The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium

[EN] Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.IRO, VMZM, HHU and PLS were supported by the Mexican National Council of Science and Technology (CONACyT) with a PhD fellowship (210085, 210100, 243380 and 219883, respectively). Work in the SDF laboratory was financed by the CONACyT grants CB-2012-177739, FC-2015-2/1061, and INFR-2015-253504, and NMM by the CONACyT grant CB-2011-165986. SDF, CF and LC acknowledge the support of the European Union FP7-PEOPLE-2009-IRSES project EVOCODE (grant no. 247587) and H2020-MSCARISE-2015 project ExpoSEED (grant no. 691109). SDF also acknowledges the Marine Biological Laboratory (MBL) in Woods Hole for a scholarship for the Gene Regulatory Networks for Development Course 2015 (GERN2015). IE acknowledges the International European Fellowship-METMADS project and the Universita degli Studi di Milano (RTD-A; 2016). Research in the laboratory of MFY was funded by NSF (grant IOS-1121055), NIH (grant 1R01GM112976-01A1) and the Paul D. Saltman Endowed Chair in Science Education (MFY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Reyes Olalde, J.; Zuñiga, V.; Serwatowska, J.; Chávez Montes, R.; Lozano-Sotomayor, P.; Herrera-Ubaldo, H.; Gonzalez Aguilera, K.... (2017). The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium. PLoS Genetics. 13(4):1-31. https://doi.org/10.1371/journal.pgen.1006726S131134Reyes-Olalde, J. I., Zuñiga-Mayo, V. M., Chávez Montes, R. A., Marsch-Martínez, N., & de Folter, S. (2013). Inside the gynoecium: at the carpel margin. Trends in Plant Science, 18(11), 644-655. doi:10.1016/j.tplants.2013.08.002Alvarez-Buylla, E. 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The AP2/ERF Transcription Factor DRNL Modulates Gynoecium Development and Affects Its Response to Cytokinin

The gynoecium is the female reproductive system in flowering plants. It is a complex structure formed by different tissues, some that are essential for reproduction and others that facilitate the fertilization process and nurture and protect the developing seeds. The coordinated development of these different tissues during the formation of the gynoecium is important for reproductive success. Both hormones and genetic regulators guide the development of the different tissues. Auxin and cytokinin in particular have been found to play important roles in this process. On the other hand, the AP2/ERF2 transcription factor BOL/DRNL/ESR2/SOB is expressed at very early stages of aerial organ formation and has been proposed to be a marker for organ founder cells. In this work, we found that this gene is also expressed at later stages during gynoecium development, particularly at the lateral regions (the region related to the valves of the ovary). The loss of DRNL function affects gynoecium development. Some of the mutant phenotypes present similarities to those observed in plants treated with exogenous cytokinins, and AHP6 has been previously proposed to be a target of DRNL. Therefore, we explored the response of drnl-2 developing gynoecia to cytokinins, and found that the loss of DRNL function affects the response of the gynoecium to exogenously applied cytokinins in a developmental-stage-dependent manner. In summary, this gene participates during gynoecium development, possibly through the dynamic modulation of cytokinin homeostasis and response

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

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

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

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

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

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