Abstract

Complex multicellular organisms evolved on Earth in an oxygen-rich atmosphere1; their tissues, including stem-cell niches, require continuous oxygen provision for efficient energy metabolism2. Notably, the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation3. Here we demonstrate, using a combination of genetic reporters and in vivo oxygen measurements, that plant shoot meristems develop embedded in a low-oxygen niche, and that hypoxic conditions are required to regulate the production of new leaves. We show that hypoxia localized to the shoot meristem inhibits the proteolysis of an N-degron-pathway4,5 substrate known as LITTLE ZIPPER 2 (ZPR2)—which evolved to control the activity of the class-III homeodomain-leucine zipper transcription factors6,7,8—and thereby regulates the activity of shoot meristems. Our results reveal oxygen as a diffusible signal that is involved in the control of stem-cell activity in plants grown under aerobic conditions, which suggests that the spatially distinct distribution of oxygen affects plant development. In molecular terms, this signal is translated into transcriptional regulation by the N-degron pathway, thereby linking the control of metabolic activity to the regulation of development in plants.

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Data availability

Accession numbers of all the Arabidopsis genes analysed in this study are listed in the text or figure legends. All numerical data used to generate the graphs displayed in this report are provided as Source Data. Seeds of transgenic lines used in this study are available from the corresponding authors upon request. The uncropped versions of all gels and blots are provided as Supplementary Fig. 1.

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Acknowledgements

This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) (Projektnummer 243440351), the Excellence Initiative of the German Federal and State Governments, Scuola Superiore Sant’Anna, the University of Pisa and the Erasmus+ programme (Z.N.V.).

Reviewer information

Nature thanks Nico Dissmeyer, Christine Foyer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Institute of Biology I, RWTH Aachen University, Aachen, Germany

    • Daan A. Weits
    • , Alicja B. Kunkowska
    • , Nicholas C. W. Kamps
    • , Katharina M. S. Portz
    • , Niko K. Packbier
    •  & Joost T. van Dongen
  2. Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy

    • Daan A. Weits
    • , Zoe Nemec Venza
    •  & Francesco Licausi
  3. Department of Stem Cell Biology, Centre for Organismal Studies, University of Heidelberg, Heidelberg, Germany

    • Christophe Gaillochet
    •  & Jan U. Lohmann
  4. Plant Molecular and Cellular Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    • Christophe Gaillochet
  5. The Freshwater Biological Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    • Ole Pedersen
  6. Biology Department, University of Pisa, Pisa, Italy

    • Francesco Licausi

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Contributions

D.A.W., J.T.v.D. and F.L. designed the experiments. D.A.W., A.B.K., N.C.W.K., K.M.S.P., N.K.P., Z.N.V., O.P. and F.L. carried out the experiments, as described in detail in Supplementary Table 8. D.A.W. and F.L. conducted the statistical analyses. C.G. and J.U.L. provided support for confocal analyses of shoot tissues. D.A.W., J.T.v.D. and F.L. wrote the manuscript with inputs by A.B.K, C.G., J.U.L. and O.P. All co-authors read and approved the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Joost T. van Dongen or Francesco Licausi.

Extended data figures and tables

  1. Extended Data Fig. 1 Low-oxygen levels in the Arabidopsis shoot apex overlap with the meristematic niche.

    a, Comparison of spatial resolution and accuracy of the custom-made Clark-type microsensor used here (red, orange and pink triangles) alongside the commercially available sensor OX10, Unisense A/S (turquoise, cyan and cobalt-blue circles). Oxygen-consuming solid medium was prepared by sodium ascorbate oxidation. The dataset shows that, in 2% agar with a steep gradient in dissolved oxygen from atmospheric equilibrium to anoxia, the two sensors show identical responses. Five independent profiles were recorded for each type of microsensor. b, Depiction of the experimental setup adopted to measure the oxygen concentration profiles in the apical-to-basal direction (left) and radial direction (right). c, Oxygen concentration profile in the apex of a four-day-old Arabidopsis seedling. The experiment was repeated twice with similar results. d, Tracking of the sensor insertion pattern reconstructed after the oxygen profile in c was taken. The SAM was visualized by confocal microscopy after FM4-64 staining of plasma membranes. e, Oxygen concentration profile obtained by inserting the Clark-type microsensor laterally through the SAM of seven-day-old Arabidopsis plants in the shoot apical region (profile 1), at the junction of cotyledon vasculature (profile 2) and below the junction (profile 3). The experiment was repeated three times with similar results. A photograph of the experimental setup is shown within the plot frame (bottom right inset). f, Position of the sensor insertion points as identified by optical microscopy (top) and localization of the CLAVATA3 (CLV3) expression domain, reported by GUS staining of plants that express pCLV3:GUS in four-day-old plants (bottom). Source Data

  2. Extended Data Fig. 2 Transcripts that belong to the core hypoxia-inducible genes are specifically enriched in shoot, but not in root, meristem tissues.

    a, Pie charts that represent the relative abundance of differentially expressed genes within the core hypoxia-inducible set1, in a comparison between the SAM and juvenile leaf tissues, and the root meristem (RM) and elongation zone and maturation zone of the root. Absolute expression levels, standard error, ratios and statistics are shown in Supplementary Tables 1 and 2 for shoots and roots, respectively. b, Schematic of SAM and root meristem cell types. c, Heat map showing the expression levels of core hypoxia-inducible genes in the SAM and root-meristem cell types depicted in b. Absolute expression levels were retrieved from the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi), and are provided in Supplementary Table 3. Source Data

  3. Extended Data Fig. 3 Low-oxygen conditions in the shoot apex induce molecular hypoxic responses.

    a, Oxygen responsiveness of a synthetic construct that consists of five repeats of the HRPE reporter (named pHRPEx5:GFP-GUS). The experiment was repeated twice with similar results. b, c, Quantification of the relative staining intensity of the pHRPEx5:GFP-GUS reporter at 21%, 10% and 5% O2 (b), and 21% and 80% O2 (c), shows that this construct effectively reports the oxygen status of tissues. One-way ANOVA followed by Tukey post hoc test (b) or two-sided t-test (c). d, RT–qPCR was used to measure the expression of the hypoxia-inducible genes PLANT CYSTEINE OXIDASE 1 (PCO1), WOUND INDUCED PROTEIN 4 (WIP4), HYPOXIA RESPONSIVE ATTENUATOR 1 (HRA1), PLANT CYSTEINE OXIDASE 2 (PCO2), HEMOGLOBIN 1 (HB1), PYRUVATE DECARBOXYLASE 1 (PDC1) and ALCOHOL DEHYDROGENASE (ADH) in leaves and shoot apices, after normoxic and hyperoxic treatments (80% O2, 6 h). The higher expression of these mRNAs in the SAM, compared to the leaf samples, was repressed by hyperoxia. A two-way ANOVA followed by Tukey post hoc test was used to assess the statistical significance of the observed differences. Source Data

  4. Extended Data Fig. 4 Hypoxic conditions are maintained in reproductive meristems.

    a, Oxygen concentration profiles obtained inserting the Clark-type microsensor in the apical-to-basal direction through the inflorescence meristem of 5-week-old Arabidopsis plants. The inset shows a photo of the actual experimental setup with the insertion of the microsensor inside the inflorescence meristem. The experiment was repeated independently three times with similar results. b, Green fluorescent signal in inflorescence and floral meristems of plants transformed with the hypoxia reporters pPCO1:GFP-GUS and pPCO2:GFP-GUS that were characterized as specifically hypoxia responsive in a previous publication24. The experiment was repeated twice with similar results. Source Data

  5. Extended Data Fig. 5 A hypoxic niche at the shoot apex is a common feature of plants.

    a, The customized Clark-type microsensor was used to measure oxygen profiles throughout the shoot apex of one-week-old S. lycopersicum var. Micro-Tom plants, in the apical-to-basal direction. These measurements show the presence of an oxygen gradient in the shoot apex of this plant species. The experiment was repeated twice with similar results. b, Photograph showing the insertion of the micro-electrode inside the tomato SAM. c, d, Overlay of oxygen profiles (c and d shown as cyan and red, respectively, in a) and micrographs of SAM tissues after FM4-64 membrane staining that show the actual penetration of the sensor. The puncture in the centre of the meristem and the concomitant accumulation of FM4-64 shows the penetration of the sensor into the tissue. The experiment was repeated four times with similar results; two examples are shown in c and d. e, Similar to Arabidopsis, the typical hypoxia-marker genes ALCOHOL DEHYDROGENASE 2 (ADH2), pyruvate decarboxylases (PDC1 and PDC3), PLANT CYSTEINE OXIDASE 2 (PCO2) and PHYTOGLOBIN 1 (HB1a) are expressed at a higher level in SAM-enriched tissues than in juvenile leaves of two-week-old plants of S. lycopersicum var. Micro-Tom. PYRUVATE DECARBOXYLASE 4 (PDC4) does not exhibit the same pattern. These results indicate that SAM cells experience hypoxic conditions. RT–qPCR was used to measure the expression of hypoxia-inducible genes (two-sided t-test; n = 4 pools of 5 plants). Source Data

  6. Extended Data Fig. 6 ZPR2-type proteins are distinguished by a variable N-terminal domain with a conserved Cys residue in the penultimate position.

    a, Multi-alignment of ZPR-type sequences from eight different angiosperm species (Aquilegia coerulea (Ac), Amborella trichopoda (Amtr), A. thaliana (At), Daucus carota (Dc), Medicago truncatula (Mt), Oryza sativa (Os), Populus trichocarpa (Pt), S. lycopersicum (Sl) and Sorghum bicolor (Sb)). At least one ZPR2-type and one ZPR3-type sequence was identified in all species that we considered. All ZPR2-type proteins have a Cys residue at the second position of the N-terminal domain. Amino acid position is displayed on top of each alignment, using A. thaliana ZPR2 as a reference. b, Molecular phylogenetic analysis by the maximum likelihood method. The tree with the highest log-likelihood (−1,500.12) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 28 protein sequences (listed in Supplementary Table 4).

  7. Extended Data Fig. 7 N-terminal tagging of proteins with a ZPR2 sequence confers degradability by the N-degron pathway.

    a, Relative mRNA level of the genotypes used for immunodetection of GFP in Fig. 2d under aerobic and hypoxic conditions (2% O2), measured by RT–qPCR. Two biological replicates from two independent lines were used in the case of the constructs with wild-type ZPR2 (35S:ZPR2-GFP) and (MAC)ZPR2-GFP lines. Four biological replicates were instead used in the case of wild-type ZPR2-GFP in the prt6 background. The effect of hypoxia treatment versus aerobic conditions was evaluated by two-way ANOVA (P value = 0.342, n = 4 pools of 3 plants). The results of the RT–qPCR analysis exclude regulation by hypoxia at the transcriptional level and—combined with the immunoblot analysis—support the existence of a control checkpoint at the post-transcriptional level. b, Relative luciferase activity of a chimeric protein that consists of the whole ZPR2 coding sequence fused to the N terminus of a firefly luciferase (ZPR2-PpLUC). This construct was transfected together with a second one, which bears a Renilla luciferase gene driven by the same 35S CaMV promoter, into Arabidopsis mesophyll protoplasts. Renilla luciferase activity was used as a normalization control. One-way ANOVA followed by Tukey post hoc test; n = 6, 4 and 5 protoplast pools for wild-type ZPR2 (MC-ZPR2), (MAC)ZPR2 (here MAC-ZPR2) and wild-type ZPR2 in prt6, respectively. c, Relative GUS activity of a gZPR2-GUS construct expressed in Arabidopsis protoplasts. A 35S:PpLuc reporter was co-transformed to equalize for transfection efficiency. The addition of an Ala residue before the Cys2 led to enhanced stability of both reporter constructs, and the expression of the wild-type ZPR2 protein fusions in the prt6 mutant background also showed enhanced protein abundance. One-way ANOVA followed by Tukey post hoc test; n = 8, 5 and 6 protoplast pools for wild-type ZPR2, (MAC)ZPR2 and wild-type ZPR2 in prt6, respectively. d, Quantification of the relative staining intensity of GUS-stained plants that express a pZPR2:ZPR2-GUS construct at 2%, 21% and 80% O2. An example of GUS staining at each oxygen concentration is shown in Fig. 3b. Images of GUS-stained plants were converted to inverted greyscale images, and the staining intensity was measured using ImageJ. Wild-type plants that were de-stained in ethanol were used to correct for the background signal. Average relative staining intensity was calculated by comparing the corrected staining intensity at each O2 concentration by the corrected staining intensity at 21% O2. These results show that ZPR2 stability in the SAM depends on oxygen availability. One-way ANOVA followed by Holm–Sidak post hoc test; n = 10, 7 and 8 plants for 2%, 21% and 80% O2, respectively. Source Data

  8. Extended Data Fig. 8 Loss of ZPR2 expression reduces the leaf initiation rate, and ectopic overexpression of ZPR2 abolishes SAM activity in an oxygen-dependent manner.

    a, Schematic of the ZPR2 gene, showing the position of two T-DNA insertions (SAIL (Syngenta Arabidopsis Insertion Library) and GABI-Kat (https://www.gabi-kat.de/) collections). The relative annealing site of the primers used in b is shown using arrows in the schematic view. b, The homozygous status of T-DNA insertions within the intron of AT3G60890 in the NASC accessions N483079 (zpr2-2, GK-866D03) and N835524 (zpr2-3, Sail_794_D11) was tested by PCR using the combinations of primers indicated in a. Genomic wild-type (Col-0 ecotype) DNA and double-distilled water were used as controls. The experiment was repeated twice with similar results. c, Amplification of the entire ZPR2 coding sequence in the wild-type, zpr2-2 and zpr2-3 genotypes. The experiment was repeated twice with similar results. d, Relative expression of ZPR2 in wild type, zpr2-2 and zpr2-3 measured by RT–qPCR. One-way ANOVA followed by Holm–Sidak post hoc test; n = 5 pools of 3 plants. e, Progression in leaf number in wild type and zpr2 T-DNA insertion mutants. The number of leaves, including cotyledons, was counted every second day from the emergence of the first pair of true leaves. Data are presented as mean and s.d.; n = 15 plants. f, Shoot phenotype of wild-type, zpr2-3 and pZPR2:ZPR2-Flag in zpr2-3 plants. At the growth condition used, the rosette of four-week-old zpr2-3 plants was smaller than that of the wild type. g, Leaf initiation rate in wild-type, zpr2-3 and pZPR2:ZPR2-Flag in zpr2-3 plants. One-way ANOVA followed by Dunn’s post hoc test; n = 41, 27, 20 and 19 plants for wild type, zpr2-3 and zpr2-3 pZPR2:ZPR2-Flag line no. 1 and zpr2-3 pZPR2:ZPR2-Flag line no. 2, respectively. h, Phenotype of wild-type Arabidopsis and 35S:ZPR2-GFP plants grown in plates containing agarized MS (half-strength) medium supplemented with 10 g l−1 sucrose under aerobic or hypoxic (2% O2) conditions for 20 days, followed by 5 days of recovery in normoxia. 35S:ZPR2-GFP plants often showed termination of meristem activity and the formation of a pin-like structure. The experiment was repeated twice with similar results. i, Percentage of shoot meristem termination (blue) or meristem progression (green) events in wild-type and 35S:ZPR2-GFP plants after five days of recovery from hypoxic growth. The number of plants that displayed either phenotype is reported in white inside the bar. The frequency of SAM termination in 35S:ZPR2-GFP plants increased after hypoxic treatments. A two-tailed Fisher’s exact test was used to compare wild-type and 35S:ZPR2-GFP plants grown in aerobic and hypoxic conditions. Source Data

  9. Extended Data Fig. 9 Repression of HD-ZIP III-target genes by ZPR2 induction and stabilization occurs in the shoot apex.

    a, Effect of ZPR2 and ZPR2–GUS on the transactivation activity of REV on the ZPR1 promoter. These data indicate that ZPR2 is not able to repress REV activity when fused with a GUS reporter protein at its C terminus. One-way ANOVA, followed by Holm–Sidak post hoc test; n = 5 protoplast pools. b, Schematic of the construct that provided oestradiol-inducible expression of ZPR2, and after protein stabilization under hypoxic conditions. c, Separate and combined effect of oestradiol (50 μM) application, for 4 h before exposure to 2% O2 for 24 h, on the expression of a GUS reporter under the control of pHEC1 (HECATE 1) and pPSK5 (PHYTOSULFOKINE 5 PRECURSOR) promoters in 6-day-old Arabidopsis seedlings that also expressed an oestradiol-inducible ZPR2 construct. Seeds of these genotypes were obtained as F1 offspring that were generated by crossing homozygous promoter:GUS lines with homozygous oestradiol-inducible ZPR2 (pMDC7:ZPR2) plants. The observation was repeated twice. A reduction in pPSK5 or pHEC1 activity by combined ZPR2 induction and hypoxia was observed in a total of 8 out of 12 and 15 out of 20 plants, respectively. d, Effect of oestradiol-mediated induction of ZPR2 and its stabilization by hypoxia on pTAA1:GUS staining in five-day-old wild-type and transgenic pMDC7:ZPR2 plants. Twenty-four hours of hypoxia, but not oestradiol treatment (50 μM), was sufficient to repress pTAA1-driven GUS expression in the wild-type background, probably via stabilization of the endogenous ZPR2 protein (2 out of 3 plants). The hypoxia treatment also inhibited expansion of the first pair of true leaves. Stimulated ZPR2 expression in the pMDC7:ZPR2 background further decreased pTAA1:GUS staining (3 out of 3 plants). This experiment was performed once. Source Data

  10. Extended Data Fig. 10 ZPR2 is required to repress HD-ZIP III-target genes.

    a, The expression of HD-ZIP III-target genes was measured using RT–qPCR in the apices of one-week-old zpr2-3 mutants and wild-type plants. These results show that the expression of five HD-ZIP III-target genes (PSK5, ATH1, ZPR1, AMP1 and POL) is significantly increased in zpr2-3 insertion mutants. Two-sided t-test; n = 10 plants. b, GUS expression under control of the MAX2 promoter in wild-type and zpr2-3-background genotypes. Restoration of wild-type background was obtained by PCR screening (for zpr2-3 insertion) and antibiotic resistance (for the pMAX2:GUS construct) in the F2 offspring of a cross between a pMAX2:GUS zpr2-3 plant and a wild-type (Col-0) plant. The observation was repeated twice. In total, the induction of pMAX2:GUS-GFP in zpr2-3 mutants was observed in 16 out of 20 plants. c, Hypothetical model describing how local hypoxia drives SAM activity by regulating HD-ZIP III transcription factors via ZPR2. Source Data

Supplementary information

  1. Supplementary Figure 1

    Full immunoblot from which the panel 2d is cropped.

  2. Reporting Summary

  3. Supplementary Data

    Supplementary Data 1: Synthetic pHRPE promoter sequence.

  4. Supplementary Tables

    This file contains Supplementary Tables 1-8 and supplementary bibliographic references.

Source data

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DOI

https://doi.org/10.1038/s41586-019-1203-6

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