Single-cell transcriptome atlas reveals spatiotemporal developmental trajectories in the basal roots of moso bamboo (Phyllostachys edulis)

Abstract

Roots are essential for plant growth and development. Bamboo is a large Poaceae perennial with 1642 species worldwide. However, little is known about the transcriptional atlas that underpins root cell-type differentiation. Here, we set up a modified protocol for protoplast preparation and report single-cell transcriptomes of 14 279 filtered single cells derived from the basal root tips of moso bamboo. We identified four cell types and defined new cell-type-specific marker genes for the basal root. We reconstructed the developmental trajectories of the root cap, epidermis, and ground tissues and elucidated critical factors regulating cell fate determination. According to in situ hybridization and pseudotime trajectory analysis, the root cap and epidermis originated from a common initial cell lineage, revealing the particularity of bamboo basal root development. We further identified key regulatory factors for the differentiation of these cells and indicated divergent root developmental pathways between moso bamboo and rice. Additionally, PheWOX13a and PheWOX13b ectopically expressed in Arabidopsis inhibited primary root and lateral root growth and regulated the growth and development of the root cap, which was different from WOX13 orthologs in Arabidopsis. Taken together, our results offer an important resource for investigating the mechanism of root cell differentiation and root system architecture in perennial woody species of Bambusoideae.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32071849), the National Key Research and Development Program of China (2021YFD2200505), and the Fundamental Research Funds of ICBR (1632021017).

Author contributions

J.G. designed the research and revised the manuscript. Z.C. and C.M. designed the research, performed experiments, analyzed data, wrote the manuscript, and revised the manuscript. X.L. performed experiments on PheWOXs and revised the manuscript. W.C., M.C., J.J., R.G. and H.F. assisted with in situ hybridization. C.W. drew a schematic of Moso bamboo basal root. J.X., Y.B., H.Z., Y.X., and Y.D. performed qPCR and plant phenotypic analysis. J.L. and S.M. provided technique support for the smooth use of the research instruments.

Data availability

All high-throughput sequencing data have been deposited in the GEO under accession number GSE229126.

Conflict of interest

The authors declare no competing interests.

References

[1] FAO. Global forest resources assessment 2010: main report. New York: Food and Agriculture Organization of the United Nations; 2010.

[2] Vorontsova MS, Clark LG, Dransfield J et al. World Checklist of Bamboos and Rattans. Kew: INBAR/Royal Botanic Gardens; 2016.

[3] Scurlock JMO, Hames B, Dayton DC . Bamboo: an overlooked biomass resource? Biomass Bioenergy. 2000; 19: 229-244.

[4] INBAR. Trade overview 2020: bamboo and rattan commodities in China. Beijing: International Bamboo and Rattan Organization. 2021.

[5] National Forestry and Grassland Administration . China Forestry Statistical Yearbook. Beijing: China Forestry Publishing House; 2020.

[6] COP15-COP27. Global innovation hub launched for transformative climate solutions. United Nations Climate Change. 2010-2022; 6-18. https://unfccc.int/news/innovation-hub-at-cop27-to-promote-transformative-climate-solutions (31 October 2022, date last accessed).

[7] BARC. The First Global Bamboo and Rattan Congress, 25-27 June 2018. http://www.barc2018.org/.

[8] BARC. The Second Global Bamboo and Rattan Congress, 6-18 November 2022. https://www.barc2022.inbar.int/#/home.

[9] Gao J . The Moso Bamboo Genome. Cham: Springer; 2021.

[10] Lou Y . Bamboo Forest’s Role in Mitigating Climate Change. Beijing. International Network for Bamboo and Rattan (INBAR); 2009.

[11] Yen TM, Lee JS . Comparing aboveground carbon sequestration between moso bamboo (Phyllostachys heterocycla) and China fir (Cunninghamia lanceolata) forests based on the allometric model . For Ecol Manag. 2011; 261: 995-1002.

[12] Zhou G, Jiang P . Density, storage and spatial distribution of carbon in Phyllostachy pubescens forest . Sci Silv Sin. 2004; 40: 20-24.

[13] Zhou F. Bamboo Forest Cultivation. Beijing: China Forestry Press; 1998.

[14] Chen M, Guo L, Ramakrishnan M et al. Rapid growth of moso bamboo (Phyllostachys edulis): cellular roadmaps, transcriptome dynamics, and environmental factors . Plant Cell. 2022; 34: 3577-3610.

[15] Raechal L, Curtis J . Root anatomy of the Bambusoideae (Poaceae). Am J Bot. 1990; 77: 475-482.

[16] Hu C, Cheng L, Wan J et al. A preliminary study of the anatomy structure of roots of bamboos. J Bamboo Res. 1990; 9: 11-21 [In Chinese].

[17] Ito R, Miyafuji H, Kasuya N . Rhizome and root anatomy of moso bamboo (Phyllostachys pubescens) observed with scanning electron microscopy . J Wood Sci. 2015; 61: 431-437.

[18] Chu C, Huang L, Wang S et al. Comparison on the anatomical structures of the roots generated from different parts of Dendrocalamus brandisii. Acta Bot Boreali-Occiden Sin. 2020; 40: 0043-0052.

[19] Zhang T, Xu Z, Shang G et al. A single-cell RNA sequencing profiles the developmental landscape of Arabidopsis root . Mol Plant. 2019; 12: 648-660.

[20] Rebouillat J, Dievart A, Verdeil JL et al. Molecular genetics of rice root development. Rice. 2009; 2: 15-34.

[21] Denyer T, Ma X, Klesen S et al. Spatiotemporal developmental trajectories in the Arabidopsis root revealed using high-throughput single-cell RNA sequencing . Dev Cell. 2019; 48: 840-852.e5.

[22] Zhang TQ, Chen Y, Liu Y et al. Single-cell transcriptome atlas and chromatin accessibility landscape reveal differentiation trajectories in the rice root. Nat Commun. 2021; 12: 2053.

[23] Liu Q, Liang Z, Feng D et al. Transcriptional landscape of rice roots at the single-cell resolution. Mol Plant. 2021; 14: 384-394.

[24] Omary M, Gil-Yarom N, Yahav C et al. A conserved superlocus regulates above- and belowground root initiation. Science. 2022; 375: eabf4368.

[25] Marand AP, Chen Z, Gallavotti A et al. A cis-regulatory atlas in maize at single-cell resolution. Cell. 2021; 184: 3041-3055.e21.

[26] Liu H, Hu D, Du P et al. Single-cell RNA-seq describes the transcriptome landscape and identifies critical transcription factors in the leaf blade of the allotetraploid peanut (Arachis hypogaea L.). Plant Biotechnol J. 2021; 19: 2261-2276.

[27] Li H, Dai X, Huang X et al. Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus. J Integr Plant Biol. 2021; 63: 1906-1921.

[28] Xie J, Li M, Zeng J et al. Single-cell RNA sequencing profiles of stem-differentiating xylem in poplar. Plant Biotechnol J. 2022; 20: 417-419.

[29] Chen Y, Tong S, Jiang Y et al. Transcriptional landscape of highly lignified poplar stems at single-cell resolution. Genome Biol. 2021; 22: 319.

[30] Wang Q, Wu Y, Peng A et al. Single-cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves. Plant Biotechnol J. 2022; 20: 2089-2106.

[31] Bai Y, Liu H, Lyu H et al. Development of a single-cell atlas for woodland strawberry (Fragaria vesca) leaves during early Botrytis cinerea infection using single cell RNA-seq . Hortic Res. 2022; 9: uhab055.

[32] Rich-Griffin C, Stechemesser A, Finch J et al. Single-cell transcriptomics: a high-resolution avenue for plant functional genomics. Trends Plant Sci. 2020; 25: 186-197.

[33] Ryu KH, Zhu Y, Schiefelbein J . Plant cell identity in the era of single-cell transcriptomics. Annu Rev Genet. 2021; 55: 479-496.

[34] Liu Z, Yu X, Qin A et al. Research strategies for single-cell transcriptome analysis in plant leaves. Plant J. 2022; 112: 27-37.

[35] Huang B, Zhuo R, Fan H et al. An efficient genetic transformation and CRISPR/Cas9-based genome editing system for moso bamboo (Phyllostachys edulis). Front Plant Sci. 2022; 13: 822022.

[36] Chen K, Hu K, Xi F et al. High-efficient and transient transformation of moso bamboo (Phyllostachys edulis) and ma bamboo (Dendrocalamus latiflorus Munro). J Plant Biol. 2023; 66: 75-86.

[37] Farmer A, Thibivilliers S, Ryu KH et al. Single-nucleus RNA and ATAC sequencing reveals the impact of chromatin accessibility on gene expression in Arabidopsis roots at the single-cell level . Mol Plant. 2021; 14: 372-383.

[38] Ryu KH, Huang L, Kang HM et al. Single-cell RNA sequencing resolves molecular relationships among individual plant cells. Plant Physiol. 2019; 179: 1444-1456.

[39] Shulse CN, Cole BJ, Ciobanu D et al. High-throughput single-cell transcriptome profiling of plant cell types. Cell Rep. 2019; 27: 2241-2247.e4.

[40] Wendrich JR, Yang BJ, Vandamme N et al. Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions. Science. 2020; 370: eaay4970.

[41] Fukaki H, Taniguchi N, Tasaka M . PICKLE is required for SOLITARY-ROOT/IAA14-mediated repression of ARF7 and ARF19 activity during Arabidopsis lateral root initiation . Plant J. 2006; 48: 380-389.

[42] Ogas J, Kaufmann S, Henderson J et al. PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis. Proc Natl Acad Sci USA. 1999; 96: 13839-13844.

[43] Mlynarova L, Nap JP, Bisseling T . The SWI/SNF chromatin-remodeling gene AtCHR12 mediates temporary growth arrest in Arabidopsis thaliana upon perceiving environmental stress . Plant J. 2007; 51: 874-885.

[44] Clowes FAL . Pattern in root meristem development in angiosperms. New Phytol. 2000; 146: 83-94.

[45] Bernier F, Berna A . Germins and germin-like proteins: plant do-all proteins. But what do they do exactly? Plant Physiol Biochem. 2001; 39: 545-554.

[46] Liu C, Fukumoto T, Matsumoto T et al. Aquaporin OsPIP1;1 promotes rice salt resistance and seed germination. Plant Physiol Biochem. 2013; 63: 151-158.

[47] Cheng Z, Hou D, Ge W et al. Integrated mRNA, microRNA transcriptome and degradome analyses provide insights into stamen development in moso bamboo. Plant Cell Physiol. 2020; 61: 76-87.

[48] Cheng Z, Ge W, Li L et al. Analysis of MADS-box gene family reveals conservation in floral organ ABCDE model of moso bamboo (Phyllostachys edulis). Front Plant Sci. 2017; 8: 656.

[49] Gao J, Zhang Y, Zhang C et al. Characterization of the floral transcriptome of moso bamboo (Phyllostachys edulis) at different flowering developmental stages by transcriptome sequencing and RNA-seq analysis . PLoS One. 2014; 9: e98910.

[50] Ge W, Zhang Y, Cheng Z et al. Main regulatory pathways, key genes and microRNAs involved in flower formation and development of moso bamboo (Phyllostachys edulis). Plant Biotechnol J. 2017; 15: 82-96.

[51] Peng Z, Zhang C, Zhang Y et al. Transcriptome sequencing and analysis of the fast growing shoots of moso bamboo (Phyllostachys edulis). PLoS One. 2013; 8: e78944.

[52] Li L, Cheng Z, Ma Y et al. The association of hormone signalling genes, transcription and changes in shoot anatomy during moso bamboo growth. Plant Biotechnol J. 2018; 16: 72-85.

[53] Zhang H, Wang H, Zhu Q et al. Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications . BMC Plant Biol. 2018; 18: 125.

[54] Yuan JL, Yue JJ, Wu XL et al. Protocol for callus induction and somatic embryogenesis in moso bamboo. PLoS One. 2013; 8: e81954.

[55] Sarkar AK, Luijten M, Miyashima S et al. Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers . Nature. 2007; 446: 811-814.

[56] De Rybel, Vassileva, Parizot B, V, B et al. A novel aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity. Curr Biol. 2010; 20: 1697-1706.

[57] Kang NY, Lee HW, Kim J . The AP2/EREBP gene PUCHI co-acts with LBD16/ASL18 and LBD18/ASL20 downstream of ARF7 and ARF19 to regulate lateral root development in Arabidopsis. Plant Cell Physiol. 2013; 54: 1326-1334.

[58] Yang Y, Zhang L, Chen P et al. UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development. EMBO J. 2020; 39: e101928.

[59] Li X, Li J, Cai M et al. Identification and evolution of the WUSCHEL-related homeobox protein family in Bambusoideae. Biomol Ther. 2020; 10: 739.

[60] Dolzblasz A, Nardmann J, Clerici E et al. Stem cell regulation by Arabidopsis WOX genes . Mol Plant. 2016; 9: 1028-1039.

[61] Haecker A, Groß-Hardt R, Geiges B et al. Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development. 2004; 131: 657-668.

[62] Hirakawa Y, Kondo Y, Fukuda H . TDIF peptide signaling regulates vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis. Plant Cell. 2010; 22: 2618-2629.

[63] Tanaka W, Pautler M, Jackson D et al. Grass meristems II: inflorescence architecture, flower development and meristem fate. Plant Cell Physiol. 2013; 54: 313-324.

[64] Romera-Branchat M, Ripoll JJ, Yanofsky MF et al. The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit . Plant J. 2013; 73: 37-49.

[65] Sakakibara K, Reisewitz P, Aoyama T et al. WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens. Development. 2014; 141: 1660-1670.

[66] Ikeuchi M, Iwase A, Ito T et al. Wound-inducible WUSCHEL-RELATED HOMEOBOX 13 is required for callus growth and organ reconnection. Plant Physiol. 2022; 188: 425-441.

[67] Zhao H, Gao Z et al. Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis). GigaScience. 2018; 7: 1-12.

[68] Butler A, Hoffman P, Smibert P et al. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018; 36: 411-420.

[69] Becht E, McInnes L, Healy J et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol. 2019; 37: 38-44.

[70] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25: 402-408.

[71] Yoo SD, Cho YH, Sheen J . Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis . Nat Protoc. 2007; 2: 1565-1572.

[72] Zhu Z, Sun B, Cai W et al. Natural variations in the MYB transcription factor MYB31 determine the evolution of extremely pungent peppers. New Phytol. 2019; 223: 922-938.

[73] Sun B, Zhou X, Chen C et al. Coexpression network analysis reveals an MYB transcriptional activator involved in capsaicinoid biosynthesis in hot peppers. Hortic Res. 2020; 7: 162.