Image_1.PDF

<p>In Arabidopsis, the gynoecium, the inner whorl of the flower, is the female reproductive part. Many tissues important for fertilization such as the stigma, style, transmitting tract, placenta, ovules, and septum, comprising the medial domain, arise from the carpel margin meristem. During gynoecium development, septum fusion occurs and tissues form continuously to prepare for a successful pollination and fertilization. During gynoecium development, cell wall modifications take place and one of the most important is the formation of the transmitting tract, having a great impact on reproductive competence because it facilitates pollen tube growth and movement through the ovary. In this study, using a combination of classical staining methods, fluorescent dyes, and indirect immunolocalization, we analyzed cell wall composition and modifications accompanying medial domain formation during gynoecium development. We detected coordinated changes in polysaccharide distribution through time, cell wall modifications preceding the formation of the transmitting tract, mucosubstances increase during transmitting tract formation, and a decrease of mannan distribution. Furthermore, we also detected changes in lipid distribution during septum fusion. Proper cell wall composition and modifications are important for postgenital fusion of the carpel (septum fusion) and transmitting tract formation, because these tissues affect plant reproductive competence.</p

AtMIKC* complexes regulate a transcriptional switch during pollen maturation

<p><b>Copyright information:</b></p><p>Taken from "MADS-complexes regulate transcriptome dynamics during pollen maturation"</p><p>http://genomebiology.com/content/8/11/R249</p><p>Genome Biology 2007;8(11):R249-R249.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2258202.</p><p></p> Of the genes downregulated in //triple mutant pollen, 83.4% exhibit peak expression at the mature pollen grain (MPG) stage of wild-type (WT) pollen development (according to Honys and Twell [11]), whereas 56.7% of the genes upregulated in this mutant peak during the immature tricellular stage (tricellular pollen [TCP]). In total, 83.5% of the upregulated genes peak during the three immature stages (unicellular microspores [UNM], BCP, and TCP). The AtMIKC* complexes contribute quite significantly to the transcriptional changes that occur during pollen maturation. We ranked all genes that were consistently called present in WT pollen according to their expression level in mature WT pollen, in descending order (the highest expressed gene received number 1). In this graph the ranking numbers of all genes upregulated and downregulated in triple mutant pollen are plotted, revealing that AtMIKC* complexes predominantly activate high-abundance and medium-abundance transcripts, while repressing low-abundance and medium-abundance transcripts. All calculations related to these graphs are included in Additional data file 2

Northern blot analysis of leaf tissue of different Arabidopsis lines containing various tagging constructs

<p><b>Copyright information:</b></p><p>Taken from "Tagging of MADS domain proteins for chromatin immunoprecipitation"</p><p>http://www.biomedcentral.com/1471-2229/7/47</p><p>BMC Plant Biology 2007;7():47-47.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2071916.</p><p></p> (A) Expression analysis of (pARC276) lines. (B) Expression analysis of (pARC277) lines. (C) Expression analysis of --tagII-FLAG-tag (pARC117) lines, -like plants are indicated with 'm' and WT-like plants with 'n'. WT, wild-type; +, line with an overexpression phenotype

Phenotypes of transgenic Arabidopsis plants with different tagging constructs

<p><b>Copyright information:</b></p><p>Taken from "Tagging of MADS domain proteins for chromatin immunoprecipitation"</p><p>http://www.biomedcentral.com/1471-2229/7/47</p><p>BMC Plant Biology 2007;7():47-47.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2071916.</p><p></p> (A) Wild-type Arabidopsis at the rosette stage, (D) at the inflorescence stage, and (G) a close-up of a flower. (B) Line with fusion construct showing an overexpression phenotype (pARC276). (C) Line with fusion construct showing a overexpression phenotype (pARC277). Rosette stage images (A-C) were taken from plants grown under the same conditions and were of the same age (bar indicates relative size). (E, H) Line with fusion construct showing an mutant phenotype (pARC308). (F, I) Line with fusion construct showing a partial -like mutant phenotype (pARC309). (J) Siliques of lines with fusion construct with either a overexpression (), mutant () phenotype, or wild-type phenotype (WT) (pARC310). (K) Arabidopsis root tip and (L) open silique with an ovule of a line expressing fusion construct (pARC310) observed by fluorescence microscopy. dz, dehiscence zone; v, valve; ov, ovule; n, nuclues; ca, carpel wall

AG and SEP3 expression analysis and chromatin immunoprecipitation (ChIP)

<p><b>Copyright information:</b></p><p>Taken from "Tagging of MADS domain proteins for chromatin immunoprecipitation"</p><p>http://www.biomedcentral.com/1471-2229/7/47</p><p>BMC Plant Biology 2007;7():47-47.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2071916.</p><p></p> Confocal Scanning Laser Microscopical (CSLM) imaging of (A) (pARC422) and (B) (pARC423) in the inflorescence. Top view (A, B) of an inflorescence with different floral bud stages (indicated by numbers). The GFP expression (green signal) is predominantly localized in the nuclei of floral meristem cells of flower buds from stage 3 onwards (comprising whorl 3 and 4 for AG, and whorl 2, 3, and 4 for SEP3, respectively). Autofluorescence is visible as red signal. (C) Anti-GFP Western blot with material from Arabidopsis WT and (pARC422) plants. Protein product is detectable in transgenic plants only. Bottom panel shows the Coomassie stained gel serving as loading control. (D) Enrichment of AG target DNA after ChAP with GFP antibody and compared with pre-immune. Quantification of target DNA was done by Real-time PCR using primers corresponding to sequences in the second intron of . FM, floral meristem, S, sepal, IM, inflorescence meristem, WT, wild-type

Overview of the gynoecium and SPT is necessary for cytokinin signaling in the young gynoecium.

<p></p><p><b>(</b></p><b>A)</b> Schematic overview and false-coloured transverse section of a stage 8 and of a stage 12 <i>Arabidopsis thaliana</i> gynoecium (pistil). The medial (M) and lateral (L) domains of the gynoecium are indicated. The CMM in the medial domain (stage 8 gynoecium; left side) is indicated and its derived structures can be seen in a stage 12 gynoecium (right side). L, lateral domain; M, medial domain. Orange, abaxial valve (abv); blue, adaxial valve (adv); white, abaxial replum (abr); pink, adaxial replum (adr); green, ovule primordium (op); red, septum primordium (sp); CMM, carpel margin meristem; septum (S); replum (R); transmitting tract (TT); ovule (O); funiculus (F). <b>(B-M)</b> Expression of the cytokinin response reporter <i>TCS</i>::<i>GFP</i> in transverse sections of gynoecia at stage 7, 8, 9, and 12 of wild-type <b>(B-E)</b>, <i>spt-2</i> <b>(F-I)</b>, and <i>35S</i>::<i>SPT</i> <b>(J-M)</b>.<b>(N-U)</b> Expression of the reporter <i>TCS</i>::<i>GFP</i> in transverse sections of gynoecia at stage 7, 8, 9, and 12, after 48 hours of 6-benzylaminopurine (BAP; a synthetic cytokinin) treatment in wild-type <b>(N-Q)</b> and <i>spt-2</i> <b>(R-U)</b>. Scale bars: 20 μm (E, I, M, Q, U), 10 μm (B-D, F-H, J-L, N-P, R-T).<p></p

The auxin transporter <i>PIN3</i> is coordinately activated by cytokinin and SPT.

<p><b>(A-C)</b> PIN3 expression in stage 9 <i>PIN3</i>::<i>PIN3-GFP</i> gynoecia that either received mock (<b>A,</b> transverse section) or BAP treatment for 48 hours (<b>B</b>, transverse section and <b>C</b>, longitudinal view). The inset in <b>(C)</b> shows a magnified view of the proliferating tissue. Arrows indicate the possible auxin flow. <b>(D-F)</b> PIN3 expression in transverse sections of stage 9 <i>PIN3</i>::<i>PIN3-GFP</i> gynoecia in <i>spt-2</i> <b>(D)</b>, <i>35S</i>::<i>SPT</i> <b>(E)</b>, and in <i>spt-2</i> treated for 48 hours with BAP <b>(F)</b>. <b>(G-J)</b> Transverse sections of stage 12 gynoecia of wild-type <b>(G, H)</b> and <i>pin3-4</i> <b>(I, J)</b>. Gynoecia phenotypes after three to four weeks of mock <b>(G, I)</b> or BAP treatment for five days <b>(H, J)</b>. Insets show a scanning electron microscopy image of the gynoecium. <b>(K)</b> Luciferase reporter assay in <i>N</i>. <i>benthamiana</i> leaves co-transformed with <i>35S</i>::<i>ARR1</i> and <i>pPIN3</i>::<i>LUC</i>. Ratio of LUC/REN activity. <b>(L)</b> ChIP experiments against the <i>PIN3</i> promoter regions (indicated by “a” and “b” in the scheme above) using an inducible <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR</i> line treated with dexamethasone or mock. <i>ACT2/7</i> served as a negative control. <b>(M)</b> Luciferase reporter assay in <i>N</i>. <i>benthamiana</i> leaves co-transformed with <i>35S</i>::<i>SPT</i> and <i>pPIN3</i>::<i>LUC</i>. Ratio of LUC/REN activity. <b>(N)</b> ChIP experiments against the <i>PIN3</i> promoter regions (indicated by “a” and “b” in the scheme above) using a <i>35S</i>::<i>SPT-HA</i> line and wild-type. <i>ACT2/7</i> served as a negative control. Error bars represent the SD for the LUC assays based on three biological replicates. ChIP results of one representative experiment is shown and the error bars represent the SD of the technical replicates. *<i>P</i> < 0.05 (LUC: Student-t test; qPCR: ANOVA). Scale bars: 10 μm (A-F), 100 μm (G-J, G-J insets). Ovule primordium (op).</p

Phenotypes of the type-B <i>arr</i> mutants and of the <i>spt</i> mutant.

<p><b>(A)</b> Mature gynoecium size of wild-type, <i>arr1</i>, <i>arr10</i>, <i>arr12</i>, <i>arr1 arr10</i>, <i>arr10 arr12</i>, <i>arr1 arr12</i>, and <i>arr1 arr10 arr12</i>. <b>(B)</b> Mature fruit size of wild-type, <i>arr1</i>, <i>arr10</i>, <i>arr12</i>, <i>arr1 arr10</i>, <i>arr10 arr12</i>, <i>arr1 arr12</i>, and <i>arr1 arr10 arr12</i>. <b>(C-F)</b> Phenotypes of the type-B <i>arr1 arr10 arr12</i> triple mutant compared to wild-type (WT): fruit length <b>(C)</b>, ovule number <b>(D)</b>, replum width <b>(E)</b>, and replum cell number <b>(F)</b>. <b>(G-I)</b> Transverse sections of stage 12 gynoecia of wild-type <b>(G)</b>, <i>arr1 arr10 arr12</i> (with transmitting tract and septum fusion defects) <b>(H)</b>, and <i>spt-2</i> <b>(I)</b>. Scale bars: 1 mm (A), 5 mm (B), 50 μm (G-I). Error bars represent SD. *<i>P</i> < 0.05 (Student-t test). Sample numbers: (C, D) WT, n = 14 and <i>arr1 arr10 arr12</i>, n = 19; (E, F) WT, n = 20 and <i>arr1 arr10 arr12</i>, n = 19.</p

SPT enables cytokinin responses during early gynoecium development and regulates type-B <i>ARR</i> gene expression.

<p><b>(A)</b> Phenotypes of wild-type, <i>arr1</i>, <i>arr10</i>, <i>arr12</i>, <i>arr1 arr10</i>, <i>arr10 arr12</i>, <i>arr1 arr12</i>, <i>arr1 arr10 arr12</i>, and <i>spt-2</i> gynoecia three to four weeks after receiving BAP treatment for five to ten days. (<b>B-E)</b> Scanning electron microscopy image of wild-type and <i>spt-2</i> stage 12 gynoecia one day after either receiving mock <b>(B, C)</b> or BAP treatment for only 48 hours <b>(D, E)</b>. Insets show a transverse section of the ovary. (<b>F</b>) Expression analysis by qRT-PCR of <i>ARR1</i>, <i>ARR10</i>, and <i>ARR12</i> in wild-type and <i>spt-12</i> dissected gynoecia. (<b>G-J</b>) <i>In situ</i> hybridization of type-B <i>ARR1</i> mRNA in wild-type <b>(G, H)</b> and <i>spt-2</i> <b>(I, J)</b> floral buds at stages 9 and 12. Arrowheads indicate the detected expression in wild-type and the absence in <i>spt-2</i>. <b>(K</b>) Luciferase reporter assay in <i>N</i>. <i>benthamiana</i> leaves co-transformed with <i>35S</i>::<i>SPT</i> and <i>pARR1</i>::<i>LUC</i>. Ratio of firefly luciferase (LUC) to Renilla luciferase (REN) activity. <b>(L)</b> ChIP experiments against the <i>ARR1</i> promoter region (indicated by “a” in the scheme above) using a <i>35S</i>::<i>SPT-HA</i> line and wild-type. <i>ACT2/7</i> served as a negative control. For the LUC assays and qRT-PCR experiments error bars represent the SD based on three biological replicates. ChIP results of one representative experiment are shown; error bars represent the SD of the technical replicates. *<i>P</i> < 0.05 (LUC: Student-t test; qRT-PCR and qPCR: ANOVA). Scale bars: 500 μm (A), 100 μm (B-E, H, J), 50 μm (insets in B-E, G, I).</p

Cytokinin signaling activates the auxin biosynthetic gene <i>TAA1</i> in a SPT-dependent manner.

<p><b>(A</b>, <b>B)</b> Expression of the translational fusion <i>TAA1</i>::<i>GFP-TAA1</i> in a transverse section of a stage 9 wild-type gynoecium that either received mock <b>(A)</b> or BAP treatment for 48 hours <b>(B)</b>. <b>(C, D)</b> Expression of the translational fusion <i>TAA1</i>::<i>GFP-TAA1</i> in a transverse section of a stage 9 <i>spt-12</i> gynoecium that received mock <b>(C)</b> or BAP treatment for 48 hours <b>(D)</b>. <b>(E)</b> Luciferase reporter assay in <i>N</i>. <i>benthamiana</i> leaves co-transformed with <i>35S</i>::<i>ARR1</i> and <i>pTAA1</i>::<i>LUC</i>. Ratio of LUC/REN activity. (<b>F</b>) ChIP experiments against the <i>TAA1</i> promoter region (indicated by “a” in the scheme above) using an inducible <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR</i> line treated with dexamethasone or mock. <i>ACT2/7</i> served as a negative control. <b>(G)</b> Luciferase reporter assay in <i>N</i>. <i>benthamiana</i> leaves co-transformed with <i>35S</i>::<i>SPT</i> and <i>pTAA1</i>::<i>LUC</i>. Ratio of LUC/REN activity. <b>(H)</b> ChIP experiments against the <i>TAA1</i> promoter region (indicated by “a” in the scheme above) using a <i>35S</i>::<i>SPT-HA</i> line and wild-type. <i>ACT2/7</i> served as a negative control. Error bars represent the SD for the LUC assays based on three biological replicates. ChIP results of one representative experiment are shown; error bars represent the SD of the technical replicates. *<i>P</i> < 0.05 (LUC: Student-t test; qPCR: ANOVA). Scale bars: 10 μm (A-D).</p