Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem

Abstract

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism

“…IDDDC is formed from dehydrodiconiferyl alcohol [G(8-5)G] by a PCBER that, in poplar, is one of the most abundant proteins present in xylem (Niculaes et al, 2014). Since the PCBER is located in the cytoplasm Vander Mijnsbrugge et al, 2000a, 2000bNiculaes et al, 2014), the data suggest that these reduced dilignols are produced intracellularly and transported into the apoplast. The spruce genome contains 10 sequences of high similarity to Populus trichocarpa PtrPCBER, several of which have good expression in developing xylem (Nystedt et al, 2013).…”

“…Also in lignifying tissues, intracellular oxidative cross-coupling of monolignols was suggested to occur based on the detection of cysteinylated 8-O-4-type dilignols in the xylem of transgenic poplar (Populus spp.) with a diminished phenylcoumaran benzylic ether reductase (PCBER) activity (Niculaes et al, 2014). The latter aryl ether dilignols are formed when Cys rather than water acts as a nucleophile in the rearomatization of quinone methides formed by the 8-O-4 cross-coupling of monolignol radicals.…”

Small Glycosylated Lignin Oligomers Are Stored in Arabidopsis Leaf Vacuoles

“…Finally, further support for intracellular coupling has recently been obtained from the comparative profiling of wild-type poplars and poplars deficient in phenylcoumaran benzylic ether reductase. In these plants, different cysteine adduct analogs of 8-O-4-dilignols have been identified (Niculaes et al, 2014). In contrast to the formation of the 8-5-and the 8-8-units following the radical coupling reactions, that of the 8-O-4-units needs an external nucleophile to rearomatize the intermediate quinone methide.…”

“…In view of intracellular combinatorial coupling, it is evident that glycosylation of the phenolic function will stabilize and/or detoxify phenolic compounds and prevent the coupling of monolignol radicals to proteins or amino acids (Cong et al, 2013;Diehl et al, 2014;Niculaes et al, 2014). However, after damage of the cell, e.g., during pathogen attack, the oligolignol glycosides stored in the vacuoles will come into contact with the apoplast, after which they may be hydrolyzed by extracellular b-glucosidases and polymerized due to the presence of reactive oxygen species and peroxidases.…”

Tricin, a Flavonoid Monomer in Monocot Lignification

“…Synthetic model compounds that would facilitate the elucidation of the role of tricin within plant cell walls are desirable as aids to be used in a mechanistic study of flavonolignin generation. (We coin the term flavonolignin to describe the racemic oligomers and polymers of monolignols that start from tricin [or other flavonoids] in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties that are presumably made in the cytoplasm [Begum et al, 2010;Niculaes et al, 2014;Dima et al, 2015]). …”