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.

A Dof-CLE circuit controls phloem organization

Nature Plants volume 8, pages 817–827 (2022)Cite this article

Subjects

Abstract

The phloem consists of sieve elements (SEs) and companion cells (CCs). Here we show that Dof-class transcription factors preferentially expressed in the phloem (phloem-Dofs) are not only necessary and sufficient for SE and CC differentiation, but also induce negative regulators of phloem development, CLAVATA3/EMBRYO SURROUNDING REGION-RELATED25 (CLE25), CLE26 and CLE45 secretory peptides. CLEs were perceived by BARELY ANY MERISTEM (BAM)-class receptors and CLAVATA3 INSENSITIVE RECEPTOR KINASE (CIK) co-receptors, and post-transcriptionally decreased phloem-Dof proteins and repressed SE and CC formation. Multiple mutations in CLE-, BAM- or CIK-class genes caused ectopic formation of SEs and CCs, producing an SE/CC cluster at each phloem region. We propose that while phloem-Dofs induce phloem cell formation, they inhibit excess phloem cell formation by inducing CLEs. Normal-positioned SE and CC precursor cells appear to overcome the effect of CLEs by reinforcing the production of phloem-Dofs through a positive feedback transcriptional regulation.

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

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Fig. 1: Phloem-Dofs are necessary and sufficient for differentiation of phloem cells.
Fig. 2: Phloem-Dof-inducible CLE peptides repress excess formation of phloem.
Fig. 3: The BAM and CIK receptor kinases are required for prevention of excess phloem formation.
Fig. 4: Phloem-Dof proteins are negatively regulated by CLE and positively regulated by phloem-Dofs.
Fig. 5: A model for CLE-mediated lateral inhibition for proper phloem cell patterning.

Data availability

Microarray data have been deposited in the Gene Expression Omnibus under accession number GSE166182. Plasmids for plant transformation and seeds produced in this study are available from P.Q. or T.K. on request. Plasmids used for protein production in insect cells are available from J.C. We used data of GSE6349 and GSE7641 downloaded from the NCBI website for initial screening of candidate genes involved in phloem development. Source data are provided with this paper.

References

  1. Heo, J.-O., Roszak, P., Furuta, K. M. & Helariutta, Y. Phloem development: current knowledge and future perspectives. Am. J. Bot. 101, 1393–1402 (2014).

    Article  PubMed  Google Scholar 

  2. Anne, P. & Hardtke, C. S. Phloem function and development-biophysics meets genetics. Curr. Opin. Plant Biol. 43, 22–28 (2018).

    Article  CAS  PubMed  Google Scholar 

  3. López-Salmerón, V., Cho, H., Tonn, N. & Greb, T. The phloem as a mediator of plant growth plasticity. Curr. Biol. 29, R173–R181 (2019).

    Article  PubMed  CAS  Google Scholar 

  4. Fukuda, H. & Ohashi-Ito, K. Vascular tissue development in plants. Curr. Top. Dev. Biol. 131, 141–160 (2019).

    Article  PubMed  Google Scholar 

  5. Rodriguez-Villalon, A. Wiring a plant: genetic networks for phloem formation in Arabidopsis thaliana roots. New Phytol. 210, 45–50 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Ruonala, R., Ko, D. & Helariutta, Y. Genetic networks in plant vascular development. Annu. Rev. Genet. 51, 335–359 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Miyashima, S. et al. Mobile PEAR transcription factors integrate positional cues to prime cambial growth. Nature 565, 490–494 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Roszak, P. et al. Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization. Science 374, eaba5531 (2021).

    Article  CAS  PubMed  Google Scholar 

  9. Wallner, E.-S. et al. Strigolactone- and karrikin-independent SMXL proteins are central regulators of phloem formation. Curr. Biol. 27, 1241–1247 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Furuta, K. M. et al. Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation. Science 345, 933–937 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Ren, S.-C. et al. CLE25 peptide regulates phloem initiation in Arabidopsis through a CLERK-CLV2 receptor complex. J. Integr. Plant Biol. 61, 1043–1061 (2019).

    CAS  PubMed  Google Scholar 

  12. Anne, P. et al. CLERK is a novel receptor kinase required for sensing of root-active CLE peptides in Arabidopsis. Development 145, dev162354 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Depuydt, S. et al. Suppression of Arabidopsis protophloem differentiation and root meristem growth by CLE45 requires the receptor-like kinase BAM3. Proc. Natl Acad. Sci. USA 110, 7074–7079 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rodriguez-Villalon, A. et al. Molecular genetic framework for protophloem formation. Proc. Natl Acad. Sci. USA 111, 11551–11556 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rodriguez-Villalon, A., Gujas, B., van Wijk, R., Munnik, T. & Hardtke, C. S. Primary root protophloem differentiation requires balanced phosphatidylinositol-4,5-biphosphate levels and systemically affects root branching. Development 142, 1437–1446 (2015).

    CAS  PubMed  Google Scholar 

  16. Graeff, M. & Hardtke, C. S. Metaphloem development in the Arabidopsis root tip. Development 148, 6 (2021).

    Article  CAS  Google Scholar 

  17. Hu, C. et al. A CLE–BAM–CIK signaling module controls root protophloem differentiation in Arabidopsis. New Phytol. https://doi.org/10.1111/nph.17791 (2021).

  18. Gujas, B. et al. A reservoir of pluripotent phloem cells safeguards the linear developmental trajectory of protophloem sieve elements. Curr. Biol. 30, 755–766.e4 (2020).

    Article  CAS  PubMed  Google Scholar 

  19. Hazak, O. et al. Perception of root‐active CLE peptides requires CORYNE function in the phloem vasculature. EMBO Rep. 18, 1367–1381 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Scacchi, E. et al. Spatio-temporal sequence of cross-regulatory events in root meristem growth. Proc. Natl Acad. Sci. USA 107, 22734–22739 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Truernit, E., Bauby, H., Belcram, K., Barthélémy, J. & Palauqui, J.-C. OCTOPUS, a polarly localised membrane-associated protein, regulates phloem differentiation entry in Arabidopsis thaliana. Development 139, 1306–1315 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Breda, A. S. et al. A cellular insulator against CLE45 peptide signaling. Curr. Biol. 29, 2501–2508.e3 (2019).

    Article  CAS  PubMed  Google Scholar 

  23. Anne, P. et al. OCTOPUS negatively regulates BIN2 to control phloem differentiation in Arabidopsis thaliana. Curr. Biol. 25, 2584–2590 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. 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  PubMed  PubMed Central  Google Scholar 

  25. Yanagisawa, S. The Dof family of plant transcription factors. Trends Plant Sci. 7, 555–560 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Zuo, J., Niu, Q.-W. & Chua, N.-H. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265–273 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Hiratsu, K., Matsui, K., Koyama, T. & Ohme-Takagi, M. Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34, 733–739 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Khan, J. A., Wang, Q., Sjölund, R. D., Schulz, A. & Thompson, G. A. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis. Plant Physiol. 143, 1576–1589 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Shaner, N. C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bonke, M., Thitamadee, S., Mähönen, A. P., Hauser, M.-T. & Helariutta, Y. APL regulates vascular tissue identity in Arabidopsis. Nature 426, 181–186 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Ito, Y. et al. Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 313, 842–845 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Hirakawa, Y. et al. Non-cell-autonomous control of vascular stem cell fate by a CLE peptide/receptor system. Proc. Natl Acad. Sci. USA 105, 15208–15213 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Stadler, R. et al. Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots. Plant J. 41, 319–331 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. De Rybel, B. et al. A bHLH complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis. Dev. Cell 24, 426–437 (2013).

    Article  PubMed  CAS  Google Scholar 

  35. Qian, P. et al. The CLE9/10 secretory peptide regulates stomatal and vascular development through distinct receptors. Nat. Plants 4, 1071–1081 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Kubo, M. Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev. 19, 1855–1860 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Schuetz, M. et al. Laccases direct lignification in the discrete secondary cell wall domains of protoxylem. Plant Physiol. 166, 798–807 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. O’Malley, R. C. et al. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell 165, 1280–1292 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Ohyama, K., Shinohara, H., Ogawa-Ohnishi, M. & Matsubayashi, Y. A glycopeptide regulating stem cell fate in Arabidopsis thaliana. Nat. Chem. Biol. 5, 578–580 (2009).

    Article  CAS  PubMed  Google Scholar 

  40. Shiu, S.-H. & Bleecker, A. B. Expansion of the receptor-like kinase/pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiol. 132, 530–543 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Corcilius, L. et al. Arabinosylation modulates the growth-regulating activity of the peptide hormone CLE40a from soybean. Cell Chem. Biol. 24, 1347–1355.e7 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Gou, X. & Li, J. Paired receptor and coreceptor kinases perceive extracellular signals to control plant development. Plant Physiol. 182, 1667–1681 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hu, C. et al. A group of receptor kinases are essential for CLAVATA signalling to maintain stem cell homeostasis. Nat. Plants 4, 205–211 (2018).

    Article  CAS  PubMed  Google Scholar 

  44. Hohmann, U., Lau, K. & Hothorn, M. The structural basis of ligand perception and signal activation by receptor kinases. Annu. Rev. Plant Biol. 68, 109–137 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci. Rep. 6, 20889 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. De Smet, I. et al. Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root. Science 322, 594–597 (2008).

    Article  PubMed  CAS  Google Scholar 

  47. Dinneny, J. R. et al. Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320, 942–945 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. Dalrymple, M. A., McGeoch, D. J., Davison, A. J. & Preston, C. DNA sequence of the herpes simplex virus type 1 gene whose product is responsible for transcriptional activation of immediate early promoters. Nucleic Acids Res. 13, 7865–7879 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kurihara, D., Mizuta, Y., Sato, Y. & Higashiyama, T. ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142, 4168–4179 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods https://doi.org/10.1038/nmeth.2019 (2012).

  51. Paciorek, T., Sauer, M., Balla, J., Wiśniewska, J. & Friml, J. Immunocytochemical technique for protein localization in sections of plant tissues. Nat. Protoc. 1, 104–107 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Wang, Z.-P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

We thank Y. Sugisawa and K. Ohashi-Ito for microarray experiments; J. Wang and S. Huang for ITC experiments; N.-H. Chua for ER8 vector, S. Takada for RPS5a-ER8, Q.-J. Chen for the CRISPR vector (pHEE401E); Y. Kondo, H. Fukuda and S. Sawa for providing CLE peptides; Y. Matsubayashi, H. Shinohara and the Arabidopsis Biological Resource Center for mutant seeds; and R. Lewis from Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (JP25113006, JP18H04837, JP19H03246, JP19K22430, JP20H04886 and JP21K19264 to T.K.; JP19K23750 and JP21K15123 to P.Q.; JPL17545 to G.W.), by National Natural Science Foundation of China (31771556) to G.W., by the Alexander von Humboldt Foundation (a Humboldt professorship to J.C.) and the Max-Planck-Gesellschaft (a Max Planck fellowship to J.C.).

Author information

Author notes

  1. Miki Zaizen-Iida

    Present address: Organismal and Evolutionary Biology Research Programme, Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland

  2. Ye Zhang

    Present address: Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, Japan

  3. These authors contributed equally: Pingping Qian, Wen Song.

Authors and Affiliations

  1. Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan

    Pingping Qian, Miki Zaizen-Iida, Sawa Kume, Guodong Wang, Ye Zhang, Kaori Kinoshita-Tsujimura & Tatsuo Kakimoto

  2. Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Wen Song & Jijie Chai

  3. Max Planck Institute for Plant Breeding Research, Cologne, Germany

    Wen Song & Jijie Chai

  4. Institute of Biochemistry, University of Cologne, Cologne, Germany

    Wen Song & Jijie Chai

  5. Cluster of Excellence in Plant Sciences (CEPLAS), Düsseldorf, Germany

    Wen Song & Jijie Chai

  6. College of Life Sciences, Shaanxi Normal University, Xi’an, China

    Guodong Wang

Authors
  1. Pingping Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wen Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miki Zaizen-Iida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sawa Kume
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guodong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ye Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kaori Kinoshita-Tsujimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jijie Chai
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Tatsuo Kakimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.Q. and T.K. conceived the core concept of the study. P.Q., W.S., M.Z.-I., S.K., G.W., Y.Z. and T.K. designed the experiments. P.Q., M.Z.-I., S.K., G.W., Y.Z., K.K.-T. and T.K. performed plant-related experiments. W.S. performed receptor-ligand binding experiments. P.Q., W.S., J.C. and T.K. wrote the paper.

Corresponding authors

Correspondence to Pingping Qian or Tatsuo Kakimoto.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Antia Rodriguez-Villalon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 Phloem-Dofs positively regulate vascular cell file numbers.

a, The spatial pattern of gene expression mediated by ER8, which was used for overexpression in this study. The GFP signal of ER8::H2B-GFP was present in all cell types in the root tip, although expression in cortex was weak after 12 hours treatment of 10 µM oestriol. b,c, Non-xylem cell file number (b) and xylem cell file number (c) in wild-type vascular tissue (pericycle is not included), ER8::Dof1.1, and ER8::Dof1.1-SRDX grown in the presence of 100 nM oestriol. d, Representative images of (b) and (c). e,f, Non-xylem cell file number in 5-day-old wild-type and dof mutant plants, and a dof-sextuple mutant line transformed with PEAR1pro::PEAR1-mNG::PEAR1ter. g,h, Xylem cell file number in 5-day-old plants of the wild-type and dof mutants, and a dof-sextuple mutant line transformed with PEAR1pro::PEAR1-mNG::PEAR1ter. i, The expression pattern of PEAR1pro::PEAR1-mNG::PEAR1ter in the phloem region of 5-day-old dof-sextuple root vasculature. b,c,e–h, Data are means ± s.e.m. Different letters indicate significant difference at adjusted P < 0.05 by two-sided Tukey’s multiple comparisons test. n, numbers of biologically independent samples. Bars, 20 µm.

Source data

Extended Data Fig. 2 Phloem-Dofs are necessary and sufficient for induction of the regulatory system for phloem development.

a, APLpro::nls3GFP. b-e, Overexpression of PEAR1 (b), PEAR2 (c), Dof3.2(d) or Dof5.3(e) in plants carrying APLpro::nls3GFP. Three-day-old plants were treated with 10 µM oestriol for 20 h. f, Dominant repression of Dof1.1-target genes by Dof1.1-SRDX inhibited the APLpro::nls3GFP expression in 5-day-old plants grown in the presence of 100 nM oestriol. In the presented plant, one pole lacks SEs and had a very faint APL signal, and the other pole possesses only PSE. g, h, Overexpression of Dof1.1 (g) or PEAR2 (h) caused ectopic GFP expression in a protophloem marker line J0701. Five-day-old plants were treated with 10 μM oestriol for 2 days. i, In control plants, NAC45pro::H2B-mRuby3 was expressed in the PSE cell file (inset, only mRuby3 channel). Overexpression of Dof1,1 caused ectopic expression of NAC45 (right), while the GFP signal of the GATA20pro::erGFP (S32) was unchanged (middle and right). Position of the cross-section view (top) is indicated with a blue line. Five-day-old plants were treated with 10 μM oestriol for 40 h. Projection: maximum projection images. Scale bars in a-f and i, 20 µm, f, 100 µm.

Extended Data Fig. 3 Overexpression of phloem-Dofs causes ectopic expression of the CC marker gene SUC2, but not xylem marker genes.

a, SUC2pro::GFP-GUS is normally expressed in CCs. b-f, Overexpression of Dof1.1, Dof2.2, PEAR1, Dof3.2, and Dof5.3 causes ectopic expression of SUC2. h-j, Overexpression of Dof2.2 does not cause ectopic expression of xylem markers, TMO5 (h), CLE9 (i) and Laccase4 (j). In (i), the outline of the root is delineated with white lines. QC, positions of the quiescent centre. a-j, Five-day-old plants were treated with 10 μM oestriol for durations described in the figure.

Extended Data Fig. 4 The effect of Dof2.2 overexpression on the CLE45 expression and effects of CLE 25, CLE26 and CLE45 peptides on phloem development and root growth.

a, TheCLE45pro::H2B-GFP was expressed specifically in the protophloem of 5-day-old plants (left, maximum projection image; middle, cross-section; positions of cross-sections are shown with a blue line) and was ectopically expressed after 40 h of Dof2.2 induction (right). b, Ectopic expression signals of CLE25pro::H2B-GFP::CLE25intron-ter were visible as early as 4 h Dof2.2 induction by oestradiol. c-f, APLpro::nls3GFP expression and the strong SR2200 staining characteristic of SEs are missing when grown on 10 nM CLE25 (d, ~700 μm from QC), CLE26 (e, ~200 μm from QC) and CLE45 (f, ~200 μm from QC) treatment as compared to APLpro::nls3GFP control (c, ~1000 μm from QC). At normal conditions, MSE differentiation starts before metaxylem differentiation. But in (d), metaxylem was differentiated but neither PSE nor MSE was differentiated. Pericycle cells are marked with orange dots. Phloem regions are encircled with dotted lines. g, h, Effects of different concentrations of CLE25, CLE26 and CLE45 on root length (g) and phloem development (judged by the presence of APLpro::nls3GFP signal and the SR2200 staining in one or two sides of the differentiation zone) (h). Two-way ANOVA for (g); peptide: F(2, 220) = 20.17, P < 0.0001; concentration: F(5, 220) = 584.2, P < 0.0001; interaction: F(10, 220) = 4.801, P < 0.0001; different letters indicate significant difference at P < 0.05 by Tukey’s test. Data are means ± s.e.m. n indicates biologically independent samples. Two-way ANOVA for (h); peptide: F(2, 214) = 13.91, P < 0.0001; concentration: F(5, 214) = 294.9, P < 0.0001; interaction: F(10, 214) = 9.015, P < 0.0001; different letters indicate significant difference at P < 0.05 by Dunn’s test. n = X biologically independent samples.

Source data

Extended Data Fig. 5 BAM and CIK are required for CLE25/26/45-mediated regulation of phloem development.

a, Effects of CLE25, CLE26 and CLE45 peptides on the root growth of bam mutants. Plants were grown for 5 days on plates containing 10 nM peptides. Data are means ± s.e.m. Two-way ANOVA; peptide: F(3, 284) = 357.4, P < 0.0001; genotype: F(3, 284) = 441.1, P < 0.0001; interaction: F(9, 284) = 66.18, P < 0.0001; different letters indicate significant difference at P < 0.05 by Tukey’s test. n, biologically independent sample number. b, Effects of CLE25 on the root growth of cik2, cik3, and the cik2/3 double mutant. Data are means ± s.e.m. Two-way ANOVA; peptide: F(1, 89) = 87.98, P < 0.0001; genotype: F(3, 89) = 42.31, P < 0.0001; interaction: F(3, 89) = 18.94, P < 0.0001; different letters indicate significant difference at P < 0.05 by Sidak’s test. n, biologically independent sample number. c, Effects of CLE25 or CLE45 (synthetic peptide, 100 nM) on phloem development. Five-day-old plants on GM plates were moved onto plates containing CLE peptides for 2 days treatment. d, Effects of CLE25 or CLE45 overexpression on phloem development. Five-day-old two individual T2 lines of ER8::CLE25 and ER8::CLE45 were treated by 10 μM oestriol for 2 days. c,d, All plants contain APLpro::nls3GFP. Percent of plants that have phloem on two sides (green) and on one side (light green), and that have no phloem (open box). The presence of phloem was determined by the presence of the APLpro::nls3GFP signal and the SR2200 staining at newly grown parts after the start of treatment. Different letters indicate significant difference at P < 0.05 by two-sided Dunn’s test. n, biologically independent sample number.

Source data

Extended Data Fig. 6 Effects of CLE25 on SE formation in the bam and cik mutants and SE cell file numbers in clv2, rpk2, and cle25/26/45 mutants.

a, Effects of CLE25 on SE formation in the bam and cik mutants. Cross-sections of the vasculatures at the meristem zone (left) and differentiation zone (right) in 8-day-old WT, bam1/3, bam1/2/3, cik2 and cik2/3 plants grown in the absence or presence of 10 nM CLE25. SE files are marked with a purple “+”, pericycle cells are marked with yellow dots. b, The SE file numbers in the wild-type and clv2, rpk2 and cle25/26/45 mutants. Data are means ± s.e.m. **Adjusted P < 0.01 (two-sided Dunnett’s multiple comparison test) relative to the wild-type. NS, not significant (adjusted P ≥ 0.05). n, biologically independent sample number.

Source data

Extended Data Fig. 7 ITC assay for receptor-ligand interaction.

Quantification of binding affinities by ITC assays. Up, CLE peptides were titrated into BAM1LRR. Bottom, CLE peptides were titrated into BAM3LRR. The binding constants (Kd values ± fitting errors) and stoichiometries (N) are indicated. The experiments were repeated three times with similar results.

Extended Data Fig. 8 Effects of CLE25 treatment on the promoter activities of PEAR1, APL, and GATA20, and the PEAR1-mNG protein level.

a, Effects of 10 nM CLE25 on the signals of the PEAR1pro::H2B-mNG::35Ster::PEAR1gene::PEAR1ter transcriptional fusion gene (top) and PEAR1pro::PEAR1-mNG::PEAR1ter translational fusion gene (bottom). b, Effects of 10 nM CLE25 on the signals of APLpro::nls3GFP. c, Effects of 10 nM CLE25 on the signals on GATA20pro::erGFP. Maximum projection images are shown. Bars, 20 µm. At least 5 plants were observed with similar results in every experiment, and we repeated similar experiments at least 3 times with similar results.

Extended Data Fig. 9 PEAR1pro::PEAR1-mNG::PEAR1ter signal is present in expanded regions in the cik2/3 mutant.

Plants grown on MS medium with 0.5% sucrose for 11 days were used. Bars, 20 µm.

Extended Data Fig. 10 Mutual- and self-regulation of phloem-Dofs.

a, Dof2.2 overexpression induces ectopic expression of Dof1.1pro::nls3GFP. b, Dof2.2 overexpression induces ectopic expression of Dof2.2pro::nls3GFP. We do not know the reason why the signal is partially in the cytoplasm. c, Dof1.1 overexpression induces ectopic expression of Dof2.3pro::H2B-mRuby3. d, Effects of Dof1.1 overexpression on Dof1.5pro::H2B-mRuby3. Bars, 20 µm. Plants were treated with 10 µM oestriol for 24 h (a, b) or 15 h (c, d). Bars, 20 µm.

Supplementary information

Supplementary Information

Supplementary figures and information.

Reporting Summary

Supplementary Table 1

Screening.

Supplementary Table 2

Microarray.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Supplementary Fig. 7

Statistical source data.

Source Data Supplementary Fig. 9

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Qian, P., Song, W., Zaizen-Iida, M. et al. A Dof-CLE circuit controls phloem organization. Nat. Plants 8, 817–827 (2022). https://doi.org/10.1038/s41477-022-01176-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-022-01176-0

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