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Amine-functionalized metal-organic framework (MOF) material is a promising CO

Secondary cell wall biosynthesis has been shown to be regulated by a suite of transcription factors. Here, we identified a new xylem vessel-specific NAC domain transcription factor, secondary wall-associated NAC domain protein5 (SND5), in Arabidopsis thaliana and studied its role in regulating secondary wall biosynthesis. We showed that the expression of SND5 and its close homolog, SND4/ANAC075, was specifically associated with secondary wall-containing cells and dominant repression of their functions severely reduced secondary wall thickening in these cells. Overexpression of SND4/5 as well as their homologs SND2/3 fused with the activation domain of the viral protein VP16 led to ectopic secondary wall deposition in cells that are normally parenchymatous. SND2/3/4/5 regulated the expression of the same downstream target genes as do the secondary wall NAC master switches (SWNs) by binding to and activating the secondary wall NAC binding elements (SNBEs). Furthermore, we demonstrated that the poplar (Populus trichocarpa) orthologs of SND2/3/4/5 also activated SNBEs and regulated secondary wall biosynthesis during wood formation. Together, these findings indicate that SND2/3/4/5 and their poplar orthologs regulate the expression of secondary wall-associated genes through activating SNBEs and they are positioned at an upper level in the SWN-mediated transcriptional network.

Several pine members of the gymnosperm-specific GT61 clades were demonstrated to be arabinosyltransferases and xylosyltransferases catalyzing the transfer of 2-O-Araf, 3-O-Araf and 2-O-Xyl side chains onto xylooligomer acceptors, indicating their possible involvement in Araf and Xyl substitutions of xylan in pine. Xylan in conifer wood is substituted at O-2 with methylglucuronic acid (MeGlcA) as well as at O-3 with arabinofuranose (Araf), which differs from xylan in dicot wood that is typically decorated with MeGlcA but not Araf. Currently, glycosyltransferases responsible for conifer xylan arabinosylation have not been identified. Here, we investigated the roles of pine glycosyltransferase family 61 (GT61) members in xylan substitutions. Biochemical characterization of four pine wood-associated GT61 members showed that they exhibited three distinct glycosyltransferase activities involved in xylan substitutions. Two of them catalyzed the addition of 2-O-α-Araf or 3-O-α-Araf side chains onto xylooligomer acceptors and thus were named Pinus taeda xylan 2-O-arabinosyltransferase 1 (PtX2AT1) and 3-O-arabinosyltransferase 1 (PtX3AT1), respectively. Two other pine GT61 members were found to be xylan 2-O-xylosyltransferases (PtXYXTs) adding 2-O-β-Xyl side chains onto xylooligomer acceptors. Furthermore, sequential reactions with PtX3AT1 and the PtGUX1 xylan glucuronyltransferase demonstrated that PtX3AT1 could efficiently arabinosylate glucuronic acid (GlcA)-substituted xylooligomers and likewise, PtGUX1 was able to add GlcA side chains onto 3-O-Araf-substituted xylooligomers. Phylogenetic analysis revealed that PtX2AT1, PtX3AT1 and PtXYXTs resided in three gymnosperm-specific GT61 clades that are separated from the grass-expanded GT61 clade harboring xylan 3-O-arabinosyltransferases and 2-O-xylosyltransferases, suggesting that they might have been recruited independently for xylan substitutions in gymnosperms. Together, our findings have established several pine GT61 members as xylan 2-O- and 3-O-arabinosyltransferases and 2-O-xylosyltransferases and they indicate that pine xylan might also be substituted with 2-O-Araf and 2-O-Xyl side chains.

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

Secondary wall deposition in xylem vessels is activated by Vascular-Related NAC Domain proteins (VNDs) that belong to a group of secondary wall NAC (SWN) transcription factors. By contrast, Xylem NAC Domain1 (XND1) negatively regulates secondary wall deposition in xylem vessels when overexpressed. The mechanism by which XND1 exerts its functions remains elusive. We employed the promoter of the fiber-specific Secondary Wall-Associated NAC Domain1 (SND1) gene to ectopically express XND1 in fiber cells to investigate its mechanism of action on secondary wall deposition. Ectopic expression of XND1 in fiber cells severely diminished their secondary wall deposition and drastically reduced the expression of SWN-regulated downstream transcription factors and secondary wall biosynthetic genes but not that of the SWN genes themselves. Transactivation analyses revealed that XND1 specifically inhibited SWN-activated expression of these downstream genes but not their MYB46-activated expression. Both the NAC domain and the C-terminus of XND1 were required for its inhibitory function and its NAC domain interacted with the DNA-binding domains of SWNs. XND1 was shown to be localized in the cytoplasm and the nucleus and its co-expression with VND6 resulted in the cytoplasmic sequestration of VND6. Furthermore, the C-terminus of XND1 was indispensable for the XND1-mediated cytoplasmic retention of VND6 and its fusion to VND6 was able to direct VND6 to the cytoplasm and render it unable to activate the gene expression. Since the XND1 gene is specifically expressed in xylem cells, these results indicate that XND1 acts through inhibiting VND functions to negatively regulate secondary wall deposition in xylem vessels.

Nitrogen-doped carbon materials with abundant defects and strong potassium adsorption ability have been recognized as potential anodes for potassium ion batteries (PIBs). However, the limited content and uncontrolled type of nitrogen-doped sites hinder the further performance improvement of PIBs. Herein, this work proposes nitrogen phosphorous co-doped hollow carbon nanofibers (PNCNFs) derived from high-energy metal-organic frameworks (MOFs) with an ultra-high nitrogen content of 19.52 wt% and a high portion of more reactive pyridinic N sites. Furthermore, the highly open architecture exploded by released gases from high-energy MOFs provides sufficient edge sites to settle the N atoms and further form pyridinic N sites induced by phosphorous dopants. The resulting PNCNFs achieve excellent potassium ion storage performance with high reversible capacity (466.2 mAh g

Xylan is the second most abundant polysaccharide in plant biomass. It is a crucial component of cell wall structure as well as a significant factor contributing to biomass recalcitrance. Xylan consists of a linear chain of β-1,4-linked xylosyl residues that are often substituted with glycosyl side chains, such as glucuronosyl/methylglucuronosyl and arabinofuranosyl residues, and acetylated at O-2 and/or O-3. Xylan from gymnosperms and dicots contains a unique reducing end tetrasaccharide sequence that is not detected in xylan from grasses, bryophytes and seedless vascular plants. Grass xylan is heavily decorated at O-3 with arabinofuranosyl residues that are frequently esterified with hydroxycinnamates. Genetic and biochemical studies have uncovered a number of genes involved in xylan backbone elongation and acetylation, xylan glycosyl substitutions and their modifications, and the synthesis of the unique xylan reducing end tetrasaccharide sequence, but some outstanding issues on the biosynthesis of xylan still remain unanswered. Here, we provide a brief overview of xylan structure and focus on discussion of the current understanding and open questions on xylan biosynthesis. Further elucidation of the biochemical mechanisms underlying xylan biosynthesis will not only shed new insights into cell wall biology but also provide molecular tools for genetic modification of biomass composition tailored for diverse end uses.

Multiple rice GT61 members were demonstrated to be xylan arabinosyltransferases (XATs) mediating 3-O-arabinosylation of xylan and the functions of XATs and xylan 2-O-xylosyltransferases were shown to be conserved in grass species. Xylan is the major hemicellulose in the cell walls of grass species and it is typified by having arabinofuranosyl (Araf) substitutions. In this report, we demonstrated that four previously uncharacterized, Golgi-localized glycosyltransferases residing in clade A or B of the rice GT61 family were able to mediate 3-O-arabinosylation of xylan when heterologously expressed in the Arabidopsis gux1/2/3 triple mutant. Biochemical characterization of their recombinant proteins established that they were xylan arabinosyltransferases (XATs) capable of transferring Araf residues onto xylohexaose acceptors, and thus they were named OsXAT4, OsXAT5, OsXAT6 and OsXAT7. OsXAT5 and the previously identified OsXAT2 were shown to be able to arabinosylate xylooligomers with a degree of polymerization of as low as 3. Furthermore, a number of XAT homologs from maize, sorghum, Brachypodium and switchgrass were found to exhibit activities catalyzing Araf transfer onto xylohexaose, indicating that they are XATs involved in xylan arabinosylation in these grass species. Moreover, we revealed that homologs of another GT61 member, xylan 2-O-xylosyltransferase (XYXT1), from these grass species could mediate 2-O-xylosylation of xylan when expressed in the Arabidopsis gux1/2/3 mutant. Together, our findings indicate that multiple OsXATs are involved in 3-O-arabinosylation of xylan and the functions of XATs and XYXTs are conserved in grass species.

Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG type consisting of repeating units of three consecutive xylosylated Glc residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1 and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1, but not AtXXT5, were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.

Contents Summary 1703 I. Introduction 1703 II. Cellulose biosynthesis 1705 III. Xylan biosynthesis 1709 IV. Glucomannan biosynthesis 1713 V. Lignin biosynthesis 1714 VI. Concluding remarks 1717 Acknowledgements 1717 References 1717 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.

AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are O-acetyltransferases acetylating fucosylated galactose residues on xyloglucan and AXY9 does not directly catalyze O-acetylation of xyloglucan but exhibits weak acetylesterase activity. Xyloglucan is a major hemicellulose that cross-links cellulose in the primary walls of dicot plants and the galactose (Gal) residues on its side chains can be mono- and di-O-acetylated. In Arabidopsis thaliana, mutations of three AXY (altered xyloglucan) genes, AXY4, AXY4L and AXY9, have previously been shown to cause a reduction in xyloglucan acetylation, but their biochemical functions remain to be investigated. In this report, we demonstrated that recombinant proteins of AXY4/XGOAT1 (xyloglucan O-acetyltransferase1), AXY4L/XGOAT2 and their close homologs from Populus trichocarpa, PtrXGOATs, displayed O-acetyltransferase activities transferring acetyl groups from acetyl CoA onto xyloglucan oligomers. Structural analysis of XGOAT-catalyzed reaction products revealed that XGOATs mediated predominantly 6-O-monoacetylation and a much lesser degree of 3-O and 4-O-monoacetylation and 4,6-di-O-acetylation of Gal residues on xyloglucan side chains. XGOATs appeared to preferentially acetylate fucosylated Gal residues with little activity toward non-fucosylated Gal residues. Mutations of the conserved amino acid residues in the GDS and DXXH motifs in AXY4/XGOAT1 resulted in a drastic reduction in its ability to transfer acetyl groups onto xyloglucan oligomers. In addition, although recombinant AXY9 was unable to transfer acetyl groups from acetyl CoA onto xyloglucan oligomers, it was catalytically active as demonstrated by its weak acetylesterase activity that was also exhibited by AXY4/XGOAT1 and AXY4L/XGOAT2. Furthermore, we showed that the AXY8 fucosidase was able to hydrolyze fucosyl residues from both non-acetylated and acetylated xyloglucan oligomers. These findings provide biochemical evidence that AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are xyloglucan O-acetyltransferases catalyzing acetyl transfer onto fucosylated Gal residues on xyloglucan side chains and the defucosylation of these acetylated side chains by apoplastic AXY8 generates side chains with acetylated, non-fucosylated Gal residues.

Xyloglucan is a major hemicellulose in plant cell walls and exists in two distinct types, XXXG and XXGG. While the XXXG-type xyloglucan from dicot species only contains O-acetyl groups on side-chain galactose (Gal) residues, the XXGG-type xyloglucan from Poaceae (grasses) and Solanaceae bears O-acetyl groups on backbone glucosyl (Glc) residues. Although O-acetyltransferases responsible for xyloglucan Gal acetylation have been characterized, the biochemical mechanism underlying xyloglucan backbone acetylation remains to be elucidated. In this study, we showed that recombinant proteins of a group of DUF231 members from rice and tomato were capable of transferring acetyl groups onto O-6 of Glc residues in cello-oligomer acceptors, indicating that they are xyloglucan backbone 6-O-acetyltransferases (XyBATs). We further demonstrated that XyBAT-acetylated cellohexaose oligomers could be readily xylosylated by AtXXT1 (Arabidopsis xyloglucan xylosyltransferase 1) to generate acetylated, xylosylated cello-oligomers, whereas AtXXT1-xylosylated cellohexaose oligomers were much less effectively acetylated by XyBATs. Heterologous expression of a rice XyBAT in Arabidopsis led to a severe reduction in cell expansion and plant growth and a drastic alteration in xyloglucan xylosylation pattern with the formation of acetylated XXGG-type units, including XGG, XGGG, XXGG, XXGG,XXGGG and XXGGG (G denotes acetylated Glc). In addition, recombinant proteins of two Arabidopsis XyBAT homologs also exhibited O-acetyltransferase activity toward cellohexaose, suggesting their possible role in mediating xyloglucan backbone acetylation in vivo. Our findings provide new insights into the biochemical mechanism underlying xyloglucan backbone acetylation and indicate the importance of maintaining the regular xyloglucan xylosylation pattern in cell wall function.

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.

Rice xylan is predominantly monoacetylated at O-2 and O-3, and 14 rice DUF231 proteins were demonstrated to be xylan acetyltransferases. Acetylated xylans are the principal hemicellulose in the cell walls of grass species. Because xylan acetylation impedes the conversion of cellulosic biomass into biofuels, knowledge on acetyltransferases catalyzing xylan acetylation in grass species will be instrumental for a better utilization of grass biomass for biofuel production. Xylan in rice (Oryza sativa) is predominantly monoacetylated at O-2 and O-3 with a total degree of acetylation of 0.19. In this report, we have characterized 14 rice DUF231 proteins (OsXOAT1 to OsXOAT14) that are phylogenetically grouped together with Arabidopsis xylan acetyltransferases ESK1 and its close homologs. Complementation analysis demonstrated that the expression of OsXOAT1 to OsXOAT7 in the Arabidopsis esk1 mutant was able to rescue its defects in 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation. Activity assay of recombinant proteins revealed that all 14 OsXOATs exhibited acetyltransferase activities capable of transferring acetyl groups from acetyl-CoA to the xylohexaose acceptor with 10 of them having high activities. Structural analysis of the OsXOAT-catalyzed products showed that the acetylated structural units consisted mainly of 2-O- and 3-O-monoacetylated xylosyl residues with a minor amount of 2,3-di-O-acetylated xylosyl units, which is consistent with the acetyl substitution pattern of rice xylan. Further kinetic studies revealed that OsXOAT1, OsXOAT2, OsXOAT5, OsXOAT6 and OsXOAT7 had high affinity toward the xylohexaose acceptor. Our results provide biochemical evidence indicating that OsXOATs are acetyltransferases involved in xylan acetylation in rice.

Wood (also termed secondary xylem) is the most abundant biomass produced by plants, and is one of the most important sinks for atmospheric carbon dioxide. The development of wood begins with the differentiation of the lateral meristem, vascular cambium, into secondary xylem mother cells followed by cell expansion, secondary wall deposition, programmed cell death, and finally heartwood formation. Significant progress has been made in the past decade in uncovering the molecular players involved in various developmental stages of wood formation in tree species. Hormonal signalling has been shown to play critical roles in vascular cambium cell proliferation and a peptide-receptor-transcription factor regulatory mechanism similar to that controlling the activity of apical meristems is proposed to be involved in the maintenance of vascular cambium activity. It has been demonstrated that the differentiation of vascular cambium into xylem mother cells is regulated by plant hormones and HD-ZIP III transcription factors, and the coordinated activation of secondary wall biosynthesis genes during wood formation is mediated by a transcription network encompassing secondary wall NAC and MYB master switches and their downstream transcription factors. Most genes encoding the biosynthesis enzymes for wood components (cellulose, xylan, glucomannan, and lignin) have been identified in poplar and a number of them have been functionally characterized. With the availability of genome sequences of tree species from both gymnosperms and angiosperms, and the identification of a suite of wood-associated genes, it is expected that our understanding of the molecular control of wood formation in trees will be greatly accelerated.

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.

Secondary walls in the form of wood and fibers are the most abundant biomass produced by vascular plants, and are important raw materials for many industrial uses. Understanding how secondary walls are constructed is of significance in basic plant biology and also has far-reaching implications in genetic engineering of plant biomass better suited for various end uses, such as biofuel production. Secondary walls are composed of three major biopolymers, i.e., cellulose, hemicelluloses and lignin, the biosynthesis of which requires the coordinated transcriptional regulation of all their biosynthesis genes. Genomic and molecular studies have identified a number of transcription factors, whose expression is associated with secondary wall biosynthesis. We comprehensively review how these secondary wall-associated transcription factors function together to turn on the secondary wall biosynthetic program, which leads to secondary wall deposition in vascular plants. The transcriptional network regulating secondary wall biosynthesis employs a multi-leveled feed-forward loop regulatory structure, in which the top-level secondary wall NAC (NAM, ATAF1/2 and CUC2) master switches activate the second-level MYB master switches and they together induce the expression of downstream transcription factors and secondary wall biosynthesis genes. Secondary wall NAC master switches and secondary wall MYB master switches bind to and activate the SNBE (secondary wall NAC binding element) and SMRE (secondary wall MYB-responsive element) sites, respectively, in their target gene promoters. Further investigation of what and how developmental signals trigger the transcriptional network to regulate secondary wall biosynthesis and how different secondary wall-associated transcription factors function cooperatively in activating secondary wall biosynthetic pathways will lead to a better understanding of the molecular mechanisms underlying the transcriptional control of secondary wall biosynthesis.

Secondary walls are mainly composed of cellulose, hemicelluloses (xylan and glucomannan) and lignin, and are deposited in some specialized cells, such as tracheary elements, fibers and other sclerenchymatous cells. Secondary walls provide strength to these cells, which lend mechanical support and protection to the plant body and, in the case of tracheary elements, enable them to function as conduits for transporting water. Formation of secondary walls is a complex process that requires the co-ordinated expression of secondary wall biosynthetic genes, biosynthesis and targeted secretion of secondary wall components, and patterned deposition and assembly of secondary walls. Here, we provide a comprehensive review of genes involved in secondary wall biosynthesis and deposition. Most of the genes involved in the biosynthesis of secondary wall components, including cellulose, xylan, glucomannan and lignin, have been identified and their co-ordinated activation has been shown to be mediated by a transcriptional network encompassing the secondary wall NAC and MYB master switches and their downstream transcription factors. It has been demonstrated that cortical microtubules and microtubule-associated proteins play important roles in the targeted secretion of cellulose synthase complexes, the oriented deposition of cellulose microfibrils and the patterned deposition of secondary walls. Further investigation of many secondary wall-associated genes with unknown functions will provide new insights into the mechanisms controlling the formation of secondary walls that constitute the bulk of plant biomass.

Xylan, a major polysaccharide in plant lignocellulosic biomass, is acetylated at O-2 and/or O-3 and its acetylation impedes the use of biomass for biofuel production. Currently, it is not known what genes encode acetyltransferases that are responsible for xylan O-acetylation. In this report, we demonstrate an essential role for the Arabidopsis gene ESKIMO1 (ESK1) in xylan O-acetylation during secondary wall biosynthesis. ESK1 expression was found to be regulated by the secondary wall master regulator SND1 (secondary wall-associated NAC domain protein1) and specifically associated with secondary wall biosynthesis. Its encoded protein was localized in the Golgi, the site of xylan biosynthesis. The esk1 mutation caused reductions in secondary wall thickening and stem mechanical strength. Chemical analyses of cell walls revealed that although the esk1 mutation did not cause apparent alterations in the xylan chain length and the abundance of the reducing end sequence, it resulted in a significant reduction in the degree of xylan acetylation. The reduced acetylation of esk1 xylan rendered it more accessible and digestible by endoxylanase, leading to generation of shorter xylooligomers compared with the wild type. Further structural analysis of xylan showed that the esk1 mutation caused a specific reduction in 2-O- and 3-O-monoacetylation of xylosyl residues but not in 2,3-di-O-acetylation or 3-O-acetylation of xylosyl residues substituted at O-2 with glucuronic acid. Consistent with ESK1's involvement in xylan O-acetylation, an activity assay revealed that the esk1 mutation led to a significant decrease in xylan acetyltransferase activity. Together, these results demonstrate that ESK1 is a putative xylan acetyltransferase required for 2-O- and 3-O-monoacetylation of xylosyl residues and indicate the complexity of the biochemical mechanism underlying xylan O-acetylation.

Xylan and xyloglucan are the two major cell wall hemicelluloses in plants, and their biosynthesis requires a steady supply of the sugar donor, UDP-xylose. UDP-xylose is synthesized through conversion of UDP-glucuronic acid (UDP-GlcA) by the activities of UDP-xylose synthase (UXS). There exist six UXS genes in the Arabidopsis thaliana genome; three of them (UXS1, UXS2 and UXS4) encode Golgi-localized enzymes and the other three (UXS3, UXS5 and UXS6) encode cytosol-localized enzymes. In this report, we investigated the contributions of these UXS genes in supplying UDP-xylose for the biosynthesis of xylan and xyloglucan. Expression analyses revealed that the six UXS genes exhibited distinct and overlapping expression patterns in different cell types of stems, root-hypocotyls and young seedlings, and that the relative enzymatic activity of UXS in the cytosol was 17 times higher than that in the Golgi. Among the six UXS genes, UXS3, UXS5 and UXS6 showed the highest expression in stems and were expressed predominantly in xylem cells and interfascicular fibers. Their predominant expression in secondary wall-forming cells was consistent with the finding that the expression of UXS3, UXS5 and UXS6 was directly activated by the secondary wall NAC master switches. Although simultaneous mutations of UXS1, UXS2 and UXS4 did not cause any apparent effects on plant growth and xylan biosynthesis, simultaneous down-regulation/mutations of UXS3, UXS5 and UXS6 led to a drastic reduction in secondary wall thickening, a severe deformation of xylem vessels, a significant decrease in xylan content without an apparent reduction in its chain length and an absence of GlcA side chains in xylan, which are reminiscent of the phenotypes of some known xylan-deficient mutants. Moreover, Immunolocalization with two xyloglucan monoclonal antibodies, LM15 and LM25, revealed a significant reduction in the amount of xylogulcan in the primary walls. These results demonstrate that the cytosol-localized UXS3, UXS5 and UXS6 play a predominant role in the supply of UDP-xylose for the biosynthesis of xylan and xyloglucan.

In this work, we present a synthesis approach for nitrogen-doped graphene-sheet-like nanostructures via the graphitization of a heteroatom polymer, in particular, polyaniline, under the catalysis of a cobalt species using multiwalled carbon nanotubes (MWNTs) as a supporting template. The graphene-rich composite catalysts (Co-N-MWNTs) exhibit substantially improved activity for oxygen reduction in nonaqueous lithium-ion electrolyte as compared to those of currently used carbon blacks and Pt/carbon catalysts, evidenced by both rotating disk electrode and Li-O(2) battery experiments. The synthesis-structure-activity correlations for the graphene nanostructures were explored by tuning their synthetic chemistry (support, nitrogen precursor, heating temperature, and transition metal type and content) to investigate how the resulting morphology and nitrogen-doping functionalities (e.g., pyridinic, pyrrolic, and quaternary) influence the catalyst activity. In particular, an optimal temperature for heat treatment during synthesis is critical to creating a high-surface-area catalyst with favorable nitrogen doping. The sole Co phase, Co(9)S(8), was present in the catalyst but plays a negligible role in ORR. Nevertheless, the addition of Co species in the synthesis is indispensable for achieving high activity, due to its effects on the final catalyst morphology and structure, including surface area, nitrogen doping, and graphene formation. This new route for the preparation of a nitrogen-doped graphene nanocomposite with carbon nanotube offers synthetic control of morphology and nitrogen functionality and shows promise for applications in nonaqueous oxygen reduction electrocatalysis for Li-O(2) battery cathodes.

Xylan is made of a linear chain of β-1,4-linked xylosyl residues, some of which are substituted with side chains, such as glucuronic acid (GlcA), methylglucuronic acid (MeGlcA) and arabinose, depending on the source of xylan. Although past studies have revealed a number of genes involved in the elongation of the xylan backbone and the addition of GlcA and arabinosyl side chains, no genes have been shown to be implicated in glucuronoxylan methylation. In this report, we investigated the roles of three Arabidopsis genes, namely GLUCURONOXYLAN METHYLTRANSFERASE1 (GXM1), GXM2 and GXM3, in xylan biosynthesis. The GXM1/2/3 genes were found to be expressed in secondary wall-forming cells and their expression was regulated by SND1, a secondary wall master transcriptional switch. Their encoded proteins were shown to be located in the Golgi, where xylan biosynthesis occurs. Chemical analysis of cell wall sugars from single and double mutants of these genes revealed that although no alterations in the amount of xylose were observed, a significant reduction in the level of MeGlcA was evident in the gxm3 single mutant and the gxm double mutants. Structural analysis of xylan demonstrated that the gxm mutations caused a specific defect in GlcA methylation on xylan without affecting the frequency of xylan substitution. Only about 10% of the GlcA residues on xylan were methylated in the gxm2/3 double mutant, whereas in the wild type 60% of the GlcA residues were methylated. Furthermore, an activity assay demonstrated that recombinant GXM proteins exhibited a methyltransferase activity capable of transferring the methyl group from S-adenosylmethionine onto GlcA-substituted xylooligomers and simultaneous mutations of GXM2/3 genes caused a loss of such a methyltransferase activity. Taken together, our results provide the first line of genetic and biochemical evidence that the three DUF579 domain-containing proteins, GXM1, GXM2 and GXM3, are methyltransferases catalyzing 4-O-methylation of GlcA side chains on xylan.

Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of β-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that mutations of two Arabidopsis family GT8 genes, GUX1 and GUX2, affect the addition of GlcA and MeGlcA to xylan, but it is not known whether they encode glucuronyltransferases (GlcATs) or indirectly regulate the GlcAT activity. In this study, we performed biochemical and genetic analyses of three Arabidopsis GUX genes to determine their roles in the GlcA substitution of xylan and secondary wall deposition. The GUX1/2/3 genes were found to be expressed in interfascicular fibers and xylem cells, the two major types of secondary wall-containing cells that have abundant xylan. When expressed in tobacco BY2 cells, the GUX1/2/3 proteins exhibited an activity capable of transferring GlcA residues from the UDP-GlcA donor onto xylooligomer acceptors, demonstrating that these GUX proteins possess xylan GlcAT activity. Analyses of the single, double and triple gux mutants revealed that simultaneous mutations of all three GUX genes led to a complete loss of GlcA and MeGlcA side chains on xylan, indicating that all three GUX proteins are involved in the GlcA substitution of xylan. Furthermore, a complete loss of GlcA and MeGlcA side chains in the gux1/2/3 triple mutant resulted in reduced secondary wall thickening, collapsed vessel morphology and reduced plant growth. Together, our results provide biochemical and genetic evidence that GUX1/2/3 are GlcATs responsible for the GlcA substitution of xylan, which is essential for normal secondary wall deposition and plant development.

The bulk of grass biomass potentially useful for cellulose-based biofuel production is the remains of secondary wall-containing sclerenchymatous fibers. Hence, it is important to uncover the molecular mechanisms underlying the regulation of secondary wall thickening in grass species. So far, little is known about the transcriptional regulatory switches responsible for the activation of the secondary wall biosynthetic program in grass species. Here, we report the roles of a group of rice and maize NAC and MYB transcription factors in the regulation of secondary wall biosynthesis. The rice and maize secondary wall-associated NACs (namely OsSWNs and ZmSWNs) were able to complement the Arabidopsis snd1 nst1 double mutant defective in secondary wall thickening. When overexpressed in Arabidopsis, OsSWNs and ZmSWNs were sufficient to activate a number of secondary wall-associated transcription factors and secondary wall biosynthetic genes, and concomitantly result in the ectopic deposition of cellulose, xylan and lignin. It was also found that the rice and maize MYB transcription factors, OsMYB46 and ZmMYB46, are functional orthologs of Arabidopsis MYB46/MYB83 and, when overexpressed in Arabidopsis, they were able to activate the entire secondary wall biosynthetic program. Furthermore, the promoters of OsMYB46 and ZmMYB46 contain secondary wall NAC-binding elements (SNBEs), which can be bound and activated by OsSWNs and ZmSWNs. Together, our results indicate that the rice and maize SWNs and MYB46 are master transcriptional activators of the secondary wall biosynthetic program and that OsSWNs and ZmSWNs activate their direct target genes through binding to the SNBE sites.

We report the genome-wide analysis of direct target genes of SND1 and VND7, two Arabidopsis thaliana NAC domain transcription factors that are master regulators of secondary wall biosynthesis in fibers and vessels, respectively. Systematic mapping of the SND1 binding sequence using electrophoretic mobility shift assay and transactivation analysis demonstrated that SND1 together with other secondary wall NACs (SWNs), including VND6, VND7, NST1, and NST2, bind to an imperfect palindromic 19-bp consensus sequence designated as secondary wall NAC binding element (SNBE), (T/A)NN(C/T) (T/C/G)TNNNNNNNA(A/C)GN(A/C/T) (A/T), in the promoters of their direct targets. Genome-wide analysis of direct targets of SND1 and VND7 revealed that they directly activate the expression of not only downstream transcription factors, but also a number of non-transcription factor genes involved in secondary wall biosynthesis, cell wall modification, and programmed cell death, the promoters of which all contain multiple SNBE sites. SND1 and VND7 directly regulate the expression of a set of common targets but each of them also preferentially induces a distinct set of direct targets, which is likely attributed to their differential activation strength toward SNBE sites. Complementation study showed that the SWNs were able to rescue the secondary wall defect in the snd1 nst1 mutant, indicating that they are functionally interchangeable. Together, our results provide important insight into the complex transcriptional program and the evolutionary mechanism underlying secondary wall biosynthesis, cell wall modification, and programmed cell death in secondary wall-containing cell types.

There exist four members of family GT43 glycosyltransferases in the Arabidopsis (Arabidopsis thaliana) genome, and mutations of two of them, IRX9 and IRX14, have previously been shown to cause a defect in glucuronoxylan (GX) biosynthesis. However, it is currently unknown whether IRX9 and IRX14 perform the same biochemical function and whether the other two GT43 members are also involved in GX biosynthesis. In this report, we performed comprehensive genetic analysis of the functional roles of the four Arabidopsis GT43 members in GX biosynthesis. The I9H (IRX9 homolog) and I14H (IRX14 homolog) genes were shown to be specifically expressed in cells undergoing secondary wall thickening, and their encoded proteins were targeted to the Golgi, where GX is synthesized. Overexpression of I9H but not IRX14 or I14H rescued the GX defects conferred by the irx9 mutation, whereas overexpression of I14H but not IRX9 or I9H complemented the GX defects caused by the irx14 mutation. Double mutant analyses revealed that I9H functioned redundantly with IRX9 and that I14H was redundant with IRX14 in their functions. In addition, double mutations of IRX9 and IRX14 were shown to cause a loss of secondary wall thickening in fibers and a much more severe reduction in GX amount than their single mutants. Together, these results provide genetic evidence demonstrating that all four Arabidopsis GT43 members are involved in GX biosynthesis and suggest that they form two functionally nonredundant groups essential for the normal elongation of GX backbone.

Wood is the most abundant biomass produced by land plants. Dissection of the molecular mechanisms underlying the transcriptional regulation of wood formation is a fundamental issue in plant biology and has important implications in tree biotechnology. Although a number of transcription factors in tree species have been shown to be associated with wood formation and some of them are implicated in lignin biosynthesis, none of them have been demonstrated to be key regulators of the biosynthesis of all three major components of wood. In this report, we have identified a group of NAC domain transcription factors, PtrWNDs, that are preferentially expressed in developing wood of poplar (Populus trichocarpa). Expression of PtrWNDs in the Arabidopsis (Arabidopsis thaliana) snd1 nst1 double mutant effectively complemented the secondary wall defects in fibers, indicating that PtrWNDs are capable of activating the entire secondary wall biosynthetic program. Overexpression of PtrWND2B and PtrWND6B in Arabidopsis induced the expression of secondary wall-associated transcription factors and secondary wall biosynthetic genes and, concomitantly, the ectopic deposition of cellulose, xylan, and lignin. Furthermore, PtrWND2B and PtrWND6B were able to activate the promoter activities of a number of poplar wood-associated transcription factors and wood biosynthetic genes. Together, these results demonstrate that PtrWNDs are functional orthologs of SND1 and suggest that PtrWNDs together with their downstream transcription factors form a transcriptional network involved in the regulation of wood formation in poplar.

Lignin is the second most abundant plant biopolymer mainly present in the secondary walls of tracheary elements and fibers in wood. Understanding how lignin is biosynthesized has long been an interest to plant biologists and will have a significant impact on tree biotechnology. Lignin is polymerized from monolignols that are synthesized through the lignin biosynthetic pathway. To make lignin, all the genes in the lignin biosynthetic pathway need to be coordinately turned on. It has been shown that a common cis-element, namely the AC element, is present in the majority of the lignin biosynthetic genes and required for their expression in lignifying cells. Important progress has been made in the identification of transcription factors that bind to the AC elements and are potentially involved in the coordinated regulation of lignin biosynthesis. The Arabidopsis MYB58 and MYB63 as well as their poplar ortholog PtrMYB28 are transcriptional activators of the lignin biosynthetic pathway, whereas the eucalyptus EgMYB2 and pine PtMYB4 transcription factors are likely Arabidopsis MYB46 orthologs involved in the regulation of the entire secondary wall biosynthetic program. It was found that the transcriptional regulation of lignin biosynthesis is under the control of the same transcriptional network regulating the biosynthesis of other secondary wall components, including cellulose and xylan. The identification of transcription factors directly activating lignin biosynthetic genes provides unprecedented tools to potentially manipulate the amount of lignin in wood and other plant products based on our needs.

It has been proposed that the transcriptional regulation of secondary wall biosynthesis in Arabidopsis is controlled by a transcriptional network mediated by SND1 and its close homologs. Uncovering all the transcription factors and deciphering their interrelationships in the network are essential for our understanding of the molecular mechanisms underlying the transcriptional regulation of biosynthesis of secondary walls, the major constituent of wood and fibers. Here, we present functional evidence that the MYB83 transcription factor is another molecular switch in the SND1-mediated transcriptional network regulating secondary wall biosynthesis. MYB83 is specifically expressed in fibers and vessels where secondary wall thickening occurs. Its expression is directly activated by SND1 and its close homologs, including NST1, NST2, VND6 and VND7, indicating that MYB83 is their direct target. MYB83 overexpression is able to activate a number of the biosynthetic genes of cellulose, xylan and lignin and concomitantly induce ectopic secondary wall deposition. In addition, its overexpression upregulates the expression of several transcription factors involved in regulation of secondary wall biosynthesis. Dominant repression of MYB83 functions or simultaneous RNAi inhibition of MYB83 and MYB46 results in a reduction in secondary wall thickening in fibers and vessels and a deformation of vessels. Furthermore, double T-DNA knockout mutations of MYB83 and MYB46 cause a lack of secondary walls in vessels and an arrest in plant growth. Together, these results demonstrate that MYB83 and MYB46, both of which are SND1 direct targets, function redundantly in the transcriptional regulatory cascade leading to secondary wall formation in fibers and vessels.

Xylan is the second most abundant polysaccharide in dicot wood. Unraveling the biosynthetic pathway of xylan is important not only for our understanding of the process of wood formation but also for our rational engineering of wood for biofuel production. Although several glycosyltransferases are implicated in glucuronoxylan (GX) biosynthesis in Arabidopsis , whether their close orthologs in woody tree species are essential for GX biosynthesis during wood formation has not been investigated. In fact, no studies have been reported to evaluate the effects of alterations in secondary wallassociated glycosyltransferases on wood formation in tree species. In this report, we demonstrate that PoGT47C, a poplar glycosyltransferase belonging to family GT47, is essential for the normal biosynthesis of GX and the normal secondary wall thickening in the wood of the hybrid poplar Populus alba × tremula . RNA interference (RNAi) inhibition of PoGT47C resulted in a drastic reduction in the thickness of secondary walls, a deformation of vessels and a decreased amount of GX in poplar wood. Structural analysis of GX using nuclear magnetic resonance (NMR) spectroscopy demonstrated that the reducing end of GX from poplar wood contains the tetrasaccharide sequence, β - D -Xyl p (1 → 3)- α - L -Rha p -(1 → 2)- α - D -Gal p A-(1 → 4)- D -Xyl p , and that its abundance was signifi cantly decreased in the GX from the wood of the GT47C-RNAi lines. The transgenic wood was found to yield more glucose by cellulase digestion than the wild-type wood, indicating that the GX reduction in wood reduces the recalcitrance of wood to cellulase digestion. Together, these results provide direct evidence demonstrating that the PoGT47C glycosyltransferase is essential for normal GX biosynthesis in poplar wood and that GX modifi cation could improve the digestibility of wood cellulose by cellulase.