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: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: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:The involvement of cytokinins (CK) in biotic stresses has been recognized, while knowledge regarding the effects of CK deficiency on plant response against pathogens is less abundant. Thus, the purpose of this study was to reveal the effects of CK deficiency on proteomics and metabolomic responses of flg22-triggered immunity. We conducted a series of histochemical assays to investigate the activity of the downstream pathways caused by flg22, such as accumulation of ROS, induction of defence genes, and callose deposition, that occurred in Arabidopsis thaliana transgenic lines overexpressing the Hordeum vulgare CKX2 gene (HvCKX2), which are therefore CK-deficient. We also used GC and LC-MS-based technology to quantify variations in stress hormone levels and metabolomic and proteomic responses in flg22-treated HvCKX2 and wild-type Arabidopsis plants. We found that CK deficiency alters the flg22-triggered plant defence response, especially through induction of callose deposition, upregulation of defence response-related proteins, increased amino acid biosynthesis, and regulation of plant photosynthesis. We also indicated that JA might be an important contributor to immune response in plants deficient in CKs. The present study offers new evidence on the fundamental role of endogenous CK in the response to pathogens, as well as the possibility of altering plant biotic tolerance by manipulating CK pools.
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