UPSC-0019
Apoplastic H2O2 effects on phenolic metabolism and gene expression in a Norway spruce lignin-forming cell culture
Lignin is a major structural biopolymer of trees and a physical barrier against pathogens. Whether peroxidases (class III) or laccases function in the last step of lignin biosynthesis, i.e., oxidize monolignols to phenolic radicals that then couple and make the lignin polymer, has been under active evaluation. In a cell suspension culture of Norway spruce (Picea abies L. Karst.), H2O2 is required for extracellular lignin production as its scavenging by potassium iodide (KI) represses lignin formation. This observation suggests that peroxidases activate monolignols in this cell culture. In order to investigate how apoplastic H2O2 level modulation affects the small molecular weight phenolic compounds and  the transcriptome, we conducted large-scale phenolic and transcriptomics analyses. In lignin-forming cultures in addition to lignin polymer, several types of di- and oligolignols were abundant in the culture medium, and various flavonoid and dilignol glycosides accumulated in the cells. After H2O2 scavenging, on the contrary, some di- and oligolignols were detected in the culture medium and some dilignol glycosides in the cells. Some dimeric structures found in the medium can be formed only by intracellular enzymes suggesting that spruce cells can transport dilignols into the apoplast. Interestingly, removal of KI restored extracellular lignin formation, enabling us to study the regulation of lignin, and di- and oligolignol formation. Scavenging of apoplastic H2O2 significantly changed gene expression, leading to a reduced carbon flux into the shikimate, monolignol and flavonoid biosynthesis pathways. Gene expression patterns of antioxidant enzyme and related genes suggest that cultured spruce cells suffer from an oxidative stress in conditions where they produce extracellular lignin, and scavenging of apoplastic H2O2 alleviates this stress. By various approaches, candidate proteins for monolignol oxidation and apoplastic H2O2 production were identified together with novel putative transcription factors and putative H2O2 receptors related to lignin biosynthesis in Norway spruce.