A Key Role for Apoplastic H₂O₂ in Norway Spruce Phenolic Metabolism

Abstract

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H 2 O 2 ) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H 2 O 2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H 2 O 2 -scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H 2 O 2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H 2 O 2 production in addition to potential H 2 O 2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism.

Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce

“…4; Supplemental Table S3). Eleven of the pathway genes detected in this study have been reported previously as induced twofold or greater under extracellular lignin-forming conditions in a cell culture of Norway spruce (Laitinen et al, 2017).…”

“…Bar 5 50 mm. 20.8) in the lignin-forming cell culture of spruce during lignin formation (Laitinen et al, 2017). Six PEROXI-DASEs, on the other hand, were more highly expressed in ray cells than in tracheids (P adj , 0.05; Supplemental Table S4).…”

“…A previous study that utilized an extracellular ligninforming cell culture of Norway spruce as a model for lignin biosynthesis had generated a gene coexpression network containing several transcription factors that were discussed as candidate regulators of monolignol biosynthesis (Laitinen et al, 2017). The present RNA-Seq data obtained using cells of developing xylem showed that out of the NAC and MYB candidate genes identified in the tissue culture study, only one NAC gene, MA_139896g0010, had a moderate expression level in rays and tracheids (normalized expression level of 2.4-2.8).…”

“…In Arabidopsis, both laccases and peroxidases have been shown to contribute to lignin biosynthesis by oxidizing monolignols to radicals that couple nonenzymatically (Berthet et al, 2011;Novo-Uzal et al, 2013;Zhao et al, 2013;Shigeto and Tsutsumi, 2016). In a Norway spruce cell culture that produces extracellular lignin (Kärkönen et al, 2002), however, peroxidases seem to have a major role in monolignol activation, as scavenging of apoplastic hydrogen peroxide efficiently hindered extracellular lignin formation (Laitinen et al, 2017). In a recent study in Norway spruce, several LACCASE genes and three PEROXIDASE genes were detected to coexpress with monolignol biosynthesis genes proposed as candidates for seasonal lignification (Jokipii-Lukkari et al, 2018).…”

“…The monolignol transport mechanism is still only partly characterized, but the data obtained indicate that ATP-binding cassette (ABC) transporters have an important role (Miao and Liu, 2010;Alejandro et al, 2012). In the cell wall, monolignols are exposed to extracellular peroxidases and laccases, the enzymes initiating polymerization of lignin by oxidizing monolignols to phenolic radicals that couple nonenzymatically (Boerjan et al, 2003;Barros et al, 2015;Laitinen et al, 2017). Until now, it has not been clear whether monolignol biosynthesis in conifers is a cell-autonomous process in xylem tracheids, the main cell type in conifer wood, or whether neighboring cells (i.e.…”