A single-nuclei transcriptome census of the Arabidopsis maturing root identifies that MYB67 controls phellem cell maturation

Abstract

The periderm provides a protective barrier in many seed plant species. The development of the suberized phellem, which forms the outermost layer of this important tissue, has become a trait of interest for enhancing both plant resilience to stresses and plant-mediated CO₂ sequestration in soils. Despite its importance, very few genes driving phellem development are known. Employing single-nuclei sequencing, we have generated an expression census capturing the complete developmental progression of Arabidopsis root phellem cells, from their progenitor cell type, the pericycle, through to their maturation. With this, we identify a whole suite of genes underlying this process, including MYB67, which we show has a role in phellem cell maturation. Our expression census and functional discoveries represent a resource, expanding our comprehension of secondary growth in plants. These data can be used to fuel discoveries and engineering efforts relevant to plant resilience and climate change.

Keywords: DAP-seq; development; differentiation; periderm; phellem; roots; snRNA-seq; suberin; transcriptome.

Figure 1.. Single-nuclei sequencing of mature Arabidopsis root identifies potential phellem nuclei populations.

Periderm development begins in the pericycle, forming phellogen cells, which differentiate into phelloderm and phellem (A). Using a GPAT5 reporter, we tracked periderm development across roots at 6–14 DAG (B). GPAT5 signal is indicated with white arrows. The images in the horizontal rectangle boxes show the cross-section, X section. The red lines in the vertical rectangle image boxes indicates the position of these digital cross-sections. Endodermal and phellem cells are stained with Nile Red; Calcofluor white is used as a counterstain. Phellem (ph); Cortex (co); Epidermis (ep); Endodermis (en). Scale bar = 20um. Single-nuclei sequencing over this period yielded a UMAP with 24 clusters (C), with suberin-related gene expression in four clusters. Clusters 21 and 13 were enriched in endodermal markers, while clusters 9 and 15 represented putative phellem nuclei. The average gene expression level for a given gene is indicated in the dot plot by the dot colour intensity. The size of each dot provides information regarding the proportion (%) of nuclei in a given cluster that showed expression of that gene (D). An additional single-nuclei experiment on the upper 1.5 cm of the root at 14 DAG further supported clusters 9 and 15 as phellem-related, with most suberin-enriched nuclei from this region clustering here (E). The schematic in panel A shows periderm development as illustrated by Wunderling et al., 2018. See also Data S1.

Figure 2.. Validation of suberising cell-type clusters.

BGAL8, a marker for cluster 13 (A), was expressed in the suberising endodermis at 14 DAG, colocalizing with Nile Red (red arrow) (B). BGAL8 was also observed in the third periderm layer, the phelloderm (asterisks indicate the periderm layers) (C). The schematic indicates root positions for each image (D). SGNH (AT5G37690), a marker for cluster 15 (E), showed expression in phellem (F) and suberised lateral root bases (G). Cluster 9 marker TLL1 (AT1G45201) (H) appeared only in mature phellem closer to the hypocotyl (I-J). The proportion of nuclei assigned to cluster 15 and 9 were plotted across the timecourse. This showed an increase over time for cluster 9, in line with it relating to a maturing cell type, while the young phellem cells fractions (cluster 15) were seen peak at 7DAG and then to flux overtime (K). Phellem (ph); Phellogen (pg); Phelloderm (pd); Cortex (co); Epidermis (ep); Endodermis (en); Pericycle (pe). Scale bar = 20um. See also Data S1 and supplementary Figure 1.

Figure 3.. Colocalization of DOT1 and SGNH as markers for early phellem development.

In the UMAP, DOT1 expression is detected in the pericycle (cluster 3) and in the young phellem-related cluster (cluster 15). This region is highlighted with a dotted line (A). These expression patterns were validated in planta using a transgenic line expressing DOT1:NLS-mCitrine. In the young root, mCitrine signal was detected in the pericycle (B) and in the mature root, signal was observed in the phellogen (C). DOT1 signal was also observe in dividing cells, which is in line with the phellogen expression pattern observed (D). The schematic shows root positions for each image (E). UMAP analysis indicated colocalization of DOT1 and SGNH in a subset of nuclei (F), confirmed using transgenic lines expressing both DOT1:NLS-mCitrine and SGNH:NLS-mScarlet. Colocalization was seen in pericycle cells just preceding developing phellem (G) and in phellem cells (H). Grey arrows indicate colocalization, white arrows indicate SGNH-only nuclei. Phellem (ph); Phellogen (pg); Cortex (co); Epidermis (ep); Endodermis (en); Pericycle (pe). Scale bar = 20 µm. See also Data S1.

Figure 4.. Key biological processes in phellem development identified through differentially expressed genes across pseudotime.

A phellem-focused UMAP was generated by subclustering pericycle, young phellem, and mature phellem nuclei. Using mature phellem as pseudotime zero, pseudotime analysis was performed in reverse with the pseudotimeline divided into equal-sized pseudobins (A). Feature plots for validated genes are shown (B). WOX4 expression identified pseudobin P4 as the likely start of phellem differentiation from the pericycle (C). DEG analysis from P4 onward highlighted the top 200 genes per bin, displayed in a heatmap (D), with bins P4-P6 representing pericycle, YPH1-YPH2 young phellem, and MPH1-MPH3 mature phellem. GO analysis of DEGs was conducted for each pseudobin (E). See also Data S2.

Figure 5.. MYB67 as a regulator of phellem development.

MYB67 is expressed within a small population of nuclei spanning two clusters relating to young phellem and maturing phellem (A and B). Phellem region length was significantly greater in two myb67 mutant alleles compared to WT roots (C) where “n” relates to the number of individual roots measured. Cell length was quantified along the length of the phellem region at 10% intervals (with 0% being at the hypocotyl junction and 90% being in some of the youngest phellem cells). For each sample at least 100 phellem cells were measured. A significant increase in phellem cell length was observed in the oldest phellem in myb67–1 (n = 15) relative to WT (n = 15) where “n” relates to number of roots phenotyped. No differences between genotypes were observed in the younger phellem cells found closer toward the root tip (D). All root phenotype data is presented as the mean ± SEM. The assigned P values are indicated by asterisks, where <0.05 = *, <0.001 = ** and <0.0001 =***. These statistical significance values were determined by two-tailed t test. See also Data S3–5 and Supplementary Figures 3–5. Transcriptional (E) and translational (F) reporter lines showed the expression of MYB67 in the phellem. Bulk RNA analysis of WT and myb67–1 roots identified 184 DEGs (fold change ≥1.5, FDR <0.05 calculated using the Benjamini-Hochberg method), visualized in a volcano plot where some key genes of interest are annotated (G). To identify which of these genes might represent direct targets of MYB67, DAP-seq was performed. This identified a likely MYB67 binding motif (H). A significant number of down-regulated DEGs were identified as direct DAP-seq targets. These are highlighted in panel G and a subset of these are shown in greater detail in panel I, which shows the DAP-seq and mRNA-seq tracks as well as the expression pattern of these genes in the context of the single nuclei UMAP. For each track the data ranges are shown in the upper left corner.