Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots

Nature Plants volume 5, pages 1033–1042 (2019)Cite this article

Subjects

Abstract

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity1, the phenotypes of the corresponding loss-of-function mutants are relatively modest, highlighting our limited understanding of the underlying transcriptional regulation. Here, we use cambium cell-specific transcript profiling followed by a combination of transcription factor network and genetic analyses to identify 62 new transcription factor genotypes displaying an array of cambial phenotypes. This approach culminated in virtual loss of cambial activity when both WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNOTTED-like from Arabidopsis thaliana 1 (KNAT1; also known as BREVIPEDICELLUS) were mutated, thereby unlocking the genetic redundancy in the regulation of cambium development. We also identified transcription factors with dual functions in cambial cell proliferation and xylem differentiation, including WOX4, SHORT VEGETATIVE PHASE (SVP) and PETAL LOSS (PTL). Using the transcription factor network information, we combined overexpression of the cambial activator WOX4 and removal of the putative inhibitor PTL to engineer Arabidopsis for enhanced radial growth. This line also showed ectopic cambial activity, thus further highlighting the central roles of WOX4 and PTL in cambium development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Identification and validation of cambium TFs during radial growth in Arabidopsis roots.
Fig. 2: A transcriptional network based on inducible overexpression analysis largely predicts the severity of cambial loss-of-function mutants.
Fig. 3: Among the TFs studied, WOX4 and KNAT1 emerged as major regulators of radial growth.
Fig. 4: Activation of positive transcriptional regulators and removal of negative regulator synergistically stimulate cambial activity.

Similar content being viewed by others

Data availability

Gene accession numbers are as follows: ARR15, AT1G74890; CYCD3;1, AT4G34160; ANT, AT4G37750; WOX14, AT1G20700; KNAT1 (also known as BP), AT4G08150; PTL, AT5G03680; SVP, AT2G22540; AGL24, AT4G24540; LBD4, AT1G31320; SCL7, AT3G50650; AGL14, AT4G11880; ATHB53, AT5G66700; ATHB5, AT5G65310; WRI3, AT1G16060; AHL11, AT3G61310; MYB47, AT1G18710; MYB95, AT1G74430; MYB87, AT4G37780; STY2, AT4G36260; RAS1, AT1G09950; AtERF019, AT1G22810; AtERF021, AT1G71450; AtERF029, AT4G25490; AtERF032, AT1G63030; AtERF043, AT4G32800; AtERF071, AT2G47520; AtERF072, AT3G16770; WOX4, AT1G46480; WOX13, AT4G35550; STM, AT1G62360; KNAT2, AT1G70510; KNAT6, AT1G23380; BOP1, AT3G57130; BOP2, AT2G41370; EDA31, AT3G10000; LBD3 (also known as ASL9), AT1G16530; LBD1, AT1G07900; SCL4, AT5G66770; SCL3, AT1G50420; SCL28, AT1G63100; ANAC015 (also known as BRN1), AT1G33280; ANAC070 (also known as BRN2), AT4G10350; SMB, AT1G79580; ANAC042 (also known as JUNGBRUNNEN 1), AT2G43000; ANAC042-like, AT3G12910; MYB3R4, AT5G11510; MYB3R1, AT4G32730; PXY (also known as TDR), AT5G61480; ERECTA/ER, AT2G26330. Raw data of Affymetrix ATH1 GeneChip (accession no. GSE125244) and RNA-seq data can be found in NCBI (accession no. PRJNA523600). Source data related to LithoGraphX analysis (Fig. 3c,d; Supplementary Fig. 11) and ANOVA analysis (Fig. 4d; Supplementary Figs. 8e and 11–13) can be found in Supplementary Datasets. The data that support the findings of this study are available from the corresponding authors upon request. Mutants generated in this study will be deposited to NASC.

Code availability

The code used for network construction and LithoGraphX analysis can be accessed at Github (https://github.com/Zhangcambium2019/Zhang2019). R-codes for boxplot, half-violin plot, median calculation, average calculation and P value calculation can be accessed at Github.

References

  1. Zhang, J., Nieminen, K., Serra, J. A. A. & Helariutta, Y. The formation of wood and its control. Curr. Opin. Plant Biol. 17, 56–63 (2014).

    Article  CAS  Google Scholar 

  2. Nieminen, K., Blomster, T., Helariutta, Y. & Mahonen, A. P. Vascular cambium development. Arabidopsis Book 13, e0177 (2015).

    Article  Google Scholar 

  3. Smetana, O. et al. High levels of auxin signalling define the stem-cell organizer of the vascular cambium. Nature 565, 485–489 (2019).

    Article  CAS  Google Scholar 

  4. Zhang, J., Elo, A. & Helariutta, Y. Arabidopsis as a model for wood formation. Curr. Opin. Biotechnol. 22, 293–299 (2011).

    Article  CAS  Google Scholar 

  5. Laux, T., Mayer, K. F. X., Berger, J. & Jurgens, G. The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Development 122, 87–96 (1996).

    CAS  PubMed  Google Scholar 

  6. Clark, S. E., Jacobsen, S. E., Levin, J. Z. & Meyerowitz, E. M. The CLAVATA and SHOOT MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis. Development 122, 1567–1575 (1996).

    CAS  PubMed  Google Scholar 

  7. Benfey, P. N. et al. Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis. Development 119, 57–70 (1993).

    CAS  PubMed  Google Scholar 

  8. Helariutta, Y. et al. The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101, 555–567 (2000).

    Article  CAS  Google Scholar 

  9. Lucas, M. et al. SHORT-ROOT regulates primary, lateral and adventitious root development in Arabidopsis. Plant Physiol. 155, 384–398 (2011).

    Article  CAS  Google Scholar 

  10. Scheres, B. et al. Mutations affecting the radial organization of the Arabidopsis root display specific defects throughout the embryonic axis. Development 121, 53–62 (1995).

    CAS  Google Scholar 

  11. DiLaurenzio, L. et al. The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86, 423–433 (1996).

    Article  CAS  Google Scholar 

  12. Ji, J. B. et al. WOX4 promotes procambial development. Plant Physiol. 152, 1346–1356 (2010).

    Article  CAS  Google Scholar 

  13. Hirakawa, Y., Kondo, Y. & Fukuda, H. TDIF peptide signaling regulates vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis. Plant Cell 22, 2618–2629 (2010).

    Article  CAS  Google Scholar 

  14. Suer, S., Agusti, J., Sanchez, P., Schwarz, M. & Greb, T. WOX4 imparts auxin responsiveness to cambium cells in Arabidopsis. Plant Cell 23, 3247–3259 (2011).

    Article  CAS  Google Scholar 

  15. Etchells, J. P., Provost, C. M., Mishra, L. & Turner, S. R. WOX4 and WOX14 act downstream of the PXY receptor kinase to regulate plant vascular proliferation independently of any role in vascular organisation. Development 140, 2224–2234 (2013).

    Article  CAS  Google Scholar 

  16. Randall, R. S. et al. AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins. Biol. Open 4, 1229–1236 (2015).

    Article  CAS  Google Scholar 

  17. Etchells, J. P., Provost, C. M. & Turner, S. R. Plant vascular cell division is maintained by an interaction between PXY and ethylene signalling. PLoS Genet. 8, e100299 (2012).

  18. Brackmann, K. et al. Spatial specificity of auxin responses coordinates wood formation. Nat. Commun. 9, 875 (2018).

  19. Birnbaum, K. et al. Cell type-specific expression profiting in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat. Methods 2, 615–619 (2005).

    Article  CAS  Google Scholar 

  20. Wagner, G. P., Pavlicev, M. & Cheverud, J. M. The road to modularity. Nat. Rev. Genet. 8, 921–931 (2007).

    Article  CAS  Google Scholar 

  21. Wang, X. W., Dalkic, E., Wu, M. & Chan, C. Gene module level analysis: identification to networks and dynamics. Curr. Opin. Biotechnol. 19, 482–491 (2008).

    Article  CAS  Google Scholar 

  22. Siligato, R. et al. MultiSite gateway-compatible cell type-specific gene-inducible system for plants. Plant Physiol. 170, 627–641 (2016).

    Article  CAS  Google Scholar 

  23. Uchida, N. & Tasaka, M. Regulation of plant vascular stem cells by endodermis-derived EPFL-family peptide hormones and phloem-expressed ERECTA-family receptor kinases. J. Exp. Bot. 64, 5335–5343 (2013).

    Article  CAS  Google Scholar 

  24. Taylor-Teeples, M. et al. An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature 517, 571–U307 (2015).

    Article  CAS  Google Scholar 

  25. Wunderling, A., Ben Targem, M., de Reuille, P. B. & Ragni, L. Novel tools for quantifying secondary growth. J. Exp. Bot. 68, 89–95 (2017).

    Article  CAS  Google Scholar 

  26. de Reuille P. B. & Ragni, L. Vascular morphodynamics during secondary growth. Methods Mol. Biol. 1544, 103–125 (2017).

  27. Guo, Y., Qin, G. J., Gu, H. Y. & Qu, L. J. Dof5.6/HCA2, a Dof transcription factor gene, regulates interfascicular cambium formation and vascular tissue development in Arabidopsis. Plant Cell 21, 3518–3534 (2009).

    Article  CAS  Google Scholar 

  28. Yordanov, Y. S., Regan, S. & Busov, V. Members of the LATERAL ORGAN BOUNDARIES DOMAIN transcription factor family are involved in the regulation of secondary growth in populus. Plant Cell 22, 3662–3677 (2010).

    Article  CAS  Google Scholar 

  29. Etchells, J. P., Mishra, L. S., Kumar, M., Campbell, L. & Turner, S. R. Wood formation in trees is increased by manipulating PXY-regulated cell division. Curr. Biol. 25, 1050–1055 (2015).

    Article  CAS  Google Scholar 

  30. Zhang, J., Serra, J. A. A. & Helariutta, Y. Wood development: growth through knowledge. Nat. Plants 1, 15060 (2015).

    Article  Google Scholar 

  31. Immanen, J. et al. Cytokinin and auxin display distinct but interconnected distribution and signaling profiles to stimulate cambial activity. Curr. Biol. 26, 1990–1997 (2016).

    Article  CAS  Google Scholar 

  32. Brady, S. M. et al. A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318, 801–806 (2007).

    Article  CAS  Google Scholar 

  33. Nawy, T. et al. Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell 17, 1908–1925 (2005).

    Article  CAS  Google Scholar 

  34. Lee, J. Y. et al. Transcriptional and posttranscriptional regulation of transcription factor expression in Arabidopsis roots. Proc. Natl Acad. Sci. USA 103, 6055–6060 (2006).

    Article  CAS  Google Scholar 

  35. Levesque, M. P. et al. Whole-genome analysis of the SHORT-ROOT developmental pathway in Arabidopsis. PLoS Biol. 4, e143 (2006).

    Article  Google Scholar 

  36. Wu, Z. J., Irizarry, R. A., Gentleman, R., Martinez-Murillo, F. & Spencer, F. A model-based background adjustment for oligonucleotide expression arrays. J. Am. Stat. Assoc. 99, 909–917 (2004).

    Article  Google Scholar 

  37. Smyth, G. K. in Bioinformatics and computational biology solutions using R and Bioconductor (eds Gentleman, R. et al.) 397–420 (Springer, 2005).

  38. Segal, E. et al. Module networks: identifying regulatory modules and their condition-specific regulators from gene expression data. Nat. Genet. 34, 166–176 (2003).

    Article  CAS  Google Scholar 

  39. Maere, S., Heymans, K. & Kuiper, M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449 (2005).

    Article  CAS  Google Scholar 

  40. Curtis, M. D. & Grossniklaus, U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 (2003).

    Article  CAS  Google Scholar 

  41. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank O. Smetana for providing the root cross-section sketches for making vector images in Fig. 1a; S. Miyashima for providing the pBI-nlsYFP-GUS vector for the cloning of pLBD4::nYFP-GUS; H. Fukuda, J. Murray, D. Smyth, U. Fischer, H. Yu, T. Mizuno, J. Lim, B. Scheres, M. Ito, S. Hepworth, V. Pautot, M. Kater and S. Turner for providing published seeds (Supplementary Table 4a,b); K. Kainulainen and M. Herpola for technical assistance; O. Smetana and L.-L. Ye for providing help on finalizing the figures and S. El-Showk for language correction. This work was supported by the Finnish Centre of Excellence in Molecular Biology of Primary Producers (Academy of Finland CoE programme 2014–2019, decision no. 271832), the Gatsby Foundation (no. GAT3395/PR3), the University of Helsinki (award no. 799992091) and the European Research Council Advanced Investigator Grant SYMDEV (no. 323052 to Y.H.); Academy of Finland (grants nos. 132376, 266431 and 271832), University of Helsinki HiLIFE fellowship to A.P.M. and National Research Foundation of Korea (nos. 2018R1A5A1023599 and 2016R1A2B2015258 to J.-Y.L.).

Author information

Author notes

  1. These authors contributed equally: G. Eswaran, J. Alonso-Serra.

Authors and Affiliations

  1. Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland

    Jing Zhang, Gugan Eswaran, Juan Alonso-Serra, Melis Kucukoglu, Jiale Xiang, Annakaisa Elo, Teddy Damén, Ari Pekka Mähönen & Ykä Helariutta

  2. Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, Finland

    Jing Zhang, Gugan Eswaran, Juan Alonso-Serra, Melis Kucukoglu, Jiale Xiang, Annakaisa Elo, Teddy Damén, Ari Pekka Mähönen & Ykä Helariutta

  3. The Sainsbury Laboratory, University of Cambridge, Cambridge, UK

    Jing Zhang, Weibing Yang, Sebastian E. Ahnert & Ykä Helariutta

  4. Production Systems, Natural Resources Institute Finland (Luke), Helsinki, Finland

    Kaisa Nieminen

  5. Samsung Genome Institute, Samsung Medical Center, Seoul, South Korea

    Je-Gun Joung

  6. Center for Genome Engineering, Institute for Basic Science, Daejeon, South Korea

    Jae-Young Yun

  7. School of Biological Sciences, Seoul National University, Seoul, South Korea

    Jung-Hun Lee & Ji-Young Lee

  8. ZMBP-Center for Plant Molecular Biology, University of Tübingen, Tübingen, Germany

    Laura Ragni

  9. Institute of Plant Sciences, University of Bern, Bern, Switzerland

    Pierre Barbier de Reuille

  10. Theory of Condensed Matter, Cavendish Laboratory, University of Cambridge, Cambridge, UK

    Sebastian E. Ahnert

Authors
  1. Jing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gugan Eswaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Juan Alonso-Serra
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Melis Kucukoglu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiale Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Weibing Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Annakaisa Elo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kaisa Nieminen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Teddy Damén
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Je-Gun Joung
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jae-Young Yun
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jung-Hun Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Laura Ragni
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Pierre Barbier de Reuille
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Sebastian E. Ahnert
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Ji-Young Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Ari Pekka Mähönen
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Ykä Helariutta
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.Z., J.-Y.L., A.P.M. and Y.H. designed the experiments. J.Z. carried out most of the experiments with help from other co-authors. K.N. constructed the pARR15::GFP line for FACS. K.N., A.E. and J.-Y.L. performed FACS and microarray hybridization. J.-Y.L. and J.-G.J. analysed the microarray data from the FACS. J.Z. generated and analysed all transgenic lines with input from A.E. on GUS reporter lines. W.Y. and J.Z. carried out the RNA in situ analysis. M.K. conducted the qRT–PCR and analysed the data for network construction. S.E.A. constructed the network and carried out perturbation analysis. J.Z. and G.E. generated combinatorial mutants. J.-Y.Y. and J.-H.L. produced the asl9 CRISPR mutant. J.Z., G.E., J.X. and T.D. performed mutant genotyping. J.Z., with assistance from J.X., G.E. and J.A.-S., characterized the mutant and overexpression line phenotypes. J.Z., G.E. and J.A.-S. conducted quantification and statistical analyses for phenotypic examination of mutants and overexpression lines. J.A.-S. ran the LithoGraphX analysis with input from G.E., L.R. and P.B.R. J.Z. performed the RNA-seq experiment and analysed the RNA-seq data with S.E.A. G.E. drew all box plot figures. J.Z., J.-Y.L., A.P.M. and Y.H. wrote the manuscript with help from the co-authors. All authors read and commented on the manuscript.

Corresponding authors

Correspondence to Ji-Young Lee, Ari Pekka Mähönen or Ykä Helariutta.

Ethics declarations

Competing interests

University of Helsinki, University of Cambridge, Natural Resources Institute Finland (Luke) and Seoul National University have filed one patent application (application no. FI0195659) on the use of constructs and methods that are described here to increase radial growth and biomass in plants, in which J.Z., G.E., J. A.-S., M.K., K.N., J.-Y.L., A.P.M. and Y.H. are listed as inventors. The remaining authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–13, Supplementary Tables 1–8, Supplementary Datasets 1–4, Supplementary References and Supplementary Note.

Reporting Summary

Supplementary Table 1

Cambium enriched gene and gene module identification in Arabidopsis roots.

Supplementary Table 2

Identification of cambium transcription factors.

Supplementary Table 3

Transcript profiling for network and the mutant phenotype prediction.

Supplementary Table 4

Plant materials, cloning vectors and primers used in this study.

Supplementary Table 5

Vascular phenotype characterization in overexpression lines.

Supplementary Table 6

Vascular phenotype characterization of mutants.

Supplementary Table 7

Differentially expressed genes in mutants.

Supplementary Table 8

Cambium genes are perturbed in mutants.

Supplementary Dataset 1

Source data for correlation analyses.

Supplementary Dataset 2

ANOVA output data.

Supplementary Dataset 3

LithoGraphX Cell classifier raw data.

Supplementary Dataset 4

RNA-seq read counts of wild-type (Col) and mutants.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Eswaran, G., Alonso-Serra, J. et al. Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots. Nat. Plants 5, 1033–1042 (2019). https://doi.org/10.1038/s41477-019-0522-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-019-0522-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing