Utilization of nitrate,nitrite and ammonium by Chlamydomonas reinhardii

In phototrophically grown Chlamydomonas cells, ammonium strongly inhibited the utilization of nitrate or nitrite. Under darkness, or in the presence of an uncoupler or inhibitor of the non-cyclic photosynthetic electron flow, the utilization of nitrate, nitrite or ammonium was suppressed. l-Methionine-d,l-sulfoximine (MSX) or azaserine, which blocks the assimilation of ammonium, inhibited the consumption of nitrate, but not nitrite, by the cells. Ammonium produced an immediate inhibition of the permease for nitrate in Chlamydomonas growing with nitrate, while ammonium-grown cells lacked this permease. The synthesis of nitrate-reductase activity was dependent on an active permease. In N-starved Chlamydomonas cells, previously treated with MSX, the permease for nitrate was insensitive to inhibition by ammonium, and a significant amount of nitrate reductase was synthetized. These cells photoproduce ammonium by reducing nitrate. Nitrogen-repleted cells, treated with MSX, actively photoproduced ammonium by reducing nitrite, but not nitrate.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-di-methyl-urea - PCCP Carbonylcyanid-p-trifluoromethoxy-phenylhydrazone - Mops 2-(N-morpholino)propanesulfonic acid - MSX l-Methionine-d,l-sulfoximine     

Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium.

Methanol is an important anaerobic substrate in industrial wastewater treatment and the natural environment. Previous studies indicate that cobalt greatly stimulates methane formation during anaerobic treatment of methanolic wastewaters. To evaluate the effect of cobalt in a mixed culture, a sludge with low background levels of cobalt was cultivated in an upflow anaerobic sludge blanket reactor. Specific inhibitors in batch assays were then utilized to study the effect of cobalt on the growth rate and activity of different microorganisms involved in the anaerobic degradation of methanol. Only methylotrophic methanogens and acetogens were stimulated by cobalt additions, while the other trophic groups utilizing downstream intermediates, H2-CO2 or acetate, were largely unaffected. The optimal concentration of cobalt for the growth and activity of methanol-utilizing methanogens and acetogens was 0.05 mg liter-1. The higher requirement of cobalt is presumably due to the previously reported production of unique corrinoid-containing enzymes (or coenzymes) by direct utilizers of methanol. This distinctly high requirement of cobalt by methylotrophs should be considered during methanolic wastewater treatment. Methylotroph methanogens presented a 60-fold-higher affinity for methanol than acetogens. This result in combination with the fact that acetogens grow slightly faster than methanogens under optimal cobalt conditions indicates that acetogens can outcompete methanogens only when reactor methanol and cobalt concentrations are high, provided enough inorganic carbon is available.     

Antibacterial substances from lichens

Crude extracts of many lichens and some lichen acids have considerable antibacterial activity, suggesting their possible value in chemotherapy and providing some justification for their folk-lore use in some countries against pulmonary disorders.     

Surface Plasmon Enhancement at a Liquid–Metal–Liquid Interface

Herein, we report the first experimental demonstration of surface plasmon enhancement at a liquid–metal–liquid interface using a pseudo-Kretschmann geometry. Pumping gold nanoparticle clusters at the interface of a p-xylene–water mixture, we were able to measure a fluorescence enhancement of three orders of magnitude in Rose Bengal at an excitation wavelength of 532 nm. The observed increase is due to the local electric field enhancement and the reduction of the fluorescence lifetime of dye molecules in the close vicinity of the metal surface. Theoretical modeling using the T-matrix method of the electric field intensity enhancement of emulated surfaces supports the experimental results. This new approach will open a new road for the study of dynamic systems using plasmonics.     

A core of three amino acids at the carboxyl‐terminal region of glutamine synthetase defines its regulation in cyanobacteria

Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein–protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site‐directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C‐terminus of Synechocystis GS. A protein–protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction.     

Larval development and growth ratios of Odonata of the Azores

To fully understand odonate life cycles, it is vital to analyse the patterns of larval growth, which are tightly associated with variations in environmental factors. However, the identification of larval instars is often difficult, especially for early development stages. We hypothesise that pond hydroperiod influences odonate larval growth, and test this idea with novel information about the environmental characteristics of 12 study ponds in Terceira Island (Azores). This study, which represents the first analysis of larval development of Odonata in the Azores, involved determining the instars of development and growth ratios. We measured the morphological characteristics of 898 Odonata larvae, and found that they significantly differed between temporary and permanent ponds. To estimate the larval instars, we used two methods: frequency distribution methods and correlation diagrams between body characteristics. The first method was the most effective, allowing the estimation of 17 instars for Anax imperator, 13 for Sympetrum fonscolombii and 10 for Ischnura hastata, as well as growth ratios between instars for head width, total length, abdomen length, wings and antenna length. Our results also suggest that a combination of the two methods is the most appropriate strategy for estimating the number of instars and growth ratios during larval development.     

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light‐dependent regulation of stromal enzymes by thioredoxin (Trx)‐catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx‐mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol‐dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx‐linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de‐epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox‐controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.     

Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii

Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins that have been described in yeast, animals, and more recently in plants. In this study, we report the molecular characterization of autophagy in the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the carboxyl-terminal conserved glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such as nutrient limitation, oxidative stress, or the accumulation of misfolded proteins in the endoplasmic reticulum caused an increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy activation. Furthermore, rapamycin-mediated inhibition of the Target of Rapamycin signaling pathway, a major regulator of autophagy in eukaryotes, results in identical effects on CrATG8 and a relocalization of this protein in Chlamydomonas cells similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to stress conditions by inducing autophagy via Target of Rapamycin signaling modulation.Protein turnover is essential for the adaptation of cells to variable environmental conditions. Similar to other eukaryotes, plants have developed two distinct mechanisms to regulate protein degradation, a selective ubiquitin/26S proteasome pathway (Vierstra, 2009) and macroautophagy (hereafter referred to as autophagy), a nonselective membrane-trafficking process (Bassham, 2009). During autophagy, a large number of cytosolic components, including entire organelles, organelle fragments, and protein complexes, are enclosed in bulk within a double-membrane structure known as the autophagosome and delivered to the vacuole/lysosome for degradation to recycle needed nutrients or degrade toxic components (Xie and Klionsky, 2007; Nakatogawa et al., 2009). The autophagosomes appear to arise from isolation membranes usually observed in close proximity to the vacuole called the preautophagosomal structure (PAS). These membranes expand and fuse to encircle portions of the cytoplasm, generating an autophagosome that is targeted to the vacuole. The outer membrane of the autophagosome then fuses with the vacuole membrane, and the remaining vesicle, known as the autophagic body, is finally released to the vacuole for its degradation (Xie and Klionsky, 2007).The evolutionary conservation of autophagy among eukaryotes indicates that structural and regulatory components of this cellular process must be also conserved. Accordingly, a significant number of autophagy-related (ATG) genes that participate in autophagy regulation and autophagosome formation have been identified, initially through genetic approaches in yeast and subsequently in higher eukaryotes, including mammals, insects, protozoa, and plants (Bassham et al., 2006; Bassham, 2007; Meijer et al., 2007). In yeast, two protein conjugation systems composed of the ubiquitin-like proteins ATG8 and ATG12 and the three enzymes ATG3, ATG7, and ATG10 play an essential role in autophagosome formation and seem to be conserved through evolution (Geng and Klionsky, 2008). ATG8 becomes modified with the lipid molecule phosphatidylethanolamine (PE) by the consecutive action of the ATG7 and ATG3 enzymes in a process mechanistically similar to ubiquitination (Ichimura et al., 2000). Prior to this modification, ATG8 must be cleaved by the Cys protease ATG4 to expose a C-terminal Gly residue that is conjugated to PE (Kirisako et al., 2000; Kim et al., 2001). ATG12 becomes covalently attached to the ATG5 protein in a conjugation reaction that is catalyzed by ATG7 and ATG10 (Mizushima et al., 1998). ATG8-PE and ATG12-ATG5 conjugates localize to autophagy-related membranes and are required for the initiation and expansion of autophagosomal membrane and hemifusion of this membrane with the vacuolar membrane (Hanada et al., 2007; Nakatogawa et al., 2007, 2009; Fujita et al., 2008; Geng and Klionsky, 2008; Xie et al., 2008).Our understanding of the autophagy process has substantially increased with the development of specific markers for autophagy. In plants, two markers for autophagosomes have been described, the monodansylcadaverine dye and GFP-ATG8 fusion protein (Yoshimoto et al., 2004; Contento et al., 2005; Thompson et al., 2005). As in other species, binding of ATG8 to autophagosomes has been used to monitor autophagy in plants. In contrast to yeast, where a single ATG8 gene is present, plants appear to contain a small gene family with several ATG8 isoforms, suggesting that autophagy is more complex in these photosynthetic organisms. For example, Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) encode nine and five ATG8 genes, respectively (Doelling et al., 2002; Hanaoka et al., 2002; Ketelaar et al., 2004; Chung et al., 2009). However, despite the high complexity of the ATG8-conjugating system in plants, important findings have been recently reported on the molecular characterization of autophagy using ATG8 as an autophagy marker in these organisms. The use of specific markers for autophagy in plants has revealed that this process is active at a basal level under normal growth and is induced upon nitrogen- or carbon-limiting conditions as well as in response to oxidative stress (Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005, 2007; Chung et al., 2009). Reverse genetic approaches have also been applied to a number of Arabidopsis ATG genes using T-DNA insertional mutants or RNA interference in order to investigate the physiological roles of autophagy in plants. The initial characterization of autophagy-deficient plants demonstrated that the ATG system is not essential under nutrient-rich conditions. However, a detailed analysis of these mutants indicated that autophagy is required for the proper response of the plant to nutrient limitation or pathogen infection. Plants lacking the ATG4, ATG5, ATG7, ATG9, or ATG10 gene display premature leaf senescence and are hypersensitive to nitrogen or carbon limitation (Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005; Phillips et al., 2008). Arabidopsis plants with reduced levels of ATG18, which is required for autophagosome formation, are more sensitive to methyl viologen treatment and accumulate high levels of oxidized proteins, demonstrating that autophagic processes participate in the response of the plant to oxidative stress (Xiong et al., 2005, 2007). Plants deficient in the autophagy genes ATG6/Beclin1, ATG3, ATG7, and ATG9 exhibit unrestricted hypersensitive response lesions in response to pathogen infection (Liu et al., 2005; Hofius et al., 2009). These findings implicate autophagy as a prosurvival mechanism to restrict programmed cell death associated with the pathogen-induced hypersensitive response in plants. Arabidopsis ATG6 has also been shown to mediate pollen germination in a manner independent of autophagy (Fujiki et al., 2007).As mentioned above, autophagy is triggered among other factors by a reduction in the availability of nutrients. This starvation signal is transmitted to the autophagic machinery by important regulatory factors, including the Ser/Thr kinases Target of Rapamycin (TOR), ATG1, and SNF1 and the phosphatidylinositol 3-kinase ATG6/Beclin1 (Diaz-Troya et al., 2008b; Bassham, 2009; Cebollero and Reggiori, 2009). TOR has been identified as a negative regulator of autophagy in yeast, mammals, and fruit flies (Diaz-Troya et al., 2008b). The pharmacological inhibition of TOR by rapamycin leads to autophagy activation through a mechanism that requires the activation of the ATG1 kinase (Kamada et al., 2000). It has been recently demonstrated in mammals and fruit flies that a rapamycin-sensitive TOR signaling complex termed TORC1 directly phosphorylates and inhibits the ATG1 kinase and its regulatory protein ATG13 (Chang and Neufeld, 2009; Hosokawa et al., 2009; Jung et al., 2009). These regulatory proteins are conserved in plants, although except for ATG6 (Liu et al., 2005), there is no direct evidence for regulation of autophagy by these signaling pathways.The unicellular green alga Chlamydomonas reinhardtii has been used as a model for the study of important cellular and metabolic processes in photosynthetic organisms (Harris, 2001). More recently, Chlamydomonas has also been proposed as a useful system for the characterization of the TOR signaling pathway in photosynthetic eukaryotes based on the finding that, unlike plants, Chlamydomonas cell growth is sensitive to rapamycin (Crespo et al., 2005; Diaz-Troya et al., 2008a). Treatment of Chlamydomonas cells with rapamycin results in a pronounced increase of vacuole size that resembles autophagy-like processes (Crespo et al., 2005). However, a role of TOR in autophagy regulation could not be demonstrated due to the absence of an autophagy marker in Chlamydomonas. Actually, no studies have been reported on any autophagy-related protein in green algae, despite the high conservation of ATG genes in Chlamydomonas (Diaz-Troya et al., 2008b).This study reports the molecular and cellular characterization of autophagy in the green alga Chlamydomonas. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) may be used as a specific autophagy marker. Nutrient limitation and cell aging trigger an autophagic response that can be traced as an increase at the level of CrATG8, the detection of modified forms of CrATG8, and a change in the cellular localization of this protein. Furthermore, we demonstrate that autophagy is inhibited by a rapamycin-sensitive TOR cascade in Chlamydomonas.     

Glyphosine Inhibits Maize Leaf Phosphoenolpyruvate Carboxylase

Glyphosine [N, N-bis-(phosphonomethyl) glycine] inhibited maizeleaf P-enolpyruvate carboxylase competitively with respect toP-enolpyruvate. The inhibition was dependent on glyphosine concentrationand pH. Glycine, but not glucose-6-phosphate, protected theenzyme from the effect of glyphosine. A related compound, glyphosate[N-(phosphonomethyl) glycine], produced little or no inhibition.P-enolpyruvate carboxylase could be one of the targets of glyphosineaction, causing growth inhibition as reported (Croft, S. M.,C. J. Arntzen, L. N. Vanderhoef and C. S. Zettinger (1974) Biochim.Biophys. Acta 335: 211-217). (Received July 10, 1986; Accepted December 4, 1986)