Local auxin biosynthesis is required for root regeneration after wounding

Abstract

The root meristem can regenerate following removal of its stem-cell niche by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized while PIN-mediated transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise temporal pattern.

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Fig. 1: Auxin biosynthesis is required for root tip regeneration.
Fig. 2: Auxin biosynthesis is required throughout the regeneration process.
Fig. 3: Local auxin synthesis by multiple sources during root tip regeneration.
Fig. 4: Stage- and tissue-specific knockdown of YUCs during regeneration.
Fig. 5: PIN-mediated polar auxin transport is not required for regeneration.
Fig. 6: Auxin synthesis and response in regenerating ERF115 dominant negative plants.
Fig. 7: Auxin is sufficient to induce regeneration in high-cut roots.

Data availability

RNA-seq data are available via GEO (series GSE145488).

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Acknowledgements

We thank J. Alonso, A. Stepanova and L. de Veylder for sharing research material, Ken-ichiro Hayashi for providing yucasin DF, M. De Martino and A. Lepar for cloning and nightly assistance, and Y. Eshed and S. Savaldi-Goldstein for comments and discussions. I.E. is supported by the Israeli Science Foundation (grant no. ISF966/17) and the Howard Hughes Medical Institute International Research Scholar Grant (grant no. 55008730).

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Authors

Contributions

R.M. and I.E. conceived and designed the study. R.M., I.C., N.G.Y., A.M. and I.E. carried out the experiments. L.F.S. generated the pGL2:iaaH line. C.V. and E.S. generated pin mutants. R.M. and I.E. wrote the paper.

Corresponding author

Correspondence to Idan Efroni.

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The authors declare no competing interests.

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Peer review informationNature Plants thanks Jose-Manuel Perez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression of sensitive auxin reporters in regenerating root tip.

a–t, Confocal images of uncut (a, k) or regenerating (b-j, l-t) pIAAmotif:mScarleti-NLS (a-j) and R2D2 (k-t), either mock (c,e,g,i,m,o,q,s) or 100 µM L-Kyn treated (d,f,h,j,n,p,r,t). Dotted vertical lines mark the protoxylem. Dashed white lines mark the auxin response peak. Scale bars are 25 µm.

Extended Data Fig. 2 Analysis of gene expression changes in regenerating roots treated with L-Kyn.

a, Number of genes whose expression was modified by L-Kyn in regenerating (Reg.) and uncut roots. b, Venn diagram of the modified genes of regenerating or uncut root meristems following 3hpc and 6hpc of 100 µM L-Kyn treatment. Genes specifically modified by L-kyn in regenerating roots and used for downstream analysis. c-d, Enriched GO terms for genes suppressed by L-Kyn treatment in regenerating root tips at 3hpc (c) and 6hpc (d) (hypergeometric test, one-sided).

Extended Data Fig. 3 Shoot-derived auxin is not required for regeneration.

a, Fluorescent stereoscope images of DR5:3xVENUS-N7 regenerating plants treated with L-Kyn and IAA supplied either to the shoot or the root. Note that while auxin can be transported from the shoot and induce lateral root formation, it does not induce auxin response at the tip. The fluorescence observed at the tip in L-Kyn and L-Kyn plants with shoot-supplied auxin is the remnant expression in the xylem. Arrowheads points to newly formed lateral roots. b, c, Confocal images of regenerating DR5:3xVENUS-N7 roots following shoot removal and treated with mock (b) or 100 µM L-Kyn (c). d, Regeneration rates of shoot-less root tips (p-values are for Tukey HSD on a logistic regression model; n=76, 80 for mock and IAA treated, respectively). Dashed white lines mark the auxin response peak. Scale bars are 25 µm.

Extended Data Fig. 4 Expression of wound-induced YUC during regeneration.

a, b, Annotated (a) and original scans (b) or ethidium bromide-stained gel electrophoresis of YUC1 and YUC4 fragments following 35 cycles of RT-qPCR amplification.

Extended Data Fig. 5 Strong lines of pYUC9:amirYUC develop pin-like terminated meristems.

a, WT floral meristem. b, pYUC9:amirYUC pin-like floral meristem.

Extended Data Fig. 6 Regeneration and tissue pattern recovery in auxin-transport deficient roots.

a–d, Confocal images of uncut (a-b) or cut (c-d) 7 DAS roots before (a,c) or after 72h of 10 µM NPA treatment (b,d). e-l, Confocal images of the ground tissue marker J0571 in uncut (e) or regenerating (f-l) roots treated with mock (f-h) or 10 µM NPA (j-l). Propidium iodide was used to stain cell walls (red). Scale bar are 25 µm.

Extended Data Fig. 7 Root meristem growth, morphology and regeneration of high-order pin mutants.

a, b, Chromatogram (a) and gel electrophoresis (b) of the mutated pin alleles used in this study with original scans provided below. c–f, 7 DAS WT (c) and pin mutants (d-f). gn, Confocal images of uncut (g-j) or regenerating roots at 72hpc (k-n) of WT (g,k) and pin mutants (h-j,l-n). oq, Confocal images of regenerating big DR5:3xVENUS-N7 root tips. Dotted vertical lines mark the protoxylem. Dashed white lines mark the auxin response peak. Propidium iodide was used to stain cell walls (grey). Scale bars are 1cm in (c–f) and 25 µm in (g-q).

Extended Data Fig. 8 Effects of disrupting auxin efflux and influx on regeneration.

ao, Confocal images of regenerating pDR5:3xVENUS-N7 (a,b,d,e,g,h,j,k,m,n) or WT (c,f,i,l,o) plants under 20 µM L-Kyn, and supplemented with 20 nM IAA (d-i), or 20nM NAA (j-o) together with 10 µM NPA (g-i,m-o). Inset in (i) shows root tip at 10 days after regeneration. pq, Confocal images of regenerating DR5:3xVENUS-N7 roots treated with 10 µM 1-NOA. Propidium iodide was used to stain cell walls (red). Scale bar are 25 µm.

Extended Data Fig. 9 Expression of auxin reporters in high-cut root tips.

ac, Confocal images of high-cut pIAAmotif:mScarleti-NLS root tips. d–f, Confocal images of DR5:3xVENUS-N7 high-cut root tips following removal of the shoot. Dotted vertical lines mark the protoxylem. Dashed white lines mark the auxin response peak. Scale bars are 25 µm.

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Matosevich, R., Cohen, I., Gil-Yarom, N. et al. Local auxin biosynthesis is required for root regeneration after wounding. Nat. Plants 6, 1020–1030 (2020). https://doi.org/10.1038/s41477-020-0737-9

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