Complex N-Glycans Are Important for Normal Fruit Ripening and Seed Development in Tomato.

Complex N-glycan modification of secretory glycoproteins in plants is still not well understood. Essential in animals, where a lack of complex N-glycans is embryo-lethal, their presence in plants seemed less relevant for a long time mostly because Arabidopsis thaliana cgl1 mutants lacking N-acetyl-glucosaminyltransferase I (GNTI, the enzyme initiating complex N-glycan maturation in the Golgi apparatus) are viable and showed only minor impairments regarding stress tolerance or development. A different picture emerged when a rice (Oryza sativa) gntI T-DNA mutant was found to be unable to reach the reproductive stage. Here, we report on tomato (Solanum lycopersicum) lines that showed severe impairments upon two RNA interference (RNAi) approaches. Originally created to shed light on the role of core α1,3-fucose and ß1,2-xylose residues in food allergy, plants with strongly reduced GNTI activity developed necrotic fruit-attached stalks and early fruit drop combined with patchy incomplete ripening. Correspondingly, semiquantitative RT-PCR of the abscission zone (az) revealed an increase of abscission markers. Also, GNTI-RNA interference (RNAi) plants were more susceptible to sporadic infection. To obtain vital tomatoes with comparable low allergenic potential, Golgi α-mannosidase II (MANII) was chosen as the second target. The resulting phenotypes were oppositional: MANII-reduced plants carried normal-looking fruits that remained attached for extended time without signs of necrosis. Fruits contained no or only few, but enlarged, seeds. Furthermore, leaves developed rolled-up rims simultaneously during the reproductive stage. Trials to cross MANII-reduced plants failed, while GNTI-reduced plants could be (back-)crossed, retaining their characteristic phenotype. This phenotype could not be overcome by ethephon or indole-3-acetic acid (IAA) application, but the latter was able to mimic patchy fruit ripening in wild-type. Phytohormones measured in leaves and 1-aminocyclopropane-1-carboxylic acid (ACC) contents in fruits showed no significant differences. Together, the findings hint at altered liberation/perception of protein-bound N-glycans, known to trigger auxin-like effects. Concomitantly, semiquantitative RT-PCR analysis revealed differences in auxin-responsive genes, indicating the importance of complex N-glycan modification for hormone signaling/crosstalk. Another possible role of altered glycoprotein life span seems subordinate, as concluded from transient expression of Arabidopsis KORRIGAN KOR1-GFP fusion proteins in RNAi plants of Nicotiana benthamiana. In summary, our analyses stress the importance of complex N-glycan maturation for normal plant responses, especially in fruit-bearing crops like tomato.

Complex-Type N-Glycans Influence the Root Hair Landscape of Arabidopsis Seedlings by Altering the Auxin Output.

Roots supply plants with nutrients and water, besides anchoring them in the soil. The primary root with its lateral roots constitutes the central skeleton of the root system. In particular, root hairs increase the root surface, which is critical for optimizing uptake efficiency. During root-cell growth and development, many proteins that are components of, e.g., the cell wall and plasma membrane are constitutively transported through the secretory system and become posttranslationally modified. Here, the best-studied posttranslational modification is protein N-glycosylation. While alterations in the attachment/modification of N-glycans within the ER lumen results in severe developmental defects, the impact of Golgi-localized complex N-glycan modification, particularly on root development, has not been studied in detail. We report that impairment of complex-type N-glycosylation results in a differential response to synthetic phytohormones with earlier and increased root-hair elongation. Application of either the cytokinin BAP, the auxin NAA, or the ethylene precursor ACC revealed an interaction of auxin with complex N-glycosylation during root-hair development. Especially in gntI mutant seedlings, the early block of complex N-glycan formation resulted in an increased auxin sensitivity. RNA-seq experiments suggest that gntI roots have permanently elevated nutrient-, hypoxia-, and defense-stress responses, which might be a consequence of the altered auxin responsiveness.

Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes.

Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry ß1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-ß1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins.

Reduction of cross-reactive carbohydrate determinants in plant foodstuff: elucidation of clinical relevance and implications for allergy diagnosis.

BACKGROUND: A longstanding debate in allergy is whether or not specific immunoglobulin-E antibodies (sIgE), recognizing cross-reactive carbohydrate determinants (CCD), are able to elicit clinical symptoms. In pollen and food allergy, ≥20% of patients display in-vitro CCD reactivity based on presence of α1,3-fucose and/or ß1,2-xylose residues on N-glycans of plant (xylose/fucose) and insect (fucose) glycoproteins. Because the allergenicity of tomato glycoallergen Lyc e 2 was ascribed to N-glycan chains alone, this study aimed at evaluating clinical relevance of CCD-reduced foodstuff in patients with carbohydrate-specific IgE (CCD-sIgE). METHODOLOGY/PRINCIPAL FINDINGS: Tomato and/or potato plants with stable reduction of Lyc e 2 (tomato) or CCD formation in general were obtained via RNA interference, and gene-silencing was confirmed by immunoblot analyses. Two different CCD-positive patient groups were compared: one with tomato and/or potato food allergy and another with hymenoptera-venom allergy (the latter to distinguish between CCD- and peptide-specific reactions in the food-allergic group). Non-allergic and CCD-negative food-allergic patients served as controls for immunoblot, basophil activation, and ImmunoCAP analyses. Basophil activation tests (BAT) revealed that Lyc e 2 is no key player among other tomato (glyco)allergens. CCD-positive patients showed decreased (re)activity with CCD-reduced foodstuff, most obvious in the hymenoptera venom-allergic but less in the food-allergic group, suggesting that in-vivo reactivity is primarily based on peptide- and not CCD-sIgE. Peptide epitopes remained unaffected in CCD-reduced plants, because CCD-negative patient sera showed reactivity similar to wild-type. In-house-made ImmunoCAPs, applied to investigate feasibility in routine diagnosis, confirmed BAT results at the sIgE level. CONCLUSIONS/SIGNIFICANCE: CCD-positive hymenoptera venom-allergic patients (control group) showed basophil activation despite no allergic symptoms towards tomato and potato. Therefore, this proof-of-principle study demonstrates feasibility of CCD-reduced foodstuff to minimize 'false-positive results' in routine serum tests. Despite confirming low clinical relevance of CCD antibodies, we identified one patient with ambiguous in-vitro results, indicating need for further component-resolved diagnosis.

Comparative analyses of Arabidopsis complex glycan1 mutants and genetic interaction with staurosporin and temperature sensitive3a.

We compare three Arabidopsis (Arabidopsis thaliana) complex glycan1 (cgl1) alleles and report on genetic interaction with staurosporin and temperature sensitive3a (stt3a). STT3a encodes a subunit of oligosaccharyltransferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the endoplasmic reticulum; cgl1 mutants lack N-acetyl-glucosaminyltransferase I activity and are unable to form complex N-glycans in the Golgi apparatus. By studying CGL1-green fluorescent protein fusions in transient assays, we show that the extra N-glycosylation site created by a point mutation in cgl1 C5 is used in planta and interferes with folding of full-length membrane-anchored polypeptides in the endoplasmic reticulum. Tunicamycin treatment or expression in the stt3a-2 mutant relieved the folding block, and migration to Golgi stacks resumed. Complementation tests with C5-green fluorescent protein and other N-glycosylation variants of CGL1 demonstrated that suppression of aberrant N-glycosylation restores activity. Interestingly, CGL1 seems to be functional also as nonglycosylated enzyme. Two other cgl1 alleles showed splicing defects of their transcripts. In cgl1 C6, a point mutation affects the 3' splice site of intron 14, resulting in frame shifts; in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T but suppressed the N-acetyl-glucosaminyltransferase I defect in cgl1 C5. Root growth assays revealed synergistic effects in double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 only. Besides demonstrating the conditional nature of cgl1 C5 in planta, our observations with loss-of-function alleles cgl1 C6 and cgl1-T in the stt3a-2 underglycosylation background prove that correct N-glycosylation is important for normal root growth and morphology in Arabidopsis.