A.R. van der Krol

Identification of the Gene Encoding the α1,3-Mannosyltransferase (ALG3) in Arabidopsis and Characterization of Downstream N-Glycan Processing

Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a mannosyltransferase gene from Arabidopsis thaliana, which is the functional homolog of the ALG3 (Dol-P-Man:Man5GlcNAc2-PP-Dol α1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a Δalg3 yeast mutant and is localized to the endoplasmic reticulum in yeast and in plants. A homozygous T-DNA insertion mutant, alg3-2, was identified in Arabidopsis with residual levels of wild-type ALG3, derived from incidental splicing of the 11th intron carrying the T-DNAs. N-glycan analysis of alg3-2 and alg3-2 in the complex-glycan-less mutant background, which lacks N-acetylglucosaminyl-transferase I activity, reveals that when ALG3 activity is strongly reduced, almost all N-glycans transferred to proteins are aberrant, indicating that the Arabidopsis oligosaccharide transferase complex is remarkably substrate tolerant. In alg3-2 plants, the aberrant glycans on glycoproteins are recognized by endogenous mannosidase I and N-acetylglucosaminyltransferase I and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3-2 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response.

Predicting sub-Golgi localization of type II membrane proteins

Abstract Motivation: Recent research underlines the importance of finegrained knowledge on protein localization. In particular, subcompartmental localization in the Golgi apparatus is important, for example, for the order of reactions performed in glycosylation pathways or the sorting functions of SNAREs, but is currently poorly understood. Results: We assemble a dataset of type II transmembrane proteins with experimentally determined sub-Golgi localizations and use this information to develop a predictor based on the transmembrane domain of these proteins, making use of a dedicated proteinstructure based kernel in an SVM. Various applications demonstrate the power of our approach. In particular, comparison with a large set of glycan structures illustrates the applicability of our predictions on a ‘glycomic’ scale and demonstrates a significant correlation between sub-Golgi localization and the ordering of different steps in glycan biosynthesis. Contact: roeland.vanham@wur.nl Supplementary information: Supplementary data are available at Bioinformatics online.

Antiphase Light and Temperature Cycles Affect PHYTOCHROME B-Controlled Ethylene Sensitivity and Biosynthesis, Limiting Leaf Movement and Growth of Arabidopsis

Light and temperature cycles influence the amplitude and phase of diurnal leaf movement rhythms by regulating ethylene biosynthesis in the petiole. In the natural environment, days are generally warmer than the night, resulting in a positive day/night temperature difference (+DIF). Plants have adapted to these conditions, and when exposed to antiphase light and temperature cycles (cold photoperiod/warm night [−DIF]), most species exhibit reduced elongation growth. To study the physiological mechanism of how light and temperature cycles affect plant growth, we used infrared imaging to dissect growth dynamics under +DIF and −DIF in the model plant Arabidopsis (Arabidopsis thaliana). We found that −DIF altered leaf growth patterns, decreasing the amplitude and delaying the phase of leaf movement. Ethylene application restored leaf growth in −DIF conditions, and constitutive ethylene signaling mutants maintain robust leaf movement amplitudes under −DIF, indicating that ethylene signaling becomes limiting under these conditions. In response to −DIF, the phase of ethylene emission advanced 2 h, but total ethylene emission was not reduced. However, expression analysis on members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase ethylene biosynthesis gene family showed that ACS2 activity is specifically suppressed in the petiole region under −DIF conditions. Indeed, petioles of plants under −DIF had reduced ACC content, and application of ACC to the petiole restored leaf growth patterns. Moreover, acs2 mutants displayed reduced leaf movement under +DIF, similar to wild-type plants under −DIF. In addition, we demonstrate that the photoreceptor PHYTOCHROME B restricts ethylene biosynthesis and constrains the −DIF-induced phase shift in rhythmic growth. Our findings provide a mechanistic insight into how fluctuating temperature cycles regulate plant growth.

Capturing hormonal and light interactions in a simulation model of shoot branching

Shoot branching, i.e. axillary bud break and subsequent branch outgrowth, is a key determinant of overall aboveground plant form. During plant development, the number of branches formed strongly influences the amount of light absorbed by the plant, and thus the plants competitive strength in terms of light capture in relation to neighbouring plants. Shoot branching is regulated by genetic and physiological factors such as hormones and assimilates. In turn, these internal factors are modulated by environmental signals such as light intensity, light spectral composition and nutrients. The internal and environmental factors are part of a complex feedback system that operates at several levels of biological organization. Here, we present a modelling approach that captures the interactions between hormones and light in the case of shoot branching. We simulated Arabidopsis individually and in a plot at different ratios of ambient red and far-red light (R:FR). A low R:FR resulted in low branching. The individual plants experienced slightly higher R:FR values and produced more branches as a result, depending on ambient R:FR. The model serves a useful scientific tool to integrate knowledge and to improve experimental approaches on shoot branching.