The petal identity gene PhDEF has a major binding and regulatory action in the epidermis
- Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, France
- Laboratoire de Biologie et Modélisation de la Cellule, École Normale Supérieure de Lyon, CNRS, UMR 5239, Inserm, U1293, Université Claude Bernard Lyon 1, France
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, France
Figures
Figure 1 with 7 supplements
Single-cell RNA-sequencing separates epidermal and mesophyll cell types in the petunia petal.
(A) Representative petunia wild-type (WT), star, wico, and phdef flowers from an upper (top picture) and side view (bottom picture). The limb and tube are indicated. Scale bar = 1 cm. Below the flower pictures, a schematic petal primordium is depicted with L1 (future epidermis) and L2 (future mesophyll) cell layers, and PhDEF expression in the WT, star, wico, and phdef primordia is indicated in orange. (B) Overview of the experimental protocol to produce petal protoplasts ready for isolation with the 10 X Genomics Chromium device. Upper picture: a WT flower cut into small fragments in the cell wall-digesting solution (scale bar = 1 cm). Bottom picture: isolated protoplasts from a WT petal after a 5 hr-long digestion process, viewed by bright-light microscopy (scale bar = 50 µm). (C) Uniform Manifold Approximation and Projection (UMAP) plot of 11,632 WT petal cells sequenced for their transcriptome, after integration from two biological replicates. Clusters of cells are color-coded and are ordered from the biggest to the smallest, based on the number of cells. (D) Dotplot of key marker genes for petal identity, stamen and carpel identity, epidermal identity, and pigmentation. The size of the dot represents the percentage of cells that express a given gene, and the color scale indicates the average expression level of the gene across all cells in a given cluster. st., stamen; ca., carpel. (E) Barplot of the number of genes enriched in the epidermis (magenta) or in the mesophyll (green), as defined by varying the log2FoldChange (log2FC) threshold and the layer specificity threshold (% of cells expressing the gene in the epidermis − % of cells expressing the gene in the mesophyll). The Kolmogorov-Smirnov two-sided test (KS test) was applied to compare the gene number distributions between epidermis- and mesophyll-enriched genes at a given layer-specificity threshold; n.s., non-significant, *p<0.05, **p<0.005, ***p<0.001.
Figure 1—figure supplement 1
Estimation of epidermal and mesophyll cell proportions from tissue sections or from petal protoplasting.
(A) Protoplasts isolated from WT mature petal. Strongly pigmented cells (like the ones pointed by the red arrowheads) are counted as cells from the adaxial epidermis, weakly pigmented cells (like the ones pointed by the blue arrowheads) are counted as cells from the abaxial epidermis, and the remaining cells (like the ones pointed by the green arrowheads) are counted as mesophyll cells. With this method, epidermal cells are underestimated since tube epidermal cells are not pigmented. Scale bar = 50 µm. (B,C) Cross-sections from WT petal limb (B) and tube (C) after toluidine blue staining, allowing to count adaxial epidermis (AdEp), abaxial epidermis (AbEp), and mesophyll (Meso) cells. Scale bar = 100 µm. (D) Dotplot and boxplot of the percentage of cells from the adaxial epidermis, abaxial epidermis, and mesophyll estimated from petal sections (tube and limb data merged) and from petal protoplast isolation. n=4 pictures (each containing on average 834 cells for protoplasts, and 486 cells for tissue sections). Student’s t-tests (two-sided) between protoplast and section cell numbers are non-significant in all cell layers.
Figure 1—figure supplement 2
Quality metrics of scRNA-Seq data.
(A) Quality and mapping metrics of the scRNA-Seq datasets produced in this study. Values are taken from the outputs of CellRanger. (B) Distribution of the number of unique features per cell (nFeature_RNA) and of the number of RNA counts per cell (nCount_RNA) for the two wild-type replicates (WT1 and WT2), the star and wico scRNA-Seq dataset.
Figure 1—figure supplement 3
Expression of selected marker genes in WT petal scRNA-Seq data.
(A–E) Dotplots of the expression of photosynthesis genes (Photosystem II subunits, A), S-phase genes (B), vasculature genes (C), histone genes (D), and epidermal (EPI) and mesophyll (MESO) markers from tobacco petals (Zhang et al., 2021) (E). Gene identifiers can be found in Supplementary file 1.
Figure 1—figure supplement 4
Barplots of the ten best Gene Ontology (GO) terms enriched for the best marker genes of each cluster of WT petal cells.
Ten most enriched GO terms (Biological Process) for the best cluster markers (with p-adjusted value <0.01 and log2FC >1, as compared to all the other clusters) after redundancy reduction with REVIGO. The color indicates the significance of the enrichment (log10(p-value)) and the length of the bar indicates the number of genes associated to this GO term (log10(Count)). The full list of GO enriched terms is provided in Supplementary file 2.
Figure 1—figure supplement 5
Expression of cluster marker genes in limb and tube WT petal tissue and corresponding UMAP plots.
(A) Relative expression level of chosen cluster markers in limb and tube tissue from WT flowers at anthesis, measured by RT-qPCR. No suitable marker could be selected for mesophyll cluster 2. n=3 biological replicates, mean ± SEM. Student’s t-test two-sided or Wilcoxon rank sum exact test (for non-parametric data distribution for KCS3 and PhTPS1), *p<0.05, **p<0.005, ***p<0.001. (B) UMAP plots of gene expression in WT scRNA-Seq data for chosen cluster markers. Average expression is shown as a color scale set by default. For each plot, the cluster for which the gene is a marker is indicated. Gene identifiers are available in Supplementary file 2. (C) In situ hybridization for the IAA14 transcript, enriched in cluster 0, in limb (up) and tube (bottom) cross-sections. Sections are oriented with the upper epidermis at the top. A colorimetric signal is detected in the upper and lower epidermis in the limb, but not in the tube. Scale bar = 50 µm.
Figure 1—figure supplement 6
Effect of removing differentially expressed genes (DEGs) by the protoplasting process on the scRNA-Seq UMAP and cell type proportions.
UMAP plots (A) and percentage of cells in each cluster (B) of WT petal scRNA-Seq data, removing DEGs by the protoplasting process at different log2FoldChange cut-offs (|log2FC|=absolute value of log2FoldChange), or keeping all genes. The number of genes removed is the following: 9985, 7646, 5966, and 4748 for |log2FC| values over 1.0, 1.5, 2.0, and 2.5, respectively. The color code for cell types is the same for the UMAP plots and the barplot, and corresponds to the cluster number. For this analysis, integration of datasets with Harmony slightly changes the UMAP shape and the identity of clusters, as compared to Figure 1C with the WT dataset with all genes; therefore, clusters are not directly comparable.
Figure 1—figure supplement 7
Barplots of the 10 best Gene Ontology (GO) terms enriched in the epidermis- and mesophyll-enriched genes.
Ten most enriched GO terms (Biological Process) for the epidermis- and mesophyll-enriched genes (selected with log2FC >0.75, specificity of expression >10% and adjusted p-value <0.01, as compared to the other layer) after redundancy reduction with REVIGO. The color indicates the significance of the enrichment (log10(p-value)), and the length of the bar indicates the number of genes associated to this GO term (log10(Count)). The full list of GO-enriched terms is provided in Supplementary file 2.
Figure 2 with 2 supplements
PhDEF regulates a different set of genes in the petal epidermis and mesophyll.
(A) Uniform Manifold Approximation and Projection (UMAP) plots of 11,632 WT, 3875 star, and 3737 wico petal cells sequenced for their transcriptome, after integration and clustering. Here, epidermal clusters (purple) and mesophyll clusters (green, without the vasculature) were merged. (B) Percentage of cells from the different epidermal and mesophyll identities identified from WT, star, and wico petal scRNA-Seq. (C) Per-cell expression levels of PhDEF and PhGLO1 in WT, star, and wico displayed on UMAP plots. (D) Dotplot of PhDEF and PhGLO1 expression level (color-coded) and percentage of cells expressing the gene (coded in the size of the dot) in the epidermis and mesophyll cells of WT, star, and wico petals. (E) Pearson’s correlation coefficient (r) plot between the pseudo-bulk transcriptomes from the mesophyll, epidermis, and vasculature cells extracted from the WT, star, and wico scRNA-Seq data. A Fisher r-to-z transformation test indicates that all correlation coefficients are significantly different (p<0.05). (F) Venn diagram of the number of epidermis (purple) and mesophyll (green) DEGs, revealing the number of epidermis- and mesophyll-specific DEGs, as well as common DEGs (i.e. differentially expressed in both layers), based on WT, star, and wico scRNA-Seq data. The percentage of activated (light gray) and repressed (dark gray) genes is also displayed. (G) Differential expression (log2FC = log2(FoldChange)) of the common DEGs in the epidermis or in the mesophyll, showing that almost all DEGs are either activated or repressed in both layers. Three genes of interest (PhDEF, PhGLO1, and PhGLO2) are displayed as color points. (H) Ten most enriched Gene Ontology (GO) terms for biological processes in epidermis-specific (left), mesophyll-specific (middle), and common (right) DEGs, after GO term redundancy reduction and sorting by p-value.
Figure 2—figure supplement 1
star flowers frequently display pigmented sectors and wico flowers frequently show pigmentation defects.
(A) Small pigmented L1-revertant sectors are visible in the petal limb of star flowers, due to dTph1 excision in the epidermis, restoring PhDEF expression in clonal sectors. Scale bar = 1 cm. (B) Two wico flowers with pigmentation defects (middle and right flowers), with a WT flower on the left for comparison. Scale bar = 1 cm.
Figure 2—figure supplement 2
Heatmap of correlation coefficients between pseudo-bulk transcriptomes from epidermis, mesophyll, and vasculature of WT, wico, and star.
Figure 3 with 2 supplements
PhDEF binds to more target genes in the epidermis than in the mesophyll.
(A) Metaplot of PhDEF binding sites in WT, wico, and star petals, with the distance from the Transcription Start Site (TSS) and the Transcription Termination Site (TTS). (B) Heatmap of the read coverage of all peaks detected in WT samples, and of corresponding regions of the genome in wico and star samples. The peaks are sorted according to the highest to lowest read coverage in WT #2, so that the same regions of the genome are in the same line in all libraries. The read coverage is color-coded, and position 0 represents the start of the peak. (C) Read coverage for PhDEF binding to the genomic regions of PhDEF and PhGLO1 in WT, star, and wico petals. Peaks that pass our pipeline for reproducibility (see Materials and methods) are displayed as thick red lines. Predicted MADS-binding sites (MADS-bs) are indicated by blue lines, while no TCP-bs were found in these sequences. Although the peak in the promoter region of PhDEF does not pass the IDR threshold, its read coverage profile strongly suggests that PhDEF binds to this position. (D) Intersection of the number of gene-associated peaks from WT, star, and wico ChIP-Seq data, and definition of the possible PhDEF binding profiles. The intersection occasionally resulted in the artificial duplication of peaks, and once corrected, this marginally changed the total number of peaks identified for each genotype in the intersection. (E) Distribution of PhDEF binding profiles across its target genes. (F–G) PhDEF binding profile over the genomic locus of AN1 (F) and EOBII (G). Predicted MADS-binding sites (MADS-bs) are indicated by blue lines, and predicted TCP-binding sites (TCP-bs) are indicated by orange lines.
Figure 3—figure supplement 1
Assessment of ChIP efficiency by qPCR, and ANOVA of PhDEF expression levels in the WT epidermis and mesophyll.
(A) Measure of ChIP enrichment in two ChIP assays (ChIP-1 and ChIP-2) for two replicates in each assay, for WT, star, and wico. The enrichment is calculated as percentage of Input, measured by qPCR on two positive control binding sites (in the PhDEF promoter, and in the Pos2 gene) and one negative control binding site (Neg1). The replicates from the first ChIP assay depicted a much better enrichment and were chosen for sequencing. (B) Barplot of the number of cells (count, y-axis) expressing PhDEF at different levels (log-normalized read counts, x-axis) in the WT epidermis (left) and in the WT mesophyll (right), extracted from the WT scRNA-Seq data. The upper panels depict all cells, and the lower panels depict only cells that express PhDEF. The mean value of PhDEF expression is 0.89 in the epidermis and 1.06 in the mesophyll (1.71 and 2.01 after removing cells that do not express PhDEF, respectively, p<2.2 e–16, Wilcoxon rank sum test, one-tailed). The coefficient of variation of PhDEF expression is 1.08 in the epidermis and 1.06 in the mesophyll (0.36 and 0.34 after removing cells that do not express PhDEF, respectively, p=0.00106 with Feltz and Miller’ asymptotic test).
Figure 3—figure supplement 2
Heatmap of the read coverage of peaks unique to a genotype.
Heatmap of the read coverage of regions corresponding to peaks uniquely detected in WT (A), wico (B), and star (C) samples. The peaks are sorted according to the highest to lowest read coverage in WT #2 (A), wico #1 (B), and star #1 (C), so that the same regions of the genome are in the same horizontal line in all libraries. The read coverage is color-coded, and position 0 represents the start of the peak.
Figure 4 with 3 supplements
Intersection between PhDEF binding and regulatory profiles, and motif enrichment under PhDEF binding sites.
(A) Distribution of PhDEF binding profiles across genes differentially expressed (DEGs) in star only, wico only, or both in star and wico (common DEGs), as compared to WT, from bulk RNA-Seq of petals at stage 8. Stars indicate a significant deviation from expected PhDEF binding profiles, either for the whole distribution (red star, Chi2 goodness-of-fit test) or an enrichment of individual categories (black star, test of equal proportions, one-sided). Binding categories with less than 5 genes were not tested for significant enrichment. (B) Distribution of PhDEF binding profiles across epidermis-specific, mesophyll-specific, or common (in both layers) DEGs, obtained from WT, star, and wico scRNA-Seq by comparing layer-specific transcriptomes. (C) Selected motifs enriched under PhDEF ChIP-Seq peaks for WT, wico, and star petals, bs = binding site. The full list of motifs detected is in Figure 4-figure supplement 2.
Figure 4—figure supplement 1
Venn diagram depicting the intersection of the ChIP-Seq, bulk RNA-Seq, and scRNA-Seq datasets.
Genes bound by PhDEF are all genes bound by PhDEF in ChIP-Seq in WT, wico, and/or star at stage 8. DEGs in RNA-Seq are DEGs in star (stage 8) and/or wico (stage 8) as compared to WT (stage 8). DEGs in scRNA-Seq are DEGs in star epidermis vs. WT epidermis and/or in wico mesophyll vs. WT mesophyll in mature petals. The overlap between genes bound by PhDEF and DEGs in RNA-Seq is significant (506 genes, p=4.73e-13, hypergeometric test), the overlap between genes bound by PhDEF and DEGs in scRNA-Seq is significant (262 genes, p=1.37e–04), and the overlap between DEGs in RNA-Seq and DEGs in scRNA-Seq is significant (274 genes, p=2.52e–17).
Figure 4—figure supplement 2
Full output of MEME-chip with motifs significantly enriched below ChIP peaks in WT, wico, and star.
MEME-chip output for WT (A), wico (B), and star (C) ChIP-Seq peaks associated with genes. The column ‘similar motifs’ corresponds to motifs automatically proposed by MEME-chip based on similarity with the JASPAR 2024 database, while the column ‘Interpretation’ is our own interpretation of the motif based on the family of transcription factors found to be similar.
Figure 4—figure supplement 3
Expression level of B- and E-class MADS-box genes and TCP genes in scRNA-Seq and bulk RNA-Seq.
(A) Dotplot of the expression of B- and E-class MADS-box genes in the WT epidermis and mesophyll. For E-class genes, the name of the A. thaliana ortholog is indicated between parentheses, from Morel et al., 2019. (B) Dotplot of the expression of TCP genes in the WT epidermis and mesophyll. The name and identifiers of P. axillaris TCP genes originate from Wanamaker et al., 2017. The TCP genes that are not displayed here were not found to be expressed. (C) Expression level of PaTCP4a in petals from WT, star, and wico, from bulk RNA-Seq at different stages (4, 8, and 12). Stars indicate a significantly higher expression in wico than in star, as computed by DESeq2 (log2FC >1, p-adj<0.01). (D) Dotplot of PaTCP4a expression in WT, star, and wico epidermis and mesophyll, from scRNA-Seq data at mature stage.
Additional files
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Supplementary file 1
Main cluster marker genes used to identify scRNA-Seq clusters.
Related to Figure 1. First sub-table: list of Petunia axillaris and Arabidopsis thaliana gene identifiers (ID) and gene names enriched in WT scRNA-Seq clusters, and used to assign cell type identity to clusters. Second sub-table: list of P. axillaris and Nicotiana attenuata (tobacco) gene identifiers (ID) and gene names, from epidermis-enriched and mesophyll-enriched genes in the tobacco petal (Zhang et al., 2021). Gene orthology was inferred by best or second best reciprocal Blast hit, as described in the Methods section. ‘This study’ indicates that the gene name had not been assigned previously in petunia.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp1-v1.xlsx
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Supplementary file 2
Gene Ontology terms enriched in the best cluster markers from WT petal cells, in the layer-enriched DEGs, and in the layer-specific DEGs.
Related to Figure 1 and Figure 2. List of enriched GO terms for the best cluster markers (with adjusted p-value <0.01 and log2FC >1, as compared to all the other clusters) of WT petal cells, in the layer-enriched DEGs (with adjusted p-value <0.01, log2FC >0.75 and specificity of expression >10%, as compared to the other layer), and in the layer-specific DEGs (differentially expressed in the star epidermis, in the wico mesophyll, or in both for the common DEGs). Each sheet contains the GO term and its description, the count and fraction of genes associated with this term in the subset and in the control (all genes expressed in the petal), and the enrichment ratio and p-value for enrichment. The terms highlighted in yellow are the ones retained after reduction of redundancy with REVIGO, and displayed on Figure 2H, S4, and S7.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp2-v1.xlsx
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Supplementary file 3
List of differentially expressed genes after WT petal protoplasting.
Related to Figure 1. Total RNA was sequenced from WT petal tissue, with or without protoplasting. Differential expression was computed with DESeq2. log2FoldChange = fold change of a given gene in protoplasted vs. crude WT petal tissue, expressed in log2 value; lfcSE = standard error of log2FoldChange; padj = p-value adjusted for multiple testing; GOterms = GO terms associated with a given gene; Annotation = automatic annotation from Li et al., 2011; Manual Annotation = manual annotation of pigmentation-related genes and transcription factors of interest for petal identity and development.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp3-v1.xlsx
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Supplementary file 4
List of layer-enriched genes and genes with layer-specific differential expression in phdef mutant cell layers.
Related to Figures 1 and 2. The content of the table is explained in the first sheet. The second sheet contains the list of layer-enriched genes (as determined with the WT scRNA-Seq data), defined as having an absolute log2(FoldChange) > 0.75 and a specificity of expression >10%; a positive log2(FoldChange) indicates that the gene is enriched in the epidermis, and a negative one that the gene is enriched in the mesophyll. The third, fourth, and fifth sheets contain respectively the lists of genes that are differentially expressed in the star epidermis, the wico mesophyll, and both. These represent potential direct and indirect targets of PhDEF in individual cell layers.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp4-v1.xlsx
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Supplementary file 5
ChIP metrics and position of all ChIP peaks detected in WT, star, and wico ChIP-Seq.
Related to Figure 3. The first sheet contains ChIP mapping metrics for each library: raw number of reads, number of unique reads, percentage of duplicated reads, percentage of mapping, number of reads after mapping quality filtering, number of mapped reads in IDR peaks, and FriP score (fraction of reads in peaks). The following sheets contain the position of the peak in the genome (Chromosome, start, end) and the peak identifier (PEAK_ID) for all genotypes and their intersections.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp5-v1.xlsx
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Supplementary file 6
Number of peaks and genes from the WT, star, and wico ChIP-Seq experiment.
Related to Figure 3. Upper table: Number and percentage of peaks associated with genes in their promoter (6 kb upstream of the TSS), terminator (6 kb downstream of the TTS), exon, or intron, in intergenic regions or unannotated regions of the genome (N/A), from the WT, star, and wico ChIP-Seq experiment. Peak enrichment was computed with a hypergeometric test, *** p<0.001. Lower table: Number and percentage of genes with 1–4 ChIP peaks detected, from the WT, star, and wico ChIP-Seq experiment.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp6-v1.xlsx
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Supplementary file 7
Position of all gene-associated ChIP peaks detected in WT, star, and wico ChIP-Seq, and their intersections.
Related to Figure 3. Each table contains the identifier of the peak (Domain), its position in the genome (Chromosome, start, end), the associated gene (as defined with the shortest distance to the peak), the position of the gene (start, end), the shortest distance of the peak to the gene, and the annotation of the peak position related to the gene (promoter, terminator, exon, or intron). The first three sheets contain the gene-associated peaks found in WT, wico, and star ChIP-Seq, and the following sheets contain the list of peaks detected in a single genotype or detected in the different intersections (WT-wico, WT-star, wico-star, WT-wico-star).
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp7-v1.xlsx
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Supplementary file 8
Analysis of enrichment of TCP-binding sites.
Related to Figure 3. TCP-binding sites were predicted with Fimo under epidermal-specific PhDEF binding peaks (Epi), mesophyll-specific (Meso), or shared peaks. Binding sites were either predicted for all peaks or for gene-associated peaks only. Control sequences were either random DNA sequences artificially generated or random sequences taken under genes from the P. axillaris genome (to compare with gene-associated peaks) or taken under random regions of the genome excluding telomeres and centromeres (to compare with all peaks). These control sequences were designed to have the same distribution of lengths and total size as PhDEF binding peaks. A Fisher’s exact test was applied to compare observed and expected binding sites.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp8-v1.xlsx
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Supplementary file 9
List of primers used in this study.
List of primers used for RT-qPCR (Figure 1-figure supplement 5) and in situ hybridization (ISH, Figure 1-figure supplement 5). The red nucleotides in primer MLY2949 indicate the T7 promoter for in vitro transcription.
- https://cdn.elifesciences.org/articles/113154/elife-113154-supp9-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/113154/elife-113154-mdarchecklist1-v1.pdf
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The petal identity gene PhDEF has a major binding and regulatory action in the epidermis
eLife 15:RP113154.
https://doi.org/10.7554/eLife.113154.2
