University of Oulu

Nickolov K, Gauthier A, Hashimoto K, Laitinen T, Väisänen E, Paasela T, Soliymani R, Kurusu T, Himanen K, Blokhina O, Fagerstedt KV, Jokipii-Lukkari S, Tuominen H, Häggman H, Wingsle G, Teeri TH, Kuchitsu K and Kärkönen A (2022) Regulation of PaRBOH1-mediated ROS production in Norway spruce by Ca2+ binding and phosphorylation. Front. Plant Sci. 13:978586. doi: 10.3389/fpls.2022.978586

Regulation of PaRBOH1-mediated ROS production in Norway spruce by Ca²⁺ binding and phosphorylation

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Author: Nickolov, Kaloian1,2; Gauthier, Adrien1,3; Hashimoto, Kenji4;
Organizations: 1Department of Agricultural Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, Finland
2Department of Ecology and Genetics, University of Oulu, Oulu, Finland
3UniLaSalle, Agro-Ecology, Hydrogeochemistry, Environments & Resources, UP 2018.C101 of the Ministry in Charge of Agriculture (AGHYLE) Research Unit CS UP 2018.C101, Mont-Saint-Aignan, France
4Department of Applied Biological Science, Tokyo University of Science, Noda, Japan
5Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, Finland
6Natural Resources Institute Finland (Luke), Production Systems, Helsinki, Finland
7Meilahti Clinical Proteomics Core Facility, Biochemistry & Dev. Biology, University of Helsinki, Biomedicum-Helsinki, Helsinki, Finland
8Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden
9Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 5 MB)
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Language: English
Published: Frontiers Media, 2022
Publish Date: 2022-10-19


Plant respiratory burst oxidase homologs (RBOHs) are plasma membrane-localized NADPH oxidases that generate superoxide anion radicals, which then dismutate to H₂O₂, into the apoplast using cytoplasmic NADPH as an electron donor. PaRBOH1 is the most highly expressed RBOH gene in developing xylem as well as in a lignin-forming cell culture of Norway spruce (Picea abies L. Karst.). Since no previous information about regulation of gymnosperm RBOHs exist, our aim was to resolve how PaRBOH1 is regulated with a focus on phosphorylation. The N-terminal part of PaRBOH1 was found to contain several putative phosphorylation sites and a four-times repeated motif with similarities to the Botrytis-induced kinase 1 target site in Arabidopsis AtRBOHD. Phosphorylation was indicated for six of the sites in in vitro kinase assays using 15 amino-acid-long peptides for each of the predicted phosphotarget site in the presence of protein extracts of developing xylem. Serine and threonine residues showing positive response in the peptide assays were individually mutated to alanine (kinase-inactive) or to aspartate (phosphomimic), and the wild type PaRBOH1 and the mutated constructs transfected to human kidney embryogenic (HEK293T) cells with a low endogenous level of extracellular ROS production. ROS-producing assays with HEK cells showed that Ca²⁺ and phosphorylation synergistically activate the enzyme and identified several serine and threonine residues that are likely to be phosphorylated including a novel phosphorylation site not characterized in other plant species. These were further investigated with a phosphoproteomic study. Results of Norway spruce, the first gymnosperm species studied in relation to RBOH regulation, show that regulation of RBOH activity is conserved among seed plants.

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Series: Frontiers in plant science
ISSN: 1664-462X
ISSN-E: 1664-462X
ISSN-L: 1664-462X
Volume: 13
Article number: 978586
DOI: 10.3389/fpls.2022.978586
Type of Publication: A1 Journal article – refereed
Field of Science: 1183 Plant biology, microbiology, virology
Funding: This work was supported by Academy of Finland (AK; decisions 251390, 256174, 283245 and 334184, SJ-L; decisions 331172 and 335972), Viikki Doctoral Programme in Molecular Biosciences (TL, EV), Integrative Life Science Doctoral Program (TL, EV), Jenny and Antti Wihuri Foundation (EV), MEXT, Japan (KK; KAKENHI grant numbers 50211884 and 25114515), JSPS, Japan (KK; KAKENHI grant number 15H01239), and Tokyo University of Science International Exchange Program (KK). Proteomic analyses were performed at the Meilahti Clinical Proteomics Core Facility, HiLIFE, supported by Biocenter Finland.
Academy of Finland Grant Number: 335972
Detailed Information: 335972 (Academy of Finland Funding decision)
Copyright information: © 2022 Nickolov, Gauthier, Hashimoto, Laitinen, Väisänen, Paasela, Soliymani, Kurusu, Himanen, Blokhina, Fagerstedt, Jokipii-Lukkari, Tuominen, Häggman, Wingsle, Teeri, Kuchitsu and Kärkönen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.