Reconstitution of Cytokinin Signaling in Rice Protoplasts
The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations...
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MDPI AG
2021-03-01
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| Series: | International Journal of Molecular Sciences |
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| author | Eunji Ga Jaeeun Song Myung Ki Min Jihee Ha Sangkyu Park Saet Buyl Lee Jong-Yeol Lee Beom-Gi Kim |
| author_facet | Eunji Ga Jaeeun Song Myung Ki Min Jihee Ha Sangkyu Park Saet Buyl Lee Jong-Yeol Lee Beom-Gi Kim |
| author_sort | Eunji Ga |
| collection | DOAJ |
| description | The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (<i>Oryza sativa</i>) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct <i>TCSn:fLUC</i>, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated <i>TCSn:fLUC</i> strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with <i>OsHP01</i>, <i>OsHP02</i>, <i>OsHP05</i>, or <i>OsHK03</i> alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice. |
| first_indexed | 2024-03-10T12:43:22Z |
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| id | doaj.art-44c79e9e7c254cb7879ab1d351c12b49 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T12:43:22Z |
| publishDate | 2021-03-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-44c79e9e7c254cb7879ab1d351c12b492023-11-21T13:39:27ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-03-01227364710.3390/ijms22073647Reconstitution of Cytokinin Signaling in Rice ProtoplastsEunji Ga0Jaeeun Song1Myung Ki Min2Jihee Ha3Sangkyu Park4Saet Buyl Lee5Jong-Yeol Lee6Beom-Gi Kim7Metabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaMetabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaMetabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaMetabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaMetabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaMetabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaMetabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaMetabolic Engineering Division, National Institute of Agricultural Sciences, RDA, Jeonju 54874, KoreaThe major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (<i>Oryza sativa</i>) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct <i>TCSn:fLUC</i>, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated <i>TCSn:fLUC</i> strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with <i>OsHP01</i>, <i>OsHP02</i>, <i>OsHP05</i>, or <i>OsHK03</i> alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.https://www.mdpi.com/1422-0067/22/7/3647riceprotoplastcytokininsignalingreconstitution |
| spellingShingle | Eunji Ga Jaeeun Song Myung Ki Min Jihee Ha Sangkyu Park Saet Buyl Lee Jong-Yeol Lee Beom-Gi Kim Reconstitution of Cytokinin Signaling in Rice Protoplasts International Journal of Molecular Sciences rice protoplast cytokinin signaling reconstitution |
| title | Reconstitution of Cytokinin Signaling in Rice Protoplasts |
| title_full | Reconstitution of Cytokinin Signaling in Rice Protoplasts |
| title_fullStr | Reconstitution of Cytokinin Signaling in Rice Protoplasts |
| title_full_unstemmed | Reconstitution of Cytokinin Signaling in Rice Protoplasts |
| title_short | Reconstitution of Cytokinin Signaling in Rice Protoplasts |
| title_sort | reconstitution of cytokinin signaling in rice protoplasts |
| topic | rice protoplast cytokinin signaling reconstitution |
| url | https://www.mdpi.com/1422-0067/22/7/3647 |
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