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Spatially expressed WIP genes control Arabidopsis embryonic root development

Nature Plants volume 8, pages 635–645 (2022)Cite this article

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Abstract

Development of plant organs is a highly organized process. In Arabidopsis, proper root development requires that distinct cell types and tissue layers are specified and formed in a restricted manner in space and over time. Despite its importance, genetic controls underlying such regularity remain elusive. Here we found that WIP genes expressed in the embryo and suspensor functionally oppose those expressed in the surrounding maternal tissues to orchestrate cell division orientation and cell fate specification in the embryonic root, thereby promoting regular root formation. The maternal WIPs act non-cell autonomously to repress root cell fate specification through SIMILAR TO RADICAL-INDUCED CELL DEATH ONE (SRO) family members. When losing all WIPs, root cells divide irregularly in the early embryo, but this barely alters their fate specification and the morphology of post-embryonic roots. Our results reveal cross-communication between the embryonic and maternal WIPs in controlling root development.

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Fig. 1: WIP genes regulate root cell division orientation.
Fig. 2: WIP1, WIP3 and/or WIP6 act non-cell autonomously to repress root cell specification.
Fig. 3: SRO family members are required for the WIP-mediated growth arrest and embryonic root development.
Fig. 4: Maternal WIPs act through SRO family members to inhibit root formation.
Fig. 5: Schematic model.

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Data availability

The RNA-seq data of wild-type and wip123456 primary root meristems have been deposited to Sequence Read Archive (PRJNA774717). All data supporting the findings of this study are available in this Article and its Supplementary Information, or from A. Bendahmane upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank M. Crespi and T. Blein for helpful discussions; B. Scheres for critical reading of the manuscript; H. Morin, C. Troadec, A. d. B. d. Granrut, all FLOCAD team members, the imaging platform and greenhouse teams at the Institute of Plant Sciences Paris-Saclay (IPS2) for technical support; and the Eurasian Arabidopsis Stock Centre (uNASC) for sharing research materials. This work was supported by the European Research Council (ERC-SEXYPARTH, 341076), the ANR (EPISEX, ANR-17-CE20-0019), and the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-40-SPS). M.V.G.R. was supported by the Intra-European Fellowships for Career Development (IEF) (Grant PIEF-GA-2012-330908).

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  1. Institute of Plant Sciences Paris-Saclay (IPS2), INRAE, CNRS, University of Paris-Saclay, University of Evry, University of Paris Cité, Gif sur Yvette, France

    Yujuan Du, Maria Victoria Gomez Roldan, Aimen Haraghi, Nawel Haili, Farhaj Izhaq, Marion Verdenaud, Adnane Boualem & Abdelhafid Bendahmane

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Contributions

Y.D. and A. Bendahmane conceptualized the project; Y.D. and A. Bendahmane developed the methodology; A. Boualem and M.V. conducted formal analysis; Y.D., M.V.G.R., A.H., N.H. and F.I. conducted the investigations; Y.D., A.H. and F.I. procured resources; Y.D. wrote the original draft; Y.D., M.V.G.R., A. Boualem, M.V. and A. Bendahmane reviewed and edited the draft; Y.D. and A. Bendahmane supervised the project; A. Bendahmane acquired funding and administered the project.

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Correspondence to Yujuan Du or Abdelhafid Bendahmane.

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Extended data

Extended Data Fig. 1 WIP genes regulate root cell division orientation.

a, Overview of wild type, wip245 (nww), wip2-4wip45 and wip2-3wip45 seedlings at 7 day-post-germination (d.p.g.). wip2-3 allele: SM_3_16705; wip2-4 allele: SM_3_23211. Scale bar: 1 cm. b-j, Images of wip245 and wip123456 embryonic roots at indicated stages. The number presented at the bottom of each image represents the counts of indicated phenotype (left) versus the total counts (right). G1: early-globular stage; G2: late-globular stage; H1: early-heart stage; H2: late-heart stage; ME: matured embryo. Magenta and blue frames: the zoom-in areas. White arrows in j indicate COL layer; white asterisks in j mark the newly formed COL cells; colored asterisks in h-i indicate possible cell division patterns from H1 to H2. Cyan: hypophysis/QC lineage; yellow: COL initial lineage; orange: COL layers; grey: delayed/failed layer formation; light purple: ground tissue initials; olive green: Epi/LRC initials; pink: vascular initials. QC: quiescent center; COL: columella; Epi: epidermis; LRC: lateral root cap. Scale bars: 50 μm. Related to Fig. 1.

Extended Data Fig. 2 Cell fate specification in wip123456 and wip245 embryonic roots.

a-e, Images of embryos, suspensors and primary roots expressing indicated reporters in wild type, wip245 and wip123456 mutants. Scale bars: 50 μm. h, Frequency and counts of the suspensors with or without DR5::GFP expression in their basal cells. Wild type, wip245, wip136 and wip123456 suspensors between globular and heart stage were sampled. Data in the frequency (the upper panel) represents mean ± s.d. from four biological replicates; sample size per replicate (n) =20. P values were calculated with two-tailed unpaired Student’s t test, mutant versus wild type: ***P < 0.005. Data in the suspensor counts (the lower panel) represents total number of examined suspensors, including but not restricting to the one used in the frequency experiments. Red lines in d,e highlight cell outlines of the wip245 hypophyseal derivatives; white arrows indicate the DR5::GFP expression in basal cells of the suspensor; white frames highlight cell outlines of the hypophyseal derivatives. Cyan dots: cells in hypophysis/QC lineage; yellow dots: cells in COL initial lineage; orange dots: cells in COL layers; grey dots: cells in delayed/failed layer formation. PR: primary root at 3 d.p.g.; QC: quiescent center; COL: columella. The experiments in a,b,f,d,e and c,g were repeated four and three times respectively, with similar results. Related to Fig. 2.

Source data

Extended Data Fig. 3 WIP1, WIP3 and WIP6 are maternally expressed.

a-d, Images of wild type, wip1 and proWIP1::cWIP1:VENUS complemented wip1 (L-12 and L-14) seeds. Scale bars: 1 mm. e-j, Images of wild type embryos, suspensors and their surrounding maternal tissues expressing indicated WIP reporters. Scale bars for e-h,j: 50 μm; scale bar for i: 1 mm. k-m, Images of wip2+/−45 siliques and developing seeds expressing indicated WIP reporters. Scale bar for k: 50 μm; scale bars for the silique panel in l,m: 1 mm; scale bars for the seed panel in l: 100 μm. n, RT-qPCR analysis of WIP1 and WIP3 transcription in wild type and wip2+/−45 siliques containing embryos between globular and heart stage. Data represents mean ± s.e.m. from three biological replicates, within each three technical repeats were included. P values were calculated with two-tailed unpaired Student’s t test, mutant versus wild type: *P < 0.05, **P < 0.01. o-q, Images of wip245 embryos and suspensors expressing indicated WIP reporters. Scale bars: 50 μm. White arrow in g: the proWIP3::gWIP3:VENUS signal. Cyan dots: cells in hypophysis/QC lineage; yellow dots: cells in COL initial lineage; orange dots: cells in COL layers. M: micropylar end; C: chalazal end; oi2: outer integument 2; oi1: outer integument 1 and ii1: inner integument 1 (endothelium); QC: quiescent center; COL: columella. The experiments in e-m and o-q were repeated three times, with similar results. Related to Fig. 2.

Source data

Extended Data Fig. 4 Embryonic expression of WIP genes promotes root formation.

a, GUS-staining of proWIP4::GUS in wild type siliques and developing seeds. Scale bar for the silique panel: 1 mm; scale bar for the seed panel: 100 μm. b, Images of wild type embryos, suspensors and primary roots expressing proWIP4::erCFP. Scale bars: 50 μm. c, Images of proWIP4::gWIP4:VENUS complemented wip245 embryonic and primary roots. Scale bar: 50 μm. d, Overview of wild type, wip245 and proWIP4::gWIP4:VENUS complemented wip245 (L-1 and L-5) seedlings at 7d.p.g.. Scale bars: 1 cm. e, Overview of wild type, wip245, proWIP4::cWIP1:VENUS complemented wip245 (L-5 and L-4) seedings at 7d.p.g.. Scale bars: 1 cm. Yellow frame and arrows: the proWIP4::gWIP4:VENUS signal in the uppermost suspensor cell; white frames highlight cell outlines of the hypophyseal derivatives. Cyan dots: cells in hypophysis/QC lineage; yellow dots: cells in COL initial lineage; orange dots: cells in COL layers. M: micropylar end; C: chalazal end; PR: primary root at 3 d.p.g.; QC: quiescent center; COL: columella. The experiments in a and b-e were repeated two and three times respectively, with similar results. Related to Fig. 2.

Extended Data Fig. 5 WIP1 inhibits plant growth via SRO family members.

a, Overview of wild type, rcd1-4, pOp6::cWIP1, 35::LhGR, DEX:WIP1, q195 and rcd1-4 DEX:WIP1 seedlings germinated on 1/2 MS medium supplemented with 30 nM DEX at 7 d.p.g.. Two biological replicates were performed. Scale bar: 1 cm. b, Left panel: overview of 35 S::LhGR, DEX:WIP1, q195 and rcd1-4 DEX:WIP1 seedlings grown on 1/2 MS medium supplemented with 30 nM DEX for 48 h. Right panel: quantification of the 48h-root growth. c, Left panel: overview of 35 S::LhGR, DEX:WIP1 and sro1 DEX:WIP1 seedlings grown on 1/2 MS medium supplemented with 30 nM DEX for 48 h. Right panel: quantification of the 48h-root growth. Black dots in b,c mark the root tip positions when the seedlings were freshly transferred, the 48-root growth was measured from the black dot to the root tip. Data represents mean ± s.e.m. from four biological replicates; sample size per replicate (n) =15. Mean value of the 48h-root growth on the mock medium is set to 100%. P values were calculated with two-tailed unpaired Student’s t test, 35 S::LhGR, q195, rcd1-4 DEX:WIP1 and sro1 DEX:WIP1 versus DEX:WIP1 respectively: ***P < 0.005. Scale bar: 1 cm. Related to Fig. 3.

Source data

Extended Data Fig. 6 RCD1 and SRO1 expression.

a, RT-qPCR analysis of RCD1 and SRO1 transcription in wild type siliques containing embryos between globular and heart stage. Data represents mean ± s.e.m. from two biological replicates, within each three technical repeats were included. P values were calculated with two-tailed unpaired Student’s t test, RCD1 versus SRO1: ***P < 0.005. b-e, Images of wild type siliques, developing seeds, embryos, suspensors and primary roots expressing indicated RCD1 and SRO1 reporters. Scale bars for b,d: 1 mm; scale bars for c,e: 50 μm. f-g, Images of proRCD1::gRCD1:VENUS complemented rcd1-4 and proSRO1::gSRO1:VENUS complemented sro1 embryos and primary roots. Scale bars: 50 μm. h, Frequency of wild type and rcd1-4sro1 roots with or without periclinally divided QC cells at indicated stages. Data represents mean ± s.d.; biological replicates (N) and sample size per replicate (n) are listed in Supplementary Table 6. P values were calculated with two-tailed unpaired Student’s t test, rcd1-4sro1 versus wild type: ***P < 0.005, P1 = 0.00098, P2 = 7.67E-05, P3 = 8.05E-05. H1: early-heart stage; H2: late-heart stage; ME: mature embryo. The experiments in b-g were repeated three times, with similar results. Related to Fig. 3.

Source data

Extended Data Fig. 7 The maternal WIPs act through SRO members to inhibit embryonic root formation.

a, mPS-PI staining of amyloplasts in wild type and rcd1-4wip245 primary roots. Scale bar: 100 μm. b, Quantification of COL layer numbers in wild type, rcd1-4wip245 and sro1wip245 mature embryos and primary roots. Data represents mean ± s.e.m.; biological replicates (N) and sample size per replicate (n) are listed in Supplementary Table 7. P values were calculated with two-tailed unpaired Student’s t test, mutant versus wild type: ***P < 0.005. c-e, sro1wip245 embryonic roots at indicated stages. The number presented at the bottom of each image represents the counts of indicated phenotype (left) versus the total counts (right). G1: early-globular stage; G2: late-globular stage; H1: early-heart stage; H2: late-heart stage; ME: matured embryo. Scale bars: 50 μm. f-h, Images of sro1wip245 embryos, suspensors and primary roots expressing indicated markers. White frames highlight cell outlines of the hypophyseal derivatives. Cyan dots: cells in hypophysis/QC lineage; yellow dots: cells in COL initial lineage; grey dots: cells in delayed/failed layer formation. Scale bars: 50 μm. Cyan: hypophysis/QC lineage; yellow: COL lineage; orange: COL layers; grey: delayed/failed layer formation; light purple: ground tissue initials; olive green: Epi/LRC initials. PR: primary root at 3 d.p.g.; QC: quiescent center; COL: columella; Epi: epidermis; LRC: lateral root cap. The experiments in a, f-g and h were repeated two, four and three times respectively, with similar results. Related to Fig. 4.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–8.

Reporting Summary

Supplementary Table

Supplementary Table 4 RNA-seq analysis of wild type and wip123456 primary root meristems. Supplementary Table 5 Frequency of embryonic roots with normal or delayed/failed layer formation. Supplementary Table 6 Frequency of wild type and rcd1-4sro1 roots with or without periclinally divided QC cells. Supplementary Table 7 Quantification of COL layer numbers. Supplementary Table 8 Source data for supplementary figures.

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Du, Y., Roldan, M.V.G., Haraghi, A. et al. Spatially expressed WIP genes control Arabidopsis embryonic root development. Nat. Plants 8, 635–645 (2022). https://doi.org/10.1038/s41477-022-01172-4

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