Flor Martínez

Suitability of Chlorella vulgaris UAM 101 for heterotrophic biomass production

The dry weight production of mixotrophic (MX), photoheterotrophic (PH) and chemoheterotrophic (QH) batch cultures of Chlorella vulgaris UAM 101 in the presence of glucose is always larger than that of the control cultures grown photoautotrophically (PA). The rates of mitochondrial respiration and of nitrate uptake are strongly stimulated in the three different models of heterotrophic growth. Glucose also promotes a marked increase of photosynthetic O2 evolution of mixotrophic cultures. There is a reduction in the protein content of mixotrophic cells, which is compensated by an increase in lipids and carbohydrates. The enhancement of the main physiological processes in the presence of glucose accounts for the heterotrophic potential of this strain.

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

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-O-methylated 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 18O-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.

Biogenesis and Ultrastructure of Carboxysomes from Wild Type and Mutants of Synechococcus sp. Strain PCC 7942

Immature inclusions representing three progressive steps of carboxysome biogenesis have been identified in Synechococcus during the period of adaptation to low-CO2 conditions: (a) ring-shaped structures, (b) electron-translucent inclusions with the shape of a carboxysome and the internal orderly arrangement of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) molecules, and (c) carboxysomes with an internal electron-translucent area, which seem to be the penultimate stage of carboxysome maturation. The ability to build up normal carboxysomes is impaired in three (M3, EK6, and D4) of four high-carbon-requiring mutants studied in this work. M3 and EK6 exhibit abundant immature electron-translucent carboxysomes but no mature ones. This finding supports the contention that an open reading frame located 7.5 kb upstream of the gene encoding the large subunit of Rubisco (altered in M3) is involved in the carboxysome composition and confirms the structural role of the small subunit of Rubisco (slightly modified in EK6) in the assembly of these structures. D4 shows few typical carboxysomes and frequent immature types, its genetic lesion affecting the apparently unrelated gene encoding a subunit of phosphoribosyl aminoamidazole carboxylase of the purine biosynthesis pathway. Revertants EK20 (EK6) and RK13 (D4) have normal carboxysomes, which means that the restoration of the ability to grow under low CO2 coincides with the proper assembling of these structures. N5, a transport mutant due to the alteration of the gene encoding subunit 2 of NADH dehydrogenase, shows an increase in the number and size of carboxysomes and frequent bar-shaped ones.

Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.

Background Cah3 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/Conclusions In 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. Significance This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Physiological alterations induced by the organophosphorus insecticide trichlorfon in Anabaena PCC 7119 grown with nitrates

Abstract The addition of trichlorfon to nitrate-containing cultures of Anabaena PCC 7119 results in a decrease in the content of all the main nitrogen compounds, and an increase in the carbohydrate fraction per unit dry weight. Photoevolution of O2 per unit chlorophyll is not affected but this activity per unit dry weight is diminished due to a decrease in photosynthetic pigments. Nitrate uptake is also lowered. Cell division and morphology are altered. All these trichlorfon-induced alterations are noticeable from the first 24 hr of treatment but inhibition of growth does not occur until the fourth day.

Effect of trichlorfon on N2-fixing cyanobacteriumAnabaena PCC 7119

The growth ofAnabaena PCC 7119 is affected by the organophosphorus insecticide trichlorfon, the inhibitory effect being dependent on the concentration of insecticide on the culture medium. The inhibition can be related to the unbalanced cell composition of trichlorfon-grown cells that have a decreased amount of phycobiliproteins, chlorophylls, and total proteins and an increased concentration of carbohydrates. An explanation for the lowered content in nitrogen compounds may lie in the inhibitory effect of the insecticide on N2 fixation. Cell cycle is also affected since cell volume increases, irregular divisions occur, and the number of cells per filament is shortened. However heterocysts frequency is not altered. The sensibility ofAnabaena PCC 7119 indicates that trichlorfon may affect negatively natural populations of cyanobacteria.

In vivo ROS and redox potential fluorescent detection in plants: Present approaches and future perspectives.

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.

Effect of aminooxyacetate, an inhibitor blocking the glycolate pathway, on the induction of a CO2-concentrating mechanism and low-CO2-inducible polypeptides in Chlamydomonas reinhardtii (Chlorophyta)

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii when cells are transferred from high (5%) to low (0.03%) CO2 concentrations. The synthesis of these polypeptides is correlated with the induction of the CO2-concentrating mechanism. Interaction between the induction of low-CO2-inducible polypeptides, the CO2-concentrating mechanism and photorespiration has been studied in wild-type C. reinhardtii with the aim of clarifying whether the glycolate pathway is involved in algal acclimation to limiting CO2 conditions. Our results showed that the induction of the 37 kDa periplasmic carbonic anhydrase and 21 kDa polypeptide under low-CO2 conditions was not observed in the presence of aminooxyacetate, an inhibitor which completely blocks glycolate metabolism. However, the induction of the 36 kDa polypeptide was not affected by this inhibitor. The presence of aminooxyacetate during the acclimation to low CO2 conditions also inhibited the increase in the photosynthe...

Chlorophyll a/b ratio and thylakoid stacking modification in response to glucose in chlorella vulgaris UAM 101

Summary Chlorophyll synthesis is not a light-dependent process in Chlorella vulgaris UAM 101. Glucose exerts a negative effect on chlorophyll synthesis that is more severe under photo- and chemoheterotrophic conditions of growth than under mixotrophic ones and in mixotrophic cultures more marked on chlorophyll b than a. The quantitative and qualitative effect of glucose on the main photosynthetic pigments is accompanied by an increase in the degree of stacking of thylakoid membranes in all cases and an increase of plastoglobuli in photoheterotrophic cells.

A 38-kilodalton low-CO2-inducible polypeptide is associated with the pyrenoid in Chlorella vulgaris

Abstract. In the green alga Chlorella vulgaris UAM 101, a CO2-concentrating mechanism (CCM) is induced when cells are transferred from high (5%) to low (0.03%) CO2 concentrations. The induction of the CCM is correlated with de-novo synthesis of several polypeptides that remain to be identified. The internal carbonic anhydrase (CA; EC 4.2.1.1) activity increased 6- to 7-fold within 6 h of acclimation to air. When crude homogenates were further separated into soluble and insoluble fractions, nearly all of the CA activity was associated with the membrane fraction. Immunoblot analysis of cell homogenates probed with antibodies raised against the 37-kDa subunit of periplasmic CA of Chlamydomonas reinhardtii showed a cross-reaction with a single 38-kDa polypeptide in both high- and low-CO2-grown cells. The up-regulation of the expression of the 38-kDa polypeptide was closely correlated with the increase in internal CA activity. Furthermore, its subcellular location was also correlated with the distribution of the activity. Immunoblot analysis of pyrenoid fractions showed that the 38-kDa polypeptide was concentrated in the pyrenoids from low-CO2-grown cells but was not present in pyrenoids from high-CO2-grown cells. In addition, immunogold labeling experiments showed that the protein was mainly associated with membranes crossing the pyrenoid, while it was absent from the pyrenoid matrix. These studies have identified a putative intracellular CA polypeptide associated with the pyrenoid in Chlorella vulgaris, suggesting that this structure may play an important role in the operation of the CCM and the acclimation to low CO2 conditions.