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Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation

Nature Plants volume 7, pages 633–643 (2021)Cite this article

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Abstract

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

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Fig. 1: PFA proteins confer pericycle characteristics.
Fig. 2: PFA mutants are defective in lateral root formation and auxin-induced cell division in the pericycle.
Fig. 3: Repression of PFB1 target genes disrupts the cell character of the pericycle.
Fig. 4: PFA and PFB proteins specifically interact in yeast and in planta.
Fig. 5: PFA and PFB proteins form a feedback loop.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and any raw data can be obtained from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

Special thanks to F. Tobe, T. Ishizuka and Y. Takiguchi for technical assistance regarding the establishment of the Y2H assay system and screening experiments, Chua N.-H. for the pER8 vector, L. Hausmann for the pLH5000 vector, Q.-J. Chen for the CRISPR vector (pHEE401E), T. Beeckman for GATA23 pro::NLS–GFP, M. Umeda for Cytrap, R. Tsien for tdTomato, S. Assmann for the BiFC vectors, E. Dafa Alla for comments on the manuscripts, and the Arabidopsis Biological Resource Centre for the Arabidopsis seeds. This work was supported by JSPS KAKENHI grant numbers 19H03246 and 19K22430 to T.K., MEXT KAKENHI grant numbers 18H04837 and 20H04886 to T.K., MEXT KAKENHI grant number 25113006 to T.K., N.M. and M.M., and a Grant-in-Aid for JSPS Fellow number 13J01792 to Y.Z.

Author information

Author notes

  1. Ye Zhang

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

  2. Takeshi Yoshizumi

    Present address: Takasaki University of Health and Welfare, Takasaki, Japan

Authors and Affiliations

  1. Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan

    Ye Zhang & Tatsuo Kakimoto

  2. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

    Nobutaka Mitsuda, Yoshimi Oshima & Masaru Ohme-Takagi

  3. Global Zero Emission Research Center, AIST, Tsukuba, Japan

    Nobutaka Mitsuda

  4. RIKEN Center for Sustainable Resource Science, Yokohama, Japan

    Takeshi Yoshizumi, Yoko Horii & Minami Matsui

  5. Greenbio Research Center, Graduate School of Science and Engineering, Saitama University, Saitama, Japan

    Masaru Ohme-Takagi

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Contributions

T.K., Y.Z. and N.M. designed the study. Y.Z. performed most of the plant experiments and some of the yeast experiments, and T.K. performed some of the experiments. Y.Z. and T.K. wrote the paper, and N.M. and M.M. commented on the manuscript. N.M. supervised the establishment of the Y2H assay system and the screening experiments. N.M., T.Y., Y.H. and M.M. made the transcription factor library. N.M., Y.O. and M.O.-T. analysed the data obtained from the Y2H assay.

Corresponding author

Correspondence to Tatsuo Kakimoto.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Tom Beeckman, Joseph Dubrovsky and Ben Scheres for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Effects of PFAs on the XPP marker expression of J0121.

a-f, Overexpression of PFA2 (b), PFA3(d), and PFA4 (f) caused ectopic expression of the xylem pole-pericycle marker GFP in the J0121 line. J0121 plants carrying PFA2, PFA3 or PFA4 under an oestrogen-inducible promoter were grown without (a, c, e) or with (b,d,f) 0.1 µM β-oestradiol for 4 (a,b), 7 (c,d), and 6 (e,f) days. Bars, 100 µm. g, PFA1 overexpression was able to induce ectopic J0121 GFP expression only in newly grown parts of the root. Two-day-old plants were transferred to the induction medium containing 0.1 μM β-oestradiol and grown vertically for 2 days. Note that ectopic GFP is present only in the distal position relative to the star (*) mark. Bar, 0.1 mm. a-g, Experiments were repeated once with similar results.

Extended Data Fig. 2 Expression patterns of PFA1, PFA2, PFA3 and PFA4.

a–d, PFA1pro::PFA-GFP. PFA1pro::PFA1-GFP is preferentially expressed in the pericycle and is not expressed in the lateral root primordia, except for some cells at the shoulder of the primordia (c and d). 6-day-old plants. e-g, PFA2 pro::H2B-tdTomato siganl is present in stage-II lateral root primordia (e), and was decreased in outer two layers of stageV and stage-VII lateral root primordia. 6-day old plants. h-k, PFA3 pro::H2B-tdTomato. Signal was present in the stele cells in the meristem (h,i) and in the mature zone (j). The signal was decreased in outer two layers of a stageV primordium (k). l-o, 6-day-old PFA4 pro::H2B-tdTomato seedlings. l,m, PFA4 was expressed in protoxylem in the root apical meristem. m, Cross section of (l). Weak signals were detected in the central cylinder. a-o, scale bars, 50 µm. a-o, Experiments were repeated once with similar results.

Extended Data Fig. 3 Overexpression of PFA2 confers the potential of auxin-induced cell division outside the pericycle.

a, Numbers of anticlinal cell divisions in the pericycle. b, Number of anticlinal cell divisions in the endodermis, cortex, and epidermis. Plants transformed with pER8::PFA2 were grown in the absence or presence of 0.1 µM β-oestradiol for 4 days, and then treated with 1 μM NAA in the absence (grey bars) or presence (red bars) of 0.1 μM β-oestradiol, respectively, for 10 hours. a,b, Mean ± standard error. Two-tailed unpaired Student’s t-tests were used to calculate the P values. n = X biologically independent plants. The experiment was repeated once with similar results.

Source data

Extended Data Fig. 4 Cumulative effects of the ectopic cell division in PFA1 overexpressing root over prolonged auxin treatment.

pER8::PFA1 plants were germinated and grown in absence (-Oest) and in presence (+Oest) of 0.1 μM β-oestradiol for 4 days. For auxin treatment, -Oest and +Oest plant were treated with an auxin, 1-naphthaleneacetic acid (NAA, 1 μM), in absence (-Oest) or in presence (+Oest) of 0.1 μM β-oestradiol, respectively, for 20 hours. Arrowheads, clusters of nuclei in endodermis, cortex, or epidermis.

Extended Data Fig. 5 Overexpression of PFA1 and PFA2 affected LRP development and lateral root emergence.

a, pER8::PFA1 plants were germinated and grown in absence (-Oest) and in presence (+Oest) of 0.1 μM β-oestradiol for 4 days. For auxin treatment, -Oest and +Oest plant were treated with an auxin, 1-naphthaleneacetic acid (NAA, 1 μM), in absence (-Oest) or in presence (+Oest) of 0.1 μM β-oestradiol, respectively, for 70 hours. Pictures showing the region of approximately 3mm-3.5 mm up from QC. b, pER8::PFA2 plants were germinated and grown in absence (-Oest) and in presence (+Oest) of 0.1 μM β-oestradiol for 4 days. For auxin treatment, -Oest and +Oest plant were treated with an auxin, 1-naphthaleneacetic acid (NAA, 1 μM), in absence (-Oest) or in presence (+Oest) of 0.1 μM β-oestradiol, respectively, for 70 hours. Pictures showing the region of approximately 3mm-3.5 mm up from QC. c, A rounded LRP-like structure formed in PFA1-overexpressors when grown in the absence of auxin treatment. pER8::PFA1 plants were grown in the absence (-Oest) or presence (+Oest) of 0.1 μM β-oestradiol for 8 days. a-c, scale bars, 100 µm.

Extended Data Fig. 6 Mutants of the PFA and PFB genes.

For the gene models, the coding regions of the exons are depicted with filled (blue or magenta) boxes (Translational start ATG is at the left ends). Regions corresponding to the bHLH domains are depicted with the magenta colour. We used T-DNA insertion mutants for PFA1, PFA2, and PFA3, for which the insertion positions are shown with yellow flags. Three T-DNA insertional mutants were crossed to generate the pfa1 2 3 triple mutant. A mutation was introduced into the PFA4 gene in the pfa1 2 3 plant to make the pfa1 2 3 4 mutant. A mutation was introduced into the PFA5 gene of a pfa1 2 3 4 plant to make pfa1 2 3 4 5. Then, a mutation was introduced into the PFA6 gene of a pfa1 2 3 4 5 plant to make pfa1 2 3 4 5 6. The pfa1 2 3 4 5 6 mutant line is maintained as PFA1(-/-) PFA2(-/-) PFA3(-/-) PFA4(-/-) PFA5(-/-) PFA6( + /-) heterozygous plants. We mutated PFB1 and PFB2 in GATA23 pro::NLS-GFP line. All mutants made by CRISPR/Cas9 method (pfa4-1. pfa5-1, pfa6-1, pfb1-1, pfb2-1) carried a frameshift mutation that generated a premature stop codon before the conserved bHLH domains.

Extended Data Fig. 7 pfa1 2 3 and pfa1 2 3 4 mutants show decreased auxin-induced cell divisions but normal lateral root formation under normal growth condition.

a, Densities of anticlinally dividing cells in the pericycle of auxin-treated pfa triple and quadruple mutants. Four-day-old plants were treated with 1 µM NAA for the indicated time and the anticlinally-dividing cells (metaphase and anaphase) were examined. b, When grown in the absence of auxin treatment, densities of the lateral root primordia and emerged lateral roots in the 8-day-old-plants of the pfa triple and quadruple mutants were not different from wildtype. a and b, Data are mean ± standard error. P values were calculated using Dunnet’s two-sided multiple comparisons test against WT was applied for each stage. n = X biologically independent plants. Experiments were repeated once with similar results.

Source data

Extended Data Fig. 8 Expression patterns of PFA5 and PFA6.

a-f, PFA5 pro::H2B-tdTomato in a root tip (a,b) and in a mature region (c-f). b, The cross section at the indicated position in (a). PFA5 signal is present in the xylem precursors. c and d, Mature regions. PFA5 is preferentially expressed in the pericycle and vasculature. e. PFA5 was expressed in the stage I primordium, but is down-regulated in later stages of lateral root primordia (f). g-j, The PFA6 pro::PFA6-GFP seedlings. g,h, Root tip. h, The cross section at the indicated position in (g). The signal is present in XPP and xylem precursor cells. i and j, Images of mature regions. The GFP signal is present in stage-I lateral root primordium (i), but is downregulated in later stages (j). Scale bars in a-c and e-j, 50 µm; in d, 10 µm. a-j, Experiments were repeated once with similar results.

Extended Data Fig. 9 Repression of PFA1 target genes impairs the pericycle function for lateral root initiation.

a, J0121 carrying pER8::PFA1-SRDX germinated and grown in the absence (-Oest) or presence (+Oest) of 0.1 µM β-oestradiol for 4-days. The GFP signal was absent when PFA1-SRDX was overexpressed. b, PFA1-SRDX inhibited formation of lateral roots (LR). Plants were germinated and grown in the absence (-Oest) or presence (+Oest) of 0.1 µM β-oestradiol for 11 days. Scale bar, 1 cm. c, Lateral root densities of PFA1-SRDX plants that were germinated and grown in the absence (control) or presence of indicated concentrations of β-oestradiol for 8 days. Tukey’s two-sided multiple comparisons test was applied for each stage. Different letters indicate statistically significant differences (p < 0.05). d, PFA1-SRDX partially impaired auxin-induced cell division in pericycle. Number of anticlinally dividing cells in PFA1-SRDX plants grown in the absence or presence of 0.1 µM β-oestradiol for 4 days and then treated with 1 µM NAA or 1 µM NAA in the absence(grey column) or presence (red column), respectively, of 0.1 µM β-oestradiol for the indicated durations. Two-tailed unpaired Student’s t-tests was used to calculate the P value. In (c) and (d), Data are presented as mean ± standard error. n = X biologically independent plants. a-d, Experiments were repeated once with similar results.

Source data

Extended Data Fig. 10 PFB2-SRDX caused loss of pericycle function for lateral root initiation.

a and b, J0121 carrying pER8::PFB2-SRDX grown in the absence (a) or presence (b) of 1 µM β-oestradiol for 4-days. The GFP signal was absent when PFB2-SRDX was overexpressed (b). c and d, pER8::PFB2-SRDX (J0121) plants grown in the absence (c) or presence (d) of 1 µM β-oestradiol for 11-days. PFB2-SRDX expressing plants lack lateral roots (d). PFB1 (e–h) or PFB2 (i–l) overexpression affected neither the J0121 GFP expression nor lateral root formation. J0121 plants carrying pER8::PFB1(e-h) or pER8::PFB2(i-l) were grown in the absence (e,g,i,k) or presence (f,h,j,l) of 0.1 µM β-oestradiol for 4-days (e,f,i,j) or 11-days (g,h,k,l). m, Densities of the lateral root primordia and emerged lateral roots in the 8-day-old pfb1 pfb2 double mutants. Mean ± standard error. Two-tailed unpaired Student’s t-tests were used to calculate the P values. n = X biologically independent samples. n-q, Expression patterns of PFB1pro::PFB1-GFP in the mature zone (n, o) and the root meristem (p, q). n, The PFB1pro::PFB1-GFP signal was present in the nuclei of the pericycle (red arrows), endodermis (orange arrow), cortex (blue arrow), and (o) in the epidermis (white arrow). Images in n and o are at different z-positions of the same root region. r-t, Expression patterns of PFB2pro::PFB2-GFP in a mature region (r) and the root meristem (t,s). r, Red arrows, the nuclei of pericycle. Orange arrows, endodermis. Bars, 100 µm (a,b,e,f,i,j,n,o,r), 5 mm (c,d,g,h,k,l) and 50 µm (n,o,s,t). a-t, Experiments were repeated once with similar results.

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Supplementary Video 1

A Cytrap plant grown in the presence of 0.1 µM β-oestradiol was overlayed with GM Phytagel containing 0.1 µM β-oestradiol and 1 µM NAA. The CYCB1.1 pro::CYCB1.1–GFP signal was acquired every 20 min. The video starts 190 min after the onset of auxin treatment.

Supplementary Video 2

A Cytrap plant transformed with pER8::PFA1 grown in the presence of 0.1 µM β-oestradiol was overlayed with GM Phytagel containing 0.1 µM β-oestradiol and 1 µM NAA. The CYCB1.1 pro::CYCB1.1–GFP signal was captured every 20 min. The video starts 190 min after the onset of auxin treatment. GFP-positive cells that can be judged to be outside the pericycle are marked with red squares.

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Zhang, Y., Mitsuda, N., Yoshizumi, T. et al. Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation. Nat. Plants 7, 633–643 (2021). https://doi.org/10.1038/s41477-021-00919-9

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