WUSCHEL-dependent chromatin regulation in maize inflorescence development at single-cell resolution

Abstract

Background: WUSCHEL (WUS) is a homeodomain transcription factor vital for stem cell proliferation in plant meristems. In maize, ZmWUS1 is expressed in the inflorescence meristem, including the central zone reservoir of stem cells. ZmWUS1 overexpression in the Barren inflorescence3 (Bif3) mutant perturbs inflorescence development due to stem cell over-proliferation.

Results: Single-cell Assay for Transposase Accessible Chromatin sequencing (scATAC-seq) shows that Bif3 alters central zone chromatin accessibility compared to normal inflorescence meristems. The CAATAATGC motif, a known homeodomain recognition site, is enriched within regions with increased chromatin accessibility in Bif3, suggesting ZmWUS1 could function as a transcriptional activator in the central zone. This motif differs from the TGAATGAA motif identified by DNA Affinity Purification sequencing (DAP-seq) of ZmWUS1, which showed low enrichment in the central zone. Conversely, regions with decreased chromatin accessibility in Bif3 are instead adjacent to AUXIN RESPONSE FACTOR genes, suggesting possible reduced auxin signaling in the Bif3 central zone.

Conclusions: This study characterized how Bif3 overexpression of ZmWUS1 influences chromatin accessibility in the central zone, reducing auxin signaling, while raising questions about differential ZmWUS1 motif usage in distinct cellular contexts.

Keywords: Cis-regulatory elements; WUSCHEL; ZmWUS1; Epigenomics; Inflorescence meristem; Maize ear; Meristem development; Single-cell ATAC-seq.

Fig. 1

Annotation of central zone nuclei with chromatin accessibility in WT and Bif3. A UMAP plot for WT and Bif3, colored to represent the 14 cell types. B Illustrations depicting the phenotypes of WT and Bif3, including longitudinal sections of the inflorescence meristem, the spikelet pair meristem, the spikelet meristem, and the floral meristem are colored to correspond with the cell types in the UMAP. Grey colors represent cell types with an unknown annotation. C–G UMAP plots highlighting nuclei exhibiting high gene body chromatin accessibility, depicted in purple, surrounding the marker genes of ZmWUS1 (C), ZmCLE7 (D), ZmOCL4 (E), ZYB15 (F), and RA1 (G). H Genome browser track displaying chromatin accessibility around marker genes for meristem-associated cells in WT and Bif3. The ranges of number of indicate CPM (counts per million) normalized Tn5 insertion number. I Gene body chromatin accessibility patterns of 30 marker genes in WT and Bif3. The dot size represents the percentage of nuclei that have chromatin accessibility around the marker genes. The values are Z-score normalized aggregated number of Tn5 insertions by cell types

Fig. 2

The intergenic chromatin accessibility profile of WT and Bif3. A A heatmap depicts the correlation of intergenic ACRs between WT and Bif3. The 2000 most variable intergenic ACRs were selected for analysis. The correlation is based on the aggregated number of Tn5 insertions by cell type between WT and Bif3. B A dot plot displays the number of differential intergenic ACRs across various cell types. The illustration provides examples of differential ACRs with either higher chromatin accessibility in WT or Bif3. The total number of differential ACRs, as well as those exhibiting higher chromatin accessibility in either Bif3 or WT, are distinctly colored. The ratio of differential ACRs is calculated by dividing the number of differential ACRs by the total number of intergenic ACRs

Fig. 3

Characteristics of differentially Accessible Chromatin Regions (differential ACRs) between WT and Bif3 by cell types. A The Position Weight Matrix (PWM) illustrates significant motifs discovered within differential ACRs in the central zone (E-value < 1). The ratio indicates the number of differential ACRs containing the motif divided by the total number of differential ACRs. The color denotes whether the differential ACR sets are increased or decreased in Bif3 in the central zone. B Box plots display the log2 fold change (log2FC) of gene body chromatin accessibility surrounding differential ACRs increased in Bif3 with the CAATAATGC motif. Log2FC was calculated by normalizing gene body chromatin accessibility of differential ACRs in Bif3 to those of WT. Rows represent individual genes, whereas columns represent cell types. Pairwise two-tailed Student’s t-test, ****FDR < 2e-16 and *FDR < 0.05. C A genome browser view illustrates chromatin accessibility in the central zone for the peaks with the CAATAATGC motif

Fig. 4

TheARFgenes are associated with decreased differential ACRs by overexpression of ZmWUS1. A Bar graph illustrates significant biological process terms identified using a Fisher’s exact test using the genes closest to the differential ACRs in the central zone (FDR < 0.05). B The left heatmap shows the logFC of differential ACRs around ARF genes, whereas the right heatmap shows the gene body chromatin accessibility of ARFs by cell types. C Chromatin accessibility in the central zone around ZmARF4 and ZmARF23. The blue bar and yellow highlighted region indicate the differential ACRs

Fig. 5

The ZmWUS1 motif identified using DAP-seq shows different activity within ACRs depending on the cell type. A A genome browser view showing the peaks from ZmWUS1 DAP-seq. The y-axis shows CPM values. B A metaplot displaying the ratio of reads within a 1-kilobase pair region surrounding the ZmWUS1 DAP-seq peaks. C Identification of the ZmWUS1 motif using a k-mer set analysis, along with PWM models of the most enriched and significant motifs. D The motif deviation of ZmOCL1 in WT and Bif3. The motif is known for Arabidopsis HDG1, which is orthologous to ZmOCL1. The box with arrows indicates the epidermis cells. E The motif deviation of ZmWUS1 in WT and Bif3. The box with arrows indicates the central zone cells

Fig. 6

A hypothetical model that could explain central zone specific ZmWUS1 cis-regulation. A Proposed model of motif usage of ZmWUS1 in central zone cells. The CAATAATGC motif was identified in the central zone cells as a potential ZmWUS1 binding site, due to its presence in the differential ACRs of the central zone, where chromatin accessibility increased in Bif3. The cell-type-specific motif usage by ZmWUS1 could be attributed to unique molecular interactions in central zone cells. B A hypothetical epidermal model. In contrast, to the central zone, ACRs in the epidermis were largely unchanged in the Bif3 mutant, suggesting that the effect of overexpressed ZmWUS1 may be minimal or absent in epidermis cells. However, the ZmWUS1 DAP-seq motif, TGAATGAA, was enriched in the epidermal ACRs. It remains unclear how ZmWUS1 might engage this motif, although one possibility is that epidermal cells acquire ZmWUS1 activity through translocation from the central zone. A structural prediction of ZmWUS1 as a homodimer binding the TGAATGAA motif is shown