but we finally obtained a fluores- in the fourth whorl (Sakai et al., 1995). Instead, SUP was cent SUP reporter just as I moved from Dartmouth to Elliot proposed to control the balance of cell proliferation between Meyerowitz’s lab at Caltech. This image of an Arabidopsis whorls 3 and 4. According to this new model, extra stamens inflorescence expressing two translational reporters for AP3 arise from whorl 3 cells that over-proliferate, while reduced (fused with a single GFP) and SUP (fused with three Venus proliferation in whorl 4 results in a loss of carpel tissue (Sakai proteins and a nuclear localization signal) was one of the et al., 1995, 2000). For more than 25 years after the isolation first images I took at Caltech; it was also the first time I man- of the sup mutant it had not been possible to discriminate aged to separate signals from GFP and YFP, which have par- between these two models. This was mostly due to limitations tially overlapping emission spectra. But most importantly, in the techniques that were used at the time, such as in situ this image clearly showed that contrary to what was previ- hybridizations or GUS reporter lines, which lack sufficient ously thought, SUP is expressed on both sides of the bound- cellular resolution and cannot not be used on live tissues. The ary between whorls 3 and 4, not just in whorl 3. SUP and image I have chosen helped solve this question. AP3 are expressed along two opposite gradients that only I first became interested in SUP during my PhD with partially overlap in whorl 3, and whorl 4 cells that express Christophe Trehin and Ioan Negrutiu at École Normale SUP in wild-type flowers at stage 5 ectopically express AP3 Supérieure de Lyon. I was studying three different mutants instead in sup mutant flowers, indicating that SUP prevents with a minor delay in the termination of floral stem cells AP3 expression in whorl 4 in a cell-autonomous manner that was manifesting through a slight increase in the num- (Prunet et al., 2017). ber of carpels and the occasional formation of extra organs Independently of the scientific significance of this image, inside the gynoecium (Prunet et al., 2008). This phenotype I love it for aesthetic reasons. One of the reasons why I stud- was correlated with a decrease in the expression of AG— ied biology in the first place is that of all sciences, it leaves the which acts as the main switch to terminate floral stem cells most room for artistic expression: observational drawing is (Lenhard et al., 2001; Lohmann et al., 2001)—in the center an integral part of the learning process. This science-meets- of the flower meristem (Prunet et al., 2008). However, the art aspect—for which the term SciArt has been coined—has combination of these three mutations resulted in a spec- long been an important driver for my work. I chose to study tacular phenotype, with the formation of an indeterminate development for my PhD because of the rich imaging pos- spiral of stamens at the center of the flower. This pheno- sibilities this field offers. I later based my postdoc research on type is also observed when combining the sup-1 mutation a confocal imaging approach for the power of the technique with the moderate loss-of-function allele ag-4 (Prunet et al., to solve developmental questions but also for the beauty of 2008). While SUP initially appeared at the margin of the the images that can be generated. I admit to spending more genetic networks I was studying, I started to increasingly time on the microscope than strictly required to answer my suspect that it was involved in the timely termination of flo- initial scientific questions, trying to get aesthetically perfect ral stem cells. images (and I consider myself lucky to work with Elliot, When I started my postdoc in Tom Jack’s lab at Dartmouth who has been very supportive of that). But then, as Samuel College, I decided to investigate the function of SUP using a H. Scudder noticed once he decided to draw his fish (‘At last live confocal imaging approach—a technique that allows us a happy thought struck me—I would draw the fish; and now to monitor the expression of multiple genes in live tissue with with surprise I began to discover new features in the creature. good cellular resolution. Our data supported the model in Just then the Professor returned. ‘That is right’, said he; ‘a which extra stamens in sup mutant flowers arise from whorl pencil is one of the best of eyes’; Scudder, 1974), carefully 4 rather than whorl 3. We observed a prolonged expression crafted images often bring to our attention interesting bio- of the stem cell marker CLAVATA3 and stem cell activator logical details that we would not have suspected otherwise. Downloaded from https://academic.oup.com/jxb/article/70/21/e6499/4962458 by DeepDyve user on 19 July 2022 My favourite flowering image | e6501 Mandel MA, Gustafson-Brown C, Savidge B, Yanofsky MF. 1992. Acknowledgements Molecular characterization of the Arabidopsis floral homeotic gene I would like to thank Tom Jack and Elliot Meyerowitz for their support. APETALA1. Nature 360, 273–277. Funding in Tom’s lab was supported by the National Science Foundation Ó’Maoiléidigh DS, Wuest SE, Rae L, et al. 2013. Control of Grant IOS-0926347; and funding in the Elliot’s laboratory was provided by reproductive floral organ identity specification in Arabidopsis by the C the Howard Hughes Medical Institute, the National Institutes of Health function regulator AGAMOUS. The Plant Cell 25, 2482–2503. Grant R01 GM104244, and the Gordon and Betty Moore Foundation Prunet N. 2017. Live confocal imaging of developing Arabidopsis flowers. through Grant GBMF3406. Journal of Visualized Experiments 122, e55156. Prunet N, Jack TP. 2014. Flower development in Arabidopsis: there is Keywords: Arabidopsis, flower development, flower meristem, boundary more to it than learning your ABCs. Methods in Molecular Biology 1110, formation, floral organ identity, APETALA3, SUPERMAN. 3–33. Prunet N, Jack TP, Meyerowitz EM. 2016. Live confocal imaging of Arabidopsis flower buds. Developmental Biology 419, 114–120. Prunet N, Morel P, Thierry AM, Eshed Y, Bowman JL, Negrutiu I, Trehin C. 2008. REBELOTE, SQUINT, and ULTRAPETALA1 function redundantly in the temporal regulation of floral meristem termination in Arabidopsis thaliana. The Plant Cell 20, 901–919. References Prunet N, Yang W, Das P, Meyerowitz EM, Jack TP. 2017. Bowman JL, Sakai H, Jack T, Weigel D, Mayer U, Meyerowitz EM. SUPERMAN prevents class B gene expression and promotes stem cell 1992. SUPERMAN, a regulator of floral homeotic genes in Arabidopsis. termination in the fourth whorl of Arabidopsis thaliana flowers. Proceedings Development 114, 599–615. of the National Academy of Sciences, USA 114, 7166–7171. Bowman JL, Smyth DR, Meyerowitz EM. 1989. Genes directing flower Sakai H, Krizek BA, Jacobsen SE, Meyerowitz EM. 2000. Regulation development in Arabidopsis. The Plant Cell 1, 37–52. of SUP expression identifies multiple regulators involved in arabidopsis Bowman JL, Smyth DR, Meyerowitz EM. 1991. Genetic interactions floral meristem development. The Plant Cell 12, 1607–1618. among floral homeotic genes of Arabidopsis. Development 112, 1–20. Sakai H, Medrano LJ, Meyerowitz EM. 1995. Role of SUPERMAN in Goto K, Meyerowitz EM. 1994. Function and regulation of the maintaining Arabidopsis floral whorl boundaries. Nature 378, 199–203. Arabidopsis floral homeotic gene PISTILLATA. Genes & Development 8, Schultz EA, Pickett FB, Haughn GW. 1991. The FLO10 gene product 1548–1560. regulates the expression domain of homeotic genes AP3 and PI in Irish VF, Sussex IM. 1990. Function of the apetala-1 gene during Arabidopsis flowers. The Plant Cell 3, 1221–1237. Arabidopsis floral development. The Plant Cell 2, 741–753. Scudder SH. 1974. In the laboratory with Agassiz. Every Saturday 16, Jack T, Brockman LL, Meyerowitz EM. 1992. The homeotic gene 369–370. APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed Smaczniak C, Immink RG, Muino JM, et al. 2012. Characterization in petals and stamens. Cell 68, 683–697. of MADS-domain transcription factor complexes in Arabidopsis flower Kaufmann K, Muiño JM, Jauregui R, Airoldi CA, Smaczniak development. Proceedings of the National Academy of Sciences, USA C, Krajewski P, Angenent GC. 2009. Target genes of the MADS 109, 1560–1565. transcription factor SEPALLATA3: integration of developmental Stewart D, Graciet E, Wellmer F. 2016. Molecular and regulatory and hormonal pathways in the Arabidopsis flower. PLoS Biology 7 , mechanisms controlling floral organ development. The FEBS Journal 283, e1000090. 1823–1830. Kaufmann K, Wellmer F, Muiño JM, et al. 2010. Orchestration of floral Wuest SE, O’Maoileidigh DS, Rae L, Kwasniewska K, Raganelli initiation by APETALA1. Science 328, 85–89. A, Hanczaryk K, Lohan AJ, Loftus B, Graciet E, Wellmer F. 2012. Lenhard M, Bohnert A, Jürgens G, Laux T. 2001. Termination of stem Molecular basis for the specification of floral organs by APETALA3 and cell maintenance in Arabidopsis floral meristems by interactions between PISTILLATA. Proceedings of the National Academy of Sciences, USA 109, WUSCHEL and AGAMOUS. Cell 105, 805–814. 13452–13457. Lohmann JU, Hong RL, Hobe M, Busch MA, Parcy F, Simon R, Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann KA, Weigel D. 2001. A molecular link between stem cell regulation and floral Meyerowitz EM. 1990. The protein encoded by the Arabidopsis homeotic patterning in Arabidopsis. Cell 105, 793–803. gene agamous resembles transcription factors. Nature 346, 35–39.
Journal of Experimental Botany – Oxford University Press
Published: Nov 18, 2019
Access the full text.
Sign up today, get DeepDyve free for 14 days.