www.sciencemag.org/content/343/6167/178/suppl/DC1
Supplementary Materials for A Spatial Accommodation by Neighboring Cells Is Required for Organ Initiation in Arabidopsis Joop E. M. Vermeer,* Daniel von Wangenheim, Marie Barberon, Yuree Lee, Ernst H. K. Stelzer, Alexis Maizel, Niko Geldner* *Corresponding author. E-mail:
[email protected] (J.E.M.V.) and
[email protected] (N.G.)
Published 10 January 2014, Science 343, 178 (2014) DOI: 10.1126/science.1245871 This PDF file includes Materials and Methods Figs. S1 to S9 Movies captions S1 to S10 References Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/content/343/6167/178/suppl/DC1) Movies S1 to S10
Materials and Methods Plant material Arabidopsis thaliana ecotype Columbia was used for all experiments. GATA23pro::NLSGFP:GUS (18) seeds were provided by T. Beeckman (VIB, Ghent, Belgium), shy2-2 (23) seeds by J. Reed (University of North Carolina, North-Carolina, USA) shy2-101 (24) seeds by H. Fukaki (Kobe University, Kobe, Japan), DR5pro::N7-3xVENUS seeds (19) by M. Bennett (University of Nottingham, Nottingham, UK). Plants were germinated on 1/2 MS (Murashige and Skoog) agar plates after 2 d in dark at 4°C. Seedlings were grown vertically at 22°C, under continuous days and were used at 7 d after shift to room temperature. Transgenic lines For generation of expression constructs, standard molecular biology procedures and Gateway Cloning Technology (Invitrogen) was used. For primer details see Table S1. For cell-type specific expression the following promoters were cloned into pGreenBasta; CASP1pro (5), PEPpro (13), PGP4pro (17). The cDNA of shy2-2 or SHY2 was cloned into the vectors with the cell type specific promoters. To generate SHY2pro::NLS3xmVENUS a 2287bp SHY2 promoter fragment (25) was cloned into pGreenBasta-NLS3xmVENUS. To generate UBQ10pro::myrpalm3xTagRFP-T, the myristoylation and palmitoylation motif of LeCDPK1 (26) was added to TagRFP-T and myrpalm3xTagRFP-T was assembled in pGreenBasta. CASP1pro::mCitrine:SYP122 was constructed by cloning the AtSYP122 (At3g52400) cDNA into pGreenBastaCASP1pro::mCITRINE. The GATA23pro::NLSGFP:GUS/UBQ10pro::EYFP:PIP1;2, SHY2pro::NLS3xmVENUS/UBQ10pro::myrpalm3xTagRFP-T, PIN1pro::PIN1:GFP/CASP1pro::CITRINE:SYP122, PIN1pro::PIN1:GFP/CASP1pro::γ-TIP:CITRINE lines were obtained by crossing. The CASP1pro::CASP1:mCherry, CASP1pro::γ-TIP-Citrine and CASP1pro::cals3m vectors were assembled using the multi-site gateway technology into pB7m34GW,3 and pH7m42GW,3 (http://gateway.psb.ugent.be). The entry clone containing cals3m(20) was kindly provided by Ykä Helariutta (University of Helsinki, Finland). Transgenic plants were generated by introduction of the plant expression constructs into an Agrobacterium tumefaciens strain GV3101 and transformation was done by floral dipping (27). Microscopy and histology Confocal laser scanning microscopy was performed on an upright Zeiss LSM 710 NLO confocal microscope. 2-Photon excitation was provided by a Ti-Sapphire Chameleon II Ultra (Coherent) and fluorescence was detected using non-descanned detection (NDD). For green and red fluorophores the following excitation and detection windows were used: GFP/mCITRINE/VENUS 960 nm, 500–550 nm; PI/TagRFP-T/mCherry/Basic Fuchsin 960 nm, 600–650 nm; aniline blue 730 nm, 500-550nm. Spectral unmixing of GFP and mCITRINE was achieved by using spectral detection combined with the online fingerprinting function of the ZEN software (Zeiss). For DR5pro::N7-3xVENUS experiments, seedlings were imaged using identical confocal settings. GUS staining was performed as described(4). Roots were cleared as described (28) or by mounting in chloral hydrate solution. Aniline blue 2
staining was performed as described (29). All plants were analyzed using differential interference contrast microscopy (Leica DM5000). Confocal images were analyzed and contrast and brightness were adjusted with the FIJI package (http://fiji.sc/Fiji) and Adobe Photoshop CS5. Light sheet fluorescence microscopy Images were collected with a Carl Zeiss C-Apochromat 40x/0.75 W objective lens. A Carl Zeiss EC Plan-Neofluar 5x/0.16 was used in the illumination path. A 3D-drift of the data was corrected using Amira (affine registration). The area of the pericycle and the endodermis cells was measured by drawing a shape around the boundaries in every time point (60 time points recorded with 5 minutes interval) using Fiji. Cell size measurements W131Y and CASP1pro::shy2-2/W131Y seedlings (5DAG) were transferred to liquid ½ MS supplemented with 10µM NAA or 10µM NPA in sterile 24-well plates (BD Biosciences). For NPA/NAA co-treatment seedlings were pre-incubated in 10µM NPA for 30 min after which an equal volume of 10µM NPA / 20µM NAA was added. After 5hr incubation at 22°C, seedlings were mounted in liquid 1/2MS. 3D image stacks were taken 6-8 cells after the appearance of protoxylem. Per treatment / genotype 25 seedlings were analyzed in three independent experiments. Pericycle cell widths were measured in the orthogonal views of the 3D-stacks using the FIJI. Statistics Statistics were performed using the R package (http://www.r-project.org).
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Figure S1. SHY2 is specifically induced in endodermal cells overlying they newly formed lateral root. SHY2 is induced in endodermal cells overlaying the lateral root primordium peaking at stage III-IV. Seedlings expressing a transcriptional reporter for SHY2, SHY2pro::NLS3xmVENUS (green) and the plasma membrane marker UBQ10pro::PM-3xtagRFP-T (magenta). Endodermal nuclei are indicated with asterisks. (A) stage I, (B) stage II, (C) stage III and (D) stage IV. (D) is represented as a frontal view with the primordium outlined with a dotted line. Images represent maximal projections and 4 single confocal sections at different depths indicated in µm. Scale bar, 20 µm. See also movies S1 to S4.
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Figure S2. The endodermis loses volume during lateral root formation. Confocal images of stage I (A), stage II (B), stage III (C) and stage IV (D) of lateral root development of plants carrying a marker for the CSD, CASP1pro::CASP1:mCherry (magenta) and the plasma membrane, UBQ10pro::EYFP:NPSN12 (green). Note that CASP1pro::CASP1:mCherry is gradually degraded and ends up in the vacuole (endodermis is indicated by asterisks). Scale bar, 20 µm.
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Figure S3. The protoplast of overlying endodermal cells stays intact during growth of the lateral root primordium into the endodermis. Projections of cross sections of the numbered positions of the original image showing a stage V lateral root primordium of a seedling expressing the plasma membrane marker CASP1pro::Citrine:SYP122 (magenta, figure 1B) to highlight the flattening and retracting protoplast during growth of the lateral root primordium through the endodermis. Accommodating endodermal cells are indicated by asterisks. Scale bar, 20 µm.
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Figure S4. The tonoplast of overlying endodermal cells gets remodeled during growth of the primordium into the endodermis. Projections of cross sections made at the numbered positions of the original image showing a stage V lateral root primordium of a seedling expressing the tonoplast marker CASP1pro::γ-TIP:Citrine (magenta, figure 1D) to highlight the flattening and remodeling of the tonoplast during growth of the lateral root primordium through the endodermis. Accommodating endodermal cells are indicated by asterisks. Scale bar, 20 µm.
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Figure S5. Specificity of used promoters. Confocal median sections of seedling roots (5DAG) expressing (A) CASP1pro::NLS3xmVENUS, (B) PEPpro::NLS3xmVENUS and (C) PGP4pro::NLS3xmVENUS. Whereas the CASP1 promoter only drives expression in the endodermis, the PGP4 promoter also shows detectable expression in the pericycle (arrows). The PEP promoter also shows very weak expression in the endodermis (arrowheads). Schematic root cross-sections show the relative activity of the used promoter fragments (arbitrary units). mVENUS signal in green, propidium iodide (magenta) is used to outline the cells. Scale bar, 20 µm.
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Figure S6. CASP1pro::shy2-2 plants do not initiate lateral roots. Staging of different developmental stages of lateral root development in Col-0 and CASP1pro::shy2-2 plants (7DAG) reveals complete absent of lateral roots in CASP1pro::shy2-2 plants.
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Figure S7. Sustained auxin treatment induces lateral root formation in CASP1pro::shy2-2 plants with severe problems to emerge. (A) Formation of lateral roots was quantified after incubation for 3 days at control, 0.1, 1 and 10 µM NAA. Histogram depicts average amount of lateral roots per cm. (B) Percentage of emerged lateral roots after 1 µM or 10 µM NAA for 3 days. (n=20). Asterisks indicate significant differences (***P
