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

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 cytokinin signaling repressors <i>AHP6</i> and <i>ARR16</i> likely block cytokinin responses in lateral tissues.

<p><b>(A-D)</b> Expression of the transcriptional reporter <i>AHP6</i>::<i>GFP</i> in transverse sections of stage 7, 8, 9, and 12 gynoecia. <b>(E, F)</b> Expression of the cytokinin response reporter <i>TCS</i>::<i>GFP</i> in transverse sections of stage 9 and 12 gynoecia in an <i>ahp6-1</i> mutant background. Arrowheads indicate the absence of GFP signal in the epidermis of the valves. <b>(G, H)</b> Phenotypes of wild-type (G) and <i>ahp6-1</i> (H) gynoecia one week after receiving BAP treatment for two weeks. <b>(I-L)</b> Expression of the transcriptional reporter <i>ARR16</i>::<i>GUS</i> (type-A <i>ARR</i>) in transverse sections of stage 7, 8, 9, and 12 gynoecia. Scale bars: 10 μm (A-C, E), 20 μm (D, F), 1 mm (G, H), 100 μm (I-L).</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

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

Model of the regulatory network in early gynoecium development integrating SPT, cytokinin signaling, auxin biosynthesis, and auxin transport.

<p>Model of the regulatory network in early gynoecium development. This regulatory network integrates the results that SPT, an important player of gynoecium development, enables cytokinin signaling in the medial domain of the young gynoecium by activating the transcription of type-B <i>ARR</i> genes (at least <i>ARR1</i> and <i>ARR12;</i> likely <i>ARR1</i> directly and <i>ARR12</i> indirectly), which proteins become active upon phosphorylation because of a phosphorelay cascade initiated when cytokinin is present, and then together activate auxin biosynthesis (<i>TAA1</i>) and transport important (<i>PIN</i>) for growth. It is likely that SPT also affects other components of the cytokinin signaling pathway (indicated by gray arrows). Solid black arrows indicate a positive regulation and a T-bar indicates a repression function, a broken black arrow indicates possible positive regulation by auxin, a double arrowhead indicates phosphorylation, purple arrows indicate possible auxin flow; CK, cytokinin; P, phosphate group.</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