Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants.

MAIN CONCLUSION: PAE8 and PAE9 have pectin acetylesterase activity and together remove one-third of the cell wall acetate associated with pectin formation in Arabidopsis leaves. In pae8 and pae9 mutants, substantial amounts of acetate accumulate in cell walls. In addition, the inflorescence stem height is decreased. Pectic polysaccharides constitute a significant part of the primary cell walls in dicotyledonous angiosperms. This diverse group of polysaccharides has been implicated in several physiological processes including cell-to-cell adhesion and pathogenesis. Several pectic polysaccharides contain acetyl-moieties directly affecting their physical properties such as gelling capacity, an important trait for the food industry. In order to gain further insight into the biological role of pectin acetylation, a reverse genetics approach was used to investigate the function of genes that are members of the Pectin AcetylEsterase gene family (PAE) in Arabidopsis. Mutations in two members of the PAE family (PAE8 and PAE9) lead to cell walls with an approximately 20 % increase in acetate content. High-molecular-weight fractions enriched in pectic rhamnogalacturonan I (RGI) extracted from the mutants had increased acetate content. In addition, the pae8 mutant displayed increased acetate content also in low-molecular-weight pectic fractions. The pae8/pae9-2 double mutant exhibited an additive effect by increasing wall acetate content by up to 37 %, suggesting that the two genes are not redundant and act on acetyl-substituents of different pectic domains. The pae8 and pae8/pae9-2 mutants exhibit reduced inflorescence growth underscoring the role of pectic acetylation in plant development. When heterologously expressed and purified, both gene products were shown to release acetate from the corresponding mutant pectic fractions in vitro. PAEs play a significant role in modulating the acetylation state of pectic polymers in the wall, highlighting the importance of apoplastic metabolism for the plant cell and plant growth.

Reduced Wall Acetylation proteins play vital and distinct roles in cell wall O-acetylation in Arabidopsis.

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.

O-acetylation of Arabidopsis hemicellulose xyloglucan requires AXY4 or AXY4L, proteins with a TBL and DUF231 domain.

In an Arabidopsis thaliana forward genetic screen aimed at identifying mutants with altered structures of their hemicellulose xyloglucan (axy mutants) using oligosaccharide mass profiling, two nonallelic mutants (axy4-1 and axy4-2) that have a 20 to 35% reduction in xyloglucan O-acetylation were identified. Mapping of the mutation in axy4-1 identified AXY4, a type II transmembrane protein with a Trichome Birefringence-Like domain and a domain of unknown function (DUF231). Loss of AXY4 transcript results in a complete lack of O-acetyl substituents on xyloglucan in several tissues, except seeds. Seed xyloglucan is instead O-acetylated by the paralog AXY4like, as demonstrated by the analysis of the corresponding T-DNA insertional lines. Wall fractionation analysis of axy4 knockout mutants indicated that only a fraction containing xyloglucan is non-O-acetylated. Hence, AXY4/AXY4L is required for the O-acetylation of xyloglucan, and we propose that these proteins represent xyloglucan-specific O-acetyltransferases, although their donor and acceptor substrates have yet to be identified. An Arabidopsis ecotype, Ty-0, has reduced xyloglucan O-acetylation due to mutations in AXY4, demonstrating that O-acetylation of xyloglucan does not impact the plant's fitness in its natural environment. The relationship of AXY4 with another previously identified group of Arabidopsis proteins involved in general wall O-acetylation, reduced wall acetylation, is discussed.

Hemicellulose biosynthesis.

One major component of plant cell walls is a diverse group of polysaccharides, the hemicelluloses. Hemicelluloses constitute roughly one-third of the wall biomass and encompass the heteromannans, xyloglucan, heteroxylans, and mixed-linkage glucan. The fine structure of these polysaccharides, particularly their substitution, varies depending on the plant species and tissue type. The hemicelluloses are used in numerous industrial applications such as food additives as well as in medicinal applications. Their abundance in lignocellulosic feedstocks should not be overlooked, if the utilization of this renewable resource for fuels and other commodity chemicals becomes a reality. Fortunately, our understanding of the biosynthesis of the various hemicelluloses in the plant has increased enormously in recent years mainly through genetic approaches. Taking advantage of this knowledge has led to plant mutants with altered hemicellulosic structures demonstrating the importance of the hemicelluloses in plant growth and development. However, while we are on a solid trajectory in identifying all necessary genes/proteins involved in hemicellulose biosynthesis, future research is required to combine these single components and assemble them to gain a holistic mechanistic understanding of the biosynthesis of this important class of plant cell wall polysaccharides.

Dissecting the role of CHITINASE-LIKE1 in nitrate-dependent changes in root architecture.

The root phenotype of an Arabidopsis (Arabidopsis thaliana) mutant of CHITINASE-LIKE1 (CTL1), called arm (for anion-related root morphology), was previously shown to be conditional on growth on high nitrate, chloride, or sucrose. Mutants grown under restrictive conditions displayed inhibition of primary root growth, radial swelling, proliferation of lateral roots, and increased root hair density. We found here that the spatial pattern of CTL1 expression was mainly in the root and root tips during seedling development and that the protein localized to the cell wall. Fourier-transform infrared microspectroscopy of mutant root tissues indicated differences in spectra assigned to linkages in cellulose and pectin. Indeed, root cell wall polymer composition analysis revealed that the arm mutant contained less crystalline cellulose and reduced methylesterification of pectins. We also explored the implication of growth regulators on the phenotype of the mutant response to the nitrate supply. Exogenous abscisic acid application inhibited more drastically primary root growth in the arm mutant but failed to repress lateral branching compared with the wild type. Cytokinin levels were higher in the arm root, but there were no changes in mitotic activity, suggesting that cytokinin is not directly involved in the mutant phenotype. Ethylene production was higher in arm but inversely proportional to the nitrate concentration in the medium. Interestingly, eto2 and eto3 ethylene overproduction mutants mimicked some of the conditional root characteristics of the arm mutant on high nitrate. Our data suggest that ethylene may be involved in the arm mutant phenotype, albeit indirectly, rather than functioning as a primary signal.

Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea.

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.

Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases.

A previously undescribed forward chemical genetic screen using hydrolases affecting the extracellular matrix is introduced. The developed screen takes advantage of the power of chemical genetics and combines it with the known substrate specificity of glycosylhydrolases, resulting in the selection of conditional mutants that exhibit structural defects in their extracellular matrix. Identification of the responsible genetic locus in those mutants significantly extends our knowledge of genes involved in the biosynthesis, metabolism, signaling, and functionality of components of the extracellular matrix. The method is exemplified by a screen of mutagenized Arabidopsis plants subjected to growth in liquid culture in the presence of a xyloglucanase, an enzyme acting on the major cross-linking glycan found in the extracellular matrix of this plant. Using this hydrolase-based screen, dozens of plant cell wall mutants (xeg mutants) were identified, leading to the identification of 23 genetic loci that affect plant cell walls. One of the identified loci is XEG113, encoding a family 77 glycosyltransferase (GT77). Detailed analysis of the wall of this mutant indicated that its extensins, structural glyocoproteins present in walls, are underarabinosylated. Xeg-113 plants exhibit more elongated hypocotyls than WT, providing genetic evidence that plant O-glycosylation--more specifically, extensin arabinosylation--is important for cell elongation.

Deep sequencing of voodoo lily (Amorphophallus konjac): an approach to identify relevant genes involved in the synthesis of the hemicellulose glucomannan.

A Roche 454 cDNA deep sequencing experiment was performed on a developing corm of Amorphophallus konjac--also known as voodoo lily. The dominant storage polymer in the corm of this plant is the polysaccharide glucomannan, a hemicellulose known to exist in the cell walls of higher plants and a major component of plant biomass derived from softwoods. A total of 246 mega base pairs of sequence data was obtained from which 4,513 distinct contigs were assembled. Within this voodoo lily expressed sequence tag collection genes representing the carbohydrate related pathway of glucomannan biosynthesis were identified, including sucrose metabolism, nucleotide sugar conversion pathways for the formation of activated precursors as well as a putative glucomannan synthase. In vivo expression of the putative glucomannan synthase and subsequent in vitro activity assays unambiguously demonstrate that the enzyme has indeed glucomannan mannosyl- and glucosyl transferase activities. Based on the expressed sequence tag analysis hitherto unknown pathways for the synthesis of GDP-glucose, a necessary precursor for glucomannan biosynthesis, could be proposed. Moreover, the results highlight transcriptional bottlenecks for the synthesis of this hemicellulose.

Simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR assay on the lightcycler instrument.

BACKGROUND: Clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C and they are, under normal circumstances, performed as separate assays. DESIGN AND METHODS: In order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler Instrument (Roche Diagnostics). RESULTS: The quantification assay was calibrated against WHO Standard 96/790. The detection limit was 30 IU/ml, the dynamic range up to 500,000,000 IU/ml. Intra- and interassay imprecisions were 1.2% and 1.9% (n = 10), respectively. The HCV RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HCV monitor test (r = 0.992; p < 0.001). CONCLUSIONS: The genotyping was performed by means of the melting temperature analysis. The concordance between our new genotyping method and the Trugene HCV 5'NC Kit was at the level of genotypes 100%. This rapid (3 h) and convenient assay is suitable for HCV genotyping, HCV detection and disease monitoring.

Aclonifen targets solanesyl diphosphate synthase, representing a novel mode of action for herbicides.

BACKGROUND: Aclonifen is a unique diphenyl ether herbicide. Despite its structural similarities to known inhibitors of the protoporphyrinogen oxidase (e.g. acifluorfen, bifenox or oxadiazon), which result in leaf necrosis, aclonifen causes a different phenotype that is described as bleaching. This also is reflected by the Herbicide Resistance Action Committee (HRAC) classification that categorizes aclonifen as an inhibitor of pigment biosynthesis with an unknown target. RESULTS: A comprehensive Arabidopsis thaliana RNAseq dataset comprising 49 different inhibitor treatments and covering 40 known target pathways was used to predict the aclonifen mode of action (MoA) by a random forest classifier. The classifier predicts for aclonifen a MoA within the carotenoid biosynthesis pathway similar to the reference compound norflurazon that inhibits the phytoene desaturase. Upon aclonifen treatment, the phytoene desaturation reaction is disturbed, resulting in a characteristic phytoene accumulation in vivo. However, direct enzyme inhibition by the herbicide was excluded for known herbicidal targets such as phytoene desaturase, 4-hydroxyphenylpyruvate dioxygenase and homogentisate solanesyltransferase. Eventually, the solanesyl diphosphate synthase (SPS), providing one of the two homogentisate solanesyltransferase substrate molecules, could be identified as the molecular target of aclonifen. Inhibition was confirmed using biochemical activity assays for the A. thaliana SPSs 1 and 2. Furthermore, a Chlamydomonas reinhardtii homolog was used for co-crystallization of the enzyme-inhibitor complex, showing that one inhibitor molecule binds at the interface between two protein monomers. CONCLUSION: Solanesyl diphosphate synthase was identified as the target of aclonifen, representing a novel mode of action for herbicides. © 2020 Society of Chemical Industry.

Two-step real-time PCR quantification of all subtypes of human immunodeficiency virus type 1 by an in-house method using locked nucleic acid-based probes.

Current HIV-1 viral-load assays are too expensive and time-consuming for small sample quantity or resource-limited setting. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. We developed an internally controlled, two-step, reverse transcription-initiated real-time PCR protocol on the LightCycler instrument achieving a favourable detection limit with an extended quantification range, detecting all HIV-1 subtypes suitable for laboratories with low sample throughput. The detection limit was found to be 100 copies/ml, the dynamic range up to 500,000,000 copies/ml. Intra and inter assay imprecision were 1.6% and 2.0% (n=10), respectively. The assay was calibrated against WHO Standard 97/656. The HIV-1 RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HIV-1 Monitor (r = 0.954; p < 0.001). This rapid (3 h) and cost effective assay is suitable for both HIV-1 detection and disease monitoring with the ability to detect and quantify all HIV-1 subtypes including O1 and O2.

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD.

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1-5 ß- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-ß1,4Araf-ß1,2Araf-ß1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its' product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

Corrigendum: Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD.

This corrects the article DOI: 10.1038/srep45341.

O-acetylation of plant cell wall polysaccharides.

Plant cell walls are composed of structurally diverse polymers, many of which are O-acetylated. How plants O-acetylate wall polymers and what its function is remained elusive until recently, when two protein families were identified in the model plant Arabidopsis that are involved in the O-acetylation of wall polysaccharides - the reduced wall acetylation (RWA) and the trichome birefringence-like (TBL) proteins. This review discusses the role of these two protein families in polysaccharide O-acetylation and outlines the differences and similarities of polymer acetylation mechanisms in plants, fungi, bacteria, and mammals. Members of the TBL protein family had been shown to impact pathogen resistance, freezing tolerance, and cellulose biosynthesis. The connection of TBLs to polysaccharide O-acetylation thus gives crucial leads into the biological function of wall polymer O-acetylation. From a biotechnological point understanding the O-acetylation mechanism is important as acetyl-substituents inhibit the enzymatic degradation of wall polymers and released acetate can be a potent inhibitor in microbial fermentations, thus impacting the economic viability of, e.g., lignocellulosic based biofuel production.

O-glycosylated cell wall proteins are essential in root hair growth.

Root hairs are single cells that develop by tip growth and are specialized in the absorption of nutrients. Their cell walls are composed of polysaccharides and hydroxyproline-rich glycoproteins (HRGPs) that include extensins (EXTs) and arabinogalactan-proteins (AGPs). Proline hydroxylation, an early posttranslational modification of HRGPs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs (which are mainly arabinosylated) and AGPs (which are mainly arabinogalactosylated). We explored the biological function of P4Hs, arabinosyltransferases, and EXTs in root hair cell growth. Biochemical inhibition or genetic disruption resulted in the blockage of polarized growth in root hairs and reduced arabinosylation of EXTs. Our results demonstrate that correct O-glycosylation on EXTs is essential for cell-wall self-assembly and, hence, root hair elongation in Arabidopsis thaliana.

OLIgo mass profiling (OLIMP) of extracellular polysaccharides.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling. An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite. Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)(1) (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself(2), and is amenable to high-throughput analyses(3). While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall. OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes(4). For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase(5, 11, 12, 13), xylan using an endo-beta-(1-4)-xylanase (6,7), or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase (8). Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined (9).