Single-nucleus transcriptome profiling unveils cell-type-specific ethylene and TOR signaling in tomato

Abstract

Plant etiolation, a critical process for seedling emergence, is regulated by ethylene and target of rapamycin (TOR) signaling pathways. However, the cell-type-specific regulation of these pathways remains poorly understood. To address this, we generated a comprehensive single-nucleus RNA transcriptome atlas of etiolated apical hooks and hypocotyls in tomato seedlings treated with the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), the TOR inhibitor Torin2, or a mock treatment. In total, we obtained high-quality gene expression profiles for 117 929 nuclei across these tissues and treatments. Our analysis identified seven major cell types within each tissue, revealing distinct cellular compositions and transcriptional programs. ACC treatment increased the proportion of epidermal cells in apical hooks, while Torin2 had limited impact on cellular composition. Differential gene expression analysis demonstrated tissue-specific sensitivity to these treatments: apical hooks exhibited extensive ACC-responsive differentially expressed genes, whereas hypocotyls were highly responsive to Torin2. Cellular responsiveness analysis uncovered divergent ethylene/auxin pathway activities, such as ACC-repressed auxin transport in hook endodermis-like cells. Dynamic trajectory analysis indicated both treatments altered cell differentiation, authenticating epidermis as the key cell type for ethylene-mediated etiolated growth. Crucially, we identified JA1 (HD-ZIP I TF) as a negative ethylene regulator enriched in epidermis, and CRISPR knockout ja1 mutants exhibited hypersensitivity to ACC. This study deciphers cell-type-specific ethylene-TOR crosstalk, providing a robust single-cell RNA sequencing framework to dissect signaling networks in crops.

Acknowledgements

We are sincerely thankful for the support provided by the China National GeneBank (CNGB). This work was supported by the National Natural Science Foundation of China (32200263 to W.H.), the Shenzhen Science and Technology Program (KQTD20230301092839007 to C.T.), and the Science and Technology Major Special Project of Shenzhen (KCXFZ20240903093900001 to W.H.)

Author contributions

W.H. and C.T. conceived and designed the experiments, and supervised the overall project. W.H., C.T., and L.Y. performed the majority of the experiments and conducted data analysis. N.H. generated the ja1 mutant lines. Q.J. and P.Z. contributed to the data analysis. W.H., L.Y., and C.T. wrote the draft manuscript. L.L., Z.C., and J.H. revised the manuscript. C.T. and W.H. finalized the manuscript. All authors have confirmed the manuscript.

Data availability

The sequencing data generated in this study have been deposited in the China National GeneBank (CNGB) Sequence Archive (CNSA) [63] with the accession number CNP0007766. Scripts used for snRNA-seq data processing in this study are available in GitHub (https://github.com/ctan2020/Tomato-hook-SC).

Conflicts of interest statement

No conflicts of interest is declared.

Supplementary material

Supplementary material is available at Horticulture Research online.

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