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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RNA-seq data are available via GEO (series GSE145488).
Heidstra, R. & Sabatini, S. Plant and animal stem cells: similar yet different. Nat. Rev. Mol. Cell Biol.15, 301–312 (2014).
Feldman, L. J. The de novo origin of the quiescent center regenerating root apices of Zea mays. Planta128, 207–212 (1976).
Sena, G., Wang, X., Liu, H.-Y., Hofhuis, H. & Birnbaum, K. D. Organ regeneration does not require a functional stem cell niche in plants. Nature457, 1150–1153 (2009).
Reinhardt, D., Frenz, M., Mandel, T. & Kuhlemeier, C. Microsurgical and laser ablation analysis of interactions between the zones and layers of the tomato shoot apical meristem. Development130, 4073–4083 (2003).
Efroni, I. A conceptual framework for cell identity transitions in plants. Plant Cell Physiol.59, 696–706 (2018).
Xu, J. et al. A molecular framework for plant regeneration. Science311, 385–388 (2006).
Efroni, I. et al. Root regeneration triggers an embryo-like sequence guided by hormonal interactions. Cell165, 1721–1733 (2016).
Heyman, J. et al. The heterodimeric transcription factor complex ERF115–PAT1 grants regeneration competence. Nat. Plants2, 16165 (2016).
Zhou, W. et al. A jasmonate signaling network activates root stem cells and promotes regeneration. Cell177, 942–956 (2019).
Weijers, D. & Wagner, D. Transcriptional responses to the auxin hormone. Annu. Rev. Plant Biol.67, 539–574 (2016).
Zhao, Y. Auxin biosynthesis and its role in plant development. Annu. Rev. Plant Biol.61, 49–64 (2010).
Swarup, R. & Péret, B. AUX/LAX family of auxin influx carriers: an overview. Front Plant Sci.3, 225 (2012).
Adamowski, M. & Friml, J. PIN-dependent auxin transport: action, regulation, and evolution. Plant Cell27, 20–32 (2015).
Grieneisen, V. A., Xu, J., Marée, A. F. M., Hogeweg, P. & Scheres, B. Auxin transport is sufficient to generate a maximum and gradient guiding root growth. Nature449, 1008–1013 (2007).
Sabatini, S. et al. An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell99, 463–472 (1999).
Petricka, J. J., Winter, C. M. & Benfey, P. N. Root development. Annu. Rev. Plant Biol.63, 563–590 (2012).
Petersson, S. V. et al. An auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA distribution and synthesis. Plant Cell21, 1659–1668 (2009).
Chen, Q. et al. Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots. Plant Cell Physiol.55, 1072–1079 (2014).
Brumos, J. et al. An improved recombineering toolset for plants. Plant Cell32, 100–122 (2020).
Brumos, J. et al. Local auxin biosynthesis is a key regulator of plant development. Dev. Cell47, 306–318 (2018).
Petrásek, J. et al. Do phytotropins inhibit auxin efflux by impairing vesicle traffic? Plant Physiol.131, 254–263 (2003).
Xu, D. et al. YUCCA9-mediated auxin biosynthesis and polar auxin transport synergistically regulate regeneration of root systems following root cutting. Plant Cell Physiol.58, 1710–1723 (2017).
Sztein, A. E., Ilić, N., Cohen, J. D. & Cooke, T. J. Indole-3-acetic acid biosynthesis in isolated axes from germinating bean seeds: the effect of wounding on the biosynthetic pathway. Plant Growth Regul.36, 201–207 (2002).
Chen, L. et al. YUCCA-mediated auxin biogenesis is required for cell fate transition occurring during de novo root organogenesis in Arabidopsis. J. Exp. Bot.67, 4273–4284 (2016).
Druege, U., Franken, P. & Hajirezaei, M. R. Plant hormone homeostasis, signaling, and function during adventitious root formation in cuttings. Front. Plant Sci.7, 381 (2016).
Tsugafune, S. et al. Yucasin DF, a potent and persistent inhibitor of auxin biosynthesis in plants. Sci. Rep.7, 13992 (2017).
He, W. et al. A small-molecule screen identifies l-kynurenine as a competitive inhibitor of TAA1/TAR activity in ethylene-directed auxin biosynthesis and root growth in Arabidopsis. Plant Cell23, 3944–3960 (2011).
Ulmasov, T., Liu, Z. B., Hagen, G. & Guilfoyle, T. J. Composite structure of auxin response elements. Plant Cell7, 1611–1623 (1995).
Lieberman-Lazarovich, M., Yahav, C., Israeli, A. & Efroni, I. Deep conservation of cis-element variants regulating plant hormonal responses. Plant Cell31, 2559–2572 (2019).
Liao, C.-Y. et al. Reporters for sensitive and quantitative measurement of auxin response. Nat. Methods12, 207–210 (2015).
Brady, S. M. et al. A high-resolution root spatiotemporal map reveals dominant expression patterns. Science318, 801–806 (2007).
Cheng, Y., Qin, G., Dai, X. & Zhao, Y. NPY1, a BTB-NPH3-like protein, plays a critical role in auxin-regulated organogenesis in Arabidopsis. Proc. Natl Acad. Sci. USA104, 18825–18829 (2007).
Blilou, I. et al. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature433, 39–44 (2005).
Verna, C. et al. Coordination of tissue cell polarity by auxin transport and signaling. eLife8, e51061 (2019).
Gil, P. et al. BIG: a calossin-like protein required for polar auxin transport in Arabidopsis. Genes Dev.15, 1985–1997 (2001).
Mellor, N. L. et al. Auxin fluxes through plasmodesmata modify root-tip auxin distribution. Development147, dev181669 (2020).
Delbarre, A., Muller, P., Imhoff, V. & Guern, J. Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta198, 532–541 (1996).
Durgaprasad, K. et al. Gradient expression of transcription factor imposes a boundary on organ regeneration potential in plants. Cell Rep.29, 453–463 (2019).
Zhao, Y. Essential roles of local auxin biosynthesis in plant development and in adaptation to environmental changes. Annu. Rev. Plant Biol.69, 417–435 (2018).
Gao, C. et al. Directionality of plasmodesmata-mediated transport in Arabidopsis leaves supports auxin channeling. Curr. Biol.30, 1970–1977 (2020).
Chen, X. et al. A simple method suitable to study de novo root organogenesis. Front. Plant Sci.5, 208 (2014).
Bustillo-Avendaño, E. et al. Regulation of hormonal control, cell reprogramming, and patterning during de novo root organogenesis. Plant Physiol.176, 1709–1727 (2018).
Xu, M. et al. Developmental functions of miR156-regulated SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes in Arabidopsis thaliana. PLoS Genet.12, e1006263 (2016).
Bahieldin, A. et al. Multifunctional activities of ERF109 as affected by salt stress in Arabidopsis. Sci. Rep.8, 6403 (2018).
Santuari, L. et al. The PLETHORA gene regulatory network guides growth and cell differentiation in Arabidopsis roots. Plant Cell28, 2937–2951 (2016).
Kareem, A. et al. PLETHORA genes control regeneration by a two-step mechanism. Curr. Biol.25, 1017–1030 (2015).
Heyman, J. et al. ERF115 controls root quiescent center cell division and stem cell replenishment. Science761, 860–864 (2013).
Heisler, M. G. et al. Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem. Curr. Biol.15, 1899–1911 (2005).
Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E. & Benfey, P. N. Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Development127, 595–603 (2000).
Stepanova, A. N. et al. TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development. Cell133, 177–191 (2008).
Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE4, e5553 (2009).
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).
The authors declare no competing interests.
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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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.
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).
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.
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.
a, WT floral meristem. b, pYUC9:amirYUC pin-like floral meristem.
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.
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). g–n, Confocal images of uncut (g-j) or regenerating roots at 72hpc (k-n) of WT (g,k) and pin mutants (h-j,l-n). o–q, 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).
a–o, 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. p–q, 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.
a–c, 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