80 research outputs found
NO3- selective mini-electrodes as a tool to investigate the NO3- traffic in Chlamydomonas reinhardtii D.
Ion selective NO3- mini-electrodes were used to measure the external NO3- concentration in C. reinhardtii liquid cultures. Electrodes were prepared using glass capillaries (1.5 mm external diameter). Capillaries were cut in 10 cm long pieces, dehydrated for 45 minutes in an oven and silanized by addition of dimethyldichlorosilane in bencene 0.1% (V/V). Once silanized, the capillaries were baked again for 30 minutes. Once cold the capillaries were backfilled with the NO3- ionophore (Fluka: 72549), which contains PVC (5.75% w/w) dissolved in tetrahydrofurane. Then, the NO3- mini-electrodes were stored in dark in a desiccator until tetrahydrofurane gets evaporated. Before use, NO3- selective mini-electrodes were backfilled with 0.1 M NaNO3 and 0.1 M KCl and connected to a high-impedance differential amplifier (WPI FD223). Mini-electrodes were calibrated in N-free Beijerinck medium, which contains 0.1 mM Cl-. In those conditions, electrodes calibration slope was 54 mV/p NO3- in the range 1 - 1000 µM NO3-. The mini-electrodes were used to continuous monitoring of the external NO3- concentration in liquid culture of different C. reinhardtii strains, incubated in N-free Beijerinck medium supplemented with 100 µM NO3Na. Previous to the assays, strains were N starved for 6 days. In the light, wild type strain uptakes NO3- at a rate of 15 nmol NO3-·106 cells-1·h-1, in the dark this rate was one third of this figure. After 5 h, the external NO3- levelled off at 10 µM in the light and around 30 µM in the dark. C. reinhardtii cells cultured in the presence of 2 mM NO3NH4 do not show significant NO3- uptake nor a mutant strain, defective in nitrate transport and having an active nitrate reductase. However, a mutant strain lacking the nitrate reductase shows an enhanced NO3- uptake rate, compared with the value obtained for the wild type in the light.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech
Impaired calcium sequestration activity in liver microsomes from fasted rats
AbstractCalcium uptake activity was assayed in liver microsomal vesicles from fed and fasted rats. This activity required ATP and was stimulated by the calcium trapping agent oxalate. The most striking feature was the low rate of calcium accumulation in liver microsomes from fasted rats. Maximal rate was inhibited up to 66 and 82% after 1 and 3 days starvation, respectively. This defective microsomal calcium handling suggests its possible involvement in the massive glycogen breakdown during starvation
Algae-Bacteria Consortia as a Strategy to Enhance H2 Production
Biological hydrogen production by microalgae is a potential sustainable, renewable and clean source of energy. However, many barriers limiting photohydrogen production in these microorganisms remain unsolved. In order to explore this potential and make biohydrogen industrially affordable, the unicellular microalga Chlamydomonas reinhardtii is used as a model system to solve barriers and identify new approaches that can improve hydrogen production. Recently, Chlamydomonas–bacteria consortia have opened a new window to improve biohydrogen production. In this study, we review the different consortia that have been successfully employed and analyze the factors that could be behind the improved H2 production
Role of Nitrate Reductase in NO Production in Photosynthetic Eukaryotes
Nitric oxide is a gaseous secondary messenger that is critical for proper cell signaling and plant survival when exposed to stress. Nitric oxide (NO) synthesis in plants, under standard phototrophic oxygenic conditions, has long been a very controversial issue. A few algal strains contain NO synthase (NOS), which appears to be absent in all other algae and land plants. The experimental data have led to the hypothesis that molybdoenzyme nitrate reductase (NR) is the main enzyme responsible for NO production in most plants. Recently, NR was found to be a necessary partner in a dual system that also includes another molybdoenzyme, which was renamed NO-forming nitrite reductase (NOFNiR). This enzyme produces NO independently of the molybdenum center of NR and depends on the NR electron transport chain from NAD(P)H to heme. Under the circumstances in which NR is not present or active, the existence of another NO-forming system that is similar to the NOS system would account for NO production and NO effects. PII protein, which senses and integrates the signals of the C–N balance in the cell, likely has an important role in organizing cell responses. Here, we critically analyze these topics
Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1
The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently
discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction
of several N-hydroxylated compounds (NHC) and nitrite. Some NHC are prodrugs containing an
amidoxime structure or mutagens such as 6-hydroxylaminopurine (HAP). We have studied this
protein in the green alga Chlamydomonas reinhardtii (crARC). Interestingly, all the ARC proteins need
the reducing power supplied by other proteins. It is known that crARC requires a cytochrome b5
(crCytb5-1) and a cytochrome b5 reductase (crCytb5-R) that form an electron transport chain from
NADH to the substrates. Here, we have investigated NHC reduction by crARC, the interaction
with its partners and the function of important conserved amino acids. Interactions among crARC,
crCytb5-1 and crCytb5-R have been studied by size-exclusion chromatography. A protein complex
between crARC, crCytb5-1 and crCytb5-R was identified. Twelve conserved crARC amino acids
have been substituted by alanine by in vitro mutagenesis. We have determined that the amino acids
D182, F210 and R276 are essential for NHC reduction activity, R276 is important and F210 is critical
for the Mo Cofactor chelation. Finally, the crARC C-termini were shown to be involved in protein
aggregation or oligomerizatio
Identification of the MAPK Cascade and its Relationship with Nitrogen Metabolism in the Green Alga Chlamydomonasreinhardtii
The mitogen activated protein kinases (MAPKs) form part of a signaling cascade through phosphorylation reactions conserved in all eukaryotic organisms. The MAPK cascades are mainly composed by threeproteins, MAPKKKs, MAPKKs and MAPKs. Some signals induce MAPKKK-mediated phosphorylation and activation of MAPKK that phosphorylate and activate MAPK. Afterward, MAPKs can act either in the cytoplasm or be imported into the nucleus to activate other proteins or transcription factors. In the green microalga Chlamydomonasreinhardtii the pathway for nitrogen (N) assimilation is well characterized, yet its regulation still has many unknown features. Nitric oxide (NO) is a fundamental signal molecule for N regulation, where nitrate reductase (NR) plays a central role in its synthesis. The MAPK cascades could be regulating N assimilation, since it has been described that the phosphorylation of NR by MAPK6 promotes NO production in Arabidopsis thaliana. We have identified the proteins involved in the MAPK cascades in Chlamydomonasreinhardtii, finding 17 MAPKs, 2 MAPKKs and 108 MAPKKKs (11 MEKK-, 94 RAF- and 3 ZIK-type) that have been structurally and phylogenetically characterized. The genetic expressions of MAPKs and the MAPKK were slightly regulated by N. However, the genetic expressions of MAPKKKs RAF14 and RAF79 showed a very strong repression by ammonium, which suggests that they may have a key role in the regulation of N assimilation, encouraging to further analyze in detail the role of MAPK cascades in the regulation of N metabolism
Chlamydomonas reinhardtii, a Reference Organism to Study Algal–Microbial Interactions: Why Can’t They Be Friends?
The stability and harmony of ecological niches rely on intricate interactions between their members. During evolution, organisms have developed the ability to thrive in different environments, taking advantage of each other. Among these organisms, microalgae are a highly diverse and widely distributed group of major primary producers whose interactions with other organisms play essential roles in their habitats. Understanding the basis of these interactions is crucial to control and exploit these communities for ecological and biotechnological applications. The green microalga Chlamydomonas reinhardtii, a well-established model, is emerging as a model organism for studying a wide variety of microbial interactions with ecological and economic significance. In this review, we unite and discuss current knowledge that points to C. reinhardtii as a model organism for studying microbial interactions
Arginine is a component of the ammonium- CYG56 signalling cascade that represses genes of the nitrogen assimilation pathway in Chlamydomonas reinhardtii
Nitrogen assimilation and metabolism are essential processes for all living organisms, yet
there is still much to be learnt on how they are regulated. The use of Chlamydomonas reinhardtii
as a model system has been instrumental not only in identifying conserved regulation
mechanisms that control the nitrogen assimilation pathway, but also in understanding how
the intracellular nitrogen status regulates metabolic processes of industrial interest such as
the synthesis of biolipids. While the genetic regulators that control the nitrogen pathway are
successfully being unravelled, other layers of regulation have received less attention. Amino
acids, for example, regulate nitrogen assimilation in certain organisms, but their role in Chlamydomonas
has not thoroughly been explored. Previous results had suggested that arginine
might repress key genes of the nitrogen assimilation pathway by acting within the
ammonium negative signalling cascade, upstream of the nitric oxide (NO) inducible guanylate
cyclase CYG56. We tested this hypothesis with a combination of genetic and chemical
approaches. Antagonising the effects of arginine with an arginine biosynthesis mutant or
with two chemical analogues released gene expression from ammonium mediated repression.
The cyg56 and related non1 mutants, which are partially insensitive to ammonium
repression, were also partially insensitive to repression by arginine. Finally, we show that
the addition of arginine to the medium leads to an increase in intracellular NO. Our data
reveal that arginine acts as a negative signal for the assimilation of nitrogen within the
ammonium-CYG56 negative signalling cascade, and provide a connection between amino
acid metabolism and nitrogen assimilation in microalgae
Characterization of a Mutant Deficient for Ammonium and Nitric Oxide Signalling in the Model System Chlamydomonas reinhardtii
The ubiquitous signalling molecule Nitric Oxide (NO) is characterized not only by the variety
of organisms in which it has been described, but also by the wealth of biological processes
that it regulates. In contrast to the expanding repertoire of functions assigned to NO, however,
the mechanisms of NO action usually remain unresolved, and genes that work within
NO signalling cascades are seldom identified. A recent addition to the list of known NO functions
is the regulation of the nitrogen assimilation pathway in the unicellular alga Chlamydomonas
reinhardtii, a well-established model organism for genetic and molecular studies that
offers new possibilities in the search for mediators of NO signalling. By further exploiting a
collection of Chlamydomonas insertional mutant strains originally isolated for their insensitivity
to the ammonium (NH4
+) nitrogen source, we found a mutant which, in addition to its
ammonium insensitive (AI) phenotype, was not capable of correctly sensing the NO signal.
Similarly to what had previously been described in the AI strain cyg56, the expression of
nitrogen assimilation genes in the mutant did not properly respond to treatments with various
NO donors. Complementation experiments showed that NON1 (NO Nitrate 1), a gene
that encodes a protein containing no known functional domain, was the gene underlying the
mutant phenotype. Beyond the identification of NON1, our findings broadly demonstrate the
potential for Chlamydomonas reinhardtii to be used as a model system in the search for
novel components of gene networks that mediate physiological responses to NO
From the Eukaryotic Molybdenum Cofactor Biosynthesis to the Moonlighting Enzyme mARC
All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue
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