Depletion of UDP-D-apiose/UDP-D-xylose synthases results in rhamnogalacturonan-II deficiency, cell wall thickening, and cell death in higher plants

D-apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of D-apiose involves UDP-D-apiose/UDP-D-xylose synthase catalyzing the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose. In this study we have analyzed the cellular effects of depletion of UDP-D-apiose/UDP-D-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-D-apiose/UDP-D-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of D-apiose as well as 2-O-methyl-L-fucose and 2-O-methyl-D-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a D-apiose deficiency.     

Partial purification and study of pollen glucuronokinase.

Glucuronokinase was purified 31-fold from pollen of Lilium longiflorum. The enzyme was inhibited by its product, α-d-glucuronate-1-P, and by UDP-d-glucuronate, and these compounds were competitive inhibitors. Inhibitor constants were 0.18 mm for d-glucuronate-1-P and 0.55 mm for UDP-d-glucuronate. These effects may have regulatory significance; both inhibitors are intermediates in the pathway by which plant cells convert myo-inositol into cell wall uronides and pentoses, and glucuronokinase is a likely step for regulation in this pathway. The enzyme exhibited considerable specificity concerning inhibitors, and an additional 22 compounds were not inhibitory. These included uronic acids other than d-glucuronate and compounds related structurally or metabolically to d-glucuronate.     

Characterization of Arabidopsis CTP:3-deoxy-D-manno-2-octulosonate cytidylyltransferase (CMP-KDO synthetase), the enzyme that activates KDO during rhamnogalacturonan II biosynthesis

In plant cells, boron (B) occurs predominantly as a borate ester associated with rhamnogalacturonan II (RG-II), but the function of this B-RG-II complex has yet to be investigated. 3-Deoxy-D-manno-2-octulosonic acid (KDO) is a specific component monosaccharide of RG-II. Mutant plants defective in KDO biosynthesis are expected to have altered RG-II structure, and would be useful for studying the physiological function of the B-RG-II complex. Here, we characterized Arabidopsis CTP:KDO cytidylyltransferase (CMP-KDO synthetase; CKS), the enzyme activating KDO as a nucleotide sugar prior to its incorporation into RG-II. Our analyses localized the Arabidopsis CKS protein to mitochondria. The Arabidopsis CKS gene occurs as a single-copy gene in the genome, and we could not obtain cks null mutants from T-DNA insertion lines. Analysis using +/cks heterozygotes in the quartet1 background demonstrated that the cks mutation rendered pollen infertile through the inhibition of pollen tube elongation. These results suggest that KDO is an indispensable component of RG-II, and that the complete B-RG-II complex is essential for the cell wall integrity of rapidly growing tissues.     

Isolation and partial characterization of apiogalacturonans from the cell wall of Lemna minor

1. A mild, reproducible extraction procedure, using 0.5% ammonium oxalate, was developed for the isolation of polysaccharides containing d-apiose from the cell wall of Lemna minor. On a dry-weight basis the polysaccharide fractions extracted with ammonium oxalate made up 14% of the material designated cell walls and contained 20% of the d-apiose originally present in the cell walls. The cell walls, as isolated, contained 83% of the d-apiose present in L. minor. 2. After extraction with ammonium oxalate, purified polysaccharides were obtained by DEAE-Sephadex column chromatography and by fractional precipitation with sodium chloride. With these procedures the material extracted at 22 degrees C could be separated into at least five polysaccharides. On a dry-weight basis two of these polysaccharides made up more than 50% of the material extracted at 22 degrees C. There was a direct relationship between the d-apiose content of the polysaccharides and their solubility in sodium chloride solutions; those of highest d-apiose content were most soluble. 3. All the polysaccharides isolated appeared to be of one general type, namely galacturonans to which were attached side chains containing d-apiose. The d-apiose content of the apiogalacturonans varied from 7.9 to 38.1%. The content of esterified d-galacturonic acid residues in all apiogalacturonans was low, being in the range 1.0-3.5%. Hydrolysis of a representative apiogalacturonan with dilute acid resulted in the complete removal of the d-apiose with little or no degradation of the galacturonan portion. 4. Treatment of polysaccharide fractions with pectinase established that those of high d-apiose content and soluble in m-sodium chloride were not degraded, whereas those of low d-apiose content and insoluble in m-sodium chloride were extensively degraded. When the d-apiose was removed from a typical pectinase-resistant polysaccharide, the remainder of the polysaccharide was readily degraded by this enzyme. 5. Periodate oxidation of representative polysaccharide fractions and apiogalacturonans and determination of the formaldehyde released showed that about 50% of the d-apiose molecules were substituted at either the 3- or the 3'-position.     

Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.     

D-apiose reductase from Aerobacter aerogenes

A strain of Aerobacter aerogenes PRL-R3 has been isolated which utilizes d-apiose as its sole source of carbon. A new enzyme, d-apiose reductase, was discovered in this strain. The enzyme was not present when the strain was grown on d-glucose. d-Apiose reductase catalyzes the nicotinamide adenine dinucleotide-dependent interconversion of d-apiose and d-apiitol. The enzyme is specific for d-apiose and d-apiitol, with a few possible exceptions. The K(m) for d-apiose is 0.02 m. The K(m) for d-apiitol is 0.01 m. The enzyme is almost completely specific for the reduced and oxidized forms of nicotinamide adenine dinucleotide. When cell-free extracts were centrifuged at 100,000 x g for 1 hr, the enzyme remained in solution. Optimal activity for the reduction of d-apiose was obtained at pH 7.5 in glycylglycine buffer, whereas for the oxidation of d-apiitol it was obtained at pH 10.5 in glycine buffer. Enzymatic reduction of d-apiose was not appreciably affected by the presence of 0.02 m ethylenediaminetetraacetate. Paper chromatography and specific spray reagents were used to identify d-apiitol and d-apiose as the products of this reversible reaction. d-Apiose and d-apiitol did not serve as substrates for ribitol dehydrogenase and d-arabitol dehydrogenase from A. aerogenes PRL-R3.     

Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis

One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana. Functional analysis revealed that this enzyme converts UDP-d-glucuronate to UDP-d-galacturonate in vitro. An expression analysis of this epimerase and its five homologs in the Arabidopsis genome by quantitative RT-PCR and promoter::GUS fusions indicated differential expression of the family members in plant tissues and expression of all isoforms in the developing pollen of A. thaliana.     

Formation of rhamnogalacturonan II-borate dimer in pectin determines cell wall thickness of pumpkin tissue

Boron (B) deficiency results in inhibition of pumpkin (Cucurbia moschata Duchesne) growth that is accompanied by swelling of the cell walls. Monomeric rhamnogalacturonan II (mRG-II) accounted for 80% to 90% of the total RG-II in B-deficient walls, whereas the borate ester cross-linked RG-II dimer (dRG-II-B) accounted for more than 80% of the RG-II in control plants. The results of glycosyl residue and glycosyl linkage composition analyses of the RG-II from control and B-deficient plants were similar. Thus, B deficiency does not alter the primary structure of RG-II. The addition of (10)B-enriched boric acid to B-deficient plants resulted within 5 h in the conversion of mRG-II to dRG-II-(10)B. The wall thickness of the (10)B-treated plants and control plants was similar. The formation and possible functions of a borate ester cross-linked RG-II in the cell walls are discussed.     

The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer

The location of the 1:2 borate-diol ester cross-link in the dimer of the plant cell wall polysaccharide rhamnogalacturonan II (RG-II) has been determined. The ester cross-links the apiofuranosyl residue of the 2-O-methyl-D-xylose-containing side chains in each of the subunits of the dimer. The apiofuranosyl residue in each of the two aceric acid-containing side chains is not esterified. The site of borate esterification is identical in naturally occurring and in in vitro synthesized dimer. Pb2+, La3+, and Ca2+ increase dimer formation in vitro in a concentration- and pH-dependent manner. Pb2+ is the most effective cation. The dimer accounts for 55% of the RG-II when the monomer (0.5 mM) is treated for 5 min at pH 3.5 with boric acid (1 mM) and Pb2+ (0.5 mM); at pH 5 the rate of conversion is somewhat slower. Hg2+ does not increase the rate of dimer formation. A cation's charge density and its ability to form a coordination complex with RG-II, in addition to steric factors, may regulate the rate and stability of dimer formation in vitro. Our data provide evidence that the structure of RG-II itself determines which apiofuranosyl residues are esterified with borate and that in the presence of boric acid and certain cations, two RG-II monomers self-assemble to form a dimer.     

Boron in plant cell walls

Boron is an essential element for higher plants, yet the primary functions remain unclear. In intact tissues of higher plants, this element occurs as both water soluble and water insoluble forms. In this review, the intracellular localisation of B and possible function of B in cell walls of higher plants are discussed. The majority of the water soluble B seems to be localised in the apoplastic region as boric acid. The water insoluble B is associated with rhamnogalacturonan II (RG-II) and the complex is ubiquitous in higher plants. In the Brassicaceae, Apiaceae, Chenopodiaceae, Asteraceae, Amaryllidaceae, and Liliaceae, nearly all the cell wall B is associated with RG-II, while in the Cucurbitaceae, only half of the cell wall B is in this complex. In duckweed, a different type of B-polysaccharide complex has been identified.Analysis of the structure of the B–RG-II complex reveals that the complex is composed of boric acid and two chains of monomeric RG-II. Boric acid does not merely bind to sugars but crosslinks two chains of pectic polysaccharide at the RG-II region through borate-diester bonding, thus forming a network of pectic polysaccharides in cell walls. The B–RG-II complex is reconstituted in vitro only by mixing monomeric RG-II and boric acid at pH 4.0. In the in vitro reconstitution, germanic acid can substitute for boric acid to some extent. The RG-II epitope, which cross reacts with the antibody toward the B-RG-II complex, is detected in walls of every cell in radish roots. The epitope is also detected in growing pollen tube cell walls, which are claimed to require B.Whilst it is now clear that boric acid links some cell wall components, it is not yet clear whether there is a structural requirement for B in cell wall function.     

Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.     

Structural characterization of red wine rhamnogalacturonan II

The pectic polysaccharide rhamnogalacturonan II (RG-II), which accounts for ˜ 20% of the ethanol-precipitable polysaccharides in red wine, has been isolated from wine polysaccharides by anion-exchange chromatography. Four fractions enriched with RG-II were obtained and the RG-II then purified to homogeneity by Concanavalin A affinity and size-exclusion chromatographies. The glycosyl-residue compositions of the four RG-IIs are similar; all the RG-IIs contain the monosaccharides (apiose, , , Kdo, Dha, and aceric acid) that are diagnostic of RG-II. The glycosyl-linkages of the neutral and acidic sugars, including aceric acid, were determined simultaneously by GC-EIMS analysis of the methylated alditol acetates generated from per-O-methylated and carboxyl-reduced RG-II. Two of the RG-IIs contain boron, most likely as a borate di-ester that cross-links two molecules of RG-II together to form a dimer. The dimer contains 3′- and 2,3,3′-linked apiosyl residues whereas the monomer contains only 3′-linked apiosyl residues which suggests that the borate di-ester is located on at least one of the apiosyl residues of RG-II. Although the wine RG-IIs all have similar structures they are not identical since they differ in the length and degree of methyl-esterification of the RG-II backbone and in the presence or absence of borate di-esters. Nevertheless, these studies show that the major structural features of wine and primary cell wall RG-II are conserved.     

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.     

Germanium does not substitute for boron in cross-linking of rhamnogalacturonan II in pumpkin cell walls

Boron (B)-deficient pumpkin (Cucurbita moschata Duchesne) plants exhibit reduced growth, and their tissues are brittle. The leaf cell walls of these plants contain less than one-half the amount of borate cross-linked rhamnogalacturonan II (RG-II) dimer than normal plants. Supplying germanium (Ge), which has been reported to substitute for B, to B-deficient plants does not restore growth or reduce tissue brittleness. Nevertheless, the leaf cell walls of the Ge-treated plants accumulated considerable amounts of Ge. Dimeric RG-II (dRG-II) accounted for between 20% and 35% of the total RG-II in the cell walls of the second to fourth leaves from Ge-treated plants, but only 2% to 7% of the RG-II was cross-linked by germanate (dRG-II-Ge). The ability of RG-II to form a dimer is not reduced by Ge treatment because approximately 95% of the monomeric RG-II generated from the walls of Ge-treated plants is converted to dRG-II-Ge in vitro in the presence of germanium oxide and lead acetate. However, dRG-II-Ge is unstable and is converted to monomeric RG-II when the Ge is removed. Therefore, the content of dRG-II-Ge and dRG-II-B described above may not reflect the actual ratio of these in muro. (10)B-Enriched boric acid and Ge are incorporated into the cell wall within 10 min after their foliar application to B-deficient plants. Foliar application of (10)B but not Ge results in an increase in the proportion of dRG-II in the leaf cell wall. Taken together, our results suggest that Ge does not restore the growth of B-deficient plants.     

Binding studies with bovine liver UPD-D-glucose dehydrogenase

The direct binding of uridine 5′-(α-d-glucopyranosyluronic acid pyrophosphate) (UDP-d-glucuronic acid) and of uridine 5′-(α-d-xylopyranosyl pyrophosphate) (UDP-d-xylose) to bovine liver UDP-d-glucose:NAD oxidoreductase (EC 1.1.1.22) has been measured by equilibrium dialysis. At saturation, the hexameric enzyme binds six molecules of UDP-d-glucuronic acid to noninteracting sites. UDP-d-xylose binds to 2 distinct classes of sites, each class binding six molecules of ligand. UDP-d-xylose is able to displace either UDP-d-glucose or UDP-d-glucuronic acid and UDP-d-glucose is able to displace UDP-d-glucuronic acid from the enzyme. It is proposed that the enzyme displays half-of-the-sites reactivity toward both substrate (UDP-d-glucose) and cosubstrate (NAD) and all-of-the-sites reactivity toward UDP-d-glucuronic acid. UDP-d-xylose is considered to bind cooperatively with high affinity to six regulatory sites and independently with lower affinity to six catalytic sites on the enzyme. Active-enzyme centrifugation studies show that UDP-d-glucose: NAD oxidoreductase is hexameric at concentrations corresponding to those used in steady-state kinetic measurements.     

Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.     

The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation

Despite a very complex structure, the sugar composition of the rhamnogalacturonan II (RG-II) pectic fraction is extremely conserved. Among its constituting monosaccharides is the seldom-observed eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo), whose phosphorylated precursor is synthesized by Kdo-8-P synthase. As an attempt to alter specifically the RG-II structure in its sugar composition and assess the consequences on the function of RG-II in cell wall and its relationship with growth, Arabidopsis null mutants were sought in the genes encoding Kdo-8-P synthase. Here, the isolation and characterization of one null mutant for the isoform 1 (AtkdsA1-S) and two distinct null mutants for the isoform 2 of Arabidopsis Kdo-8-P synthase (AtkdsA2-V and AtkdsA2-S) are described. Evidence is provided that AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity, and that it accounts for 75% of the mRNAs to be translated into Kdo-8-P synthase. Furthermore, this predominant expression of AtKDSA2 over AtKDSA1 was confirmed by quantification of the cytosolic Kdo content in the mutants, in a variety of ecotypes. The inability to identify a double knockout mutant originated from pollen abortions, due to the inability of haploid pollen of the AtkdsA1- AtkdsA2- genotype to form an elongated pollen tube properly and perform fertilization.     

Cloning and expression of a UDP-glucuronic acid decarboxylase gene in rice

A cDNA fragment was cloned from rice immature seeds by the RT-PCR method. The deduced amino acid sequence of the cDNA showed a high degree of identity with UDP-d-glucuronic acid decarboxylase (UXS) from other plants and was most similar to the soluble UXS from Arabidopsis. The recombinant protein, expressed in an Escherichia coli system, catalysed the conversion of UDP-d-glucuronic acid to UDP-d-xylose, confirming that the gene encoded UXS. The uxs gene was expressed in mature, harvested rice seeds as well as in immature seeds 14 d post-anthesis, suggesting that the uxs gene is necessary at the beginning of the germination period. This is the first report of the cloning of the uxs gene from monocots.     

Presence of rhamnogalacturonan II in the juices produced by enzymatic liquefaction of Agave pulquero stem (Agave mapisaga)

Rhamnogalacturonan II (RG-II), a small complex pectic polysaccharide is released from Agave pulquero stems (Agave mapisaga), after the production period of aguamiel. RG-II was obtained by treatment with two commercial liquefying enzyme preparations, it was isolated by size-exclusion chromatography and characterized. RG-IIs contains diagnostic sugars such as apiose, 2-O-methyl-l-fucose, 2-O-methyl-d-xylose, aceric acid, Kdo and Dha. Glycosyl-linkage compositions of the Agave pulquero RG-II like structures are similar to the theoretical model described through sycamore RG-II structure. The presence of 3′-linked apiose indicates that the obtained juice from Agave pulquero plant contains the free RG-II dimer. Thus, when pectinolytic enzyme preparations are used to process Agave pulquero (A. mapisaga), RG-II is released as one of the main soluble polysaccharide fraction.