Floral transition should be strictly regulated because it is one of the most critical developmental processes in plants. Arabidopsis terminal flower 2 (tfl2) mutants show an early-flowering phenotype that is relatively insensitive to photoperiod, as well as several other pleiotropic phenotypes. We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FLOWERING LOCUS T (FT) gene, a floral pathway integrator. Molecular cloning of TFL2 showed that it encodes a protein with homology to heterochromatin protein 1 (HP1) of animals and Swi6 of fission yeast. TFL2 protein localizes in subnuclear foci and expression of the TFL2 gene complemented yeast swi6(-) mutants. These results suggested that TFL2 might function as an HP1 in Arabidopsis: Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the floral homeotic genes APETALA3, PISTILLATA, AGAMOUS and SEPALLATA3. The pleiotropic phenotype of the tfl2 mutant could reflect the fact that TFL2 represses the expression of multiple genes. Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis.

Proteins decorated with arabinogalactan (AG) have important roles in cell wall structure and plant development, yet the structure and biosynthesis of this polysaccharide are poorly understood. To facilitate the analysis of biosynthetic mutants, water-extractable arabinogalactan proteins (AGPs) were isolated from the leaves of Arabidopsis (Arabidopsis thaliana) plants and the structure of the AG carbohydrate component was studied. Enzymes able to hydrolyze specifically AG were utilized to release AG oligosaccharides. The released oligosaccharides were characterized by high-energy matrix-assisted laser desorption ionization-collision-induced dissociation mass spectrometry and polysaccharide analysis by carbohydrate gel electrophoresis. The Arabidopsis AG is composed of a b-(1→3)-galactan backbone with b-(1→6)-D-galactan side chains. The b-(1→6)-galactan side chains vary in length from one to over 20 galactosyl residues, and they are partly substituted with single a-(1→3)-Larabinofuranosyl residues. Additionally, a substantial proportion of the b-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via b-(1→6)-linkages. The b-(1→6)-galactan side chains are occasionally substituted with a-L-fucosyl. In the fucose-deficient murus1 mutant, AGPs lack these fucose modifications. This work demonstrates that Arabidopsis mutants in AGP structure can be identified and characterized. The detailed structural elucidation of the AG polysaccharides from the leaves of Arabidopsis is essential for insights into the structure-function relationships of these molecules and will assist studies on their biosynthesis.

UDP-sugars, activated forms of monosaccharides, are synthesized through de novo and salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDPsugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDPGal, UDP-glucuronic acid, UDP-L-arabinose, and UDPxylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5-7.5, and at 45°C in the presence of 2 mM Mg 2؉ . The apparent K m values for Glc 1-phosphate and L-arabinose 1-phosphate were 0.34 and 0.96 mM, respectively. PsUSP cDNA was cloned by reverse transcriptase-PCR. PsUSP appears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues from Arabidopsis and rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed in Escherichia coli catalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDPsugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.It is highly probable that the relative amounts and the architecture of cell wall polysaccharides and of the sugar moieties of glycolipids and glycoproteins in higher plants are regulated by the level of nucleotide sugars available, as well as by the levels of glycosyltransferases that incorporate monosaccharide units from respective nucleotide sugars into the polymers. Activated nucleotide sugars that serve as glycosyl donors for these polymers in higher plants are generated through de novo pathways, in which various UDP-and GDP-sugars are produced through sequential interconversions from UDP-Glc and GDP-Man as the starting substrates (1). In the salvage pathways, alternative routes to synthesize nucleotide sugars, free monosaccharides released during the degradation of polysaccharides and glycoconjugates are first phosphorylated by the action of monosaccharide kinases, then converted into nucleotide sugars by the action of pyrophosphorylases in the presence of the respective nucleotide triphosphates as co-substrates. Pyrophosphorylases principally catalyze both the following forward (synthesis of NDP-sugars) and reverse (pyrophospholysis) reactions (1): monosaccharides 1-phosphates ϩ NTP % NDP-sugars ϩ PP i , where NTP and NDP are nucleoside triphosphates and diphosphates, respectively. Var...

Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that selectively bind to arabinogalactan proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish (Raphanus sativus) root AGP was not reduced after enzyme treatment to remove a-L-arabinofuranosyl and b-glucuronosyl residues and b-1,6-galactan side chains, it was completely lost after degradation of the b-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia (Acacia senegal) AGPs, increased rather than decreased during the repeated degradation of b-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, b-1,3-galactooligosaccharides longer than b-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides cross linked to hydrazine beads detected an interaction of b-1,3-galactooligosaccharides longer than b-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for b-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for b-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.Arabinogalactan proteins (AGPs) are a type of plant proteoglycans consisting of a Hyp-rich core protein and large arabinogalactan (AG) moieties (Fincher et al., 1983;Nothnagel, 1997). Although there are many molecular species of AGP differentiated by their core proteins, the AG moieties commonly comprise b-1,3-galactan main chains and b-1,6-galactan side chains, to which L-Ara and other auxiliary sugars, such as GlcA, 4-O-methyl-GlcA, L-Fuc, L-Rha, and Xyl, are attached (Fincher et al., 1983;Nothnagel, 1997;Seifert and Roberts, 2007). A commercial product of AGPs prepared from the acacia (Acacia senegal) tree is known as gum arabic and utilized as a food stabilizer. In the Japanese herbal remedy Juzen-Taiho-To, AGs from Astragalus membranaceus are the active ingredient (Majewska-Sawka and Nothnagel, 2000;Kiyohara et al., 2002). In intact plants, AGPs are implicated in various physiological events and serve as extracellular constituents and signaling molecules. For instance, an AGP from stylar transmitting tissue attracts pollen tub...

SUMMARYArabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globularstage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.

These authors contributed equally to this work. SUMMARYWe have characterized a b-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 b-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to b-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the b-1,6-and b-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-b-1,6-Gal and b-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 b-1,3-and b-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.

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An exo-␤-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-␤-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward ␤-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze ␤-1,4-linked galacto-oligosaccharides, ␤-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze ␤-1,3-galactosyl galactosaminide, ␤-1,3-galactosyl glucosaminide, or ␤-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal ␤-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from ␤-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl ␤-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass ␤-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and ␤-1,3-glucan, although it bound ␤-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a ␤-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.Arabinogalactan proteins (AGPs) 1 are a family of complex proteoglycans widely distributed in plants. They are found in the extracellular matrix associated with the plasma membrane and cell wall (1, 2). Although their precise functions are unknown, AGPs have been implicated in diverse developmental roles, including differentiation, cell-cell recognition, and embryogenesis (3-5). AGPs are characterized by large amounts of carbohydrate components rich in galactose (sugars in the present study are D series unless otherwise designated) and Larabinose, and protein components rich in hydroxyproline, serine, threonine, alanine, and glycine (1). The carbohydrate moieties of AGPs have a common structure consisting of a ␤-1,3-galactosyl backbone to which side chains of ␤-1,6-galactosyl residues are attached through O-6. L-Arabinose and lesser amounts of other auxiliary sugars, such as glucuronic acid, L-rhamnose, and L-fucose, are attached to t...