| 1. |
- Giummarella, Nicola, et al.
(författare)
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Nativity of lignin carbohydrate bonds substantiated by biomimetic synthesis
- 2019
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 70:20, s. 5591-5601
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Tidskriftsartikel (refereegranskat)abstract
- The question of whether lignin is covalently linked to carbohydrates in native wood, forming what is referred to as lignin–carbohydrate complexes (LCCs), still lacks unequivocal proof. This is mainly due to the need to isolate lignin from woody materials prior to analysis, under conditions leading to partial chemical modification of the native wood polymers. Thus, the correlation between the structure of the isolated LCCs and LCCs in situ remains open. As a way to circumvent the problematic isolation, biomimicking lignin polymerization in vivo and in vitro is an interesting option. Herein, we report the detection of lignin–carbohydrate bonds in the extracellular lignin formed by tissue-cultured Norway spruce cells, and in modified biomimetic lignin synthesis (dehydrogenation polymers). Semi-quantitative 2D heteronuclear singular quantum coherence (HSQC)-, 31P -, and 13C-NMR spectroscopy were applied as analytical tools. Combining results from these systems, four types of lignin–carbohydrate bonds were detected; benzyl ether, benzyl ester, γ-ester, and phenyl glycoside linkages, providing direct evidence of lignin–carbohydrate bond formation in biomimicked lignin polymerization. Based on our findings, we propose a sequence for lignin–carbohydrate bond formation in plant cell walls.
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| 2. |
- Laitinen, Teresa, et al.
(författare)
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A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism
- 2017
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Ingår i: Plant Physiology. - : American Society of Plant Biologists. - 0032-0889 .- 1532-2548. ; 174:3, s. 1449-1475
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Tidskriftsartikel (refereegranskat)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 (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 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 H2O2-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 H2O2 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 H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism.
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| 3. |
- Nickolov, Kaloian, et al.
(författare)
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Regulation of PaRBOH1-mediated ROS production in Norway spruce by Ca2+ binding and phosphorylation
- 2022
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Ingår i: Frontiers in Plant Science. - : Frontiers Media S.A.. - 1664-462X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- 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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