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Robust organ size requires robust timing of initiation orchestrated by focused auxin and cytokinin signalling

Nature Plants volume 6, pages 686–698 (2020)Cite this article

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Abstract

Organ size and shape are precisely regulated to ensure proper function. The four sepals in each Arabidopsis thaliana flower must maintain the same size throughout their growth to continuously enclose and protect the developing bud. Here we show that DEVELOPMENT RELATED MYB-LIKE 1 (DRMY1) is required for both timing of organ initiation and proper growth, leading to robust sepal size in Arabidopsis. Within each drmy1 flower, the initiation of some sepals is variably delayed. Late-initiating sepals in drmy1 mutants remain smaller throughout development, resulting in variability in sepal size. DRMY1 focuses the spatiotemporal signalling patterns of the plant hormones auxin and cytokinin, which jointly control the timing of sepal initiation. Our findings demonstrate that timing of organ initiation, together with growth and maturation, contribute to robust organ size.

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Fig. 1: Mutations in the DRMY1 gene lead to increased variation in sepal size and shape.
Fig. 2: Sepal initiation is variably delayed in the drmy1-2 mutant.
Fig. 3: Sepals with delayed initiation in drmy1-2 mutants remain smaller throughout development.
Fig. 4: Focused auxin and cytokinin signalling are required for robust sepal initiation.
Fig. 5: Spatiotemporal patterns of auxin and cytokinin signalling regulate the timing of sepal initiation.

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Data availability

All other data are available in the main text, the Extended Data figures, the Supplementary Data or the Source Data.

RNA-seq data are available at NCBI BioProject PRJNA564625. Individual RNA-seq read sets are archived in SRA under the following accession numbers: WT replicate 1, SRX6821462; WT replicate 2, SRX6821463; WT replicate 3, SRX6821464; drmy1-2 replicate 1, SRX6821465; drmy1-2 replicate 2, SRX6821466; and drmy1-2 replicate 3, SRX6821467. Gene information is available under the following accession numbers: DRMY1, AT1G58220; AHP6, AT1G80100; TIR1, AT3G62980; AFB1, AT4G03190; AFB2, AT3G26810; AFB3, AT1G12820; WOL, AT2G01830; and PIN1, AT1G73590. Source Data for Figs. 1–4 and Extended Data Figs. 1–7 are provided with the paper.

Change history

  • 07 October 2020

    In the version of this Article originally published, Supplementary Data Table 1 and all the Source Data files were linked to the incorrect documents; this has now been corrected.

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Acknowledgements

We thank F. Besnard, A. Bretscher, J. Cammarata, K. Harline, J. McGory and B. V. L. Vadde for comments on the manuscript. We thank X. Zhu for drawing the anatomical diagrams by hand. We thank E. Meyerowitz and A. Garda for sharing the seeds for DR5::3XVENUS-N7/PIN1::GFP (Ler). We thank T. Vernoux and G. Brunoud for sharing the seeds for pTCS::GFP (Col), pPIN1::PIN1-GFP (Col) and TCS DR5 (Col). We thank F. Kateryna and F. Zhao for teaching the protocol for PIN1 immunolocalization. We thank F. Besnard for assisting with phyllotaxy measurement. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under award no. R01GM134037 (A.H.K.R), Human Frontier Science Program grant no. RGP0008/2013 (A.B., O.H., A.H.K.R., C.-B.L. and R.S.S.), Weill Institute startup funding (E.M.S. and A.H.K.R.) and Cornell Graduate School travel grant program (M.Z.). We thank Cornell University Biotechnology Resource Center for their sequencing service (supported by NIH grant no. 1S10OD010693-01). This work made use of the Cornell Center for Materials Research-shared SEM facilities, which are supported through the NSF MRSEC programme (no. DMR-1719875). We thank the PLATIM facility of SFR Biosciences (UMS3444/CNRS, US8/Inserm, ENS de Lyon, UCBL) for the use of microscopes. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or other funding agencies.

Author information

Author notes

  1. Richard S. Smith

    Present address: John Innes Centre, Norwich, UK

Authors and Affiliations

  1. Weill Institute for Cell and Molecular Biology and School of Integrative Plant Science, Section of Plant Biology, Cornell University, Ithaca, NY, USA

    Mingyuan Zhu, Weiwei Chen, Lilan Hong, Zhou Wang & Adrienne H. K. Roeder

  2. Key Laboratory of Horticulture Science for Southern Mountains Regions of Ministry of Education, College of Horticulture and Landscape Architecture, Southwest University, Beibei, China

    Weiwei Chen

  3. Academy of Agricultural Sciences of Southwest University, State Cultivation Base of Crop Stress Biology for Southern Mountainous Land of Southwest University, Beibei, China

    Weiwei Chen

  4. Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, INRIA, Lyon, France

    Vincent Mirabet, Simone Bovio, Olivier Hamant & Arezki Boudaoud

  5. Lycée Auguste et Louis Lumière, Lyon, France

    Vincent Mirabet

  6. Institute of Nuclear Agricultural Sciences, Key Laboratory of Nuclear Agricultural Sciences of Ministry of Agriculture and Zhejiang Province, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

    Lilan Hong

  7. Department of Comparative Development and Genetics, Max Planck Institute for Plant Breeding Research, Cologne, Germany

    Soeren Strauss & Richard S. Smith

  8. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Erich M. Schwarz

  9. Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Japan

    Satoru Tsugawa

  10. Department of Mathematics, Stockholm University, Stockholm, Sweden

    Chun-Biu Li

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Contributions

Conception and design for experiments was carried out by M.Z., A.H.K.R, V.M., A.B., O.H., R.S.S. and C.-B.L. Isolation of the vos2 (drmy1-2) mutant was performed by L.H. and A.H.K.R. Phenotypic analysis of the vos2 (drmy1-2) mutant was done by M.Z. and Z.W. Variability of organ shape analysis was carried out by C.-B.L. SEM was performed by M.Z. and A.H.K.R. Live imaging and analysis were done by M.Z. and W.C. AFM was carried out by M.Z and S.B. MorphoGraphX plug-in development for DR5 and TCS quantification was performed by S.S. and R.S.S. Computational analysis of spatial and temporal variability of growth was done by S.T. and C.-B.L. RNA-seq analysis was carried out by E.M.S., M.Z. and A.H.K.R. M.Z. and A.H.K.R. wrote the manuscript. Revising and editing of the manuscript was done by M.Z., W.C., V.M., L.H., S.B., S.S., E.M.S., S.T., R.S.S, C.-B.L., O.H., A.B. and A.H.K.R.

Corresponding authors

Correspondence to Arezki Boudaoud or Adrienne H. K. Roeder.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Michalis Barkoulas 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 drmy1-2 floral organs have enhanced sepal size and shape variability.

a, DRMY1 mutations have little effect on the floral organ number. Numbers of sepals, petals, stamens and carpels were quantified for both WT and the drmy1-2 mutant. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of organ number, WT versus drmy1-2 petal: 1.14E-03; WT versus drmy1-2 stamen number: 2.14E-08). Measure of centre: mean. Error bars: standard error of the mean. n = 30 flowers. b, Quantification of the mean sepal area of the four sepals from an individual flower. Sequential flowers along the main branch of the stem (flower number on the x-axis) were measured at stage 14. Three replicates were included for both WT (blue) and drmy1-2 (red). Original individual sample curves can be found in Source Data. The mean of the 3 replicates are presented as thick blue and red lines with the SD as partially translucent background. c, Quantification of the sepal shape variability for outer, inner and lateral sepals. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of shape variability, WT versus drmy1-2 inner sepal: 2.70E-02; WT versus drmy1-2 lateral sepal: 1.00E-07) Measure of centre: mean. Error bars: standard error of the mean. n = 60 for both WT and drmy1-2 10th to 25th flowers along the main branch. d, What at first appeared to be two sepals initiated at the inner sepal position of the drmy1-2 flower (left panel) fused to form a single sepal with a split tip at later time points of the live imaging (right panel). Red arrowheads: initiating sepals. Red arrow: the fused sepal. Scale bar: 20 μm. e, Coefficient of variation (CV) calculated for the areas of the outer, inner and lateral sepals. Sepals from different flowers were pooled together. n = 48 flowers. f, Average CV calculated for the areas of all petals in each single flower for WT and drmy1-2. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of CV for petal area within individual flowers, WT versus drmy1-2: 1.27E-03). Measure of centre: mean. Error bars: standard error of the mean. n = 20 flowers for both WT and drmy1-2.

Source data

Extended Data Fig. 2 DRMY1 is required for sepal size robustness.

a, Sequencing of DRMY1 transcripts from the drmy1-2 mutant verified that splicing defects occur. DRMY1 transcripts were reverse transcribed and amplified from RNA extracted from the drmy1-2 mutant and inserted into pENTR/D-TOPO for sequencing. Black shading: nucleotides remaining in the transcript after the splicing; Gray shading: nucleotides spliced out. Orange capital letter: exon. Purple lower-case letter: intron in the WT DRMY1 transcript. Red arrowhead indicates one base pair shift. b, qRT-PCR measuring the expression of DRMY1 in WT and the drmy1-2 mutant using two pairs of primers: one before the mutation site and the other across the mutation site. The expression level in WT quantified with the primers before the mutation site was set to 1 using the Delta-delta-CT method. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of expression fold change, WT versus drmy1-2 before mutation: 3.31E-03; WT versus drmy1-2 across mutation: 2.01E-04). Measure of centre: mean. Error bars: standard error of the mean. n = 3 biological replicates. c–h, Inflorescences of WT (c), drmy1-1 (d), drmy1-2 (e), F1 of the cross between drmy1-1 and drmy1-2 for allelism test (f), T3 plants of drmy1-2 transformed with pDRMY1::DRMY1 (g), and T3 plants of drmy1-2 transformed with pDRMY1::DRMY1-mCitrine (h). Orange arrows: smaller sepals in individual flowers. Note, open flower buds indicate unequal sepal sizes. Scale bars: 0.5 mm. n = 3 inflorescences. i,j, Transcriptional (i, pDRMY1::3XVENUS-N7, nuclear localized gray signal) and translational (j, pDRMY1::DRMY1-mCitrine, gray) DRMY1 reporter expression patterns are similar. Cell walls were stained with PI in i and plasma membranes were fluorescently labeled with pUBQ10::mCherry-RCI2A in j. Both DRMY1 reporters are expressed in the inflorescence meristem, floral meristems, and initiating floral organs, with stronger expression in the periphery. Scale bars: 20 μm. n = 3 inflorescences.

Source data

Extended Data Fig. 3 Cell wall stiffness increases in the drmy1-2 mutant.

a, Graph of flower radius used to assess developmental stage based on flower size. The radii of flowers without sepal primordia (SP = 0), with only the outer sepal primordium (SP = 1), or with outer and inner sepal primordium (SP > 1) were measured for wild-type and drmy1-2 inflorescences. The critical size threshold for outer sepal initiation is specified with a yellow dashed line. Note this size is the same for wild type and drmy1-2, indicating the stage of outer sepal initiation is not affected in drmy1-2. In contrast, the critical size threshold for inner sepal initiation (orange dashed lines), is larger for drmy1-2 than wild type, consistent with delayed inner sepal initiation. In the violin plots, the black line represents the median and the individual data points are shown. n = 46 flowers for WT SP = 0; n = 21 for WT SP = 1; n = 45 for WT SP > 1; n = 53 for drmy1-2 SP = 0; n = 63 for drmy1-2 SP = 1; n = 33 for drmy1-2 SP > 1. b, The average apparent elastic modulus calculated from AFM measurements of the flowers is significantly higher for the drmy1-2 mutant. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of apparent elastic modulus, WT versus drmy1-2: 6.81E-04). Measure of centre: mean. Error bars: standard errors of the mean. n = 11 samples measured for both WT and drmy1-2. c, Cell shrinkage heatmap after osmotic treatment in WT and drmy1-2. Group of cells were segmented together for area comparison. Red in the heatmap represents less shrinkage, thus stiffer cell wall. Scale bar: 50 μm. n = 3 flowers. d, Average shrinkage ratio after osmotic treatment further confirms that cells undergo less shrinkage in the drmy1-2 mutant, indicating the cell walls are stiffer. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of shrinkage ratio, WT versus drmy1-2: 5.56E-08). Measure of centre: mean. Error bars: standard errors of the mean. n = 161 cell groups for WT and n= 129 cell groups for drmy1-2. e, Bar graph of GO terms that are overrepresented (against a genome wide frequency) among genes more strongly expressed in drmy1-2 inflorescences than in WT inflorescences. Genes used for this GO term analysis are listed in the ‘Upreg. in drmy1-2, padj < 0.1’ table of Supplementary Data 1. f, Bar graph of GO terms that are overrepresented (against a genome wide frequency) among genes more strongly expressed in WT inflorescences than in drmy1-2 inflorescences. Genes used for this GO term analysis are listed in the ‘Downreg. in drmy1-2, padj < 0.1’ table of Supplementary Data 1. For both (e) and (f), a subset of significant GO terms was selected for each graph (Fisher test with Yekutieli multi-test adjustment, significance level 0.05 using the AgriGo 2.0 website). The percentage of differentially expressed genes in drmy1-2 versus WT associated the GO term is shown with black bars. Genome (gray) reports the frequency of genes associated with that term in the Arabidopsis genome, which would be the frequency expected by chance for a randomly selected subset of genes. The percentage of genes was calculated as the number of genes associated with that term divided by the total number of genes. n = 3 biological replicates.

Source data

Extended Data Fig. 4 Sepal cell growth is slower in the drmy1-2 mutant.

a, b, 24-hour late stage (from stage 6 to stage 9) cellular growth heatmap for both WT (a) and drmy1-2 (b) outer sepals. Relative growth rate is defined as final cell size divided by initial cell size. Segmented cells outlined in yellow. Note the outer sepal base is at the bottom of the image and its tip points up. Scale bars: 20 μm. c, Growth curves of the late stage average cellular growth for both WT and drmy1-2. *: Flower stage 9 extends over multiple 24-hour intervals. Measure of centre: mean. Error bars represent standard error of the mean. n = 3 flowers. d, 36-hour cell division heatmap for both WT and drmy1-2. The total number of cells derived from one progenitor is represented in the heatmap with 1 meaning no divisions. Throughout sepal development, the drmy1-2 sepal cells undergo fewer divisions than WT. Scale bars: 20 μm. e, Confocal images of sepals from individual flowers (shown in Fig. 3a,b) after 11 days of live imaging. Area variability was quantified by the coefficient of variation (CV). Two lateral primordia fused to form the left drmy1-2 lateral sepal. Scale bar: 100 μm. Sepals from 1 flower are shown here, representing 3 live imaging series. f, The outer sepal area plotted as a function of the inner sepal area in individual flowers. Each color represents a pool of three plants and each point is for one flower (using the same dataset as Fig. 1d and Extended Data Fig. 1b). In addition to plotting the values, we use a Gaussian kernel to estimate their probability density function. We represent the probability density function with labelled contour lines shaping the density. We use a bandwith factor of 0.5, chosen to make the data distribution easier to read. n = 167 WT flowers and n = 148 drmy1-2 flowers. g, The mean drmy1-2 sepal area divided by WT mean sepal area ratio for each sepal type. Sepals from different flowers were pooled together (using flowers 10–25 from the same dataset as Fig. 1d and Extended Data Fig. 1b). n = 48 flowers.

Source data

Extended Data Fig. 5 Spatiotemporal averaging is not affected in the drmy1-2 mutant at early stage.

a, The maximal principal direction of growth (PDGmax, white line) of WT sepal cells calculated for 24-hour and 48-hour intervals. For 24-hour intervals, the PDGmax shows both spatial and temporal variations in WT. Cell outlines are shown in cyan. Over the whole 48-hour interval these variations average out such that the PDGmax are oriented vertically along the major growth axis of the sepal. One cell showing good temporal averaging is highlighted with blue boxes and magnified in insets. The PDGmax are visualized on the earlier time point. n = 3 live imaging series. Scale bar: 20 μm. b, The maximal principal direction of growth (PDGmax, white line) of the drmy1-2 sepal cells calculated for 24-hour and 48-hour intervals. The PDGmax also shows similar spatial and temporal variations in the drmy1-2 situation. One cell showing good temporal averaging is again highlighted with red boxes at different time points, indicating that temporal averaging of growth direction is not affected by DMRY1 mutations. The PDGmax are visualized on the earlier time point. n = 3 live imaging series. Scale bar: 20 μm. c, Graph plotting the average spatial variability of the growth rates (Varea) for sepal epidermal cells during sepal development. Blue curves are for WT sepals and red curves are for drmy1-2 sepals. Measure of centre: mean. Error bars represent standard error. n = 3 flowers. d, Graph plotting the average temporal variability of the growth rates (Darea) for sepal epidermal cells during the development of sepals. Blue curves are for WT sepals and red curves are for drmy1-2 sepals. Measure of centre: mean. Error bars represent standard error. n = 3 flowers.

Extended Data Fig. 6 Auxin signaling is suppressed and more diffuse while cytokinin signaling expands and is enhanced in drmy1-2 mutants.

a, Confocal imaging of the DR5 auxin response reporter (white) in the whole inflorescence of WT and the drmy1-2 mutant. p35S::mCherry-RCI2A: red, for plasma membrane. Scale bar: 20 μm. n = 10 inflorescences. b, Confocal imaging of the TCS cytokinin signaling reporter (gray) in whole inflorescences of WT and the drmy1-2 mutant. Chlorophyll autofluorescence: red. Scale bar: 20 μm. n = 10 inflorescences. c, Confocal imaging of PIN1 immunolocalization experiments to show PIN1 accumulation in inflorescences and flowers of WT and drmy1-2. PIN1 exhibits polar localization in drmy1-2 similar to wild type; however, it forms more convergence points in flowers. Blue/Red arrowheads: PIN1 convergence points. Inset: Same images with increased brightness to show the morphology of the flowers. Scale bars: 20 μm. n = 3 inflorescences. d, Confocal imaging of pPIN1::PIN1-GFP to show PIN1 accumulation in the inflorescences and flowers of WT and drmy1-2. Again, PIN1 forms abnormal convergence points in drmy1-2. Blue/Red arrowheads: PIN1 convergence points. Scale bars: 50 μm for IM and 20 μm for flowers. n = 3 inflorescences. e, Images of whole plants for WT and drmy1-2, showing the bushiness and short stature of drmy1-2. Scale bar: 2 cm. n = 3 plants. f, Confocal images of root meristems for WT and drmy1-2. The regions specified by yellow arrowheads indicate the meristematic zone. Scale bar: 50 μm. n = 3 root tips. g, Photograph of 10-day old seedlings for WT and drmy1-2, showing drmy1-2 has shorter roots and fewer lateral roots. Scale bar: 1 cm. n = 3 plates. h, Confocal images of inflorescence meristems for WT and drmy1-2 (Top view). Yellow dashed circles indicate how meristem sizes were measured in i. Scale bar: 50 μm. n = 10 inflorescence meristems. i, Quantification of inflorescence meristem sizes for WT and drmy1-2. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of inflorescence meristem sizes, WT versus drmy1-2: 2.01E-07). Measure of centre: mean. Error bars represent standard error of the mean. n = 10 inflorescence meristems. j, Histograms of divergence angles between siliques for WT and drmy1-2, showing the enhanced variability in phyllotaxy observed in drmy1-2 mutants. 137° is expected for spiral phyllotaxy observed in wild type.

Source data

Extended Data Fig. 7 Cytokinin treatment mimics the drmy1-2 mutant.

a, b, Confocal imaging of the whole inflorescences of WT (a) and drmy1-2 (b) cultured in mock conditions or 5 μm BAP (synthetic cytokinin) for 6 days. p35S::mCitrine-RCI2A: gray, for plasma membrane. Phenotypes quantified in Extended Data Fig. 7c. Blue or red arrowheads: flowers with obvious delayed sepal initiation phenotypes. Scale bars: 50 μm. c, Graph characterizing the proportions of flower phenotypes observed after 5 µM BAP treatment for 6 days. Normal phenotype (N, black) is defined as similar to wild type. Mildly affected phenotype (M, grey) is similar to drmy1-2. Severely affected phenotype (S, silver) is more severe than drmy1-2. n = 23 (N: 23/23, M: 0/23, S: 0/23) flowers from 7 inflorescences for mock treated wild type; n = 42 (N: 4/42, M: 35/42, S: 3/42) flowers from 12 inflorescences for mock treated drmy1-2; n = 47 (N: 2/47, M: 14/47, S: 31/47) flowers from 16 inflorescences for BAP treated wild type; and n = 37 (N: 0/37, M: 3/37, S: 34/37) flowers from 18 inflorescences for BAP treated drmy1-2. A one-sided Kolmogorov-Smirnov test was used to compare the distributions of different situations. p-value for WT mock versus drmy1-2 mock: 7.73 E-14; p-value for WT mock versus WT BAP: 1.67 E-16; p-value for WT BAP versus drmy1-2 mock: 4.53 E-9; p-value for WT BAP versus drmy1-2 BAP: 7.55 E-3; p-value for drmy1-2 mock versus drmy1-2 BAP: 1.95E-15. d, Graph of flower radius to assess developmental stage based on flower size. The radii of flowers without sepal primordia (SP = 0) or with only the outer sepal primordium (SP = 1) were measured for wild-type and drmy1-2 inflorescences cultured in either mock or 5 µM BAP for 6 days. The critical size threshold for outer sepal initiation is specified with a yellow dashed line. Note this size is the same for wild type mock and wild type BAP samples, indicating the stage of outer sepal initiation is not affected by BAP treatment. In contrast, for BAP treated drmy1-2, this characteristic size is more variable, consistent with the strongly enhanced phenotype. In the violin plots, the black line represents the median and the individual data points are shown. WT Mock SP = 0: n = 5; WT Mock SP = 1: n = 12; drmy1-2 Mock SP = 0: n = 11; drmy1-2 Mock SP = 1: n = 14; WT BAP SP = 0: n = 19; WT BAP SP = 1: n = 19; drmy1-2 BAP SP = 0: n = 19; drmy1-2 BAP SP = 1: n = 19. e, 5 µM BAP treatment on the DR5 auxin signaling reporter (white) for 3 days. p35S::mCherry-RCI2A: red, for plasma membrane; Red arrowhead: indicates the same flower before and after the BAP treatment. Scale bar: 50 μm. Note the DR5 signal becomes more diffuse after cytokinin treatment. n = 3 inflorescences. f, 5 µM BAP treatment on PIN1-GFP (cyan) auxin efflux carrier for 2 days. Red arrowhead: indicates the same flower before and after the BAP treatment; Scale bar: 50 μm. PIN1-GFP appears to form additional convergence points similar to drmy1-2. n = 3 inflorescences. g, Long-term treatment of flowers with 5 µM BAP causes severe sepal size defects. Wild-type inflorescences were cultured for 6 days on mock or BAP media, dissected to reveal flowers with initiating sepals, and further cultured for 14 days to examine the effects on sepal size. n = 3 flowers. h, The sepal area distribution for mature WT, drmy1-2, ahp6, drmy1-2 ahp6, and tir1-1 afb1-1 afb2-1 afb3-1 (tir1afb1-2-3 for short) sepals. The boxes extend from the lower to upper quartile values of the data and the whiskers extend past 1.5 of the interquartile range. Outliers are indicated with small dots. Sepals from different flowers were pooled together. n = 35 flowers. Wild-type and drmy1-2 data was subsampled from that shown in Extended Data Fig. 1b. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of sepal area, WT versus drmy1-2: 2.70E-33; WT versus ahp6: 1.42E-38; WT versus drmy1-2 ahp6: 9.91E-25; WT versus tir1 abf1-1 afb2-1 afb3-1: 1.22E-39). i, Average coefficient of variation (CV) calculated for the areas of all sepals in each single flower for WT, drmy1-2, ahp6, drmy1-2 ahp6, and tir1-1 afb1-1 afb2-1 afb3-1. n = 35 flowers. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of CV, WT versus drmy1-2: 4.99E-14; WT versus ahp6: 1.75E-13; WT versus drmy1-2 ahp6: 8.06E-16; WT versus tir1 abf1-1 afb2-1 afb3-1: 3.31E-32). Measure of centre: mean. Error bars represent standard error of the mean.

Source data

Extended Data Fig. 8 BAP treatment functions through cytokinin signaling.

a, 5 µM BAP treatment on the TCS cytokinin signaling reporter (gray) for 24 hours. Control showing that cytokinin treatment enhances TCS reporter expression. Chlorophyll autofluorescence: red. Scale bars: 50 μm. n = 3 inflorescences for each treatment. b, 5 µM BAP treatment on the cytokinin receptor mutant wol-1 for 4 days. Control showing that mutation of the cytokinin receptor (wol-1) abrogates delayed sepal initiation in response to cytokinin. Lower left flower removed during imaging. Cell walls stained with PI: gray. Scale bar: 50 μm. n = 3 inflorescences. c, NAA (auxin) treatment in a gradient of concentration on the TCS cytokinin signaling reporter (gray) for 24 hours. Auxin treatment did not enhance TCS reporter expression. Control showing that the induction of TCS reporter expression is specific to cytokinin treatment. Chlorophyll autofluorescence: red. Scale bars: 20 μm. n = 3 inflorescences for each treatment.

Extended Data Fig. 9 Cellular growth remains heterogeneous and randomly oriented for the drmy1-2 inner sepals.

a, b, Cumulative 18-hour cellular growth heatmap for both WT (a) and drmy1-2 (b) floral meristems. White dashed boxes highlight the bands of cells with slower growth rate which specify the boundary. They are always adjacent to the fast growth regions at the periphery, where sepals initiate. Segmented cells outlined in yellow. Three replicates are shown. Note that the drmy1-2 replicate 1 grows relatively normally. Scale bars: 10 μm. c,d, 18-hour cellular growth anisotropy heatmap for the same WT (c) and drmy1-2 (d) floral meristems. Growth anisotropy was calculated by dividing the cell stretch at the maximum direction by the cell stretch at the minimum direction. Cyan indicates higher growth anisotropy while black indicates lower growth anisotropy. White lines within the cells shows the maximum principle directions of growth. The initiating regions have higher anisotropy with the periphery part showing longitudinal growth and the boundary parts showing latitudinal growth. Three replicates are shown. Scale bars: 10 μm. e, Side views of the floral meristems at the last time points with outer sepals on the right and inner sepals on the left. The morphology of outer sepals was used for staging and appears equivalent in all samples. Arrowheads: the initiation/bulging of sepals from floral meristems. Scale bars: 10 μm. n = 3 flowers shown. R = replicate. Heat map and anisotropy are visualized on the later time point.

Supplementary information

Supplementary Information

Supplementary methods, Tables 1–3, captions for Supplementary Videos 1–7 and caption for Supplementary Data Table 1.

Reporting Summary

Supplementary Video 1

Live imaging every 6 h of a WT flower expressing a plasma membrane marker (35S::mCitrine-RCI2A, pLH13, white) to track sepal initiation events. Scale bars, 20 μm. n = 3 flowers.

Supplementary Video 2

Live imaging every 6 h of a drmy1-2 flower expressing a plasma membrane marker (35S::mCitrine-RCI2A, pLH13, white) to track sepal initiation events. Scale bars, 20 μm. n = 3 flowers.

Supplementary Video 3

Live imaging every 6 h of a WT flower with both DR5 (red) and TCS (green) to track the spatiotemporal distribution of auxin and cytokinin responses. Purple, chlorophyll autofluorescence for flower morphology. Scale bars, 10 μm. n = 3 flowers.

Supplementary Video 4

Live imaging every 6 h of a WT flower with DR5 (white) and plasma membrane marker (p35S::mCherry-RCI2A, red) to track the spatiotemporal distribution of auxin responses. Scale bars, 10 μm. n = 3 flowers.

Supplementary Video 5

Live imaging every 6 h of a WT flower with TCS (green) to track the spatiotemporal distribution of cytokinin responses. Red, chlorophyll autofluorescence for flower morphology. Scale bars, 10 μm. n = 3 flowers.

Supplementary Video 6

Live imaging every 6 h of a drmy1-2 flower with DR5 (white) and plasma membrane marker (p35S::mCherry-RCI2A, red) to track the spatiotemporal distribution of auxin responses. Scale bars, 10 μm. n = 3 flowers.

Supplementary Video 7

Live imaging every 6 h of a drmy1-2 flower with TCS (green) to track the spatiotemporal distribution of cytokinin responses. Red, chlorophyll autofluorescence for flower morphology. Scale bars, 20 μm. n = 3 flowers.

Supplementary Data Table 1

Gene annotations and RNA-seq data for WT versus drmy1-2 inflorescences (n = 3 biological replicates).

Source data

Source Data Fig. 1

Whole-population sepal area (n = 400) for Fig. 1c. CV for Fig. 1d with graphs of individual replicates. Sepal area for flowers along the main branches for Fig 1d (n = 3 for WT and drmy1-2). The last six sheets are also the source data for ED Figs. 1b and 4f.

Source Data Fig. 2

Time interval between sepal initiation events for Fig. 2e,f.

Source Data Fig. 3

Cellular growth rate quantified from the live-imaging series for Fig. 3e. Sepal area for 10th to 25th flowers along the main branches for Fig. 3f (n = 3 for WT and drmy1-2). The last three sheets are also the source data for ED Figs. 1e and 4g.

Source Data Fig. 4

Quantification of DR5 and TCS signal in flowers (n = 10, for Fig. 4c,d).

Source Data Extended Data Fig. 1

Quantification of floral organ numbers for ED Fig. 1a. Quantification of sepal shape variability for ED Fig. 1c. Quantification of petal area for ED Fig. 1f.

Source Data Extended Data Fig. 2

qRT–PCR raw results for DRMY1 expression for ED Fig. 2b.

Source Data Extended Data Fig. 3

Quantification of flower radius versus sepal initiation events for ED Fig. 3a. Quantification of AFM measurement for ED Fig. 3b. Quantification of osmotic treatment for ED Fig. 3d. GO term analysis for upregulated genes in the drmy1 mutant for ED Fig. 3e. GO term analysis for downregulated genes in the drmy1 mutant for ED Fig. 3f.

Source Data Extended Data Fig. 4

Quantification of cellular growth rate for stages 6–9 sepal development for ED Fig 4c.

Source Data Extended Data Fig. 6

Quantitation of inflorescence meristem size for ED Fig. 6i. Quantification of divergence angles between siliques along the main branches for ED Fig. 6j.

Source Data Extended Data Fig. 7

Characterization of flower morphology for WT and drmy1-2 flowers grown under mock or BAP treatment conditions for ED Fig. 7c. Quantification of flower radius versus sepal initiation events under mock or BAP treatment conditions for ED Fig. 7d. Quantification of sepal areas for WT, drmy1-2, ahp6, drmy1-2 ahp6 and tir1afb1-2-3 for ED Fig. h,i.

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Zhu, M., Chen, W., Mirabet, V. et al. Robust organ size requires robust timing of initiation orchestrated by focused auxin and cytokinin signalling. Nat. Plants 6, 686–698 (2020). https://doi.org/10.1038/s41477-020-0666-7

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