Flavan-3-ols in Norway Spruce: Biosynthesis, Accumulation, and Function in Response to Attack by the Bark Beetle-Associated Fungus Ceratocystis polonica        

Abstract

Proanthocyanidins (PAs) are common polyphenolic polymers of plants found in foliage, fruit, bark, roots, rhizomes, and seed coats that consist of flavan-3-ol units such as 2,3-trans-(+)-catechin and 2,3-cis-(–)-epicatechin. Although the biosynthesis of flavan-3-ols has been studied in angiosperms, little is known about their biosynthesis and ecological roles in gymnosperms. In this study, the genes encoding leucoanthocyanidin reductase, a branch point enzyme involved in the biosynthesis of 2,3-trans-(+)-flavan-3-ols, were identified and functionally characterized in Norway spruce (Picea abies), the most widespread and economically important conifer in Europe. In addition, the accumulation of flavan-3-ols and PAs was investigated in Norway spruce saplings after wounding or inoculation with the fungal pathogen Ceratocystis polonica, which is vectored by bark beetles (Ips typographus) and is usually present during fatal beetle attacks. Monomeric and dimeric flavan-3-ols were analyzed by reverse-phase high-pressure liquid chromatography, while the size and subunit composition of larger PAs were characterized using a novel acid hydrolysis method and normal phase chromatography. Only flavan-3-ol monomers with 2,3-trans stereochemistry were detected in spruce bark; dimeric and larger PAs contained flavan-3-ols with both 2,3-trans and 2,3-cis stereochemistry. Levels of monomers as well as PAs with a higher degree of polymerization increased dramatically in spruce bark after infection by C. polonica. In accordance with their role in the biosynthesis of 2,3-trans-(+)-flavan-3-ols, transcript abundance of Norway spruce LEUCOANTHOCYANIDIN REDUCTASE genes also increased significantly during fungal infection. Bioassays with C. polonica revealed that the levels of 2,3-trans-(+)-catechin and PAs that are produced in the tree in response to fungal infection inhibit C. polonica growth and can therefore be considered chemical defense compounds.

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection

“…The recovery of epicatechin after hydrolysis of PAs indicates that epicatechin might contribute to the extension of PA chains. Similar mechanisms also were observed in grape and Norway spruce (Bogs et al, 2005;Hammerbacher et al, 2014).…”

“…Studies also demonstrated that the biosynthesis of flavan-3-ols and PAs increased after infection by fungal endophytes in poplar (Pfabel et al, 2012) as well as after infection by pathogenic fungi in other plant species, such as bilberry (Vaccinium myrtillus; Koskimäki et al, 2009) and Fagus crenata (Yamaji and Ichihara, 2012). Increased accumulation of flavan-3-ols also has been recorded in Norway spruce (Picea abies) during infection by necrotrophic fungi (Danielsson et al, 2011;Hammerbacher et al, 2014). Therefore, a range of plants, including poplar, respond to pathogen attack by accumulating both monomeric flavan-3-ols and PAs.…”

“…Especially in woody plant species, catechin and PAs accumulate upon pathogen infection and are thought to represent antimicrobial defenses (Barry et al, 2002;Miranda et al, 2007;Danielsson et al, 2011;Hammerbacher et al, 2014;Nemesio-Gorriz et al, 2016). In support of this hypothesis, pretreatment of roots with catechin induced systemic resistance in shoots against the bacterial pathogen Pseudomonas syringae (Prithiviraj et al, 2007).…”

“…Flavan-3-ols differ structurally from other flavonoids by having a nearly saturated C ring with an additional hydroxyl group on the 3-position, which as a chiral center gives rise to cis-and trans-forms of the basic PA-forming units, 2,3-trans-(+)-catechin or 2,3-cis-(2)-epicatechin. The structural diversity of PAs is increased further by the hydroxylation patterns of the B-ring (monohydroxylation, dihydroxylation, or trihydroxylation) and the degree of polymerization (up to more than 100 flavan-3-ol units; Ferreira and Slade, 2002;Hammerbacher et al, 2014).…”

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism

“…For the shikimate pathway and aromatic amino acid pathways (according to Maeda and Dudareva, 2012): ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; CM, chorismate mutase; CS, chorismate synthase; DAHP synthase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; DHD-SDH, 3-dehydroquinate dehydratase-shikimate dehydrogenase; DHQS, 3-dehydroquinate synthase; EPSP synthase, 5-enolpyruvylshikimate 3-phosphate synthase; HPP-AT, 4-hydroxyphenylpyruvate aminotransferase; PDH, prephenate dehydrogenase; PDT, prephenate dehydratase; PPA-AT, prephenate aminotransferase; PPY-AT, phenylpyruvate aminotransferase; SK, shikimate kinase. For the flavonoid pathway (according to Hammerbacher et al, 2014): ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone-3-hydroxylase; F39H, flavonol-39-hydroxylase; F395H, flavonol-39,59-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase. For the general phenylpropanoid pathway: CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; C3H, p-coumaroyl shikimate 3-hydroxylase; C4H, cinnamate 4-hydroxylase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; PAL, Phe ammonia lyase; 4CL, 4-coumarate-CoA ligase.…”