Contents Summary1703I.Introduction1703II.Cellulose biosynthesis1705III.Xylan biosynthesis1709IV.Glucomannan biosynthesis1713V.Lignin biosynthesis1714VI.Concluding remarks1717Acknowledgements1717References1717 Summary Secondary walls are synthesized in specialized cells, such as tracheary elements and fibers, and their remarkable strength and rigidity provide strong mechanical support to the cells and the plant body. The main components of secondary walls are cellulose, xylan, glucomannan and lignin. Biochemical, molecular and genetic studies have led to the discovery of most of the genes involved in the biosynthesis of secondary wall components. Cellulose is synthesized by cellulose synthase complexes in the plasma membrane and the recent success of in vitro synthesis of cellulose microfibrils by a single recombinant cellulose synthase isoform reconstituted into proteoliposomes opens new doors to further investigate the structure and functions of cellulose synthase complexes. Most genes involved in the glycosyl backbone synthesis, glycosyl substitutions and acetylation of xylan and glucomannan have been genetically characterized and the biochemical properties of some of their encoded enzymes have been investigated. The genes and their encoded enzymes participating in monolignol biosynthesis and modification have been extensively studied both genetically and biochemically. A full understanding of how secondary wall components are synthesized will ultimately enable us to produce plants with custom‐designed secondary wall composition tailored to diverse applications.

Xylan is a major hemicellulose in the secondary walls of vessels and fibers, and its acetylation is essential for normal secondary wall assembly and properties. The acetylation of xylan can occur at multiple positions of its backbone xylosyl residues, including 2-O-monoacetylation, 3-O-monoacetylation, 2,3-di-O-acetylation and 3-O-acetylation of 2-O-glucuronic acid (GlcA)-substituted xylosyl residues, but the biochemical mechanism controlling the regiospecific acetylation of xylan is largely unknown. Here, we present biochemical characterization of a group of Arabidopsis thaliana DUF231-containing proteins, namely TBL28, ESK1/TBL29, TBL30, TBL3, TBL31, TBL32, TBL33, TBL34 and TBL35, for their roles in catalyzing the regiospecific acetylation of xylan. Acetyltransferase activity assay of recombinant proteins demonstrated that all of these proteins possessed xylan acetyltransferase activities catalyzing the transfer of acetyl groups from acetyl-CoA onto xylooligomer acceptors albeit with differential specificities. Structural analysis of their reaction products revealed that TBL28, ESK1, TBL3, TBL31 and TBL34 catalyzed xylan 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation with differential positional preference, TBL30 carried out 2-O- and 3-O-monoacetylation, TBL35 catalyzed 2,3-di-O-acetylation, and TBL32 and TBL33 mediated 3-O-acetylation of 2-O-GlcA-substituted xylosyl residues. Furthermore, mutations of the conserved GDS and DXXH motifs in ESK1 were found to result in a complete loss of its acetyltransferase activity. Together, these results establish that these nine DUF231-containing proteins are xylan acetyltransferases mediating the regiospecific acetylation of xylan and that the conserved GDS and DXXH motifs are critical for their acetyltransferase activity.

We demonstrate a 15-fold enhancement of solid-state NMR signals via dynamic nuclear polarization (DNP) based on a stable, naturally occurring radical in a protein: the flavin mononucleotide (FMN) semiquinone of flavodoxin. The linewidth of flavodoxin’s EPR signal suggests that the dominant DNP mechanism is the solid effect, consistent with the field-dependent DNP enhancement profile. The magnitude of the enhancement as well as the bulk-polarization build-up time constant (τB) with which it develops are dependent on the isotopic composition of the protein. Deuteration of the protein to 85 % increased the nuclear longitudinal relaxation time T1n and τB by factors of five and seven, respectively. Slowed dissipation of polarization can explain the two-fold higher maximal enhancement than that obtained in proteated protein, based on the endogenous semiquinone. In contrast, the long τB of TOTAPOL-based DNP in non-glassy samples was not accompanied by a similarly important long T1n, and in this case the enhancement was greatly reduced. The low concentrations of radicals occurring naturally in biological systems limit the magnitude of DNP enhancement that is attainable by this means. However, our enhancement factors of up to 15 can nonetheless make an important difference to the feasibility of applying solid-state NMR to biochemical systems. We speculate that DNP based on endogenous radicals may facilitate MAS NMR characterization of biochemical complexes and even organelles, and could also serve as a source of additional structural and physiological information.

Mannans are hemicellulosic polysaccharides commonly found in the primary and secondary cell walls of land plants, and their mannosyl residues are often acetylated at O-2 and O-3. Currently, little is known about the genes responsible for the acetylation of mannans. In this report, we investigated the roles of a subgroup of DUF231 proteins including 11 from Arabidopsis thaliana and one from Amorphophallus konjac in mannan acetylation. Acetyltransferase activity assays of their recombinant proteins revealed that four Arabidopsis DUF231 proteins possessed an enzymatic activity capable of transferring acetyl groups from acetyl-CoA onto the mannohexaose acceptor, and thus were named mannan O-acetyltransferases (MOAT1, MOAT2, MOAT3 and MOAT4). Their close homolog from A. konjac (named AkMOAT1) also exhibited mannan O-acetyltransferase activity. Structural analysis of the MOAT-catalyzed reaction products demonstrated that these MOATs catalyzed 2-O- and 3-O-monoacetylation of mannosyl residues, an acetyl substitution pattern similar to that of Arabidopsis glucomannan. Site-directed mutagenesis showed that mutations of the conserved residues in the GDS and DXXH motifs of MOAT3 abolished its acetyltransferase activity, indicating the essential roles of these motifs in its activity. In addition, simultaneous RNA interference (RNAi) inhibition of the expression of the four Arabidopsis MOAT genes led to a drastic reduction in the degree of acetyl substitutions in glucomannan, further corroborating their role in glucomannan acetylation. Together, these results present the first lines of biochemical and genetic evidence demonstrating that these four Arabidopsis DUF231 members and their close A. konjac homolog are mannan O-acetyltransferases.

Whereas low-temperature (-78 °C) reaction of the lithium dithiolene radical 1 with boron bromide gives the dibromoboron dithiolene radical 2 , the parallel reaction of 1 with (C H ) BCl (0 °C) affords the dicyclohexylboron dithiolene radical 3 . Radicals 2 and 3 were characterized by single-crystal X-ray diffraction, UV/Vis, and EPR spectroscopy. The nature of these radicals was also probed computationally. Under mild conditions, 3 undergoes unexpected thiourea-mediated B-C bond activation to give zwitterion 4, which may be regarded as an anionic dithiolene-modified carbene complex of the sulfenyl cation RS (R=cyclohexyl).