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Hunting Monolignol Transporters: Membrane Proteomics and Biochemical Transport Assays with Membrane Vesicles Of Journal of Experimental Botany, Vol. 71, No. 20 pp. 6379–6395, 2020 doi:10.1093/jxb/eraa368 Advance Access Publication 10 August 2020 This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) RESEARCH PAPER Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce Downloaded from https://academic.oup.com/jxb/article/71/20/6379/5890496 by Umea University Library user on 05 February 2021 Enni Väisänen1,2,*, Junko Takahashi1,3, , Ogonna Obudulu3,7, , Joakim Bygdell4, , Pirkko Karhunen5, Olga Blokhina1, , Teresa Laitinen2, Teemu H. Teeri2, , Gunnar Wingsle3, , Kurt V. Fagerstedt1, , and Anna Kärkönen2,6,*, 1 Viikki Plant Science Centre, Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, 00014 Helsinki, Finland 2 Viikki Plant Science Centre, Department of Agricultural Sciences, University of Helsinki, 00014 Helsinki, Finland 3 Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre (UPSC), Swedish University of Agricultural Sciences, 90183 Umeå, Sweden 4 Department of Chemistry, Computational Life Science Cluster (CLiC), Umeå University, 90187 Umeå, Sweden 5 Department of Chemistry, University of Helsinki, 00014 Helsinki, Finland 6 Natural Resources Institute Finland (Luke), Production Systems, Plant Genetics, 00790 Helsinki, Finland 7 Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, 40530 Gothenburg, Sweden * Correspondence: [email protected] or [email protected] Received 21 January 2020; Editorial decision 28 July 2020; Accepted 2 August 2020 Editor: Qiao Zhao, Chinese Academy of Sciences, China Abstract Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue- cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tono- plast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters. Keywords: Lignin biosynthesis, monolignol transport, plasma membrane, proteomics, transporter proteins. © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 6380 | Väisänen et al. Introduction Lignification of plant cell walls is a developmental process in In addition, transport of dilignols seems possible. The pres- water-transporting xylem cells and support-giving scleren- ence of conjugates of sinapyl alcohol and p-coumaric acid in chyma cells. In addition, it can be an induced as a protective grass lignin suggests that there is a system that transports dimers response against stresses. In lignification, hydroxycinnamic al- into the apoplast, since a transferase making these conjugates cohols (monolignols) form a polymer in the cell wall. The bio- exists in the cytoplasm (Hatfield et al., 2008, 2009). It seems synthetic process of lignification includes the production of possible that the first step of polymerization, dimerization, oc- monolignols through the monolignol biosynthetic pathway, a curs partly in the cytosol, and the dimer is transported to the branch of the phenylpropanoid pathway. Next, monolignols cell wall and incorporated into the growing polymer. are secreted into the cell wall, oxidized to radicals, and the rad- In the current study, membrane proteomic analysis of Downloaded from https://academic.oup.com/jxb/article/71/20/6379/5890496 by Umea University Library user on 05 February 2021 icals are polymerized into lignin, a complex network of link- developing xylem with active lignification and lignin-forming ages between monolignols (Perkins et al., 2019). The process is tissue-cultured cells (Kärkönen et al., 2002) of Norway spruce mediated by a wide variety of enzymes in multiple locations. (Picea abies) was conducted. As a comparison, the membrane The plasma membrane (PM) serves the plant cell as a con- proteome of developing phloem, which does not lignify (except trolling boundary between the cell wall and the cytoplasm. stone cells), was investigated. Microsomal vesicles (microsomal This boundary has many roles, including maintaining cellular fraction; MF) are a heterogeneous mixture of cellular mem- ionic homeostasis, creating concentration gradients, and thus branes. Partially enriched PM vesicles (upper phase fraction; regulating biochemical reactions, and detecting and mediating UP) (Kärkönen et al., 2014) were prepared from MF material signals between the apoplast and the symplast. In addition, the and their proteomes were analyzed by liquid chromatography- PM is important for cell wall biosynthesis because cellulose mass spectrometry (LC-MS/MS). To take a closer look at is produced directly at the PM. The biosynthesis of lignin is membrane-localized, putatively lignification-related proteins, separated in space: monolignol biosynthesis takes place in the protein identifications were compared with several published cytoplasm and lignin polymerization occurs in the cell wall. gene expression studies (Nystedt et al., 2013; Jokipii-Lukkari Thus, monolignols require transportation through the PM et al., 2017; Laitinen et al., 2017; Blokhina et al., 2019), and can- to the site of polymerization. According to biochemical data, didates for monolignol transport and other lignification-related transport of monolignols through the PM involves enzym- processes were identified. Biochemical monolignol transport atic transporters (Miao and Liu, 2010; Alejandro et al., 2012). assays with multiple candidate transport forms showed that MF Still, passive diffusion has a likely role in aglycone transport prepared from developing xylem, but not those from developing (Boija and Johansson, 2006; Vermaas et al., 2019), whereas phloem, had a H+-gradient-dependent, secondary active, Golgi-mediated transport does not seem probable (Kaneda tonoplast-localized transport of coniferin and p-coumaryl al- et al., 2008). cohol glucoside. A strikingly similar, active tonoplast-localized The transport mechanism of monolignols has remained as coniferin and p-coumaryl alcohol glucoside transport was de- a mystery (Perkins et al., 2019). Even the transported form tected in MF prepared from non-lignifying control material, is not yet known. The most favored candidates, monolignol consisting of tissue-cultured Bright Yellow-2 (BY-2) cells of to- glucosides (such as coniferyl alcohol glucoside, or coniferin), bacco (Nicotiana tabacum). This observation questions whether are non-toxic, non-reactive, and readily detectable mol- this transporter is dedicated to lignin biosynthesis. In addition, ecules in the developing secondary xylem of many gymno- MF isolated from the lignin-forming cultured cells had a trans- sperms (Morikawa et al., 2010; Terashima et al., 2016) porter able to transport coniferin, but its affinity for coniferin and some angiosperms (e.g. Magnolia; Tsuji et al., 2004). was much lower than those in xylem or BY-2 cells. In combin- Coniferin β-glucosidases, which have been detected in, for ation with the proteomic data, correlation analyses using ex- example, developing xylem of lodgepole pine (Pinus contorta; isting Norway spruce RNA-seq data were utilized to identify Dharmawardhana et al., 1995), cleave the sugar moiety, leaving candidate monolignol transporters. the free alcohol form ready to react with peroxidases/laccases and the growing lignin polymer. Results using coniferyl al- cohol and coniferin in assays with membrane vesicles prepared Materials and methods from aerial tissues of Arabidopsis thaliana (Miao and Liu, 2010) suggest a role for PM-located ABC transporter(s) in coniferyl Plant material alcohol transport. In addition, Alejandro et al. (2012) pro- In each of the years 2011, 2013, and 2014, an approximately 40-year- posed that a G-type ABC transporter, AtABCG29, functions old Norway spruce (Picea abies [L.] Karst., clone E8504) tree, grown in in p-coumaryl alcohol transport at the PM in Arabidopsis root Ruotsinkylä,
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