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Assembly of Arundinella anomala genome to facilitate single-cell resolved functional and developmental characterization of C4 distinctive cells

Nature Plants volume 12, pages 88–106 (2026) Cite this article

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Abstract

C4 plants operate a highly efficient photosynthetic CO2 concentrating mechanism. However, C4 photosynthesis represented by maize is based on the typical Kranz-type leaf anatomy, which involves complex regulation of vascular development coupling with metabolic distribution. To explore the possibility of using alternative C4 leaf anatomy as reference for engineering C3 crops, we sequenced, assembled and annotated the genome of Arundinella anomala, a C4 grass with variant Kranz anatomy and interveinal distinctive cells (DC). Following single-cell level transcriptomes for comparative analyses between C4 bundle sheath and DC cells, genetic and metabolic support for the intensified C4 function of DC cells were observed, including increased cyclic photosynthetic electron transport, carbon fixation and starch synthesis. Further, the mechanism involving SHORT-ROOT (SHR) and auxin to trigger independent development or proliferation of DC cells was explored. Notably, spaced distribution of DC-like cells can be achieved in rice leaves by inducing the expression of ZmSHR1. This work laid a foundation for introducing functional DC-like cells among the intervascular mesophyll cells of C3 grass leaves, and provided resources and strategies for engineering C4 traits into C3 crops.

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Fig. 1: The leaf anatomy and genome assembly of A. anomala.
The alternative text for this image may have been generated using AI.
Fig. 2: Generation of an A. anomala leaf single-cell gene expression atlas and the expression profile of marker genes.
The alternative text for this image may have been generated using AI.
Fig. 3: Identification of the BS and DC cell populations.
The alternative text for this image may have been generated using AI.
Fig. 4: Accelerated carbon fixation and starch accumulation in DC.
The alternative text for this image may have been generated using AI.
Fig. 5: Increased ROS accumulation and cyclic electron transport process in DC.
The alternative text for this image may have been generated using AI.
Fig. 6: Auxin induced development of multiplied DCs and increased expression of SHR genes in A. anomala.
The alternative text for this image may have been generated using AI.
Fig. 7: ‘DC cells’ may be induced in rice leaves by controlled expression of ZmSHR1 gene.
The alternative text for this image may have been generated using AI.

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Data availability

The genome sequence data of A. anomala are deposited in NCBI under BioProject PRJNA1282067. The genome assembly and annotation result files are available via Figshare at https://doi.org/10.6084/m9.figshare.30374623 (ref. 99). The scRNA-seq and LCM RNA-seq datasets have been deposited in the Sequence Read Archive under the accessions PRJNA1282080 and PRJNA1283371. Source data are provided with this paper.

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Acknowledgements

We thank J. Wu for kindly providing the A. anomala species and field-grown picture, Y. Pu for providing the A. setosa species and E. Wang, J. Wang and A. Fleming for fruitful discussions. We also thank C. Zhao and J. Yang for helping with the single-cell experiment, G. Chen for helping with the metabolite analysis, Y. Mai for helping with flow cytometry, W. Cai and L. Zhu for technical support with microscopy, and F. Yuan and Q. Gao for technical support with plant growth in the phytotron. This work was supported by the Biological Breeding-National Science and Technology Major Project (2023ZD04072) awarded to P.W. and by the Key Laboratory of Plant Carbon Capture, Chinese Academy of Sciences.

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Author notes

  1. These authors contributed equally: Hong Su, Yan Li, Yonghe Chen.

Authors and Affiliations

  1. Key Laboratory of Plant Carbon Capture, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Hong Su, Yonghe Chen, Rui Zhang & Peng Wang

  2. University of Chinese Academy of Sciences, Beijing, China

    Hong Su & Yonghe Chen

  3. National Center for Gene Research, State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Yan Li, Hengyun Lu, Bin Han & Qiang Zhao

  4. State Key Laboratory for Crop Genetics & Germplasm Enhancement and Utilization, Nanjing Agricultural University, Nanjing, China

    Wentao Dong

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Contributions

H.S., Q.Z. and P.W. designed the study. Y.L., Q.Z., H.L. and H.S. performed genome assembly and annotation. Y.C. and W.D. observed the ZmSHR1 DEX-induction lines of rice. R.Z. performed qRT–PCR experiments. H.S. worked on all the other experiments. H.S., Y.L., B.H., Q.Z. and P.W. analysed the data and wrote the paper.

Corresponding authors

Correspondence to Qiang Zhao or Peng Wang.

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Competing interests

Patent applications related to this work have been submitted by W.D., Y.C. and P.W. The other authors declare no competing interests.

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Nature Plants thanks Pinghua Li, Jisen Zhang 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 Comparison between A. anomala and A. setosa.

a,b, Cross-sections showing the leaf anatomy of A. anomala (a) with DC (indicated by red arrows) and A. setosa (b) without DC. c, Image of 3 weeks old seedlings. d,e, The overview of chromosomes from A. anomala (d) and A. setosa (e). Scale bar, 10 μm.

Extended Data Fig. 2 Comparative analysis of gene families among Arundinella anomala and six other C4 grasses.

a, Venn diagram showing the distribution of gene families among Arundinella anomala, Paspalum vaginatum, Saccharum spontaneum, Miscanthus sinensis, Sorghum bicolor, Andropogon gerardi, and Zea mays. The central number indicates gene families shared by all six species, while numbers in each petal represent unique gene families for each species. b, KEGG pathway enrichment analysis of gene families specific to A. anomala. The x-axis represents the number of genes enriched in each pathway. Pathways are grouped by major KEGG categories, with “Metabolism” being the most enriched category among the A. anomala-specific gene families.

Extended Data Fig. 3 Subgenomes of A. anomala.

a, Estimated insertion times of LTR-RTs in A subgenome and B subgenome of A. anomala. The X- and Y-axes indicate the insertion times and the density of intact LTR-RTs at each time, respectively. b, Multiple sequence alignment of randomly selected centromeric monomer sequences identified from A. anomala genome. Two distinct types of centromeric repeat units were detected: a 103 bp monomer (bottom) and a 137 bp monomer (top). The 137 bp unit represents an extended variant formed by a 34 bp insertion within the 103 bp sequence. c, The stacked bar chart shows the relative abundance of the 103 bp (blue) and 137 bp (orange) centromeric monomers identified in the centromeric regions of each chromosome. The Y-axis indicates the percentage of each repeat type within the centromeric region of each chromosome. d, Distribution of the estimated synonymous substitution rates (Ks) for syntenic conserved gene pairs between the A and B subgenomes. e, Distribution of the estimated lineage specific ratios of nonsynonymous substitution rates to synonymous substitution rates for syntenically conserved genes between the A and B subgenomes. Ks: synonymous nucleotide substitution rate. Ka: nonsynonymous substitution rates.

Extended Data Fig. 4 Gene expression profiles along maize leaf gradients.

The expression of maize homologues of A. anomala genes in Fig. 2d, e was observed from https://www.bar.utoronto.ca/.

Extended Data Fig. 5 Expression profile of selected genes in clusters of interest.

The representative UMAP plot on the bottom right shows 6 selected cell clusters as a subset from Fig. 2h. Colour scales in the UMAP plots indicate gene expression levels in individual cells.

Extended Data Fig. 6 Phylogenetic analysis of genes encoding C4 enzymes and their non-C4 isoforms.

Phylogenetic and expression (UMAP plots) analysis of NADP-ME (a), NADP-MDH (b), PPDK (c), PEPC (d) and CA (e) were presented. Seita indicates Setaria italica, Aa indicates Arundinella anomala, Sobic indicates Sorghum bicolor, Zm indicates Zea mays, Os indicates Oryza sativa, and AT indicates Arabidopsis thaliana. Genes in the light red clade encode proteins in the C4 pathway. Tree scale: 0.1. Colour scales in the UMAP plots indicate gene expression levels in individual cells. f, Expression profiles of C4-related homologs identified in A. anomala. Gene pairs were classified into five categories according to subgenome expression patterns across five tissues (aerial, ear, leaf, sheath, stem): A_dominant, B_dominant, balanced, A_only, and B_only. Heatmap colors represent normalized TPM values, with grey indicating gene loss. Red-labeled genes denote C4 core functional homologs.

Extended Data Fig. 7 KEGG Pathway enrichment analysis of DEGs in BS and DC cells.

The horizontal coordinate represents the enrichment score for each pathway, while the vertical coordinate displays pathway names. Dot colors indicate the significance of enrichment (redder hues denote more significant P-values), and dot sizes correspond to the number of enriched genes per pathway.

Extended Data Fig. 8 Higher expression levels of ROS scavenging (a) and photorespiration (b) related genes in DC relative to BS cells.

Data presented in parallel by LCM RNA-seq data (heat map) and scRNA-seq data (dot plot). For heat map, color scale shows the fold change of gene expression in DC relative to that in BS. Dot size, proportion of cluster cells expressing a given gene; Dot colour, the average expression level. Asterisks indicate more than 1.5 folds of gene expression in DC relative to BS from both datasets.

Extended Data Fig. 9 Auxin treatment induces abnormal development of vascular tissue and DCs in A. anomala leaves.

a-d, Representative images of leaf frozen sections under 2,4-D treatments at concentrations of 0 mg/L (a), 0.06 mg/L (b), 0.08 mg/L (c) and 0.1 mg/L (d). Red triangles indicate abnormal veins or DCs. Images in thin red rectangle frames are the enlargements of the thick red rectangle framed regions above. Scale bar, 100 μm. This experiment was independently repeated three times and similar results were observed. e-f, Leaf traits quantification of A. anomala under different concentrations of auxin treatments. In e, red arrows show the trends of frequency changes of “single DC”, “2DC”, or “>=3DC” events relative to the total number of vein events plus DC events, in response to varying auxin concentrations. Sample sizes for each group were n = 8 (0 mg/L), n = 12 (0.06 mg/L), n = 10 (0.08 mg/L), and n = 9 (0.1 mg/L), respectively. In f, sample sizes for each group were n = 17 (0 mg/L), n = 9 (0.06 mg/L), n = 10 (0.08 mg/L), and n = 5 (0.1 mg/L), respectively. Error bars in e and f represent means ± SD. Seedlings were subjected to auxin treatment since germination, and were sampled for observation after 2 weeks’ growth.

Extended Data Table 1 Additional genome information of A. anomala
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Statistical source data for Figs. 2, 6 and 7 and Extended Data Figs. 6 and 9.

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Su, H., Li, Y., Chen, Y. et al. Assembly of Arundinella anomala genome to facilitate single-cell resolved functional and developmental characterization of C4 distinctive cells. Nat. Plants 12, 88–106 (2026). https://doi.org/10.1038/s41477-025-02183-7

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