Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.

Glycine betaine is mostly known as an osmoprotectant. It is involved in the osmotic adaptation of eukaryotic and bacterial cells, and accumulates up to 1 M inside cells subjected to an osmotic upshock. Since, like other osmolytes, it can act as a protein stabilizer, its thermoprotectant properties were investigated. In vitro, like protein chaperones such as DnaK, glycine betaine and choline protect citrate synthase against thermodenaturation, and stimulate its renaturation after urea denaturation. In vivo, the internal concentration of glycine betaine is neither increased nor decreased after heat shock (this contrasts with a massive increase after osmotic upshock). However, even in exponential-phase bacteria grown in usual minimal salts media, the internal glycine betaine concentration attains levels (around 50 mM) which can protect proteins against thermodenaturation in vitro. Furthermore, glycine betaine and choline restore the viability of a dnaK deletion mutant at 42 SC, suggesting that glycine betaine not only acts as a thermoprotectant in vitro, but also acts as a thermoprotectant for Escherichia coli cells in vivo.

Monolignol glucosides are thought to be implicated in the lignin biosynthesis pathway as storage and/or transportation forms of cinnamyl alcohols between the cytosol and the lignifying cell walls. The hydrolysis of these monolignol glucosides would involve b-glucosidase activities. In Arabidopsis (Arabidopsis thaliana), in vitro studies have shown the affinity of b-GLUCOSIDASE45 (BGLU45) and BGLU46 for monolignol glucosides. BGLU45 and BGLU46 genes are expressed in stems. Immunolocalization experiments showed that BGLU45 and BGLU46 proteins are mainly located in the interfascicular fibers and in the protoxylem, respectively. Knockout mutants for BGLU45 or BGLU46 do not have a lignin-deficient phenotype. Coniferin and syringin could be detected by ultra-performance liquid chromatography-mass spectrometry in Arabidopsis stems. Stems from BGLU45 and BGLU46 mutant lines displayed a significant increase in coniferin content without any change in coniferyl alcohol, whereas no change in syringin content was observed. Other glucosylated compounds of the phenylpropanoid pathway were also deregulated in these mutants, but to a lower extent. By contrast, BGLU47, which is closely related to BGLU45 and BGLU46, is not implicated in either the general phenylpropanoid pathway or in the lignification of stems and roots. These results confirm that the major in vivo substrate of BGLU45 and BGLU46 is coniferin and suggest that monolignol glucosides are the storage form of monolignols in Arabidopsis, but not the direct precursors of lignin.