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Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration

Nature Plants volume 7, pages 1453–1460 (2021)Cite this article

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Abstract

In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.

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Fig. 1: Single-cell RNA-seq analysis of callus.
Fig. 2: WOX5/7 promote auxin production in callus.
Fig. 3: WOX5/7 promote cytokinin signalling in callus.
Fig. 4: Overexpression of WOX5 or WOX7 promotes shoot regeneration.

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Data availability

The sequence data can be accessed at the Arabidopsis Genome Initiative (https://www.arabidopsis.org/) under the following accession numbers: WOX11 (AT3G03660), WOX5 (AT3G11260), WOX7 (AT5G05770), PLT1 (AT3G20840), PLT2 (AT1G51190), SCR (AT3G54220), ARR5 (AT3G48100), ARR7 (AT1G19050), ARR12 (AT2G25180), ARR2 (AT4G16110), WUS (AT2G17950), JKD (AT5G03150), TAA1 (AT1G70560), TAR2 (AT4G24670), KCS6 (AT1G68530), PDF1 (AT2G42840), ATML1 (AT4G21750), BDG1 (AT1G64670), PIN2 (AT5G57090), SCZ (AT1G46264), NPY4 (AT2G23050), HAN (AT3G50870), SMXL3 (AT3G52490), TMO5 (AT3G25710), WAT1 (AT1G75500), ANT (AT4G37750), ATHB8 (AT4G32880), 4CL1 (AT1G51680), PXY (AT5G61480), bHLH068 (AT4G29100), TAN1 (AT3G05330) and HIK (AT1G18370). The RNA-seq and single-cell RNA-seq data have been deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the accession numbers GSE156990, GSE178354 and GSE156991. The RNA-seq and single-cell RNA-seq data can be accessed using the online tool (http://xulinlab.cemps.ac.cn/), and gene IDs can be used to search for gene expression patterns. The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank ABRC, T. Xu and J.-W. Wang for providing mutant seeds and marker lines. This work was supported by grants from the National Natural Science Foundation of China (no. 31630007), the Strategic Priority Research Program of the Chinese Academy of Sciences (no. XDB27030103), Youth Innovation Promotion Association CAS (no. 2014241) and the National Key Laboratory of Plant Molecular Genetics to L.X.

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Authors and Affiliations

  1. National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Ning Zhai & Lin Xu

  2. University of Chinese Academy of Sciences, Beijing, China

    Ning Zhai

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N.Z. and L.X. designed the research, analysed the data and wrote the article. N.Z. performed the experiments.

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Correspondence to Lin Xu.

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Peer review information Nature Plants thanks Kalika Prasad, Pil Joon Seo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–9, legend of Video 1, legends of Tables 1–7 and references.

Reporting Summary

Supplementary Video 1

WOX5 expression pattern in callus on CIM.

Supplementary Tables

Supplementary Table 1. List of cell clusters 0 to 9. Supplementary Table 2. GO analysis of cell cluster 2. Supplementary Table 3. List of genes in RNA-seq analysis of Col-0 and wox5-1 wox7-1 calli. Supplementary Table 4. List of genes in RNA-seq analysis of Col-0 and plt1-21 plt2-21 calli. Supplementary Table 5. List of genes regulated by WOX5/7 and PLT1/2. Supplementary Table 6. List of primers used in this study. Supplementary Table 7. List of genes related to the cell cycle.

Source data

Source Data Fig. 2

Unprocessed western blots for Fig.2b.

Source Data Fig. 3

Unprocessed western blots for Fig.3e.

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Zhai, N., Xu, L. Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration. Nat. Plants 7, 1453–1460 (2021). https://doi.org/10.1038/s41477-021-01015-8

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