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Transcriptional and imprinting complexity in Arabidopsis seeds at single-nucleus resolution

Nature Plants volume 7, pages 730–738 (2021)Cite this article

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Abstract

Seeds are a key life cycle stage for many plants. Seeds are also the basis of agriculture and the primary source of calories consumed by humans1. Here, we employ single-nucleus RNA-sequencing to generate a transcriptional atlas of developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the primary site of gene imprinting in flowering plants, mediates the relationship between the maternal parent and the embryo2. We identify transcriptionally uncharacterized nuclei types in the chalazal endosperm, which interfaces with maternal tissue for nutrient unloading3,4. We demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell-cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. Thus, imprinting is spatially and temporally heterogeneous. Increased paternal expression in the chalazal region suggests that parental conflict, which is proposed to drive imprinting evolution, is fiercest at the boundary between filial and maternal tissues.

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Fig. 1: Distinct nuclei types in Arabidopsis endosperm.
Fig. 2: Identification of clusters by in situ hybridization analysis.
Fig. 3: Imprinting heterogeneity in endosperm.

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Data availability

All sequencing data generated in this study have been deposited to the NCBI Gene Expression Omnibus with accession number GSE157145.

Code availability

Scripts used in analysis have been deposited to Github at https://github.com/clp90/endosperm_snRNAseq_2021.

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Acknowledgements

We thank the Massachusetts Institute of Technology (MIT) BioMicro Center and the Whitehead Institute Genome Technology Core and Flow Cytometry Core Facility for research assistance, and F. Lafontaine for valuable input on statistical methods. This research was funded by NIH R01 GM112851 and NSF MCB 1453459 grants to M.G., NSF Graduate Research Fellowship and Abraham Siegel Fellowship to C.L.P., and NSF IOS 1812116 to R.A.P.

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Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Colette L. Picard, Rebecca A. Povilus, Ben P. Williams & Mary Gehring

  2. Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, MA, USA

    Colette L. Picard

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Mary Gehring

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Contributions

M.G. and C.L.P. conceived the project. C.L.P. and R.A.P. conducted experiments. C.L.P., R.A.P. and B.P.W. analysed data with input from M.G. C.L.P. and M.G. wrote the manuscript with edits from R.A.P and B.P.W.

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Correspondence to Mary Gehring.

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Extended data

Extended Data Fig. 1 Seed developmental stages assayed, FANS profiles, and impact on endosperm enrichment.

(a) Summary of seed developmental stages in the different genotypes and timepoints assayed. Number of seeds imaged for each bar shown at top. Scale bar 100 μm. (b) FANS sorting profiles of Col x Cvi (CxV) seeds at 2 DAP (sorted 09/26/17), 3 DAP (08/10/17), 4 DAP (11/16/17) and 5 DAP (11/14/17). The 2 DAP sample was processed on a different FACS machine than the other three samples. (c) Percent of allelic reads that were derived from the maternally inherited allele, for nuclei assigned as embryo, endosperm, and seed coat (see methods). Median, interquartile range and upper-/lower-adjacent values (1.5*IQR) indicated by center line, box, and whiskers of each boxplot, respectively. (d) Percent of nuclei per batch (96-well plate) assigned to endosperm. Nuclei from later timepoints, as well as from the 6C peak, are more likely to correspond to endosperm than nuclei from earlier timepoints or from the 3C peak.

Extended Data Fig. 2 Clustering of all 1437 high-quality nuclei in the dataset.

(a) Heatmap of SC3 clustering of all 1437 nuclei. Genotype, FANS peak, prep method (see ‘Seed nuclei FANS’), sequencing type, % maternal (percent of allelic reads derived from maternal allele), and seed age also shown. (b) Partitioning of the variance in CPM values for the 22,950 expressed genes in the dataset over the 1437 nuclei samples, according to tissue, peak, genotype and DAP, using the R package ‘variancePartition’53. Median, interquartile range and upper-/lower-adjacent values (1.5*IQR) indicated by center line, box, and whiskers within each violin plot. (c) Same as (b), over the 1096 Col x Cvi and Cvi x Col 4 DAP samples only. In this group, prep and sequencing type are less confounded with sources of biological variation (for example all washed samples are either Col x Cvi or Cvi x Col 4 DAP, so prep is confounded with genotype and DAP in the full dataset), so their contribution to the variation could be more reliably estimated. (d) Average expression of marker genes for various seed compartments (globular and heart stage)9,52 for nuclei in each cluster. Size indicates the average percent of nuclei with > 0 counts, color indicates average log2(CPM) for all nuclei with CPM > 0.

Extended Data Fig. 3 Characterization of seed coat nuclei.

(a) SC3 clustering of 4 DAP seed coat nuclei. (b) Average expression of LCM seed tissue markers9,52, over seed coat clusters. Dot color: average log2(CPM); dot size: average percent nuclei with CPM > 0. (c) Cell cycle phase by cluster. (d) Average expression of genes specific to particular seed coat cell layers54,55,56 across nuclei clusters. Schematic of seed coat cell layers, from ii1 (the endothelium, innermost) to oi2 (epidermis, outermost); layers where expression was observed in indicated study highlighted green. Red star: significantly higher expression in cluster (permutation test, p < 0.05). (e) Top 5 GO terms for significantly upregulated genes in each cluster. (f) Cluster identities and characteristics; false-colored Col x Cvi (left) and Cvi x Col (right) seed images. EB = embryo, EN = endosperm, CSC = chalazal seed coat. Inset: the five seed coat cell layers.

Extended Data Fig. 4 Heatmaps of the 5 most significantly enriched GO terms among genes upregulated (top) and downregulated (bottom) in each seed coat cluster.

Significant terms are flagged in left heatmap, while average expression ‘enrichment score’ across all genes associated with GO term is shown at right. Average includes any genes associated with the GO-term that are not significantly up/downregulated in the indicated cluster, so average may not reflect expectations. Full lists of significant GO-terms, and specific lists of genes in each significant GO-term that are up/downregulated in cluster, are in Supplementary Data 2. Order of rows and columns same for left and right heatmaps.

Extended Data Fig. 5 Heatmaps of the 5 most significantly enriched GO terms among genes upregulated (top) and downregulated (bottom) in each endosperm cluster.

Significant terms, p < 0.005, are flagged in left heatmap, while average expression ‘enrichment score’ across all genes associated with GO term is shown at right. Average includes any genes associated with the GO-term that are not significantly up/downregulated in the indicated cluster; so average may not reflect expectations. Full lists of significant GO-terms, and specific lists of genes in each significant GO-term that are up/downregulated in cluster, are in Supplementary Data 2. Order of rows/columns same for left and right heatmaps.

Extended Data Fig. 6 In situ hybridization analysis for additional cluster-specific transcripts.

(a) Expression data for four additional marker genes used for RNA in situ hybridization experiments, across endosperm and seed coat clusters. (b) In situ hybridization (purple signal) results for two micropylar/peripheral clusters. AT4G11080 is most notably expressed in peripheral and micropylar endosperm and in the embryo. AT1G09380 is most notably expressed in the micropylar endosperm and seed coat. In gene summaries, expression indicated by hatched pattern indicates variable expression in that zone among seeds. (c) In situ hybridization results for two additional chalazal endosperm transcripts not shown in Fig. 2: AT4G13380 is predominantly expressed in the chalazal cyst, while AT1G44090 is predominantly expressed in the chalazal nodules. (b-c) Black arrowheads indicate sites of transcript accumulation; white arrowheads indicate examples of sites without transcripts. Number of seeds with expression in specific zones relative to the number of seeds examined is shown in bottom left of panels; expression in one zone does not exclude expression in other zones. Seeds were from three independent controlled pollination events, collected together. For all antisense probes, in situ experiment was performed at least twice, except for AT4G11080, which was performed once. Both sense and antisense probe images shown. Scale bars = 25 μm.

Extended Data Fig. 7 Cell cycle is a source of variability among endosperm clusters.

(a) t-SNE projection and trajectory analysis of 1,309 nuclei in the dataset, based on expression of a manually curated list of 22 cell cycle-dependent marker genes. Dotted line represents cell cycle trajectory from G0 -> G1 -> S -> G2 -> M. (b) Average expression of six of the 22 marker genes used in analysis shown in (a), with nuclei ordered according to their linear projection onto the cell cycle trajectory, starting from G0 (left) to M (right). Moving averages were calculated using a sliding window of 200 data points. (c) Percent of nuclei in each phase of the cell cycle that were sorted from the 3C or 6C FANS peak (d) Distribution of nuclei among cell cycle phases in seed coat and endosperm. Endosperm data are further divided into peripheral, micropylar, and chalazal; the chalazal region is also divided into the cyst and nodules. (e) Distribution of nuclei among cell cycle phases for each of the endosperm clusters.

Extended Data Fig. 8 Statistical power and accuracy of imprinting model under various simulated conditions.

(a) Percent of simulations (out of 200) where the null hypothesis of no parental bias was rejected, for simulations with varied total expression and log2(m/p) ratio (r). Simulations mimicked degree of maternal skew in the Col x Cvi data, so ‘unbiased’ simulations had r = 1.5. Twelve values of total expression were tested: 0.01, 0.05, 0.1, 0.15, 0.25, 0.5, 0.75, 1.0, 1.5, 3.5, 15, and 50. The 1st, 25th, 50th, 75th and 99th percentiles for total expression in the Col x Cvi dataset are 0.033, 0.21, 0.58, 1.57 and 15.4, respectively. Blue lines indicate paternal bias, red indicate maternal bias. (b) Effect of number of observations (nuclei) in simulations on power to reject H0adj. Highly expressed and highly biased genes can be detected even with as few as 5 observations. Blue lines indicate tests for paternal bias, red indicate tests for maternal bias.

Extended Data Fig. 9 Expression of chromatin-related genes.

(a) Sperm ChIP-seq profiles from28 over non-imprinted genes, all PEGs, chalazal PEGs and non-chalazal PEGs. (b) Heatmap of expression enrichment scores (ES) across endosperm nuclei clusters, for 464 chromatin-related genes with variable expression across the clusters. Inset: subset of genes enriched in chalazal nodules, cyst, or both (top); subset of genes with depleted expression in chalazal endosperm, grouped by expression pattern (bottom). Not all genes in highlighted region in left plot shown. (c) Heatmap of expression ES for the full 4 DAP endosperm + seed coat dataset, over cell cycle phases. 227 chromatin-related genes with variation across cell cycle shown. Color bar same as (b). (d) Expression ES in endosperm vs. seed coat for 553 chromatin-related genes. Color bar same as (b). (e) Average expression profiles across the endosperm clusters for four genes shown in (b) (see arrows). Stars indicate clusters with significantly enriched expression based on a permutation test. CPM = counts per million.

Supplementary information

Supplementary Information

Supplementary text, Methods, refs. 57–83 and Figs. 1–16.

Reporting Summary

Supplementary Data 1

Characteristics of all snRNA-seq libraries generated in this study.

Supplementary Data 2

Summary of all genes significantly differentially expressed among the Col,Cvi 4 DAP endosperm and seed coat clusters.

Supplementary Data 3

Cell cycle trajectory analysis and cell cycle phase assignments for all nuclei.

Supplementary Data 4

Imprinting analysis results and lists of MEGs and PEGs for Col,Cvi 4 DAP endosperm data.

Supplementary Data 5

Expression and imprinting variability across nuclei clusters and cell cycle.

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Picard, C.L., Povilus, R.A., Williams, B.P. et al. Transcriptional and imprinting complexity in Arabidopsis seeds at single-nucleus resolution. Nat. Plants 7, 730–738 (2021). https://doi.org/10.1038/s41477-021-00922-0

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