|JAGGED controls growth anisotropyand coordination between cell sizeand cell cycle during plant organogenesis.|
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|PMID: 22902754 Owner: NLM Status: MEDLINE|
|BACKGROUND: In all multicellular organisms, the links between patterning genes, cell growth, cell cycle, cell size homeostasis, and organ growth are poorly understood, partly due to the difficulty of dynamic, 3D analysis of cell behavior in growing organs. A crucial step in plant organogenesis is the emergence of organ primordia from the apical meristems. Here, we combined quantitative, 3D analysis of cell geometry and DNA synthesis to study the role of the transcription factor JAGGED (JAG), which functions at the interface between patterning and primordium growth in Arabidopsis flowers.
RESULTS: The floral meristem showed isotropic growth and tight coordination between cell volume and DNA synthesis. Sepal primordia had accelerated cell division, cell enlargement, anisotropic growth, and decoupling of DNA synthesis from cell volume, with a concomitant increase in cell size heterogeneity. All these changes in growth parameters required JAG and were genetically separable from primordium emergence. Ectopic JAG activity in the meristem promoted entry into S phase at inappropriately small cell volumes, suggesting that JAG can override a cell size checkpoint that operates in the meristem. Consistent with a role in the transition from meristem to primordium identity, JAG directly repressed the meristem regulatory genes BREVIPEDICELLUS and BELL 1 in developing flowers.
CONCLUSIONS: We define the cellular basis for the transition from meristem to organ identity and identify JAG as a key regulator of this transition. JAG promotes anisotropic growth and is required for changes in cell size homeostasis associated with accelerated growth and the onset of differentiation in organ primordia.
|Katharina Schiessl; Swathi Kausika; Paul Southam; Max Bush; Robert Sablowski|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2012-08-16|
|Title: Current biology : CB Volume: 22 ISSN: 1879-0445 ISO Abbreviation: Curr. Biol. Publication Date: 2012 Oct|
|Created Date: 2012-10-12 Completed Date: 2013-04-30 Revised Date: 2013-07-12|
Medline Journal Info:
|Nlm Unique ID: 9107782 Medline TA: Curr Biol Country: England|
|Languages: eng Pagination: 1739-46 Citation Subset: IM|
|Copyright © 2012 Elsevier Ltd. All rights reserved.|
|Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.|
|APA/MLA Format Download EndNote Download BibTex|
Cell Cycle / genetics*
Cell Cycle Proteins / genetics, metabolism*
Flowers / cytology*, growth & development*, metabolism
Gene Expression Regulation, Developmental
Gene Expression Regulation, Plant
Homeodomain Proteins / genetics, metabolism
Meristem / cytology, genetics, metabolism
Transcription Factors / genetics, metabolism
|BB/F005571/1//Biotechnology and Biological Sciences Research Council; BB/J004588/1//Biotechnology and Biological Sciences Research Council|
|0/Arabidopsis Proteins; 0/BEL1 protein, Arabidopsis; 0/Cell Cycle Proteins; 0/Homeodomain Proteins; 0/JAGGED protein, Arabidopsis; 0/Transcription Factors|
|Curr Biol. 2012 Oct 9;22(19):R838-40
Journal ID (nlm-ta): Curr Biol
Journal ID (iso-abbrev): Curr. Biol
Publisher: Cell Press
© 2012 ELL & Excerpta Medica.
Received Day: 4 Month: 5 Year: 2012
Revision Received Day: 15 Month: 6 Year: 2012
Accepted Day: 6 Month: 7 Year: 2012
pmc-release publication date: Day: 09 Month: 10 Year: 2012
Print publication date: Day: 09 Month: 10 Year: 2012
Volume: 22 Issue: 19
First Page: 1739 Last Page: 1746
PubMed Id: 22902754
Publisher Id: CURBIO9741
|JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis|
|Robert Sablowski1∗||Email: firstname.lastname@example.org|
1Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
2School of Computing Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
|∗Corresponding author email@example.com
3Present address: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark
A fundamental question in biology is how the activity of regulatory genes acting within cells is translated into the shape and size of macroscopic organs. In plants, growth is based only on increased cell number and cell size, in contrast to animals, in which cell migration and cell death also play important roles. In spite of this simplifying feature, understanding the link between regulatory genes and the growth and shape of plant organs is still a considerable challenge. In theory, a complete understanding of growth would require information about rates, anisotropy, and directions of growth, and how these vary spatially and over time . So far, these parameters have not been associated experimentally to specific regulatory genes, partly because of the difficulty of obtaining quantitative, dynamic, and three-dimensional (3D) information about organ growth. This situation has begun to change with new methods to analyze and model the dynamics of plant tissue growth in 3D [2, 3].
Shoot organs are initiated at the periphery of apical meristems, which continuously produce new cells to replenish those recruited into organ primordia . These primordia develop into leaves during vegetative growth and into floral buds during the reproductive phase of development. Each bud contains its own floral meristem, which produces floral organ primordia in concentric whorls, with sepal primordia emerging first, followed by petal, stamen, and carpel primordia, after which the floral meristem is terminated. The spatial pattern of primordium initiation around the meristem (phyllotaxis) results from regulated transport of the phytohormone auxin . Local auxin maxima induce primordium initiation, associated with repression of regulatory genes that maintain meristem activity, such as the homeodomain proteins SHOOT MERISTEMLESS (STM)  and BREVIPEDICELLUS (BP) [7, 8].
After primordia have been initiated, the growth of shoot organs is conventionally divided in two phases: primary morphogenesis, based mostly on cell proliferation, and secondary morphogenesis, based mostly on endoreduplication and cell expansion [9, 10]. Although multiple genes have been identified that control the final size and shape of plant organs, it remains largely unknown how these genes coordinate cell proliferation and expansion to determine organ shape and size [9, 11]. One of the regulators of the proliferative phase of organ growth in Arabidopsis is JAGGED (JAG), which encodes a zinc finger transcription factor expressed during the emergence of all shoot organs, but not in the meristem [12, 13]. In leaves, jag mutations cause serrated margins, and in floral organs cause reduced growth preferentially in the distal region. Based on reduced expression of histone H4 in jag-1 petals, JAG function has been linked to cell proliferation during organ growth . JAG is a direct target of the floral homeotic genes AGAMOUS and SEPALATA3, which are key patterning genes during floral bud development [14, 15]. Therefore, JAG appears to function at the interface between patterning genes and organ growth.
To understand the changes in cell behavior that mediate the effects of JAG on organ growth, we used recently developed methods for dynamic analysis of cell geometry and established a protocol for combined 3D analysis of cell geometry and patterns of DNA synthesis. Our analysis shows that cells in the meristem and early floral organ primordia have different growth regimes, and that JAG is a key regulator of the transition between these growth regimes. One of the unanticipated functions of JAG was to change the coordination between cell growth and cell cycle during organ development.
Previous work on jag mutants focused on macroscopic phenotypes in leaves and floral organs at relatively late stages of development; defects in early organogenesis have been observed but have not been characterized in detail [12, 13]. We focused our analysis on the growth of sepal primordia, which are the first to emerge from the floral meristem and are therefore readily accessible for imaging.
To select buds at equivalent developmental stages in the wild-type (WT) and jag-1, we relied on the phyllotactic position of the buds around the inflorescence meristem. The spiral pattern of bud emergence was unaltered in jag-1 compared to the WT (see Figure S1 available online); based on the rate of production of mature flowers in jag-1 and in the WT, the frequency of bud initiation was also unchanged (data not shown). The size of the floral meristem was comparable in WT and jag-1 buds at the same position, whereas growth of the sepal primordia was clearly inhibited in the mutant buds (Figure S1). Therefore the jag-1 mutation specifically affects growth of the sepal primordia in early floral buds.
To understand the cellular basis for the growth defects in jag-1 buds, we compared the growth dynamics of the meristem and sepal primordia in the WT and in jag-1, using the 3D live imaging approach MARS (multiangle image acquisition, 3D reconstruction and cell segmentation) . Sepal primordia started emerging in buds at position 9–10 in the phyllotactic spiral, so these were selected and imaged at 24 hr intervals over 2 days (Figures 1A, 1C, and 1E). We digitally marked groups of epidermal cells (sectors) in the initial bud and manually tracked the descendants of each cell (clone) over time (Figures 1B, 1D, and 1F). Within each virtual sector, we analyzed changes in cell number, cell volumes, total sector volume, and growth isotropy (i.e., the ratio between growth along the minimal and maximal growth axes) (Figure 2). We only considered isotropy of growth parallel to the epidermal surface, because the thickness of the epidermal cells remained uniform (data not shown).
As expected from the JAG expression pattern [12, 13] (Figure S1), growth of the floral meristem was comparable in the WT and in jag-1, so we only present the analysis of the WT meristem. Growth of sectors within the floral meristem was isotropic and sector growth was due primarily to increased cell numbers, whereas cell volumes and heterogeneity in cell and clone volumes remained approximately constant (Figures 1A, 1B, and 2). In contrast, sectors in the adaxial epidermis of the WT sepal primordium showed anisotropic growth, with maximum growth along the apical-basal axis; virtual sectors grew faster than in the meristem due to a combination of increased cell proliferation and increased cell volumes; the sepal primordium also showed increasing heterogeneity in cell volumes (Figures 1C, 1D, and 2). Strikingly, growth in the adaxial epidermis of the jag-1 sepal primordium was similar to that of the meristem by any parameter used (cell proliferation rate, changes in cell volume, heterogeneity in cell and clone volumes, growth isotropy) (Figures 1E, 1F, and 2A–2D). Similar results were obtained in three independent live imaging experiments (Figure S2).
We conclude that the cellular basis for tissue growth is different between the meristem and the early primordium. This change in growth regime depends on JAG and is necessary for proper primordium growth but is not required for primordium emergence.
Based on our observations that JAG promoted both cell proliferation and an increase in average cell volume, we next studied how cell cycle and cell volume are coordinated during meristem and primordium growth. Because it has been shown that inhibition of DNA synthesis stops meristem growth, whereas inhibition of M phase entry does not prevent meristem and primordium growth , we focused on the S phase as the growth-limiting step of the cell cycle. To monitor simultaneously cell volume and progression through S phase, we combined methods for high-resolution imaging of cell walls in deep tissues  with labeling of newly synthesized DNA using the nucleotide analog 5-ethynyl-2′-deoxyuridine (EdU)  (Figure 3A) and then applied the 3D segmentation step of MARS  to the cell wall images of flower buds (Figure 3B). By analyzing cells in the epidermal layer of the floral meristem and of the adaxial epidermis of sepal primordia, we were able to relate the data to the live imaging analysis shown in Figures 1 and 2.
In the floral meristem of both the WT and the jag-1 mutant, cell volumes ranged from approximately 100 to 200 μm3 and EdU-labeled cells had a larger median volume than unlabeled cells (Figures 3E and 3F). This suggests that cell growth in the meristem occurs mostly before S phase and that DNA synthesis is initiated in cells that reach a threshold volume, as seen, for example, in budding yeast . In accordance with the live imaging results, cell volumes in the WT primordium varied over a wider range of volumes (approximately 100–300 μm3) (Figure 3G). Different to the results in the meristem, EdU labeling did not correlate with cell volume in the primordium (Figure 3G). In contrast, the jag-1 primordium once again was similar to the meristem, with a clear correlation between cell volume and DNA synthesis (Figure 3H). Consistent results were obtained for each genotype using buds at positions 10–12 in the phyllotactic spiral (data not shown); Figure 3 compares a WT bud at stage 10 with a jag-1 bud at position 12 to compensate for the slower growth of jag-1 primordia and ensure that the different cell behavior was not related simply to primordium size. In summary, cell volume and entry into S phase are coordinated in meristem cells and the transition to organ primordium identity included a JAG-dependent decoupling of S phase entry from cell volume.
The results above suggested that in primordia, JAG might override a mechanism that coordinates S phase entry and cell size or that JAG activates a mechanism that increases cell volume independently of the cell cycle, for example, by increasing vacuolar volume. Electron microscopy, however, indicated that vacuoles do not contribute significantly to cell volume in the adaxial epidermis of emerging sepal primordia (Figure 3). To test whether JAG is sufficient to decouple S phase entry from cell volume, and whether this is necessarily associated with an increase in cell volumes, we activated JAG ectopically in the floral meristem.
Sustained ectopic expression of JAG severely disrupts flower development ; therefore, we used transient activation of JAG in the floral meristem. For this, we generated plants (35S::JAG:GR) that expressed ubiquitously a fusion between JAG and the rat glucocorticoid receptor steroid-binding domain, which renders transcription factors dexamethasone-dependent . Transient treatment of 35S:JAG-GR jag-2 inflorescences with 10 μM dexamethasone rescued organ growth at both early and late developmental stages, confirming that the JAG-GR fusion provided inducible JAG function (Figures 4A–4D). We then monitored cell volumes and S phase entry in the floral meristem of 35S::JAG-GR buds at position 10–13 after incubation in media with or without 10 uM dexamethasone. Meristem cells of mock-treated buds showed again the coordination between cell volume and S phase entry described above (Figure 4E). After JAG-GR was activated, coordination between cell volume and S phase entry was lost in meristem cells, as seen before for WT sepal primordia (Figure 3). In contrast to the WT primordia, however, the median cell size in the meristem decreased in response to JAG activation, because cells entered S phase at abnormally small volumes (Figure 4F). Thus the JAG-induced decoupling of S phase entry from cell volume does not require a concomitant increase in median cell volumes. These results suggest that cells in the floral meristem have the potential to enter S phase at small volumes but are normally prevented from doing so by a mechanism that can be overridden by JAG.
The results above revealed that cells in the jag mutant primordium behave like meristem cells in several ways: rate of growth, growth isotropy, cell enlargement, cell size homogeneity, and coordination between cell size and entry into S phase. This raised the question whether JAG antagonizes genes required for meristem development and whether these genes would be ectopically expressed in jag mutant primordia.
One of the key meristem regulators is STM, which encodes a KNOX-type homeodomain protein required for the establishment and maintenance of all shoot meristems . BP is a close STM homolog that directs development of the stem and flower pedicels  but can also assume the role of STM in meristem maintenance . Both the STM and BP proteins have been reported to function as heterodimers with TALE homeodomain proteins, of which BELL 1 (BEL1) is the best characterized, with roles in inflorescence meristem and ovule development [21, 22]. We therefore tested the regulatory effect of JAG on STM, BP, and BEL1. In the case of STM, repression by JAG-GR suggested that it might be a target of JAG, however, RNA in situ hybridization did not show changes in STM expression in the jag-2 mutant (data not shown). In contrast, we saw that BP and BEL1 were targeted both by ectopic and endogenous JAG, as detailed below.
Both BP and BEL1 were significantly downregulated 4 hr after activation of JAG-GR throughout inflorescence apices with dexamethasone (Figure 5A). Downregulation by JAG-GR still occurred in the presence of cycloheximide, showing that repression of BP and BEL1 did not involve intermediate steps that required protein synthesis . Chromatin immunoprecipitation (ChIP) was then used to test whether JAG-GR directly interacted with BP and BEL1 in a dexamethasone-dependent way. In both cases, reproducible binding was seen to specific regions upstream of the starting codon (Figures 5B and 5C).
To verify that BP and BEL1 are regulated by endogenous JAG, we compared expression in WT and jag-2 young floral buds with RNA in situ hybridization (Figure 6). For BP, the known expression pattern in flower pedicels and stems  was comparable in WT and jag-2 plants (Figures 6A–6D). In addition, jag-2 buds showed ectopic expression at the base of organ primordia, both in emerging sepals (Figures 6A and 6B) and in the emerging carpel primordia of stage 6 buds  (Figures 6C and 6D). For BEL1, strong expression in ovules, which serve as a control for the known expression pattern, was similar in WT and in jag-2 (Figures 6G and 6H). The low levels of BEL1 expression reported in floral meristems  were not detected by in situ hybridization in our conditions, either in WT or jag-2 buds (Figures 6E and 6F). In sections through the same inflorescence apices, however, clear expression of BEL1 was seen in the sepal primordia of jag-2 buds, but not in the WT controls (Figures 6E and 6F). qRT-PCR independently confirmed that the levels of BP and BEL1 messenger RNA (mRNA) were increased in jag-1 inflorescences apices relative to the WT (Figure 5A).
Ectopic BP expression causes leaf and floral defects reminiscent of those seen in the jag mutants , prompting us to test the functional consequences of ectopic BP expression in jag. The jag-1 bp double mutant, however, showed additive phenotypes, with the stem and pedicel defects seen in bp combined with the same organ development defects seen in jag-1, both at early and late stages (Figure S4). Therefore the organ growth defects seen in jag mutants are not caused primarily by ectopic BP expression.
We conclude that direct repression of BP and BEL1 by JAG provides molecular evidence that JAG has a role in promoting the transition from meristem to primordium identity, in accordance with the changes in cellular parameters described above. However, the function of JAG in organ growth is likely to involve more than repression of meristem regulators.
The aim of this work was to understand how growth regulators direct the growth of organ primordia at the cellular level. Our analysis revealed that JAG is required for the transition from meristem to primordium growth patterns, including a shift from isotropic to anisotropic growth, increased cell proliferation and cell enlargement. Unexpectedly, we also observed a JAG-dependent loss of coordination between cell volume and DNA synthesis, associated with increased heterogeneity in cell sizes.
In accordance with a role for JAG in the transition from meristem to primordium identity, we show that JAG represses at least two meristem regulatory genes, BP and BEL1. It has been shown that the growth defects of jag-2 sepals and petals are strongly enhanced by mutations in ASYMMETRIC LEAVES 1 and 2 (AS1 and AS2), which are known repressors of BP . Combined with the genetic interaction between jag-2 and as1/2, the direct interaction of JAG-GR with BP and BEL1 and the ectopic expression of these genes in jag-2 primordia reveal JAG as a novel member of the gene regulatory network that controls the transition from meristem to primordium identity.
In contrast to the regulatory network, the cellular basis for the transition from meristem to primordium identity has been less well characterized. Primordium emergence involves changes to the cell wall structure that facilitate cell expansion [28, 29]. Measurements of the geometry of surface cells have previously shown that the initiation of leaf primordia in Anagalis and floral buds in Arabidopsis is accompanied by increased growth rates and increased growth anisotropy [30, 31]. Analysis of the surface geometry of Arabidopsis sepal primordia at stages later than those reported here also revealed strong growth anisotropy and cell size heterogeneity . However, it has been unclear to what extent the mechanisms involved in the emergence of primordia from the meristem are the same required for subsequent primordium growth and morphogenesis. We showed that in the jag mutant, sepal primordia still emerged and were physically distinguishable from the meristem, but their cells continued to grow in the same way as the meristem cells. Therefore, the changes in cell behavior that underpin primordium growth are genetically separable from those required for primordium emergence.
One of the consequences of JAG function in sepal primordia was increased cell size heterogeneity. In unicellular organisms, uniform cell sizes are maintained by cell size checkpoints [19, 33]. In multicellular organisms, the control of cell size homeostasis is much less well understood. It has been proposed that a cell size checkpoint operates to maintain a minimal cell size prior to mitosis to ensure daughter cell viability but that external, developmental inputs often override cell autonomous size checkpoints [19, 34, 35]. Our results suggest that a checkpoint for cell size operates during G1-S progression in the meristem and is deactivated in the primordium in a JAG-dependent way. Although we reveal JAG as an upstream regulator of cell size homeostasis during plant development, it remains to be seen whether JAG directly controls cell-autonomous mechanisms that couple cell growth and cell cycle or whether intermediate regulatory genes and signals are involved.
Regardless of the mechanism, there are several reasons why cell size and cell cycle may need to be coupled in the meristem, but not in sepal primordia. Cell sizes influence auxin transport through tissues , so irregularity of cell sizes could affect auxin-dependent meristem patterning. Conversely, increased variability in cell size may be required during differentiation in the primordium; the finding that cell size controls transcriptional responses  raises the possibility that changes in cell size may not only be a consequence but also a cause of differentiation. Additionally, a mechanism that triggered cell division at a threshold volume might impose a growth penalty that would be acceptable in the meristem but incompatible with the fast growth rates seen in organ primordia. Such a growth penalty could result from variation in growth rate during the cell cycle. For example, slower growth in M phase has been widely observed  and could be caused in plants by rearrangements of the cytoskeleton, which has a key role not only in mitosis but also in cell wall expansion . Mathematical modeling will be required to explore these hypotheses further.
Arabidopsis thaliana Landsberg-erecta (L-er) was used as the WT; jag-1 , originally in Columbia (Col) background, was backcrossed three times into L-er; jag-2  was originally L-er. The 35S:JAG:GR construct was generated in the binary vector pCGN1547 as described , transformed into L-er by floral dip  and selected for complementation after crossing to jag-2. For expression analysis and ChIP, plants were grown on soil in 16 hr light, 20°C/8 hr dark, 18°C cycles. For quantitative 3D imaging, plants were grown on soil at 20°C in short days (10 hr light/14 hr dark) for 4 weeks and subsequently transferred to 16°C, continuous light during flowering.
For time-lapse imaging, inflorescence apices were prepared and imaged as described . For combined mPS-PI (modified pseudo-Schiff-propidium iodide)  and EdU  imaging, inflorescence tips were dissected and buds larger than 0.5 mm were removed. The dissected apices were grown for 45 hr in sterile GM medium  at 16°C under continuous light; for JAG-GR activation, the media also contained 10 μM dexamethasone (from 10 mM stock in ethanol) or 0.1% ethanol for mock treatment. Apices were then transferred to the same medium supplemented with 10 μM EdU (Invitrogen) and grown for another 3 hr, followed by 15 min each in 15%, 30%, 50%, 70%, 85%, 95%, and 100% ethanol. After 16 hr in ethanol and further dissection leaving only the inflorescence meristem and buds up to position 15–16, the samples were rehydrated through the same ethanol series and incubated at 37°C overnight in alpha-amylase (Sigma) 0.3 mg/mL in phosphate buffer 20 mM pH7.0, 2 mM NaCl, 0.25 mM CaCl2. All subsequent steps were at room temperature with gentle shaking: first, the apices were rinsed in water and incubated for 1 hr in solution containing 10 μM Alexa 488-azide (Invitrogen) and 100 mM Tris pH 8.5; this was followed by 30 min in solution contining 10 μM Alexa 488-azide, 100 mM Tris, 1 mM CuSO4, 100 mM ascorbic acid, pH 8.5; the apices were subsequently washed three times in water, treated 30 min in 1% periodic acid, washed twice in water, and incubated 2 hr in Schiff-PI reagent . The samples were finally cleared with chloral hydrate solution and mounted in Hoyer’s medium , before imaging with a Zeiss 510 Meta confocal microscope with excitation at 488 nm and emission filters set to 572–625 nm for propidium iodide and 505–600 nm for EdU.
Image alignment and segmentation was performed with mars-alt version 1 (http://openalea.gforge.inria.fr/doc/vplants/vtissue/doc/_build/html/user/mars_alt_v1/index.html) . For segmentation of mPS-PI images, the EMPILER and SEGMENTATION scripts of mars_alt v1 were used; zviewer and zfuse (mars_alt v1) were used to overlap segmented images with EdU images and to mark selected cells. A custom Python script was used to edit segmented images and retain only selected cells. Segmented images from MARS were imported into Matlab (MathWorks) and cell volumes were calculated based on the number of voxels per cell using custom Matlab scripts. Measurement of growth isotropy in epidermal sectors was based on growth tensors as described , using projections of the sectors onto a plane minimizing the sum of squared distances. Statistical analysis was performed with RCommander (http://socserv.mcmaster.ca/jfox/Misc/Rcmdr/). Custom scripts are available upon request.
For image display, MARS image files (.inr.gz) were imported into ImageJ64 (http://rsbweb.nih.gov/ij/download.html) using LOCI (http://loci.wisc.edu/bio-formats/imagej) and displayed in 3D using the 3D Viewer plugin (http://rsb.info.nih.gov/ij/plugins/) (Figures 1, 3C, 3D, and 4); projections of virtual sectors were also created with 3D viewer and cell clones were tracked manually and colored with Adobe Photoshop CS4 (Adobe Systems). Figure 3A was created with the ImageJ 3D Viewer plugin and Figure 3B with zfuse (mars-alt v1). Photoshop CS4 was used for final editing of the images (cropping, sizing, brightness and contrast).
For JAG-GR activation, inflorescences were dipped once into 0.015% Silwet L-77 (De Sangosse) 0.1% ethanol solution and supplemented with dexamethasone and cycloheximide as described in Figure 5. After 4 hr in daylight, inflorescence apices (only unopened flower buds) of 12 plants were collected per sample in three biological replicates per treatment. RNA was extracted using the RNEasy plant mini kit (QIAGEN), treated with Ambion® DNA-free (Invitrogen) and reverse transcribed using oligo (dT) 12–18 (Invitrogen), Superscript III reverse transcriptase (Invitrogen) and RNasin RNase Inhibitor (Promega) according to the manufacturers’ instructions. Quantitative PCR was performed in technical triplicates using primers BEL1-F, BEL1-R, BP-F, BP-R, STM-F, and STM-R (Supplemental Information) with the LightCycler 480 System and SYBR Green I (Roche). Data were normalized to ACTIN2 (amplified with primers ACT2-F, ACT2-R; Supplemental Information) as described .
JAG-GR was activated as described above and ChIP was as described , except for the following modifications: 70–80 inflorescence apices were used per sample; fixation buffer was modified to pH 8.5 and phenylmethylsulfonyl fluoride (PMSF) used at 0.1 mM; after grinding in liquid nitrogen, 300–500 mg tissue powder was resuspended in 700 μl of lysis buffer, modified to contain 0.1 mM PMSF and two tablets of protease inhibitor cocktail complete Mini, EDTA-free (Roche) added per 50 ml of buffer; the supernatant after sonication was precleared for 2 hr with 25 μl Dynabeads Protein A beads (Invitrogen) equilibrated in lysis buffer with 1 mg/ml BSA and 20 μg/ml sonicated salmon sperm, then incubated with 2 μl GR-antibodies (AB3580, Abcam) per 100 μl lysate at 4°C, overnight; 15 μl of equilibrated Dynabeads Protein A beads were added per 100 μl lysate and incubated at 4°C for 4 hr, before proceeding with washes and decrosslinking as described . Q-PCR was as described above using primers BEL3947-F/BEL3947-R, BELUTR-F/BELUTR, BP2609-F/BP2609-R, and BP1064-F/BP1064-R (Supplemental Information).
BEL-1 complementary DNA (cDNA) (nt 442–2077) and BP cDNA (full length) were cloned in pBluescript KS(–). Probes were transcribed in vitro from linearized plasmids using the DIG RNA Labeling kit (Roche) and T7 RNA Polymerase (Roche). In situ hybridization was performed as described  and imaged using a Leica DM 6000 microscope.
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Sequence data can be found in the Arabidopsis Genome Initiative database under the accession numbers JAG (AT1G68480), BP (AT4G08150), BEL-1(AT5G41410), and ACT2 (AT3G18780).
Document S1. Figures S1–S4, Table S1, and Supplemental Experimental Procedures
We thank Christophe Godin, Eric Moscardi, and Leonardo Alves Jr. for assistance with setting up MARS; Jan Traas and Pradeep Das for advice on live imaging and criticism; Sue Bunewell for help with electron microscopy; Carolyn Ohno for jag-2 seeds; the Nottingham Arabidopsis Stock Centre (NASC) for jag-1; Pauline Haleux for advice on statistics; and Nicolas Arnaud and Susana Sauret-Gueto for critical reading. K.S. received a Marie-Curie fellowship (237909); the work was supported by BBSRC grants BB/F005571/1 and BB/J004588/1 and the John Innes Foundation.
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