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GhBES1 mediates brassinosteroid regulation of leaf size by activating expression of GhEXO2 in cotton (Gossypium hirsutum)

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Abstract

Key message

We proposed a working model of BR to promote leaf size through cell expansion. In the BR signaling pathway, GhBES1 affects cotton leaf size by binding to and activating the expression of the E-box element in the GhEXO2 promoter region.

Abstract

Brassinosteroid (BR) is an essential phytohormone that controls plant growth. However, the mechanisms of BR regulation of leaf size remain to be determined. Here, we found that the BR deficient cotton mutant pagoda1 (pag1) had a smaller leaf size than wild-type CRI24. The expression of EXORDIUM (GhEXO2) gene, was significantly downregulated in pag1. Silencing of BRI1-EMS-SUPPRESSOR 1 (GhBES1), inhibited leaf cell expansion and reduced leaf size. Overexpression of GhBES1.4 promoted leaf cell expansion and enlarged leaf size. Expression analysis showed GhEXO2 expression positively correlated with GhBES1 expression. In plants, altered expression of GhEXO2 promoted leaf cell expansion affecting leaf size. Furthermore, GhBES1.4 specifically binds to the E-box elements in the GhEXO2 promoter, inducing its expression. RNA-seq data revealed many down-regulated genes related to cell expansion in GhEXO2 silenced plants. In summary, we discovered a novel mechanism of BR regulation of leaf size through GhBES1 directly activating the expression of GhEXO2.

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All datasets generated for this study are included in the article/Supplementary Files.

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Acknowledgements

We thank **n Li and Peng Huo (Zhengzhou Research Center, Institute of Cotton Research of CAAS, Zhengzhou) for their technical assistance.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31971987), Creative Research Groups of China (31621005), Natural Science Foundation of Henan (212300410093) and State Key Laboratory of Cotton Biology Open Fund (CB2020A02).

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Author notes

  1. Shengdong Li, and Kun **ng have contributed equally to this work.

Authors and Affiliations

  1. Zhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Henan, 450001, Zhengzhou, China

    Shengdong Li, Guoquan Chen, Le Liu, Mengzhen Guo, Zhao Liu & Zuoren Yang

  2. State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, 455000, Henan, China

    Kun **ng, Ghulam Qanmber, Yan Hou, Lili Lu & Zuoren Yang

  3. College of Chemistry, Zhengzhou University, Henan, 450001, Zhengzhou, China

    Lingbo Qu

  4. State Key Laboratory of Cotton Biology (Hebei Base), Hebei Agricultural University, Baoding, 071001, Hebei, China

    Kun **ng

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  1. Shengdong Li
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Contributions

All authors contributed to the study conception and design. SL: Experimentation, writing. KX: Methodology, formal analysis, Writing & editing. GQ: Writing & editing. GC: Conceptualization, writing – review & editing. LL: Software, data curation. MG: Formal analysis. YH: Review & editing. LL: Methodology, data curation. ZL: Supervision. ZY: Supervision.

Corresponding authors

Correspondence to Zhao Liu or Zuoren Yang.

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Supplementary Information

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11103_2022_1313_MOESM1_ESM.tif

Graphical representation of average cell area, number of cells per leaf area, and total number of cortical cells. (A) The average area of cells in the selected area in the leaf of pag1 and WT (CRI24) plants. (B) The average area of cells in the selected area in the leaf of overexpression GhBES1.4, GhBES1-RNAi, and WT (CRI24) plants. (C) The average area of cells in the selected area in the leaf of VIGS-GhEXO2 and vector control plants. (D) The number of cells per leaf area in the selected area in the leaf of pag1 and WT (CRI24) plants (20，000 μm 2). (E) The number of cells per leaf area in the selected area in the leaf of overexpression GhBES1.4, GhBES1-RNAi, and WT (CRI24) plants (20，000 μm 2). (F) The number of cells per leaf area in the selected area in the leaf of VIGS-GhEXO2 and vector control plants (20，000 μm 2). (G) The total number of cortical cells in the leaf of pag1 and WT (CRI24) plants. (H) The total number of cortical cells in the leaf of overexpression GhBES1.4, GhBES1-RNAi, and WT (CRI24) plants. (I) The total number of cortical cells in the leaf of VIGS-GhEXO2 and vector control plants. Student’s t-test: ** P<0.05，***P<0.001. Supplementary file1 (TIF 17631 kb)

11103_2022_1313_MOESM2_ESM.tif

(A) Heatmap of expression of DEGs related to hormone-related pathway in pag1 cotyledon transcriptome data. (B) Heatmap of DEG expression associated with cell expansion-related pathways in GhBES1.4 overexpression cotyledon transcriptome data. Supplementary file2 (TIF 3320 kb)

11103_2022_1313_MOESM3_ESM.tif

(A) Multiple sequence alignment of GhEXO2 and Arabidopsis EXO proteins showed the presence of the conserved Phi_1 domain and a signal peptide region in all observed EXO proteins. (B) Phylogenetic analysis of GhEXO genes and Arabidopsis EXO genes. Supplementary file3 (TIF 20380 kb)

11103_2022_1313_MOESM4_ESM.tif

Identification of GhBES1 transgenic plants. (A) qRT-PCR validation of GhBES1.2, GhBES1.4, and GhBES1.5 transcript levels in RNAi plants. (B) PCR validation of GhBES1-pBI121 vector in RNAi plants. Primer sequences are provided in Table S2. (C) qRT-PCR validation of GhBES1.4 transcript levels in overexpressed plants. (D) PCR validation of GhBES1-pCAMBIA2300 vector in GhBES1.4 overexpression plants. Student’s t-test: *P<0.05, **P<0.01,***P<0.001. Supplementary file4 (TIF 9890 kb)

Phenotype of GhBES1.4 overexpression (A) and GhBES1.4-RNAi (B) plant. Supplementary file5 (TIF 16964 kb)

11103_2022_1313_MOESM6_ESM.tif

(A) Microscopic imaging analysis for cell elongation in the stem of GhEXO2 silenced and control plants. (B) Graphical representation of the number of cells in the selected area in the stem of GhEXO2 silenced and control plants. Each experiment was performed with three biological repeats and the error bars indicate the standard deviation among these replicates. Student’s t-test: ***P<0.001. Supplementary file6 (TIF 7008 kb)

11103_2022_1313_MOESM7_ESM.tif

Relative expression patterns of cell elongation genes. qRT-PCR analysis of selected genes (from RNA-seq data) related to cell elongation (GhKCS, GhEXPA4, GhEXPA8s, GhIRX15-L, and GhLNG1) and BR biosynthesis (GhCPD and GhDWF4) was conducted. Each experiment was performed in three biological repeats and the error bar indicated the standard deviation among these replicates. Supplementary file7 (TIF 10603 kb)

Results of RNA-seq data analysis and gene expression data. Supplementary file8 (XLSX 17709 kb)

All primer sequences used in this study. Supplementary file9 (DOCX 21 kb)

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Li, S., **ng, K., Qanmber, G. et al. GhBES1 mediates brassinosteroid regulation of leaf size by activating expression of GhEXO2 in cotton (Gossypium hirsutum). Plant Mol Biol 111, 89–106 (2023). https://doi.org/10.1007/s11103-022-01313-5

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