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Generation and Characterization of a Specific Polyclonal Antibody against Arabidopsis thaliana Phytochrome-Interacting Factor 3

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Abstract

Phytochrome-interacting factors (PIFs) are a basic helix-loop-helix family of transcriptional regulators that maintain skotomorphogenesis and suppress photomorphogenesis. PIFs are regulated by plant photoreceptors, especially phytochromes. In general, PIFs physically interact with phytochromes, and this interaction induces PIF’s phosphorylation and subsequent degradation, contributing to the initiation of photomorphogenic development. Among the eight members of PIF (PIF1 to PIF8) reported in Arabidopsis thaliana, PIF3 is the first discovered member and plays central roles in de-etiolation and chlorophyll biosynthesis. More recently, PIF3 has been also reported to regulate hormone signaling and cold tolerance in plants. Although PIF3 protein shows dynamic behaviors in plants, its study has been limited due to the lack of an authentic PIF3 antibody. In this study, we produced polyclonal antibodies using inclusion bodies and characterized the PIF3 antibody based on specificity and sensitivity. In addition, we investigated PIF3 phosphorylation and degradation during phytochrome-mediated light signaling in plants. Furthermore, we successfully performed in vitro protein–protein interaction and co-immunoprecipitation assays between phytochrome B (phyB) and PIF3 using the antibody. Therefore, we obtained an authentic PIF3 antibody that could be used as a valuable tool to study the multi-faceted functions of PIF3.

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Acknowledgements

This research was supported under the framework of international cooperation program (NRF Grant no. 2019K2A9A1A06100097 to J.-I.K), and by the Basic Science Research program (NRF Grant no. 2018R1A6A3A11045293 to Y.-J.H.) managed by the National Research Foundation of Korea, and in part by the Next-Generation BioGreen21 Program from Rural Development Administration, Republic of Korea (TAGC Grant no. PJ01325301). We would like to thank Editage (www.editage.co.kr) for English language editing.

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YJH and JIK designed the project, WYK produced the polyclonal antibody, and DMC and JYC performed the experiments. YJH, DMC, JYC and JIK analyzed the data and discussed about the results. YJH and JIK wrote the paper, and all authors approved the manuscript.

Corresponding authors

Correspondence to Yun-Jeong Han or Jeong-Il Kim.

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Choi, DM., Cho, JY., Kim, WY. et al. Generation and Characterization of a Specific Polyclonal Antibody against Arabidopsis thaliana Phytochrome-Interacting Factor 3. J. Plant Biol. 64, 181–191 (2021). https://doi.org/10.1007/s12374-021-09302-9

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