Electrochemical oxo-functionalization of cyclic alkanes and alkenes using nitrate and oxygen.

Direct functionalization of C(sp3)-H bonds allows rapid access to valuable products, starting from simple petrochemicals. However, the chemical transformation of non-activated methylene groups remains challenging for organic synthesis. Here, we report a general electrochemical method for the oxidation of C(sp3)-H and C(sp2)-H bonds, in which cyclic alkanes and (cyclic) olefins are converted into cycloaliphatic ketones as well as aliphatic (di)carboxylic acids. This resource-friendly method is based on nitrate salts in a dual role as anodic mediator and supporting electrolyte, which can be recovered and recycled. Reducing molecular oxygen as a cathodic counter reaction leads to efficient convergent use of both electrode reactions. By avoiding transition metals and chemical oxidizers, this protocol represents a sustainable oxo-functionalization method, leading to a valuable contribution for the sustainable conversion of petrochemical feedstocks into synthetically usable fine chemicals and commodities.

Pectin Synthesis and Pollen Tube Growth in Arabidopsis Involves Three GAUT1 Golgi-Anchoring Proteins: GAUT5, GAUT6, and GAUT7.

The major cell wall pectic glycan homogalacturonan (HG) is crucial for plant growth, development, and reproduction. HG synthesis occurs in the Golgi and is catalyzed by members of the galacturonosyltransferase (GAUT) family with GAUT1 being the archetypal and best studied family member. In Arabidopsis suspension culture cells and tobacco leaves, the Golgi localization of Arabidopsis GAUT1 has been shown to require protein-protein interactions with its homolog GAUT7. Here we show that in pollen tubes GAUT5 and GAUT6, homologs of GAUT7, also target GAUT1 to the Golgi apparatus. Pollen tube germination and elongation in double homozygous knock-out mutants (gaut5 gaut6, gaut5 gaut7, and gaut6 gaut7) are moderately impaired, whereas gaut5 -/- gaut6 -/- gaut7 +/- triple mutant is severely impaired and male infertile. Amounts and distributions of methylesterified HG in the pollen tube tip were severely distorted in the double and heterozygous triple mutants. A chimeric protein comprising GAUT1 and a non-cleavable membrane anchor domain was able to partially restore pollen tube germination and elongation and to reverse male sterility in the triple mutant. These results indicate that GAUT5, GAUT6, and GAUT7 are required for synthesis of native HG in growing pollen tubes and have critical roles in pollen tube growth and male fertility in Arabidopsis.

A Glycosyltransferase from Nicotiana alata Pollen Mediates Synthesis of a Linear (1,5)-α-L-Arabinan When Expressed in Arabidopsis.

The walls of Nicotiana alata pollen tubes contain a linear arabinan composed of (1,5)-α-linked arabinofuranose residues. Although generally found as a side chain on the backbone of the pectic polysaccharide rhamnogalacturonan I, the arabinan in N. alata pollen tubes is considered free, as there is no detectable rhamnogalacturonan I in these walls. Carbohydrate-specific antibodies detected arabinan epitopes at the tip and along the shank of N. alata pollen tubes that are predominantly part of the primary layer of the bilayered wall. A sequence related to ARABINAN DEFICIENT1 (AtARAD1), a presumed arabinan arabinosyltransferase from Arabidopsis (Arabidopsis thaliana), was identified by searching an N alata pollen transcriptome. Transcripts for this ARAD1-like sequence, which we have named N. alata ARABINAN DEFICIENT-LIKE1 (NaARADL1), accumulate in various tissues, most abundantly in the pollen grain and tube, and encode a protein that is a type II membrane protein with its catalytic carboxyl terminus located in the Golgi lumen. The NaARADL1 protein can form homodimers when transiently expressed in Nicotiana benthamiana leaves and heterodimers when coexpressed with AtARAD1 The expression of NaARADL1 in Arabidopsis led to plants with more arabinan in their walls and that also exuded a guttation fluid rich in arabinan. Chemical and enzymatic characterization of the guttation fluid showed that a soluble, linear α-(1,5)-arabinan was the most abundant polymer present. These results are consistent with NaARADL1 having an arabinan (1,5)-α-arabinosyltransferase activity.

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

BACKGROUND: Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. RESULTS: The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. CONCLUSIONS: LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

SnRK1 from Arabidopsis thaliana is an atypical AMPK.

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic ß and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, ß(3) and ßγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique ßγ subunit and one of three non-catalytic ß subunits: ß(1), ß(2) or the plant-specific ß(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical ßγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the ß(1), ß(2) and ßγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

In vitro grown pollen tubes of Nicotiana alata actively synthesise a fucosylated xyloglucan.

Nicotiana alata pollen tubes are a widely used model for studies of polarized tip growth and cell wall synthesis in plants. To better understand these processes, RNA-Seq and de novo assembly methods were used to produce a transcriptome of N. alata pollen grains. Notable in the reconstructed transcriptome were sequences encoding proteins that are involved in the synthesis and remodelling of xyloglucan, a cell wall polysaccharide previously not thought to be deposited in Nicotiana pollen tube walls. Expression of several xyloglucan-related genes in actively growing pollen tubes was confirmed and xyloglucan epitopes were detected in the wall with carbohydrate-specific antibodies: the major xyloglucan oligosaccharides found in N. alata pollen grains and tubes were fucosylated, an unusual structure for the Solanaceae, the family to which Nicotiana belongs. Finally, carbohydrate linkages consistent with xyloglucan were identified chemically in the walls of N. alata pollen grains and pollen tubes grown in culture. The presence of a fucosylated xyloglucan in Nicotiana pollen tube walls was thus confirmed. The consequences of this discovery to models of pollen tube growth dynamics and more generally to polarised tip-growing cells in plants are discussed.

Glycan profiling of plant cell wall polymers using microarrays.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)(1,2). However, understanding the biosynthesis and function of these components remains challenging. Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall(3). Furthermore, their composition is also modified during development(1), or in response to environmental cues(4). In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis(5). However, relatively few of the biosynthetic genes have been functionally characterized (4,5). Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types(6). Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained(7). Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification. Monoclonal antibodies (mAbs)(8,9) have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously(9,10,11). Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)(10,11) that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems(12) and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

The dynamics of plant cell-wall polysaccharide decomposition in leaf-cutting ant fungus gardens.

The degradation of live plant biomass in fungus gardens of leaf-cutting ants is poorly characterised but fundamental for understanding the mutual advantages and efficiency of this obligate nutritional symbiosis. Controversies about the extent to which the garden-symbiont Leucocoprinus gongylophorus degrades cellulose have hampered our understanding of the selection forces that induced large scale herbivory and of the ensuing ecological footprint of these ants. Here we use a recently established technique, based on polysaccharide microarrays probed with antibodies and carbohydrate binding modules, to map the occurrence of cell wall polymers in consecutive sections of the fungus garden of the leaf-cutting ant Acromyrmex echinatior. We show that pectin, xyloglucan and some xylan epitopes are degraded, whereas more highly substituted xylan and cellulose epitopes remain as residuals in the waste material that the ants remove from their fungus garden. These results demonstrate that biomass entering leaf-cutting ant fungus gardens is only partially utilized and explain why disproportionally large amounts of plant material are needed to sustain colony growth. They also explain why substantial communities of microbial and invertebrate symbionts have evolved associations with the dump material from leaf-cutting ant nests, to exploit decomposition niches that the ant garden-fungus does not utilize. Our approach thus provides detailed insight into the nutritional benefits and shortcomings associated with fungus-farming in ants.

Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple.

The dihydrochalcone phlorizin (phloretin 2'-glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP-glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [(3)H]-UDP-glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP-glucose co-migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterization of a UDP-glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.

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.

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles.

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials.

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.

High-throughput mapping of cell-wall polymers within and between plants using novel microarrays.

We describe here a methodology that enables the occurrence of cell-wall glycans to be systematically mapped throughout plants in a semi-quantitative high-throughput fashion. The technique (comprehensive microarray polymer profiling, or CoMPP) integrates the sequential extraction of glycans from multiple organs or tissues with the generation of microarrays, which are probed with monoclonal antibodies (mAbs) or carbohydrate-binding modules (CBMs) with specificities for cell-wall components. The profiles generated provide a global snapshot of cell-wall composition, and also allow comparative analysis of mutant and wild-type plants, as demonstrated here for the Arabidopsis thaliana mutants fra8, mur1 and mur3. CoMPP was also applied to Physcomitrella patens cell walls and was validated by carbohydrate linkage analysis. These data provide new insights into the structure and functions of plant cell walls, and demonstrate the potential of CoMPP as a component of systems-based approaches to cell-wall biology.