Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense

In Arabidopsis and other Brassicaceae, the enzyme myrosinase (beta-thioglucoside glucohydrolase, TGG) degrades glucosinolates to produce toxins that deter herbivory. A broadly applicable selection for meiotic recombination between tightly linked T-DNA insertions was developed to generate Arabidopsis tgg1tgg2 double mutants and study myrosinase function. Glucosinolate breakdown in crushed leaves of tgg1 or tgg2 single mutants was comparable to that of wild-type, indicating redundant enzyme function. In contrast, leaf extracts of tgg1tgg2 double mutants had undetectable myrosinase activity in vitro, and damage-induced breakdown of endogenous glucosinolates was apparently absent for aliphatic and greatly slowed for indole glucosinolates. Maturing leaves of myrosinase mutants had significantly increased glucosinolate levels. However, developmental decreases in glucosinolate content during senescence and germination were unaffected, showing that these processes occur independently of TGG1 and TGG2. Insect herbivores with different host plant preferences and feeding styles varied in their responses to myrosinase mutations. Weight gain of two Lepidoptera, the generalist Trichoplusia ni and the facultative Solanaceae-specialist Manduca sexta, was significantly increased on tgg1tgg2 double mutants. Two crucifer-specialist Lepidoptera had differing responses. Whereas Plutella xylostella was unaffected by myrosinase mutations, Pieris rapae performed better on wild-type, perhaps due to reduced feeding stimulants in tgg1tgg2 mutants. Reproduction of two Homoptera, Myzus persicae and Brevicoryne brassicae, was unaffected by myrosinase mutations.     

Isomeric analysis of oligomannosidic N-glycans and their dolichol-linked precursors

Oligomannosidic (OM) N-glycans occur as a mixture of isomers, which at early stages of glycosidase trimming also comprise structures with one to three glucose residues. A complementary set of isomers is generated during the biosynthesis of the lipid-linked precursor. Here, we demonstrate the remarkable capacity of liquid chromatography (LC) with porous graphitic carbon and mass spectrometric detection for the determination of OM isomers. Protein-linked N-glycans were released enzymatically from samples with known isomer composition such as kidney bean proteins and ribonuclease B. Lipid-linked oligosaccharides were obtained by a direct mild acid hydrolysis of microsomes thus avoiding biphasic partitioning. A parallel analysis of pyridylaminated glycans by amide-silica and reversed-phase high-performance LC, the application of branch-specific α-mannosidases and work with ALG mutant plants led to the assignment of the relative retention times of the isomers occurring during the degradation of the Glc(3)Man(9)GlcNAc(2) precursor oligosaccharide to Man(5)GlcNAc(2) and beyond. A tightly woven net of evidence supports these assignments. Noteworthy, this isomer assignment happens in the course of a comprehensive analysis of all types of a sample's N-glycans.     

拟南芥芥子酶基因TGG6是花特异表达的假基因

芥子酶是一类催化硫代葡萄糖苷水解的同工酶．TGG6是在拟南芥中新发现的芥子酶基因．从拟南芥几个不同生态型中克隆了他G6基因的全长核基因和cDNA片段．序列分析结果表明,所有供斌的生态型的他G6基因都与拟南芥第3个芥子酶基因彤G3类似,在编码区存在1个以上移码突变,不能编码完整多肽．生态型Col-0第10个内含子的剪切边界还发生了缺失,导致内含子不能被切除．初步确定TGG6是一个假基因．然而,RT-PCR结果却表明TGG6在花器中特异性表达,说明TGG6在进化的某个阶段可能是有功能的基因,由于某种原因。该基因在进化过程中被失活．     

Discovery and structural characterization of fucosylated oligomannosidic N-glycans in mushrooms

L-fucose is a common constituent of Asn-linked glycans in vertebrates, invertebrates, and plants, but in fungal glycoproteins, fucose has not been found so far. However, by mass spectrometry we detected N-glycans and O-glycans containing one to six deoxyhexose residues in fruit bodies of several basidiomycetes. The N-glycans of chanterelles (Cantharellus cibarius) contained a deoxyhexose chromatographically identical to fucose and sensitive to α-L-fucosidase. Analysis of individual glycan species by tandem MS, glycosidase digestion, and finally (1)H NMR revealed the presence of L-fucose in α1,6-linkage to an α1,6-mannose of oligomannosidic N-glycans. The substitution by α1,6-mannose of α1,2-mannosyl residues of the canonical precursor structure was yet another hitherto unknown modification. No indication for the occurrence of yet other modifications, e.g. bisecting N-acetylglucosamine, was seen. Besides fucosylated N-glycans, short O-linked mannan chains substituted with fucose were present on chanterelle proteins. Although undiscovered so far, L-fucose appears to represent a prominent feature of protein-linked glycans in the fungal kingdom.     

Beta-N-acetylhexosaminidases HEXO1 and HEXO3 are responsible for the formation of paucimannosidic N-glycans in Arabidopsis thaliana

Most plant glycoproteins contain substantial amounts of paucimannosidic N-glycans instead of their direct biosynthetic precursors, complex N-glycans with terminal N-acetylglucosamine residues. We now demonstrate that two β-N-acetylhexosaminidases (HEXO1 and HEXO3) residing in different subcellular compartments jointly account for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. Total N-glycan analysis of hexo knock-out plants revealed that HEXO1 and HEXO3 contribute equally to the production of paucimannosidic N-glycans in roots, whereas N-glycan processing in leaves depends more heavily on HEXO3 than on HEXO1. Because hexo1 hexo3 double mutants do not display any obvious phenotype even upon exposure to different forms of abiotic or biotic stress, it should be feasible to improve the quality of glycoprotein therapeutics produced in plants by down-regulation of endogenous β-N-acetylhexosaminidase activities.     

Nucleotide variation at the myrosinase-encoding locus, TGG1, and quantitative myrosinase enzyme activity variation in Arabidopsis thaliana

The Arabidopsis thaliana TGG1 gene encodes thioglucoside glucohydrolase (myrosinase), an enzyme catalysing the hydrolysis of glucosinolate compounds. The enzyme is involved in plant defence against some insect herbivores, and is present in species of the order Capparales (Brassicales). Nucleotide variation was surveyed by sequencing c. 2.4 kb of the TGG1 locus in a sample of 28 worldwide A. thaliana accessions, and one Arabidopsis lyrata ssp. lyrata individual. Myrosinase activity was quantified for 27 of these same A. thaliana accessions, plus five additional others. Overall, estimated nucleotide diversity in A. thaliana was low compared to other published A. thaliana surveys, and the frequency distribution was skewed toward an excess of low-frequency variants. Furthermore, comparison to the outgroup species A. lyrata demonstrated that A. thaliana exhibited an excess of high-frequency derived variants relative to a neutral equilibrium model, suggesting a selective sweep. A. thaliana accessions differed significantly in total myrosinase activity, but analysis of variance detected no statistical evidence for an association between quantitative enzyme activity and alleles at the TGG1 myrosinase-encoding locus. We thus conclude that other, unsurveyed factors primarily affect the observed myrosinase activity levels in this species. The pattern of nucleotide variation was consistent with a model of positive selection but might also be compatible with a completely neutral model that takes into account the metapopulation behaviour of this highly inbreeding species which experienced a relatively recent worldwide expansion.     

A functional OGG1 homologue from Arabidopsis thaliana

One of the major mutagenic base lesions in DNA caused by exposure to reactive oxygen species is 7,8-dihydro-8-oxoguanine (8-oxoG). Genes coding for DNA repair enzymes that recognise 8-oxoG have been reported in bacteria, yeast, mammals and plants. The prokaryotic and eukaryotic genes are functional homologues but differ in their primary sequence. We have cloned, sequenced, and expressed a new Arabidopsis thaliana cDNA that shows sequence homology to the eukaryotic genes coding for 8-oxoG DNA N-glycosylases (OGG1). The 40.3-kDa enzyme it encodes (AtOGG1) introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxoG residue. In addition, AtOGG1 can form a Schiff base with 8-oxoG in the presence of NaBH₄, suggesting that it is a bifunctional DNA N-glycosylase. Furthermore, expression of AtOGG1 in an Escherichia coli strain that is deficient in the repair of 8-oxoG in DNA suppresses its spontaneous-mutator phenotype. Thus, we have demonstrated that AtOGG1 is not only a structural but also a functional eukaryotic OGG1 homologue.     

Peroxidase activity of annexin 1 from Arabidopsis thaliana

On the basis of earlier reports suggesting that annexin A1 from Arabidopsis thaliana (AnnAt1) participates in limiting the excessive levels of reactive oxygen species during oxidative burst in plants, we examined the sensitivity of recombinant AnnAt1 to hydrogen peroxide and its peroxidase activity. Purified recombinant protein remains mostly alpha-helical and binds to lipids in a calcium-dependent manner. Upon oxidation recombinant AnnAt1 exhibits a tendency to form dimers in vitro. AnnAt1 is also sensitive to the presence of reducing agents, suggesting that AnnAt1 is a redox sensor in plant cells. Moreover, using two independent methods we found that AnnAt1 displayed peroxidase activity which is probably related to the presence of a heme-binding domain within AnnAt1, as present in other peroxidases. Indeed, site-directed mutagenesis within this domain resulted in a complete abrogation of the activity of AnnAt1. Furthermore, this activity was found to be sensitive to the phosphorylation state of the protein.     

Solution structure of thioredoxin h1 from Arabidopsis thaliana

Present in virtually every species, thioredoxins catalyze disulfide/dithiol exchange with various substrate proteins. While the human genome contains a single thioredoxin gene, plant thioredoxins are a complex protein family. A total of 19 different thioredoxin genes in six subfamilies has emerged from analysis of the Arabidopsis thaliana genome. Some function specifically in mitochondrial and chloroplast redox signaling processes, but target substrates for a group of eight thioredoxin proteins comprising the h subfamily are largely uncharacterized. In the course of a structural genomics effort directed at the recently completed A. thaliana genome, we determined the structure of thioredoxin h1 (At3g51030.1) in the oxidized state. The structure, defined by 1637 NMR-derived distance and torsion angle constraints, displays the conserved thioredoxin fold, consisting of a five-stranded beta-sheet flanked by four helices. Redox-dependent chemical shift perturbations mapped primarily to the conserved WCGPC active-site sequence and other nearby residues, but distant regions of the C-terminal helix were also affected by reduction of the active-site disulfide. Comparisons of the oxidized A. thaliana thioredoxin h1 structure with an h-type thioredoxin from poplar in the reduced state revealed structural differences in the C-terminal helix but no major changes in the active site conformation.     

Molecular cloning and functional expression of beta1, 2-xylosyltransferase cDNA from Arabidopsis thaliana

The transfer of xylose from UDP-xylose to the core beta-linked mannose of N-linked oligosaccharides by beta1,2-xylosyltransferase (XylT) is a widespread feature of plant glycoproteins which renders them immunogenic and allergenic in man. Here, we report the isolation of the Arabidopsis thaliana XylT gene, which contains two introns and encodes a 60.2 kDa protein with a predicted type II transmembrane protein topology typical for Golgi glycosyltransferases. Upon expression of A. thaliana XylT cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited XylT activity in vitro. Furthermore, the recombinant enzyme displayed XylT activity in vivo in the insect cells, as judged by the acquired cross-reaction of cellular glycoproteins with antibodies against the beta1,2-xylose epitope. The cloned XylT cDNA as well as the recombinant enzyme are essential tools to study the role of beta1,2-xylose in the immunogenicity and allergenicity of plant glycoproteins at the molecular level.     

Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth

Late embryogenesis abundant (LEA) proteins constitute a set of proteins widespread in the plant kingdom that show common physicochemical properties such as high hydrophilicity and high content of small amino acid residues such as glycine, alanine, and serine. Typically, these proteins accumulate in response to water deficit conditions imposed by the environment or during plant normal development. In this work, we show that the over-expression in Escherichia coli of proteins of the LEA 2 and the LEA 4 families from Arabidopsis thaliana leads to inhibition of bacterial growth and that this effect is dependent on discrete regions of the proteins. Our data indicate that their antimicrobial effect is achieved through their interaction with intracellular targets. The relevance of the cationic nature and the predicted structural organization of particular protein domains in this detrimental effect on the bacteria growth process is discussed.     

The carotenase AtCCD1 from Arabidopsis thaliana is a dioxygenase

Apocarotenoids resulting from the oxidative cleavage of carotenoids serve as important signaling and accessory molecules in a variety of biological processes. The enzymes catalyzing these reactions are referred to as carotenases or carotenoid oxygenases. Whether they act according to a monooxygenase mechanism, requiring two oxygens from different sources, or a dioxygenase mechanism is still a topic of controversy. In this study, we utilized the readily available beta-apo-8'-carotenal as a substrate for the heterologously expressed AtCCD1 protein from Arabidopsis thaliana to investigate the oxidative cleavage mechanism of the 9,10 double bond of carotenoids. Beta-ionone and a C(17)-dialdehyde were detected as products by gas and liquid chromatography-mass spectrometry as well as NMR analysis. Labeling experiments using H(2)(18)O or (18) O(2) showed that the oxygen in the keto-group of beta-ionone is derived solely from molecular dioxygen. When experiments were performed in an (18)O(2)-enriched atmosphere, a substantial fraction of the C(17)-dialdehyde contained labeled oxygen. The results unambiguously demonstrate a dioxygenase mechanism for the carotenase AtCCD1 from A. thaliana.     

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: K_m (Ala)=1.53 mM, K_m (2-oxoglutarate)=0.18 mM, k_cat=124.6 s⁻¹, k_cat/K_m=8.1 × 10⁴ M⁻¹·s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated K_m values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.     

Role of oligomannosidic N-glycans in the proliferation,adhesion and signalling of C6 glioblastoma cells

The potential role of glycoprotein N-glycans in the proliferation and adhesion of C6 glioblastoma cells was investigated using a set of N-glycosylation inhibitors (tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin, swainsonine), and traffic (monensin). It was observed that both the proliferative and adhesive properties of C6 cells were dependent upon the expression at the cell surface of glycoproteins with oligomannosidic and hybrid type N-glycans, whereas the absence of N-glycans (tunicamycin) or the presence of glucosyl-oligomannosides (deoxynojirimycin and castanospermine) and the absence of glycoproteins at the cell surface (monensin) reduced both the proliferative and adhesive properties of C6 cells. Studies of the classical elements of signalling pathways indicated that the different inhibitors have a low impact on tyrosine phosphorylations and oncogene product expression (except the ras oncogene product), except on phosphorylations on other residues. An endogenous soluble lectin (CSL; J. Neurochem. 49 (1987) 1250), specific for oligomannosidic and hybrid type N-glycans, was present and externalised by the cells through a pinching-off of large intracellular vesicles, a mechanism that was not blocked by monensine; in contrast with the externalisation of its glycoprotein ligands. The inhibitory effect of anti-CSL Fab fragments on adhesion indicates that the polyvalent CSL acts as a bridging molecule for a family of surface glycoproteins expressed at the surface of C6 cells. The inhibitory effect of the same Fab fragments on the proliferation indicates that CSL is a mitogen for these cells, possibly involved in clustering its surface glycoprotein ligands. A mechanism for the loss of contact inhibition is discussed based on the over-expression of CSL ligands in C6 glioblastoma cells relative to normal cells.     

Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana.

The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U.mg-1. The native enzyme (approximately 450 kDa) consists of 11-13 subunits (38 kDa). The temperature optimum was determined to be 35 degrees C and a pH optimum of 9 was found. Thus, recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus. The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA. The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases. Aliphatic nitriles are the most effective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile. In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenylpropionitrile is increased by a factor of 330, and the Km value is reduced by a factor of 23. With the exception of fluoro, substituents in the alpha position to the nitrile function completely inhibit the hydrolysis.     

Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana.

Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.