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Characterization of BRASSINOSTEROID F-BOX Proteins BRFPs that Regulate BRASSINOSTEROID-INSENSITIVE 2 Kinase

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Abstract

BRASSINOSTEROIDS (BRs) play important roles in regulating plant growth and development. The Arabidopsis GSK3-like kinase BIN2, a major negative regulator of BR signaling, plays diverse roles in plant growth and developmental processes. In a previous study, based on the relationship between BIN2 and its substrate, we reported the possibility that BIN2 protein stability is controlled by BRASSINOSTEROID F-BOX Protein 1 and 2, BRF1 and BRF2, (BRF1/2, renamed BRFP1/2). The aim of the study was to characterize the functions of the F-box proteins BRFP1 and BRFP2, and to confirm their relationship with the BIN2 proteins and the role of the BR signaling for plant growth and development. BRFP1-overexpressing (BRFP1-FLAG) plants displayed better growth compared to the wild-type Col-0 plants in contrast to brfp1/2 mutants. BRFP1 plants were sensitive to BL, the most active BR, as opposed to the brfp1/2 mutants. We confirmed that BRFP1 overexpression suppressed the dwarf BIN2-HA phenotype by reducing BIN2 protein levels through BRFP1–BIN2 interactions in vivo. These results suggest the possibility that the stability of BIN2 is regulated by BRFP1. Altogether, results of our finding propose that the F-box proteins BRFP1/2 are involved in BR signaling for regulating plant growth.

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Acknowledgements

This work was done with financial assistance of Prof. Sunghwa Choe and grant from the National Research Foundation of Korea (NRF; grant No. 2013R1A1A2059445 to Y.J.J).

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Author notes

  1. Yu Jeong Jeong and June Seung Lee contributed equally.

Authors and Affiliations

  1. School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, 08826, Korea

    Yu Jeong Jeong

  2. Department of Life Science, Chung-Ang University, Seoul, 06974, Korea

    June Seung Lee

  3. Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul, 05006, Korea

    Dong-Gwan Kim

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  1. Yu Jeong Jeong
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Contributions

YJJ designed and performed the experiments. YJJ, JSL and DGK analyzed the data and wrote the manuscript.

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Correspondence to Yu Jeong Jeong.

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Supplementary Information

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Fig. S1

Comparison of protein expression levels of BRFP1-FLAG/BIN2-HA and BIN2-HA plants. (A) Western blot analysis result using anti-HA. Total protein extracts from the 1-week-old seedling of the transgenic plant shown in Fig. 1(E), grown in ½ MS media without MG132. (B) The intensities of protein bands in (A) were quantified with reference to actin control bands using ImageJ analysis program. (C) Western blot analysis result using anti-Flag antibody. Total protein extracts from the 1-week-old seedling of the transgenic plant shown in Fig. 1(E), grown in ½ MS media.Supplementary file1 (TIF 54 KB)

Fig. S2

Measurement of hypocotyl length in 6-day-old BRFP1-overexpressing and brfp1/2 seedlings. Error bars indicate SD (n ≥20). Asterisks indicate P < 0.001 (*) in Student’s t-test analysis.Supplementary file2 (TIF 10 KB)

Fig. S3

Expression profiles of Arabidopsis BRFP1 (AT5G62970) obtained from the Klepikova Arabidopsis Atlas and Arabidopsis Embryo of eFP browser database. Data were collected a high resolution map of the Arabidopsis thaliana developmental (A) and embryo (B) expression profile based on publicly available transcriptome databases at eFP browser (microarray).Supplementary file3 (TIF 371 KB)

Fig. S4

Expression profiles of Arabidopsis BRFP2 (AT5G56690) obtained from the Klepikova Arabidopsis Atlas and Arabidopsis Embryo of eFP browser database. Data were collected a high resolution map of the Arabidopsis thaliana developmental (A) and embryo (B) expression profile based on publicly available transcriptome databases at eFP browser (microarray).Supplementary file4 (TIF 306 KB)

Fig. S5

Expression profiles of Arabidopsis BRFP1 (AT5G62970) obtained from organs and developmental stages of the TraVA. Data were collected a high resolution map of the Arabidopsis thaliana organs and developmental stages expression profile based on publicly available RNA-seq databases at TRAVA (RNA-seq). Read counts for BRFP1 among all samples.Supplementary file5 (TIF 1048 KB)

Fig. S6

Expression profiles of Arabidopsis BRFP2 (AT5G56690) obtained from organs and developmental stages of TraVA. Data were collected a high resolution map of the Arabidopsis thaliana organs and developmental stages expression profile based on publicly available RNA-seq databases at TRAVA (RNA-seq). Read counts for BRFP2 among all samples.Supplementary file6 (TIF 426 KB)

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Jeong, Y.J., Lee, J.S. & Kim, DG. Characterization of BRASSINOSTEROID F-BOX Proteins BRFPs that Regulate BRASSINOSTEROID-INSENSITIVE 2 Kinase. J. Plant Biol. 65, 53–63 (2022). https://doi.org/10.1007/s12374-021-09335-0

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