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

Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants

A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas[W]

Nitrate assimilation genes in Chlamydomonas are negatively modulated by ammonium through a pathway that involves NO, cGMP, and calcium. This work shows that similar mechanisms might be operating in plants

Functional Genomics of the Regulation of the Nitrate Assimilation Pathway in Chlamydomonas

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype

Scheme representing the involvement of arginine in the ammonium-<i>cyg56</i> signalling cascade.

<p>Grey circles represent signalling molecules. Dark blue boxes represent proteins, and light blue boxes represent genes. The scheme shows that a rise in intracellular ammonium leads to a rise in arginine and NO through mechanisms that remain to be characterized. NO promotes CYG56 activity by binding to its heme domain, resulting in an increase of cGMP levels that repress <i>NIA1</i> and <i>AMT1</i>.<i>1</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196167#pone.0196167.ref011" target="_blank">11</a>].</p

Application of arginine leads to an increase in DAF-FM DA fluorescence.

<p>Cells of the 704 strain were left to grow for several days in medium containing 8 mM NH₄⁺ until they reached the exponential growth phase. Cells were then washed and transferred to nitrogen-free medium where they were exposed for one hour to different nitrogen sources as indicated on the figure. Background fluorescence due to autofluoresence was determined in a control without DAF-FM DA and was subtracted to the total fluorescence measured in the samples. (<b>A</b>). DAF-FM DA fluorescence measured after exposure to different concentrations of arginine. (<b>B</b>). DAF-FM DA fluorescence measured after exposure to 10 mM of Arginine and 10 mM of NO₂^-. Error bars represent the standard error from at least three independent measurements in (A), and at least seven independent measurements from two experiments in (B). Statistical differences between nitrogen treatments in (B) was determined with a one way ANOVA followed by multiple group comparison with the Tukey test. There was a significant effect of the nitrogen source on DAF-FM DA fluorescence (P<0.001, α = 0.05), and letters indicate the different statistical groups.</p

Arginine represses <i>NIA1</i> expression by acting within the <i>CYG56</i> pathway.

<p>Arylsulfatase (ARS) activity in the presence or absence of arginine was determined in the parental strain 704, and in the <i>cyg56</i> and <i>non1</i> mutants. NIA1::ARS activity was measured after three and four days of growth on solid medium supplied with 4 mM NO₃^– and with different concentrations of arginine as indicated on the figure. (<b>A</b>). Growth control showing that the cells grew normally in the different conditions used in this experiment. (<b>B</b>). ARS test after three days of growth. (<b>C</b>). ARS test after four days of growth. The three strains 704, <i>cyg56</i> and <i>non1</i> bear a copy of the <i>ARS</i> gene fused to the <i>NIA1</i> promoter. ARS activity in the presence of arginine reveals that the <i>NIA1</i> promoter is not fully sensitive to arginine-mediated repression.</p