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 glucuronic acid (GlcA)/methylglucuronic acid (MeGlcA) 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 remains 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 patterns 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.

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.

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.

Rice ß-glucosidase Os12BGlu38 is required for synthesis of intine cell wall and pollen fertility.

Glycoside hydrolase family1 ß-glucosidases play a variety of roles in plants, but their in planta functions are largely unknown in rice (Oryza sativa). In this study, the biological function of Os12BGlu38, a rice ß-glucosidase, expressed in bicellular to mature pollen, was examined. Genotype analysis of progeny of the self-fertilized heterozygous Os12BGlu38 T-DNA mutant, os12bglu38-1, found no homozygotes and a 1:1 ratio of wild type to heterozygotes. Reciprocal cross analysis demonstrated that Os12BGlu38 deficiency cannot be inherited through the male gamete. In cytological analysis, the mature mutant pollen appeared shrunken and empty. Histochemical staining and TEM showed that mutant pollen lacked intine cell wall, which was rescued by introduction of wild-type Os12BGlu38 genomic DNA. Metabolite profiling analysis revealed that cutin monomers and waxes, the components of the pollen exine layer, were increased in anthers carrying pollen of os12bglu38-1 compared with wild type and complemented lines. Os12BGlu38 fused with green fluorescent protein was localized to the plasma membrane in rice and tobacco. Recombinant Os12BGlu38 exhibited ß-glucosidase activity on the universal substrate p-nitrophenyl ß-d-glucoside and some oligosaccharides and glycosides. These findings provide evidence that function of a plasma membrane-associated ß-glucosidase is necessary for proper intine development.

Pigmentation and Flavonoid Metabolite Diversity in Immature 'Fuji' Apple Fruits in Response to Lights and Methyl Jasmonate.

Artificial pigmentation of apple fruits has been intensely evaluated to generate less pigmented red apples, which are profitable because of the changes in fruit quality. In this study, we analyzed the diversity of flavonoids and the patterns of flavonoid metabolic gene expression under light irradiation with or without methyl jasmonate (MeJA) treatment in immature (S1) and color-turning (S2) staged 'Fuji' apples. Further, we assessed the metabolic regulation at the gene level between anthocyanin and flavonol in light-responsive apple skins. UV-B exposure within 3 days was found to significantly stimulate anthocyanin accumulation in apple skin compared to other light exposure. S1 skin was more sensitive to UV-B and MeJA treatment, in the aspect of indaein accumulation. The enhancement of apple pigmentation following treatment with adequate levels of UV-B and MeJA was maximized at approximately 72 h. Red (range from 4.25 to 17.96 µg·g-1 DW), blue (range from 4.59 to 9.17 µg·g-1 DW) and UV-A (range from 3.98 to 19.12 µg·g-1 DW) lights contributed to the induction of idaein content. Most genes related to the flavonoid pathways increased their expression under UV-B exposure, including the gene expression of the transcription factor, MdMYB10, a well-known upstream factor of flavonoid biosynthesis in apples. The boosted upregulation of MdMYB10, MdCHS, MdF3H MdLDOX, and MdUFGT genes due to MeJA in UV-B was found and may contribute the increase of idaein. UV-A and UV-B caused higher quercetin glycoside content in both S1 and S2 apple skins than longer wavelengths, resulting in significant increases in quercetin-3-O-galactoside and quercetin-3-O-glucoside. These results suggest that the application of adequate UV-B with MeJA in less-pigmented postharvest apples will improve apple color quality within a short period.

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.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar.

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1-sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development.

Root hairs are important for absorption of nutrients and water from the rhizosphere. The Root Hair Defective-Six Like (RSL) Class II family of transcription factors is expressed preferentially in root hairs and has a conserved role in root hair development in land plants. We functionally characterized the seven members of the RSL Class II subfamily in the rice (Oryza sativa) genome. In root hairs, six of these genes were preferentially expressed and four were strongly expressed. Phenotypic analysis of each mutant revealed that Os07g39940 plays a major role in root hair formation, based on observations of a short root hair phenotype in those mutants. Overexpression (OX) for each of four family members in rice resulted in an increase in the density and length of root hairs. These four members contain a transcription activation domain and are targeted to the nucleus. They interact with rice Root Hairless1 (OsRHL1), a key regulator of root hair development. When heterologously expressed in epidermal cells of Nicotiana benthamiana leaves, OsRHL1 was predominantly localized to the cytoplasm. When coexpressed with each of the four RSL Class II members, however, OsRLH1 was translocated to the nucleus. Transcriptome analysis using Os07g39940-OX plants revealed that 86 genes, including Class III peroxidases, were highly up-regulated. Furthermore, reactive oxygen species levels in the root hairs were increased in Os07g39940-OX plants but were drastically reduced in the os07g39940 and rhl1 mutants. Our results demonstrate that RSL Class II members function as essential regulators of root hair development in rice.

Physiological Importance of Pectin Modifying Genes During Rice Pollen Development.

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

Molecular dissection of a rice microtubule-associated RING finger protein and its potential role in salt tolerance in Arabidopsis.

Although a number of RING E3 ligases in plants have been demonstrated to play key roles in a wide range of abiotic stresses, relatively few studies have detailed how RING E3 ligases exert their cellular actions. We describe Oryza sativa RING finger protein with microtubule-targeting domain 1 (OsRMT1), a functional RING E3 ligase that is likely involved in a salt tolerance mechanism. Functional characterization revealed that OsRMT1 undergoes homodimer formation and subsequently autoubiquitination-mediated protein degradation under normal conditions. By contrast, OsRMT1 is predominantly found in the nucleus and microtubules and its degradation is inhibited under salt stress. Domain dissection of OsRMT1 indicates that the N-terminal domain is required for microtubule targeting. Bimolecular fluorescence complementation analysis and degradation assay revealed that OsRMT1-interacted proteins localized in various organelles were degraded via the ubiquitin (Ub)/26S proteasome-dependent pathway. Interestingly, when OsRMT1 and its target proteins were co-expressed in N. benthamiana leaves, the protein-protein interactions appeared to take place mainly in the microtubules. Overexpression of OsRMT1 in Arabidopsis resulted in increased tolerance to salt stress. Our findings suggest that the abundance of microtubule-associated OsRMT1 is strictly regulated, and OsRMT1 may play a relevant role in salt stress response by modulating levels of its target proteins.

Positive regulation of rice RING E3 ligase OsHIR1 in arsenic and cadmium uptakes.

The metalloid arsenic (As) and the heavy metal cadmium (Cd) are ubiquitously found at low concentrations in the earth. High concentrations of these elements in the soil and crops are severely dangerous to human health. We attempted to retrieve the RING E3 ubiquitin ligase gene for regulating As and Cd uptakes via the ubiquitin 26S proteasome system. Semi-quantitative reverse transcription polymerase chain reaction was conducted for a total of 47 Oryza sativa RING finger protein (OsRFP) genes to assess their expression patterns when exposed to As and Cd treatments. We identified one gene Oryza sativa heavy metal induced RING E3 ligase 1 (OsHIR1), which was significantly upregulated with both treatments. A yeast hybrid screen and a bimolecular fluorescence complementation assay showed that OsHIR1 clearly interacts with 5 substrate proteins, including tonoplast intrinsic protein 4;1 (OsTIP4;1) in the plasma membrane. In addition, OsHIR1 strongly degraded the protein level of OsTIP4;1 via the ubiquitin 26S proteasome system. Heterogeneous overexpression of OsHIR1 in Arabidopsis exhibited As- and Cd-insensitive phenotypes and resulted in decreased As and Cd accumulation in the shoots and roots, relative to the control. Herein, we report the novel finding that the OsHIR1 E3 ligase positively regulates OsTIP4;1 related to As and Cd uptakes.

The rice RING E3 ligase, OsCTR1, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in Arabidopsis.

Plant growth under low water availability adversely affects many key processes with morphological, physiological, biochemical and molecular consequences. Here, we found that a rice gene, OsCTR1, encoding the RING Ub E3 ligase plays an important role in drought tolerance. OsCTR1 was highly expressed in response to dehydration treatment and defense-related phytohormones, and its encoded protein was localized in both the chloroplasts and the cytosol. Intriguingly, the OsCTR1 protein was found predominantly targeted to the cytosol when rice protoplasts transfected with OsCTR1 were treated with abscisic acid (ABA). Several interacting partners were identified, which were mainly targeted to the chloroplasts, and interactions with OsCTR1 were confirmed by using biomolecular fluorescence complementation (BiFC). Interestingly, two chloroplast-localized proteins (OsCP12 and OsRP1) interacted with OsCTR1 in the cytosol, and ubiquitination by OsCTR1 led to protein degradation via the Ub 26S proteasome. Heterogeneous overexpression of OsCTR1 in Arabidopsis exhibited hypersensitive phenotypes with respect to ABA-responsive seed germination, seedling growth and stomatal closure. The ABA-sensitive transgenic plants also showed improvement in their tolerance against severe water deficits. Taken together, our findings lend support to the hypothesis that the molecular functions of OsCTR1 are related to tolerance to water-deficit stress via ABA-dependent regulation and related systems.

Design of high-performance entangling logic in silicon quantum dot systems with Bayesian optimization.

Device engineering based on computer-aided simulations is essential to make silicon (Si) quantum bits (qubits) be competitive to commercial platforms based on superconductors and trapped ions. Combining device simulations with the Bayesian optimization (BO), here we propose a systematic design approach that is quite useful to procure fast and precise entangling operations of qubits encoded to electron spins in electrode-driven Si quantum dot (QD) systems. For a target problem of the controlled-X (CNOT) logic operation, we employ BO with the Gaussian process regression to evolve design factors of a Si double QD system to the ones that are optimal in terms of speed and fidelity of a CNOT logic driven by a single microwave pulse. The design framework not only clearly contributes to cost-efficient securing of solutions that enhance performance of the target quantum operation, but can be extended to implement more complicated logics with Si QD structures in experimentally unprecedented ways.

Distinguishing Affective Temperament Profiles in Major Depressive Disorder and Bipolar Disorder Through the Short Version of TEMPS-A: Cross-Sectional Study Using Latent Profile Analysis.

OBJECTIVE: This study aimed to elucidate the distinct response patterns exhibited by patients diagnosed with bipolar disorder (BD) and those with major depressive disorder (MDD) through the application of the short version of the Temperament Evaluation of Memphis, Pisa, Paris, and San Diego Autoquestionnaire (TEMPS-A-SV). METHODS: A total of 2,458 participants consisting of patients with MDD (n=288), BD (BD I, n=111; BD II, n=427), and control group (n=1,632) completed the TEMPS-A-SV. The response patterns of the participants were classified into distinct profiles using latent profile analysis. The study further examined the impact of covariates such as age, sex, and diagnostic group on derived latent profile memberships. RESULTS: The following three latent profiles were identified: High Affective Temperament Group (17.86%), Low Affective Temperament Group (41.25%), and Middle Affective Temperament Group (40.89%). Compared with the patient group with MDD and BD, the control group was more likely to belong in the Low Affective Temperament Group, which showed a higher score on hyperthymic temperament than the Middle Affective Temperament Group. Furthermore, compared with the patients with BD, the MDD patients were more likely to be in the Low Affective Temperament Group rather than the Middle Affective Temperament Group. CONCLUSION: These results indicate that different affective temperaments exist between patients with MDD and BD. Attempting to classify response patterns using the TEMPS-A-SV can help diagnose MDD and BD correctly.

Harpins, multifunctional proteins secreted by gram-negative plant-pathogenic bacteria.

Harpins are glycine-rich and heat-stable proteins that are secreted through type III secretion system in gram-negative plant-pathogenic bacteria. Many studies show that these proteins are mostly targeted to the extracellular space of plant tissues, unlike bacterial effector proteins that act inside the plant cells. Over the two decades since the first harpin of pathogen origin, HrpN of Erwinia amylovora, was reported in 1992 as a cell-free elicitor of hypersensitive response (HR), diverse functional aspects of harpins have been determined. Some harpins were shown to have virulence activity, probably because of their involvement in the translocation of effector proteins into plant cytoplasm. Based on this function, harpins are now considered to be translocators. Their abilities of pore formation in the artificial membrane, binding to lipid components, and oligomerization are consistent with this idea. When harpins are applied to plants directly or expressed in plant cells, these proteins trigger diverse beneficial responses such as induction of defense responses against diverse pathogens and insects and enhancement of plant growth. Therefore, in this review, we will summarize the functions of harpins as virulence factors (or translocators) of bacterial pathogens, elicitors of HR and immune responses, and plant growth enhancers.

Arabidopsis family GT43 members are xylan xylosyltransferases required for the elongation of the xylan backbone.

Xylan is the second most abundant polysaccharide in plant biomass targeted for biofuel production. Therefore, it is imperative to understand the biochemical mechanism underlying xylan biosynthesis. Although previous genetic studies have identified several genes implicated in xylan biosynthesis, biochemical proof of any of their encoded proteins as a xylan xylosyltransferase (XylT) responsible for xylan backbone biosynthesis is still lacking. In this study, we investigated the enzymatic activities of two Arabidopsis thaliana GT43 members, IRX9 (Irregular Xylem9) and IRX14, which have been genetically shown to be non-redundantly involved in the elongation of the xylan backbone. IRX9 and IRX14, alone or simultaneously, were heterologously expressed in tobacco BY2 cells, and microsomes isolated from the transgenic BY2 cells were tested for XylT activity using xylotetraose (Xyl(4)) as an acceptor and UDP-[(14)C]xylose as a donor. It was found that although microsomes with expression of IRX9 or IRX14 alone exhibited little incorporation of radiolabeled xylose, a high level of incorporation of radiolabeled xylose onto Xyl(4) was conferred by microsomes with co-expression of IRX9 and IRX14. Further analysis using fluorescent anthranilic acid-labeled xylotetraose (Xyl(4)-AA) as an acceptor revealed that up to five ß-(1,4)-linked xylosyl residues were able to be transferred onto Xyl(4)-AA by microsomes with co-expression of IRX9 and IRX14. Furthermore, it was shown that xylooligomers ranging from Xyl(3)-AA to Xyl(6)-AA could all be used as acceptors for the xylosyl transfer by microsomes with co-expression of IRX9 and IRX14. Together, these findings provide the first biochemical evidence that IRX9 and IRX14 are xylosyltransferases that operate cooperatively in the elongation of the xylan backbone.

Arabidopsis GUX proteins are glucuronyltransferases responsible for the addition of glucuronic acid side chains onto xylan.

Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of ß-(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.

Three Arabidopsis DUF579 domain-containing GXM proteins are methyltransferases catalyzing 4-o-methylation of glucuronic acid on xylan.

Xylan is made of a linear chain of ß-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.

Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar.

Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.

The four Arabidopsis reduced wall acetylation genes are expressed in secondary wall-containing cells and required for the acetylation of xylan.

Xylan is one of the major polysaccharides in cellulosic biomass, and understanding the mechanisms underlying xylan biosynthesis will potentially help us design strategies to produce cellulosic biomass better suited for biofuel production. Although a number of genes have been shown to be essential for xylan biosynthesis, genes involved in the acetylation of xylan have not yet been identified. Here, we report the comprehensive genetic and functional studies of four Arabidopsis REDUCED WALL ACETYLATION (RWA) genes and demonstrate their involvement in the acetylation of xylan during secondary wall biosynthesis. It was found that the RWA genes were expressed in cells undergoing secondary wall thickening and their expression was regulated by SND1, a transcriptional master switch of secondary wall biosynthesis. The RWA proteins were shown to be localized in the Golgi, where xylan biosynthesis occurs. Analyses of a suite of single, double, triple and quadruple rwa mutants revealed a significant reduction in the secondary wall thickening and the stem mechanical strength in the quadruple rwa1/2/3/4 mutant but not in other mutants. Further chemical and structural analyses of xylan demonstrated that the rwa1/2/3/4 mutations resulted in a reduction in the amount of acetyl groups on xylan. In addition, the ratio of non-methylated to methylated glucuronic acid side chains was altered in the rwa1/2/3/4 mutant. Together, our results demonstrate that the four Arabidopsis RWA genes function redundantly in the acetylation of xylan during secondary wall biosynthesis.