Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans.
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ABSTRACT:
SUBMITTER: Strasser R
PROVIDER: S-EPMC1134966 | biostudies-other | 2005 Apr
REPOSITORIES: biostudies-other
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Project description:Background and Aim:The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. Methods:In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. Key Results:ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. Conclusions:The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.
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Project description:We investigated the existence of the transgenerational memory of iron (Fe) deficiency stress, in Arabidopsis thaliana. Plants were grown under Fe deficiency/sufficiency, and so were their offspring. The frequency of somatic homologous recombination (SHR) events, of DNA strand breaks as well as the expression of the transcription elongation factor TFIIS-like gene increase when plants are grown under Fe deficiency. However, SHR frequency, DNA strand break events, and TFIIS-like gene expression do not increase further when plants are grown for more than one generation under the same stress, and furthermore, they decrease back to control values within two succeeding generations grown under control conditions, regardless of the Fe deficiency stress history of the mother plants. Seedlings produced from plants grown under Fe deficiency evolve more oxygen than control seedlings, when grown under Fe sufficiency: however, this trait is not associated with any change in the protein profile of the photosynthetic apparatus and is not transmitted to more than one generation. Lastly, plants grown for multiple generations under Fe deficiency produce seeds with greater longevity: however, this trait is not inherited in offspring generations unexposed to stress. These findings suggest the existence of multiple-step control of mechanisms to prevent a genuine and stable transgenerational transmission of Fe deficiency stress memory, with the tightest control on DNA integrity.
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Project description:Many plants synchronize their life cycles in response to changing seasons and initiate flowering under favourable environmental conditions to ensure reproductive success. To confer a robust seasonal response, plants use diverse genetic programmes that integrate environmental and endogenous cues and converge on central floral regulatory hubs. Technological advances have allowed us to understand these complex processes more completely. Here, we review recent progress in our understanding of genetic and molecular mechanisms that control flowering in Arabidopsis thaliana.
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Project description:Studying the interaction between the hemibiotrophic bacterium Pseudomonas syringae pv. tomato DC3000 and Arabidopsis thaliana has shed light onto the various forms of mechanisms plants use to defend themselves against pathogen attack. While a lot of emphasis has been put on investigating changes in protein expression in infected plants, only little information is available on the effect infection plays on the plants N-glycan composition. To close this gap in knowledge, total N-glycans were enriched from P. syringae DC3000-infected and mock treated Arabidopsis seedlings and analyzed via MALDI-TOF-MS. Additionally, fluorescently labelled N-glycans were quantified via HPLC-FLD. N-glycans from infected plants were overall less processed and displayed increased amounts of oligomannosidic N-glycans. As multiple peaks for certain oligomannosidic glycoforms were detected upon separation via liquid chromatography, a porous graphitic carbon (PGC)-analysis was conducted to separate individual N-glycan isomers. Indeed, multiple different N-glycan isomers with masses of two N-acetylhexosamine residues plus 8, 9 or 10 hexoses were detected in the infected plants which were absent in the mock controls. Treatment with jack bean α-mannosidase resulted in incomplete removal of hexoses from these N-glycans, indicating the presence of glucose residues. This hints at the accumulation of misfolded glycoproteins in the infected plants, likely because of endoplasmic reticulum (ER) stress. In addition, poly-hexose structures susceptible to α-amylase treatment were found in the DC3000-infected plants, indicating alterations in starch metabolism due to the infection process.
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Project description:There is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid ?-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, half-maximal inhibitory concentration of isofagomine and thermal stability of the cgl-derived GCase were comparable with those of imiglucerase, a commercially available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose terminated. Additional purification was required to remove ?15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including the use of a null cgl mutant.
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Project description:Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are important for the maintenance of genomic stability. Telomeres were considered as typical heterochromatic regions, but in light of recent results, this view should be reconsidered. Asymmetrically located cytosines in plant telomeric DNA repeats may be substrates for a DNA methyltransferase enzyme and indeed, it was shown that these repeats are methylated. Here, we analyse the methylation of telomeric cytosines and the length of telomeres in Arabidopsis thaliana methylation mutants (met 1-3 and ddm 1-8), and in their wild-type siblings that were germinated in the presence of hypomethylation drugs. Our results show that cytosine methylation in telomeric repeats depends on the activity of MET1 and DDM1 enzymes. Significantly shortened telomeres occur in later generations of methylation mutants as well as in plants germinated in the presence of hypomethylation drugs, and this phenotype is stably transmitted to the next plant generation. A possible role of compromised in vivo telomerase action in the observed telomere shortening is hypothesized based on telomere analysis of hypomethylated telomerase knockout plants. Results are discussed in connection with previous data in this field obtained using different model systems.
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Project description:BACKGROUND:Complex I (CI or NADH:ubiquinone oxidoreductase) deficiency is the most frequent cause of mitochondrial respiratory chain defect. Successful attempts to rescue CI function by introducing an exogenous NADH dehydrogenase, such as the NDI1 from Saccharomyces cerevisiae (ScNDI1), have been reported although with drawbacks related to competition with CI. In contrast to ScNDI1, which is permanently active in yeast naturally devoid of CI, plant alternative NADH dehydrogenases (NDH-2) support the oxidation of NADH only when the CI is metabolically inactive and conceivably when the concentration of matrix NADH exceeds a certain threshold. We therefore explored the feasibility of CI rescue by NDH-2 from Arabidopsis thaliana (At) in human CI defective fibroblasts. RESULTS:We showed that, other than ScNDI1, two different NDH-2 (AtNDA2 and AtNDB4) targeted to the mitochondria were able to rescue CI deficiency and decrease oxidative stress as indicated by a normalization of SOD activity in human CI-defective fibroblasts. We further demonstrated that when expressed in human control fibroblasts, AtNDA2 shows an affinity for NADH oxidation similar to that of CI, thus competing with CI for the oxidation of NADH as opposed to our initial hypothesis. This competition reduced the amount of ATP produced per oxygen atom reduced to water by half in control cells. CONCLUSIONS:In conclusion, despite their promising potential to rescue CI defects, due to a possible competition with remaining CI activity, plant NDH-2 should be regarded with caution as potential therapeutic tools for human mitochondrial diseases.
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Project description:In all eukaryotes N-glycosylation is the most prevalent protein modification of secretory and membrane proteins. Although the N-glycosylation capacity and the individual steps of the N-glycan processing pathway have been well studied in the model plant Arabidopsis thaliana, little attention has been paid to the characterization of the glycosylation status of individual proteins. We report here the structural analysis of all N-glycans present on the endogenous thioglucoside glucohydrolases (myrosinases) TGG1 and TGG2 from A. thaliana. All nine glycosylation sites of TGG1 and all four glycosylation sites of TGG2 are occupied by oligomannosidic structures with Man?GlcNAc? as the major glycoform. Analysis of the oligomannosidic isomers from wild-type plants and mannose trimming deficient mutants by liquid chromatography with porous graphitic carbon and mass spectrometry revealed that the N-glycans from both myrosinases are processed by Golgi-located ?-mannosidases.
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Project description:The minor light-harvesting complexes CP24, CP26, and CP29 have been proposed to play a key role in the zeaxanthin (Zx)-dependent high light-induced regulation (NPQ) of excitation energy in higher plants. To characterize the detailed roles of these minor complexes in NPQ and to determine their specific quenching effects we have studied the ultrafast fluorescence kinetics in knockout (ko) mutants koCP26, koCP29, and the double mutant koCP24/CP26. The data provide detailed insight into the quenching processes and the reorganization of the Photosystem (PS) II supercomplex under quenching conditions. All genotypes showed two NPQ quenching sites. Quenching site Q1 is formed by a light-induced functional detachment of parts of the PSII supercomplex and a pronounced quenching of the detached antenna parts. The antenna remaining bound to the PSII core was also quenched substantially in all genotypes under NPQ conditions (quenching site Q2) as compared with the dark-adapted state. The latter quenching was about equally strong in koCP26 and the koCP24/CP26 mutants as in the WT. Q2 quenching was substantially reduced, however, in koCP29 mutants suggesting a key role for CP29 in the total NPQ. The observed quenching effects in the knockout mutants are complicated by the fact that other minor antenna complexes do compensate in part for the lack of the CP24 and/or CP29 complexes. Their lack also causes some LHCII dissociation already in the dark.
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Project description:A mutant of Arabidopsis thaliana (L.) Heyn. (a small plant in the crucifer family) that lacks glycine decarboxylase activity owing to a recessive nuclear mutation has been isolated on the basis of a growth requirement for high concentrations of atmospheric CO2. Mitochondria isolated from leaves of the mutant did not exhibit glycine-dependent O2 consumption, did not release 14CO2 from [14C]glycine, and did not catalyse the glycine-bicarbonate exchange reaction that is considered to be the first partial reaction associated with glycine cleavage. Photosynthesis in the mutant was decreased after illumination under atmospheric conditions that promote partitioning of carbon into intermediates of the photorespiratory pathway, but was not impaired under non-photorespiratory conditions. Thus glycine decarboxylase activity is not required for any essential function unrelated to photorespiration. The photosynthetic response of the mutant in photorespiratory conditions is probably caused by an increased rate of glyoxylate oxidation, which results from the sequestering of all readily transferable amino groups in a metabolically inactive glycine pool, and by a depletion of intermediates from the photosynthesis cycle. The rate of release of 14CO2 from exogenously applied [14C]glycollate was 14-fold lower in the mutant than in the wild type, suggesting that glycine decarboxylation is the only significant source of photorespiratory CO2.
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