Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-Omethylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of 18 O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants. Molecular & Cellular

BackgroundCah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions.Results/ConclusionsIn the present work we demonstrate that after transfer to low CO2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5–6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.SignificanceThis is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Chlorella vulgaris strain UAM 101 has been isolated from the effluent of a sugar refinery. This alga requires glucose to achieve maximal growth rate even under light saturating conditions. The growth rate of cultures grown on light + CO2 + glucose (3.16 per day) reaches the sum of those grown on light + CO2 (1.95 per day) and on dark + glucose (1.20 per day). Unlike other Chlorella strains, uptake of glucose (about 2 micromoles per milligram dry weight per hour) was induced to the same extent in the light and dark and was not photosensitive. The rate of dark respiration was not affected by light and was strongly stimulated by the presence of glucose (up to about 40% in 4 hours). The rate of photosynthetic 02 evolution was measured as a function of the CO2 concentration. These experiments were conducted with cells which experienced different concentrations of CO2 or glucose during growth. The maximal photosynthetic rate was inhibited severely by growing the cells in the presence of glucose. A rather small difference in the apparent photosynthetic affinity for extracellular inorganic carbon (from 10-30 micromolar) was found between cells grown under low and high CO2. Growth with glucose induced a reduction in the apparent affinity (45 micromolar) even though cells had not been provided with CO2. Experiments performed at different pH values indicate CO2 as the major carbon species taken from the medium by Chlorella vulgaris UAM 101. We (9) have previously described the heterotrophic potential of the algal population from a sugar refinery wastewater environment. The most abundant alga in this habitat, Chlorella vulgaris UAM 101, exhibited a relatively low rate of growth when cultured photoautotrophically. Its growth rate, however, was strongly stimulated when the medium was supplemented with some of the organic compounds normally present in its habitat.The present paper reports the effect of light on the growth ofthis strain, as well as special characteristics ofglucose uptake and photosynthetic and respiratory performance in light that are related to the glucose stimulation of growth. MATERIALS AND METHODS OrganismChlorella vulgaris UAM 101 is a wild strain that we have isolated from the wastewater effluent of a sugar refinery '

Reactive oxygen species (ROS) are metabolic by-products in aerobic organisms including plants. Endogenously produced ROS act as cellular messengers and redox regulators involved in several plant biological processes, but excessive accumulation of ROS cause oxidative stress and cell damage. Understanding ROS signalling and stress responses requires precise imaging and quantification of local, subcellular and global ROS dynamics with high selectivity, sensitivity, and spatiotemporal resolution. Several fluorescent vital dyes have been tested so far, which helped to provide relevant spatially resolved information of oxidative stress dynamics in plants subjected to harmful environmental conditions. However, certain plant characteristics, such as high background fluorescence of plant tissues in vivo and antioxidant mechanisms, can interfere with ROS detection. The development of improved small-molecule fluorescent dyes and protein-based ROS sensors targeted to subcellular compartments will enable in vivo monitoring of ROS and redox changes in photosynthetic organisms.

No abstract