Abstract
Key message
WOX5 has a potential in activating cytokinin signaling and shoot regeneration, in addition to its role in pluripotency acquisition. Thus, overexpression of WOX5 maximizes plant regeneration capacity during tissue culture.
In vitro plant regeneration involves two steps: callus formation and de novo shoot organogenesis. The WUSCHEL‐RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor is known to be mainly expressed during incubation on callus-inducing medium (CIM) and involved in pluripotency acquisition in callus, but whether WOX5 also affects de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) remains unknown. Based on the recent finding that WOX5 promotes cytokinin signaling, we hypothesized that ectopic expression of WOX5 beyond CIM would further enhance overall plant regeneration capacity, because intense cytokinin signaling is particularly required for shoot regeneration on SIM. Here, we found that overexpression of the WOX5 gene on SIM drastically promoted de novo shoot regeneration from callus with the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, negative regulators of cytokinin signaling. The enhanced shoot regeneration phenotypes were indeed dependent on cytokinin signaling, which were partially suppressed in the progeny derived from crossing WOX5-overexpressing plants with cytokinin-insensitive 35S:ARR7 plants. The function of WOX5 in enhancing cytokinin-dependent shoot regeneration is evolutionarily conserved, as conditional overexpression of OsWOX5 on SIM profoundly enhanced shoot regeneration in rice callus. Overall, our results provide the technical advance that maximizes in vitro plant regeneration by constitutively expressing WOX5, which unequivocally promotes both callus pluripotency and de novo shoot regeneration.
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References
Dai X, Liu Z, Qiao M, Li J, Li S, Xiang F (2017) ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression. J Integr Plant Biol 59:747–758. https://doi.org/10.1111/jipb.12567
Fan M, Xu C, Xu K, Hu Y (2012) LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration. Cell Res 22:1169–1180. https://doi.org/10.1038/cr.2012.63
Higuchi M, Pischke MS, Mahonen AP, Miyawaki K, Hashimoto Y, Seki M, Kobayashi M, Shinozaki K, Kato T, Tabata S, Helariutta Y, Sussman MR, Kakimoto T (2004) In planta functions of the Arabidopsis cytokinin receptor family. Proc Natl Acad Sci USA 101:8821–8826. https://doi.org/10.1073/pnas.0402887101
Jung YJ, Lee HJ, Kim JH, Kim DH, Kim HK, Cho Y-G, Bae S, Kang KK (2019) CRISPR/Cas9-targeted mutagenesis of F3′H, DFR and LDOX, genes related to anthocyanin biosynthesis in black rice (Oryza sativa L.). Plant Biotechnol Rep 13:521–531. https://doi.org/10.1007/s11816-019-00579-4
Kareem A, Durgaprasad K, Sugimoto K, Du Y, Pulianmackal AJ, Trivedi ZB, Abhayadev PV, Pinon V, Meyerowitz EM, Scheres B, Prasad K (2015) PLETHORA genes control regeneration by a two-step mechanism. Curr Biol 25:1017–1030. https://doi.org/10.1016/j.cub.2015.02.022
Kim JY, Yang W, Forner J, Lohmann JU, Noh B, Noh YS (2018) Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis. EMBO J 37. https://doi.org/10.15252/embj.201798726
Lee K, Park OS, Go JY, Yu J, Han JH, Kim J, Bae S, Jung YJ, Seo PJ (2021) Arabidopsis ATXR2 represses de novo shoot organogenesis in the transition from callus to shoot formation. Cell Rep 37:109980. https://doi.org/10.1016/j.celrep.2021.109980
Lee K, Park OS, Seo PJ (2017) Arabidopsis ATXR2 deposits H3K36me3 at the promoters of LBD genes to facilitate cellular dedifferentiation. Sci Signal 10. https://doi.org/10.1126/scisignal.aan0316
Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T (1998) Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95:805–815. https://doi.org/10.1016/s0092-8674(00)81703-1
Meng WJ, Cheng ZJ, Sang YL, Zhang MM, Rong XF, Wang ZW, Tang YY, Zhang XS (2017) Type-B ARABIDOPSIS RESPONSE REGULATORs specify the shoot stem cell niche by dual regulation of WUSCHEL. Plant Cell 29:1357–1372. https://doi.org/10.1105/tpc.16.00640
Park J, Bae S, Kim JS (2015) Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31:4014–4016. https://doi.org/10.1093/bioinformatics/btv537
Pi L, Aichinger E, van der Graaff E, Llavata-Peris CI, Weijers D, Hennig L, Groot E, Laux T (2015) Organizer-derived WOX5 signal maintains root columella stem cells through chromatin-mediated repression of CDF4 expression. Dev Cell 33:576–588. https://doi.org/10.1016/j.devcel.2015.04.024
Ren B, Liang Y, Deng Y, Chen Q, Zhang J, Yang X, Zuo J (2009) Genome-wide comparative analysis of type-A Arabidopsis response regulator genes by overexpression studies reveals their diverse roles and regulatory mechanisms in cytokinin signaling. Cell Res 19:1178–1190. https://doi.org/10.1038/cr.2009.88
Salvi E, Rutten JP, Di Mambro R, Polverari L, Licursi V, Negri R, Dello Ioio R, Sabatini S, Ten Tusscher K (2020) A self-organized PLT/Auxin/ARR-B network controls the dynamics of root zonation development in Arabidopsis thaliana. Dev Cell 53(431–443):e423. https://doi.org/10.1016/j.devcel.2020.04.004
Sugimoto K, Jiao Y, Meyerowitz EM (2010) Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell 18:463–471. https://doi.org/10.1016/j.devcel.2010.02.004
Zhai N, Xu L (2021) Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration. Nat Plants 7:1453–1460. https://doi.org/10.1038/s41477-021-01015-8
Zhang W, Swarup R, Bennett M, Schaller GE, Kieber JJ (2013) Cytokinin induces cell division in the quiescent center of the Arabidopsis root apical meristem. Curr Biol 23:1979–1989. https://doi.org/10.1016/j.cub.2013.08.008
Zhang Y, Jiao Y, Liu Z, Zhu YX (2015) ROW1 maintains quiescent centre identity by confining WOX5 expression to specific cells. Nat Commun 6:6003. https://doi.org/10.1038/ncomms7003
Zhang TQ, Lian H, Zhou CM, Xu L, Jiao Y, Wang JW (2017) A two-step model for de novo activation of WUSCHEL during plant shoot regeneration. Plant Cell 29:1073–1087. https://doi.org/10.1105/tpc.16.00863
Acknowledgements
We thank Dr. Thomas Laux (Albert-Ludwigs-University Freiburg, Germany) for kindly providing 35S:WOX5-GR and wox5-1 seeds.
Funding
This work was supported by the Next-Generation New Plant Breed Technology Program (PJ01653002) provided by the Rural Development Administration and by the Basic Science Research (NRF-2021R1I1A1A01054378 to K.L.; NRF-2022R1A2B5B02001266 to P.J.S.) and Basic Research Laboratory (NRF-2022R1A4A3024451) programs funded by the National Research Foundation of Korea.
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PJS and KL conceived and designed the study. KL, JHK, and OSP conducted the experiments. YJJ analyzed the data. PJS, YJJ, and KL wrote the manuscript. All authors read and approved the manuscript.
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Communicated by Youn-Il Park.
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Lee, K., Kim, J.H., Park, OS. et al. Ectopic expression of WOX5 promotes cytokinin signaling and de novo shoot regeneration. Plant Cell Rep 41, 2415–2422 (2022). https://doi.org/10.1007/s00299-022-02932-4
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DOI: https://doi.org/10.1007/s00299-022-02932-4