Molecular cloning and expression of an encoding galactinol synthase gene (AnGolS1) in seedling of Ammopiptanthus nanus.

Based on the galactinol synthase (AnGolS1) fragment sequence from a cold-induced Suppression Subtractive Hybridization (SSH) library derived from Ammopiptanthus nanus (A. nanus) seedlings, AnGolS1 mRNA (including the 5' UTR and 3' UTR) (GenBank accession number: GU942748) was isolated and characterized by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). A substrate reaction test revealed that AnGolS1 possessed galactinol synthase activity in vitro and could potentially be an early-responsive gene. Furthermore, quantitative real-time PCR (qRT-PCR) indicated that AnGolS1 was responded to cold, salts and drought stresses, however, significantly up-regulated in all origans by low temperatures, especially in plant stems. In addition, the hybridization signals in the fascicular cambium were strongest in all cells under low temperature. Thus, we propose that AnGolS1 plays critical roles in A. nanus low-temperature stress resistance and that fascicular cambium cells could be involved in AnGolS1 mRNA transcription, galactinol transportation and coordination under low-temperature stress.

Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis.

The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged -323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.

A monogalactosyldiacylglycerol synthase found in the green sulfur bacterium Chlorobaculum tepidum reveals important roles for galactolipids in photosynthesis.

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.

Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling.

Plant family 1 UDP-dependent glycosyltransferases (UGTs) catalyze the glycosylation of a plethora of bioactive natural products. In Arabidopsis thaliana, 120 UGT encoding genes have been identified. The crystal-based 3D structures of four plant UGTs have recently been published. Despite low sequence conservation, the UGTs show a highly conserved secondary and tertiary structure. The sugar acceptor and sugar donor substrates of UGTs are accommodated in the cleft formed between the N- and C-terminal domains. Several regions of the primary sequence contribute to the formation of the substrate binding pocket including structurally conserved domains as well as loop regions differing both with respect to their amino acid sequence and sequence length. In this review we provide a detailed analysis of the available plant UGT crystal structures to reveal structural features determining substrate specificity. The high 3D structural conservation of the plant UGTs render homology modeling an attractive tool for structure elucidation. The accuracy and utility of UGT structures obtained by homology modeling are discussed and quantitative assessments of model quality are performed by modeling of a plant UGT for which the 3D crystal structure is known. We conclude that homology modeling offers a high degree of accuracy. Shortcomings in homology modeling are also apparent with modeling of loop regions remaining as a particularly difficult task.

Type A and type B monogalactosyldiacylglycerol synthases are spatially and functionally separated in the plastids of higher plants.

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. In Arabidopsis, the three MGDG synthases are classified into type A (atMGD1) and type B MGD isoforms (atMGD2 and atMGD3). atMGD1 is an inner envelope membrane-associated protein of chloroplasts and is responsible for the bulk of galactolipid biosynthesis in green tissues. MGD1 function is indispensable for thylakoid membrane biogenesis and embryogenesis. By contrast, type B atMGD2 and atMGD3 are localized in the outer envelopes and have no important role in chloroplast biogenesis or plant development under nutrient-sufficient conditions. These type B MGD genes are, however, strongly induced by phosphate (Pi) starvation and are essential for alternative galactolipid biosynthesis during Pi starvation. MGD1 gene expression is up-regulated by light and cytokinins. By contrast, Pi starvation-dependent expression of atMGD2/3 is suppressed by cytokinins but induced through auxin signaling pathways. These growth factors may control the functional sharing of the inner envelope pathway by atMGD1 and the outer envelope pathway by atMGD2/3 according to the growth environment.

Requirement for a core 1 galactosyltransferase in the Drosophila nervous system.

Mucin type O-glycosylation is a widespread modification of eukaryotic proteins, but its functional requirements remain incompletely understood. It is initiated by the attachment of N-acetylgalactosamine (GalNAc) to Ser or Thr residues, and then elongated by additional sugars. We have examined requirements for mucin-type glycosylation in Drosophila by characterizing the expression and phenotypes of core 1 galactosyltransferases (core 1 GalTs), which elongate O-GalNAc by adding galactose in a beta1,3 linkage. Drosophila encode several putative core 1 GalTs, each expressed in distinct patterns. CG9520 (C1GalTA) is expressed in the amnioserosa and central nervous system. A null mutation in C1GalTA is lethal, and mutant animals exhibit a striking morphogenetic defect in which the ventral nerve cord is greatly elongated and the brain hemispheres are misshapen. Lectin staining and blotting experiments confirmed that C1GalTA contributes to the synthesis of Gal-beta1,3-GalNAc in vivo. Our results identify a role for mucin-type O-glycosylation during neural development in Drosophila.

Identification of a novel group of putative Arabidopsis thaliana beta-(1,3)-galactosyltransferases.

To begin biochemical and molecular studies on the biosynthesis of the type II arabinogalactan chains on arabinogalactan-proteins (AGPs), we adopted a bioinformatic approach to identify and systematically characterise the putative galactosyltransferases (GalTs) responsible for synthesizing the beta-(1,3)-Gal linkage from CAZy GT-family-31 from Arabidopsis thaliana. These analyses confirmed that 20 members of the GT-31 family contained domains/motifs typical of biochemically characterised beta-(1,3)-GTs from mammalian systems. Microarray data confirm that members of this family are expressed throughout all tissues making them likely candidates for the assembly of the ubiquitously found AGPs. One member, At1g77810, was selected for further analysis including location studies that confirmed its presence in the Golgi and preliminary enzyme substrate specificity studies that demonstrated beta-(1,3)-GalT activity. This bioinformatic/molecular study of CAZy GT-family-31 was validated by the recent report of Strasser et al. (Plant Cell 19:2278-2292, 2007) that another member of this family (At1g26810; GALT1) encodes a beta-(1,3)-GalT involved in the biosynthesis of the Lewis a epitope of N-glycans in Arabidopsis thaliana.

Amino-acid substitution in the disordered loop of blood group B-glycosyltransferase enzyme causes weak B phenotype.

BACKGROUND: Few studies have investigated the reaction kinetics and interactions with nucleotide donor and acceptor substrates of mutant human ABO glycosyltransferases. Previous work identified a B(w) allele featuring a 556G>A polymorphism giving rise to a weak B phenotype. This polymorphism is predicted to cause a M186V amino-acid mutation within a highly conserved series of 16 amino acids present both in both blood group A- and blood group B-synthesizing enzymes. These residues are known as the disordered loop because their location cannot be determined in the crystal structure of the enzyme. Another patient has been identified with a 556G>A B(w) allele and the kinetics of the resulting mutant glycosyltransferase were studied. STUDY DESIGN AND METHODS: Serologic testing with murine and human reagents, amplification of the coding regions of exons 6 and 7, and DNA sequencing were performed with standard protocols. Enzyme kinetic studies utilized a model of human GTB M186V expressed in Escherichia coli with radiolabeled UDP-galactose and UDP-N-acetylgalactosamine as donor substrates and synthetic H-disaccharide as acceptor following standard protocols. RESULTS: The patient's red blood cells demonstrated a weak, but not mixed-field, B phenotype. Kinetic studies on the mutant enzyme revealed diminished activity (k(cat) = 0.15 per sec with UDP-galactose compared to 5.1 per sec for wild-type GTB) and elevated K(m) values for all substrates. CONCLUSION: This enzyme with a mutation in the disordered loop produces weak B antigen expression because of greatly decreased enzyme activity and reduced affinity for B-donor and acceptor substances.

DGD2, an arabidopsis gene encoding a UDP-galactose-dependent digalactosyldiacylglycerol synthase is expressed during growth under phosphate-limiting conditions.

The galactolipid digalactosyldiacylglycerol (DGDG), one of the main chloroplast lipids in higher plants, is believed to be synthesized by the galactolipid:galactolipid galactosyltransferase, which transfers a galactose moiety from one molecule of monogalactosyldiacylglycerol (MGDG) to another. Here, we report that Arabidopsis as well as other plant species contain two genes, DGD1 and DGD2, encoding enzymes with DGDG synthase activity. Using MGDG and UDP-galactose as substrates for in vitro assays with DGD2 we could for the first time measure DGDG synthase activity of a heterologously expressed plant cDNA. UDP-galactose, but not MGDG, serves as the galactose donor for DGDG synthesis catalyzed by DGD2, providing clear evidence for the existence of a UDP-galactose-dependent DGDG synthase in higher plants. In in vitro assays, DGD2 was capable of galactosylating DGDG, resulting in the synthesis of an oligogalactolipid tentatively identified as trigalactosyldiacylglycerol. DGD2 mRNA expression in leaves was very low but was strongly induced during growth under phosphate-limiting conditions. This induction correlates with the previously described increase in DGDG during phosphate deprivation. Therefore, in contrast to DGD1, which is responsible for the synthesis of the bulk of DGDG found in chloroplasts, DGD2 apparently is involved in the synthesis of DGDG under specific growth conditions.

Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana.

In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.

Identification and characterization of an UDP-Gal: N-acetyllactosaminide alpha-1,3-D-galactosyltransferase in calf thymus.

Calf thymus was found to contain a high activity of a galactosyltransferase, which transfers galactose from UDP-galactose to asialo-alpha 1-acid glycoprotein and N-acetyllactosamine. By means of competition and acceptor-specificity studies the enzyme could be distinguished from an N-acetylglucosaminide beta-1,4-galactosyltransferase and an N-acetylgalactosamine-protein beta-1,3-galactosyltransferase, which in addition occur in calf thymus, as well as from the blood-group-B-associated alpha-galactosyltransferase. Identification of the products revealed that the enzyme accomplishes an alpha 1 leads to 3 linkage resulting in a terminal Gal(alpha 1 leads to 3)Gal(beta 1 leads to 4)GlcNAc sequence. The enzyme is membrane-bound and is activated by Triton X-100. It shows optimal activity over a broad pH range (5.5-7.0) and has a pronounced requirement for Mn2+ ions (Km = 6.1 mM) for its action. It is suggested that the alpha-1,3-galactosyltransferase functions in the biosynthesis of calf thymocyte cell-surface glycoconjugates including glycoproteins.

Two alpha-3-D-galactosyltransferases in rabbit stomach mucosa with different acceptor substrate specificities.

Homogenates of rabbit stomach mucosa were examined for enzymes catalysing the transfer of D-galactose from UDP-D-galactose to various low-molecular-weight acceptors of known structure. Treatment of the products with alpha and beta-D-galactosidases revealed that D-galactose was transferred in both alpha and beta-anomeric linkages. The beta-D-galactosyltransferase used N-acetylglucosamine and compounds containing terminal nonreducing beta-N-acetylglucosaminyl residues as acceptor substrates. The compounds accepting D-galactose in alpha-anomeric linkage had unsubstituted terminal non-reducing beta-D-galactosyl units or a fucose substituent on the carbon-2 position of a subterminal beta-D-galactosyl unit. Methylation analysis of the products formed with N-acetyllactosamine [beta-D-Galp(1 leads to 4)D-GlcNAcp] and 2'fucosyllactose [alpha-L-Fucp(1 leads to 2)-beta-D-Galp(1 leads to 4)D-Glcp] revealed that D-galactose was transferred to the carbon-3 position of the beta-D-galactosyl residue in both of these acceptor substrates. Competition experiments with the two substrates indicated that the transfer of D-galactose was catalysed in each case by a different alpha-3-D-galactosyltransferase. Differences were also observed in the solubility properties of the enzymes: the alpha-3-D-galactosyltransferase using acceptor substrates with unsubstituted beta-D-galactosyl residues was more readily soluble both in the presence and absence of detergents than the transferase using beta-D-galactosyl residues substituted at carbon-2 with L-fucose. These findings demonstrate that rabbit stomach mucosa has two distinct alpha-3-D-galactosyltransferases: one, which is more tightly membrane-bound, resembles the human B-gene-specified transferase in its acceptor specificity, and the second, which is a more soluble enzyme, transfers D-galactose to the same positional linkage in unsubstituted beta-D-galactosyl residues.