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

Abstract

Complex <i>N</i>-glycan modification of secretory glycoproteins in plants is still not well understood. Essential in animals, where a lack of complex <i>N</i>-glycans is embryo-lethal, their presence in plants seemed less relevant for a long time mostly because <i>Arabidopsis thaliana cgl1</i> mutants lacking <i>N</i>-acetyl-glucosaminyltransferase I (GNTI, the enzyme initiating complex <i>N</i>-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 (<i>Oryza sativa</i>) <i>gntI</i> T-DNA mutant was found to be unable to reach the reproductive stage. Here, we report on tomato (<i>Solanum lycopersicum</i>) lines that showed severe impairments upon two RNA interference (RNAi) approaches. Originally created to shed light on the role of <i>core</i> α1,3-fucose and β1,2-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, <i>GNTI</i>-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 <i>N</i>-glycans, known to trigger auxin-like effects. Concomitantly, semiquantitative RT-PCR analysis revealed differences in auxin-responsive genes, indicating the importance of complex <i>N</i>-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 <i>Nicotiana benthamiana</i>. In summary, our analyses stress the importance of complex <i>N-</i>glycan maturation for normal plant responses, especially in fruit-bearing crops like tomato.