WUSCHEL-RELATED HOMEOBOX genes are crucial for normal vascular organization and wood formation in poplar.

Vascular cambium in tree species is a cylindrical domain of meristematic cells that are responsible for producing secondary xylem (also called wood) inward and secondary phloem outward. The poplar (Populus trichocarpa) WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family members, PtrWUSa and PtrWOX13b, were previously shown to be expressed in vascular cambium and differentiating xylem cells in poplar stems, but their functions remain unknown. Here, we investigated roles of PtrWUSa, PtrWOX13b and their close homologs in vascular organization and wood formation. Expression analysis showed that like PtrWUSa and PtrWOX13b, their close homologs, PtrWUSb, PtrWUS4a/b and PtrWOX13a/c, were also expressed in vascular cambium and differentiating xylem cells in poplar stems. PtrWUSa also exhibited a high level of expression in developing phloem fibers. Expression of PtrWUSa fused with the dominant EAR repression domain (PtrWUSa-DR) in transgenic poplar caused retarded growth of plants with twisted stems and curled leaves and a severe disruption of vascular organization. In PtrWUSa-DR stems, a drastic proliferation of cells occurred in the phloem region between vascular cambium and phloem fibers and they formed islands of ectopic vascular tissues or phloem fiber-like sclerenchyma cells. A similar proliferation of cells was also observed in PtrWUSa-DR leaf petioles and midveins. On the other hand, overexpression of PtrWOX4a-DR caused ectopic formation of vascular bundles in the cortical region, and overexpression of PtrWOX13a-DR and PtrWOX13b-DR led to a reduction in wood formation without affecting vascular organization in transgenic poplar plants. Together, these findings indicate crucial roles of PtrWUSa and PtrWOX13a/b in regulating vascular organization and wood formation, which furthers our understanding of the functions of WOX genes in regulating vascular cambium activity in tree species.

A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.

MAIN CONCLUSION: A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Biochemical characterization of rice xylan biosynthetic enzymes in determining xylan chain elongation and substitutions.

Grass xylan consists of a linear chain of ß-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or (methyl)glucuronic acid [(Me)GlcA] residues and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remain elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted xylohexaose acceptors, they transferred little GlcA residues onto Araf-substituted xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution pattern of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.

Acetylation of homogalacturonan and rhamnogalacturonan-I is catalyzed by a suite of trichome birefringence-like proteins.

Plant cell wall polysaccharides, including xylan, mannan, xyloglucan, and pectins, are often acetylated and members of the domain of unknown function 231 (DUF231)/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases mediating the acetylation of xylan, mannan, and xyloglucan. However, little is known about the O-acetyltransferases responsible for pectin acetylation. In this report, we biochemically characterized a suite of Arabidopsis DUF231/TBL proteins for their roles in pectin acetylation. We generated 24 TBL recombinant proteins in mammalian cells and demonstrated that 10 of them were able to transfer acetyl groups from acetyl-CoA onto the pectins homogalacturonan (HG) or rhamnogalacturonan-I (RG-I), and thus were named pectin O-acetyltransferase 1 to 10 (POAT1 to 10). It was found that POAT2,4,9,10 specifically acetylated HG and POAT5,6 acetylated RG-I, whereas POAT1,3,7,8 could act on both HG and RG-I. The acetylation of HG and RG-I by POATs was further corroborated by hydrolysis with pectin acetylesterases and by nuclear magnetic resonance spectroscopy. In addition, mutations of the conserved GDS and DXXH motifs in POAT3 and POAT8 were shown to lead to a loss of their ability to acetylate HG and RG-I. Furthermore, simultaneous RNA interference downregulation of POAT1,3,6,7,8 resulted in reduced cell expansion, impaired plant growth, and decreased pectin acetylation. Together, our findings indicate that these POATs are pectin O-acetyltransferases involved in acetylation of the pectin polysaccharides HG and RG-I.

SPOTTED-LEAF7 targets the gene encoding ß-galactosidase9, which functions in rice growth and stress responses.

ß-Galactosidases (Bgals) remove terminal ß-D-galactosyl residues from the nonreducing ends of ß-D-galactosidases and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite the many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) ß-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast 1-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical ß-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

An Arabidopsis family GT106 glycosyltransferase is essential for xylan biosynthesis and secondary wall deposition.

MAIN CONCLUSION: We have demonstrated that the Arabidopsis FRA9 (fragile fiber 9) gene is specifically expressed in secondary wall-forming cells and essential for the synthesis of the unique xylan reducing end sequence. Xylan is made of a linear chain of ß-1,4-linked xylosyl (Xyl) residues that are often substituted with (methyl)glucuronic acid [(Me)GlcA] side chains and may be acetylated at O-2 and/or O-3. The reducing end of xylan from gymnosperms and dicots contains a unique tetrasaccharide sequence consisting of ß-D-Xylp-(1 → 3)-α-L-Rhap-(1 → 2)-α-D-GalpA-(1 → 4)-D-Xylp, the synthesis of which requires four different glycosyltransferase activities. Genetic analysis in Arabidopsis thaliana has so far implicated three glycosyltransferase genes, FRA8 (fragile fiber 8), IRX8 (irregular xylem 8) and PARVUS, in the synthesis of this unique xylan reducing end sequence. Here, we report the essential role of FRA9, a member of glycosyltransferase family 106 (GT106), in the synthesis of this sequence. The expression of the FRA9 gene was shown to be induced by secondary wall master transcriptional regulators and specifically associated with secondary wall-forming cells, including xylem and fiber cells. T-DNA knockout mutation of the FRA9 gene caused impaired secondary cell wall thickening in leaf veins and a severe arrest of plant growth. RNA interference (RNAi) downregulation of FRA9 led to a significant reduction in secondary wall thickening of fibers, a deformation of xylem vessels and a decrease in xylan content. Structural analysis of xylanase-released xylooligomers revealed that RNAi downregulation of FRA9 resulted in a diminishment of the unique xylan reducing end sequence and complete methylation of xylan GlcA side chains, chemotypes reminiscent of those of the fra8, irx8 and parvus mutants. Furthermore, two FRA9 close homologs from Populus trichocarpa were found to be wood-associated functional orthologs of FRA9. Together, our findings uncover a member of the GT106 family as a new player involved in the synthesis of the unique reducing end sequence of xylan.

Outstanding questions on xylan biosynthesis.

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 ß-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.

Independent recruitment of glycosyltransferase family 61 members for xylan substitutions in conifers.

MAIN CONCLUSION: 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-ß-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.

Identification of xylan arabinosyl 2-O-xylosyltransferases catalyzing the addition of 2-O-xylosyl residue onto arabinosyl side chains of xylan in grass species.

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.

Cell wall biology of the moss Physcomitrium patens.

The moss Physcomitrium (previously Physcomitrella) patens is a non-vascular plant belonging to the bryophytes that has been used as a model species to study the evolution of plant cell wall structure and biosynthesis. Here, we present an updated review of the cell wall biology of P. patens. Immunocytochemical and structural studies have shown that the cell walls of P. patens mainly contain cellulose, hemicelluloses (xyloglucan, xylan, glucomannan, and arabinoglucan), pectin, and glycoproteins, and their abundance varies among different cell types and at different plant developmental stages. Genetic and biochemical analyses have revealed that a number of genes involved in cell wall biosynthesis are functionally conserved between P. patens and vascular plants, indicating that the common ancestor of mosses and vascular plants had already acquired most of the biosynthetic machinery to make various cell wall polymers. Although P. patens does not synthesize lignin, homologs of the phenylpropanoid biosynthetic pathway genes exist in P. patens and they play an essential role in the production of caffeate derivatives for cuticle formation. Further genetic and biochemical dissection of cell wall biosynthetic genes in P. patens promises to provide additional insights into the evolutionary history of plant cell wall structure and biosynthesis.

Functional analysis of GT61 glycosyltransferases from grass species in xylan substitutions.

MAIN CONCLUSION: 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.

A Single Xyloglucan Xylosyltransferase Is Sufficient for Generation of the XXXG Xylosylation Pattern of Xyloglucan.

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.

Xylem vessel-specific SND5 and its homologs regulate secondary wall biosynthesis through activating secondary wall NAC binding elements.

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.

XND1 Regulates Secondary Wall Deposition in Xylem Vessels through the Inhibition of VND Functions.

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.

A Group of O-Acetyltransferases Catalyze Xyloglucan Backbone Acetylation and Can Alter Xyloglucan Xylosylation Pattern and Plant Growth When Expressed in Arabidopsis.

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.

Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

Evolutionary origin of O-acetyltransferases responsible for glucomannan acetylation in land plants.

Mannans are an abundant cell wall polysaccharide in bryophytes, seedless vascular plants and gymnosperms. A previous study has shown that mannan acetylation in Arabidopsis and konjac is mediated by mannan O-acetyltransferases belonging to the Domain of Unknown Function (DUF) 231 family. However, little is known about the acetylation patterns of mannans in bryophytes and seedless vascular plants, and the evolutionary origin of mannan O-acetyltransferases in land plants has not yet been studied. Phylogenetic analysis of the DUF231 family revealed that DUF231 members were present in the charophycean green algae and evolved to form overlapped and divergent phylogenetic groups in different taxa of land plants. Acetyltransferase activity assays of recombinant proteins demonstrated that a number of group II DUF231 members from moss, Selaginella, pine, spruce, rice and poplar were mannan 2-O- and 3-O-acetyltransferases, whereas the two group I DUF231 members from the alga Klebsormidium nitens were not. Structural analysis of mannans from moss and Selaginella showed they were composed of mannosyl and glucosyl residues and the mannosyl residues were acetylated at O-2 and O-3. These findings indicate that although the DUF231 genes originated in algae, their recruitment as mannan O-acetyltransferases probably occurred in bryophytes, and the biochemical functions of these O-acetyltransferases are evolutionarily conserved throughout land plants.

Secondary cell wall 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.

Members of the DUF231 Family are O-Acetyltransferases Catalyzing 2-O- and 3-O-Acetylation of Mannan.

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.

Xyloglucan O-acetyltransferases from Arabidopsis thaliana and Populus trichocarpa catalyze acetylation of fucosylated galactose residues on xyloglucan side chains.

MAIN CONCLUSION: 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.