CASANOVA: Permutation inference in factorial survival designs.

We propose inference procedures for general factorial designs with time-to-event endpoints. Similar to additive Aalen models, null hypotheses are formulated in terms of cumulative hazards. Deviations are measured in terms of quadratic forms in Nelson-Aalen-type integrals. Different from existing approaches, this allows to work without restrictive model assumptions as proportional hazards. In particular, crossing survival or hazard curves can be detected without a significant loss of power. For a distribution-free application of the method, a permutation strategy is suggested. The resulting procedures' asymptotic validity is proven and small sample performances are analyzed in extensive simulations. The analysis of a data set on asthma illustrates the applicability.

High-sensitivity cardiac troponin I after coronary artery bypass grafting for post-operative decision-making.

AIMS: Current troponin cut-offs suggested for the post-operative workup of patients following coronary artery bypass graft (CABG) surgery are based on studies using non-high-sensitive troponin assays or are arbitrarily chosen. We aimed to identify an optimal cut-off and timing for a proprietary high-sensitivity cardiac troponin I (hs-cTnI) assay to facilitate post-operative clinical decision-making. METHODS AND RESULTS: We performed a retrospective analysis of all patients undergoing elective isolated CABG at our centre between January 2013 and May 2019. Of 4684 consecutive patients, 161 patients (3.48%) underwent invasive coronary angiography after surgery, of whom 86 patients (53.4%) underwent repeat revascularization. We found an optimal cut-off value for peak hs-cTnI of >13 000 ng/L [>500× the upper reference limit (URL)] to be significantly associated with repeat revascularization within 48 h after surgery, which was internally validated through random repeated sampling with 1000 iterations. The same cut-off also predicted 30-day major adverse cardiovascular events and all-cause mortality after a median follow-up of 3.1 years, which was validated in an external cohort. A decision tree analysis of serial hs-cTnI measurements showed no added benefit of hs-cTnI measurements in patients with electrocardiographic or echocardiographic abnormalities or haemodynamic instability. Likewise, early post-operative hs-cTnI elevations had a low yield for clinical decision-making and only later elevations (at 12-16 h post-operatively) using a threshold of 8000 ng/L (307× URL) were significantly associated with repeat revascularization with an area under the curve of 0.92 (95% confidence interval 0.88-0.95). CONCLUSION: Our data suggest that for hs-cTnI, higher cut-offs than currently recommended should be used in the post-operative management of patients following CABG.

Which test for crossing survival curves? A user's guideline.

BACKGROUND: The exchange of knowledge between statisticians developing new methodology and clinicians, reviewers or authors applying them is fundamental. This is specifically true for clinical trials with time-to-event endpoints. Thereby, one of the most commonly arising questions is that of equal survival distributions in two-armed trial. The log-rank test is still the gold-standard to infer this question. However, in case of non-proportional hazards, its power can become poor and multiple extensions have been developed to overcome this issue. We aim to facilitate the choice of a test for the detection of survival differences in the case of crossing hazards. METHODS: We restricted the review to the most recent two-armed clinical oncology trials with crossing survival curves. Each data set was reconstructed using a state-of-the-art reconstruction algorithm. To ensure reproduction quality, only publications with published number at risk at multiple time points, sufficient printing quality and a non-informative censoring pattern were included. This article depicts the p-values of the log-rank and Peto-Peto test as references and compares them with nine different tests developed for detection of survival differences in the presence of non-proportional or crossing hazards. RESULTS: We reviewed 1400 recent phase III clinical oncology trials and selected fifteen studies that met our eligibility criteria for data reconstruction. After including further three individual patient data sets, for nine out of eighteen studies significant differences in survival were found using the investigated tests. An important point that reviewers should pay attention to is that 28% of the studies with published survival curves did not report the number at risk. This makes reconstruction and plausibility checks almost impossible. CONCLUSIONS: The evaluation shows that inference methods constructed to detect differences in survival in presence of non-proportional hazards are beneficial and help to provide guidance in choosing a sensible alternative to the standard log-rank test.

The TaCslA12 gene expressed in the wheat grain endosperm synthesizes wheat-like mannan when expressed in yeast and Arabidopsis.

Mannan is a class of cell wall polysaccharides widespread in the plant kingdom. Mannan structure and properties vary according to species and organ. The cell walls of cereal grains have been extensively studied due to their role in cereal processing and to their beneficial effect on human health as dietary fiber. Recently, we showed that mannan in wheat (Triticum aestivum) grain endosperm has a linear structure of ß-1,4-linked mannose residues. The aim of this work was to study the biosynthesis and function of wheat grain mannan. We showed that mannan is deposited in the endosperm early during grain development, and we identified candidate mannan biosynthetic genes expressed in the endosperm. The functional study in wheat was unsuccessful therefore our best candidate genes were expressed in heterologous systems. The endosperm-specificTaCslA12 gene expressed in Pichia pastoris and in an Arabidopsis thaliana mutant depleted in glucomannan led to the production of wheat-like linear mannan lacking glucose residues and with moderate acetylation. Therefore, this gene encodes a mannan synthase and is likely responsible for the synthesis of wheat endosperm mannan.

A cautionary tale on using imputation methods for inference in matched-pairs design.

MOTIVATION: Imputation procedures in biomedical fields have turned into statistical practice, since further analyses can be conducted ignoring the former presence of missing values. In particular, non-parametric imputation schemes like the random forest have shown favorable imputation performance compared to the more traditionally used MICE procedure. However, their effect on valid statistical inference has not been analyzed so far. This article closes this gap by investigating their validity for inferring mean differences in incompletely observed pairs while opposing them to a recent approach that only works with the given observations at hand. RESULTS: Our findings indicate that machine-learning schemes for (multiply) imputing missing values may inflate type I error or result in comparably low power in small-to-moderate matched pairs, even after modifying the test statistics using Rubin's multiple imputation rule. In addition to an extensive simulation study, an illustrative data example from a breast cancer gene study has been considered. AVAILABILITY AND IMPLEMENTATION: The corresponding R-code can be accessed through the authors and the gene expression data can be downloaded at www.gdac.broadinstitute.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Factorial analyses of treatment effects under independent right-censoring.

This paper introduces new effect parameters for factorial survival designs with possibly right-censored time-to-event data. In the special case of a two-sample design, it coincides with the concordance or Wilcoxon parameter in survival analysis. More generally, the new parameters describe treatment or interaction effects and we develop estimates and tests to infer their presence. We rigorously study their asymptotic properties and additionally suggest wild bootstrapping for a consistent and distribution-free application of the inference procedures. The small sample performance is discussed based on simulation results. The practical usefulness of the developed methodology is exemplified on a data example about patients with colon cancer by conducting one- and two-factorial analyses.

Clinical outcome after decompression of intraneural peroneal ganglion cyst and its morphologic correlation to postoperative nerve ultrasound.

OBJECTIVE: Intraneural ganglion cysts are rare and benign mucinous lesions that affect peripheral nerves, most frequently the common peroneal nerve (CPN). The precise pathophysiological mechanisms of intraneural ganglion cyst development remain unclear. A well-established theory suggests the spread of mucinous fluid along the articular branch of the peroneal nerve as the underlying mechanism. Clinical outcome following decompression of intraneural ganglion cysts has been demonstrated to be excellent. The aim of this study was to evaluate the correlation between clinical outcome and ultrasound-detected morphological nerve features following decompression of intraneural ganglion cysts of the CPN. METHODS: Data were retrospectively analyzed from 20 patients who underwent common peroneal nerve ganglion cyst decompression surgery at the Universität Ulm/Günzburg Neurosurgery Department between October 2003 and October 2017. Postoperative clinical outcome was evaluated by assessment of the muscular strength of the anterior tibial muscle, the extensor hallucis longus muscle, and the peroneus muscle according to the Medical Research Council grading system. Hypesthesia was measured by sensation testing. In all patients, postoperative morphological assessment of the peroneal nerve was conducted between October 2016 and October 2017 using the iU22 Philips Medical ultrasound system at the last routine follow-up appointment. Finally, the correlations between morphological changes in nerve ultrasound and postoperative clinical outcomes were evaluated. RESULTS: During the postoperative ultrasound scan an intraneural hypoechogenic ring structure located at the medial side of the peroneal nerve was detected in 15 (75%) of 20 patients, 14 of whom demonstrated an improvement in motor function. A regular intraneural fasicular structure was identified in 3 patients (15%), who also reported recovery. In 1 patient, a recurrent cyst was detected, and 1 patient showed intraneural fibrosis for which recovery did not occur in the year following the procedure. Two patients (10%) developed neuropathic pain that could not be explained by nerve ultrasound findings. CONCLUSIONS: The results of this study demonstrate significant recovery from preoperative weakness after decompression of intraneural ganglion cysts of the CPN. A favorable clinical outcome was highly correlated with an intraneural hypoechogenic ring-shaped structure on the medial side of the CPN identified during a follow-up postoperative ultrasound scan. These study results indicate the potential benefit of ultrasound scanning as a prognostic tool following decompression procedures for intraneural ganglion cysts of the CPN.

A wild bootstrap approach for the Aalen-Johansen estimator.

We suggest a wild bootstrap resampling technique for nonparametric inference on transition probabilities in a general time-inhomogeneous Markov multistate model. We first approximate the limiting distribution of the Nelson-Aalen estimator by repeatedly generating standard normal wild bootstrap variates, while the data is kept fixed. Next, a transformation using a functional delta method argument is applied. The approach is conceptually easier than direct resampling for the transition probabilities. It is used to investigate a non-standard time-to-event outcome, currently being alive without immunosuppressive treatment, with data from a recent study of prophylactic treatment in allogeneic transplanted leukemia patients. Due to non-monotonic outcome probabilities in time, neither standard survival nor competing risks techniques apply, which highlights the need for the present methodology. Finite sample performance of time-simultaneous confidence bands for the outcome probabilities is assessed in an extensive simulation study motivated by the clinical trial data. Example code is provided in the web-based Supplementary Materials.

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans.

BACKGROUND: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. RESULTS: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. CONCLUSIONS: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

Engineering of plant cell walls for enhanced biofuel production.

The biomass of plants consists predominately of cell walls, a sophisticated composite material composed of various polymer networks including numerous polysaccharides and the polyphenol lignin. In order to utilize this renewable, highly abundant resource for the production of commodity chemicals such as biofuels, major hurdles have to be surpassed to reach economical viability. Recently, major advances in the basic understanding of the synthesis of the various wall polymers and its regulation has enabled strategies to alter the qualitative composition of wall materials. Such emerging strategies include a reduction/alteration of the lignin network to enhance polysaccharide accessibility, reduction of polymer derived processing inhibitors, and increases in polysaccharides with a high hexose/pentose ratio.

A ß-glucuronosyltransferase from Arabidopsis thaliana involved in biosynthesis of type II arabinogalactan has a role in cell elongation during seedling growth.

We have characterized a ß-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana that is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to the Carbohydrate Active Enzyme database glycosyltransferase family 14 (GT14). The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoris transferred glucuronic acid (GlcA) to ß-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to ß-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the ß-1,6- and ß-1,3-galactan present in type II AG. Two allelic T-DNA insertion mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild-type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-ß-1,6-Gal and ß-1,3-Gal, indicating an in vivo role of AtGlcAT14A in synthesis of those structures in type II AG. Moreover, a relative increase in the levels of 3-, 6- and 3,6-linked galactose (Gal) and reduced levels of 3-, 2- and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation in AtGlcAT14A results in a relative increase of the longer and branched ß-1,3- and ß-1,6-galactans. This increase of galactosylation in the mutants is most likely caused by increased availability of the O6 position of Gal, which is a shared acceptor site for AtGlcAT14A and galactosyltransferases in synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role for the glucuronosyltransferase in the biosynthesis of type II AG, with a biological role during seedling growth.

Solution-state 2D NMR spectroscopy of plant cell walls enabled by a dimethylsulfoxide-d6/1-ethyl-3-methylimidazolium acetate solvent.

Lignocellulosic biomass is composed of the polysaccharides cellulose and hemicellulose and the polyphenol lignin. Many current methods for analyzing the structure of lignocelluloses involve a sequential extraction of the material and subsequent analysis of the resulting fractions, which is labor-intensive and time-consuming. The work presented here assesses the dissolution of whole lignocellulosic material, focusing on biomass derived from the perennial bioenergy grass Miscanthus. The solvent dimethylsulfoxide (DMSO)-d6 containing 1-ethyl-3-methylimidazolium acetate ([Emim]OAc) was able to dissolve lignocellulosic material completely and gave high-resolution 2D heteronuclear single quantum coherence (HSQC) NMR spectra of the entire array of wall polymers. Extrapolated time-zero HSQC was applied using DMSO-d6/[Emim]OAc-d14 and enabled quantitative analysis of structural traits of lignocellulose components.

XAX1 from glycosyltransferase family 61 mediates xylosyltransfer to rice xylan.

Xylan is the second most abundant polysaccharide on Earth and represents an immense quantity of stored energy for biofuel production. Despite its importance, most of the enzymes that synthesize xylan have yet to be identified. Xylans have a backbone of ß-1,4-linked xylose residues with substitutions that include α-(1→2)-linked glucuronosyl, 4-O-methyl glucuronosyl, and α-1,2- and α-1,3-arabinofuranosyl residues. The substitutions are structurally diverse and vary by taxonomy, with grass xylan representing a unique composition distinct from dicots and other monocots. To date, no enzyme has yet been identified that is specific to grass xylan synthesis. We identified a xylose-deficient loss-of-function rice mutant in Os02g22380, a putative glycosyltransferase in a grass-specific subfamily of family GT61. We designate the mutant xax1 for xylosyl arabinosyl substitution of xylan 1. Enzymatic fingerprinting of xylan showed the specific absence in the mutant of a peak, which was isolated and determined by (1)H-NMR to be (ß-1,4-Xyl)(4) with a ß-Xylp-(1→2)-α-Araf-(1→3). Rice xax1 mutant plants are deficient in ferulic and coumaric acid, aromatic compounds known to be attached to arabinosyl residues in xylan substituted with xylosyl residues. The xax1 mutant plants exhibit an increased extractability of xylan and increased saccharification, probably reflecting a lower degree of diferulic cross-links. Activity assays with microsomes isolated from tobacco plants transiently expressing XAX1 demonstrated xylosyltransferase activity onto endogenous acceptors. Our results provide insight into grass xylan synthesis and how substitutions may be modified for increased saccharification for biofuel generation.

Arabidopsis thaliana AtUTr7 encodes a golgi-localized UDP-glucose/UDP-galactose transporter that affects lateral root emergence.

Nucleotide sugar transporters (NSTs) are antiporters comprising a gene family that plays a fundamental role in the biosynthesis of complex cell wall polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have observable phenotypes, the biological function(s) of most NSTs in cell wall biosynthesis and assembly have remained elusive. Here, we report the characterization of AtUTr7 from Arabidopsis (Arabidopsis thaliana (L.) Heynh.), which is homologous to multi-specific UDP-sugar transporters from Drosophila melanogaster, humans, and Caenorhabditis elegans. We show that AtUTr7 possesses the common structural characteristics conserved among NSTs. Using a green fluorescent protein (GFP) tagged version, we demonstrate that AtUTr7 is localized in the Golgi apparatus. We also show that AtUTr7 is widely expressed, especially in the roots and in specific floral organs. Additionally, the results of an in vitro nucleotide sugar transport assay carried out with a tobacco and a yeast expression system suggest that AtUTr7 is capable of transferring UDP-Gal and UDP-Glc, but not a range of other UDP- and GDP-sugars, into the Golgi lumen. Mutants lacking expression of AtUTr7 exhibited an early proliferation of lateral roots as well as distorted root hairs when cultivated at high sucrose concentrations. Furthermore, the distribution of homogalacturonan with a low degree of methyl esterification differed in lateral root tips of the mutant compared to wild-type plants, although additional analytical procedures revealed no further differences in the composition of the root cell walls. This evidence suggests that the transport of UDP-Gal and UDP-Glc into the Golgi under conditions of high root biomass production plays a role in lateral root and root hair development.

Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells.

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

Comprehensive compositional analysis of plant cell walls (Lignocellulosic biomass) part I: lignin.

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation(1) and other products such as biocomposite materials(7). Plant biomass remains one of the greatest untapped reserves on the planet(4). It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin(6) and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant(4). Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization(3), and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units(5). The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II(2).

Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part II: carbohydrates.

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation(2) and other products such as biocomposite materials(6). Plant biomass remains one of the greatest untapped reserves on the planet(4). It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin(5) and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation(5). Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant(4). Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose(7). The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I(3).

Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growth.

Cellulose synthase-like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell-wall polymers. The CSL D sub-family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in-depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map-based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub-family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C- or N-terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype-rescued transgenic plants expressing OsCSLD4-GUS under the control of its own promoter. Two phenotype-altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell-wall formation and plant growth.

Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis.

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis.

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.