Characterization of a Mutant Deficient for Ammonium and Nitric Oxide Signalling in the Model System Chlamydomonas reinhardtii

Abstract

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.

Fig 1. Identification of genes co-regulated with CYG56.

(A) Pairwise correlations between CYG56 expression levels and the expression levels of candidate genes for ammonium signalling. The squared correlation coefficient (R²) and the p values were determined with the Pearson test. 1 indicates a perfect correlation, as illustrated by the correlation of CYG56 expression with itself (black bar). 0 indicates the absence of correlation. The three most significant correlations are indicated in dark grey. (B) Scatter plots showing the data distribution of the three most significant correlations detected in (A). The scatter plot showing CYG56 expression levels plotted against 258.90CG expression illustrates a negative result. (C) Expression of candidate genes in the 54.10 mutant. 54.10 was grown in four nitrogen contexts and harvested at four times points per condition (see Methods), and mean relative expression levels were calculated and presented using the same rationale than in a previous report [28]. Each mean was determined with the 16 data points so that it would reflect the general behaviour of a gene in the mutant and be robust to occasional misregulation patterns of a gene in a particular condition. A threefold cut off (shaded area) is used to highlight the most significant misregulation patterns. CYG56 and CDP1 are shown for comparison, and 258.90CG as an illustration of a negative control. The genes analysed for the first time in this study are shown in bold characters.

Fig 2. Phenotype of the 42.49 mutant.

(A) Arylsulfatase (ARS) activity in the parental strain 704 and in the 42.49 mutant after four days on solid medium containing either 4 mM NO₃^– as the sole nitrogen source, or NO₃^– supplemented with NH₄⁺ at the indicated concentrations. Both 704 and 42.49 strains bear a copy of the ARS gene fused to the NIA1 promoter, so that ARS activity in the presence of NH₄⁺ reveals that the promoter is not fully sensitive to NH₄⁺ repression. (B) NIA1 transcript abundance was quantified by qRT PCR in 704 and 42.49 strains after 3 and 6 hours in medium containing 4 mM NO₃^– + 1 mM NH₄⁺. The data were obtained from three technical replicates of two biological samples, and the error bars represent the standard deviation. (C) NR activity was determined in cell extracts of 704 and 42.49 strains in the same conditions than in (B). One mU of enzyme activity corresponds to the reduction of 1 nmol of substrate per minute. These results are representative of three independent biological replicates. (D) The residual concentration of NH₄⁺ that remained in the medium was determined in parallel to the NR activity assay described in (C). (E) In an independent experiment from (B), transcript abundance of NIA1, NRT2.1, AMT1.1, AMT1.2 and NIT2 was determined in 704 (wt) and 42.49 after 6 hours in medium containing NO₃⁻4 mM and NH₄⁺ 1 mM. Results are expressed in % relative to the wild type. The presence of NH₄⁺ was checked but not quantified.

Fig 3. NON1 (candidate gene 42.49CG1) is the gene underlying the AI phenotype of the 42.49 mutant.

(A) Position of the insertion in the 42.49 mutant. The insertion of the pSI104 plasmid took place at position 1775805 on chromosome 16, in the second intron of the NON1 sequence (accession number Cre16.g655050). The two next downstream genes on the chromosome are also represented (accession numbers Cre16.g665100 and Cre16.g655150). Cre16.g665100 corresponds to the second designated candidate gene in this region (42.49CG2). Grey and black boxes indicate exons and UTRs, respectively. The grey arrows mark the start and orientation of the coding sequences. The black arrow indicates the position and orientation of the insert. The grey shaded area starting at the pSI104 position represents the deletion caused by the insertion, and the fading effect illustrates that the right border of the insertion has not been identified. The position of the 7.8 Kb fragment subcloned from BAC 33I20 and used for complementation is represented by a grey line. (B) 42.49CG1 expression was quantified in the wild type strain 704 grown in standard media containing 4 mM of NO₃^- (light grey) or 8 mM of NH₄⁺ (dark grey). Samples were harvested 30 minutes, 1 hour, 3 hours and 24 hours after induction in the two conditions. The means were calculated based on data from three technical replicates of two biological samples. Error bars represent the standard deviation. (C) Complementation of the AI phenotype with the NON1 gene. The 42.49 mutant was transformed with a plasmid containing the NON1 genomic DNA sequence, and NON1 and NIA1 transcripts were quantified by qRT PCR in the selected lines after 6 hours in medium containing NO₃⁻4 mM and NH₄⁺ 1 mM. Various lines were resistant to the antibiotic but did not express NON1 (S2C Fig), and were used as negative controls. The histogram shows mean NIA1 expression levels in positive transformants (n = 7) and negative controls (n = 5). Error bars represent the standard deviation. *** indicates p ≤ 10⁻¹⁵ with a Student t test (α = 0.05). (D) Graphical output of a BLAST analysis highlighting the conservation of the N terminal part of the NON1 protein with proteins of other algae. Sequence ID numbers of proteins from the different organisms are (from top to bottom): XP_002950714.1 (Volvox carteri), XP_005847655.1 (Chlorella variabilis), XP_005849673.1 (Chlorella variabilis), XP_005645512.1 (Coccomyxa subellipsoidea), KIY97900.1 (Monoraphidium neglectum), XP_002501227.1 (Micromonas sp. RCC299), XP_003062310.1 (Micromonas pusilla CCMP1545). Numbers indicate amino acid positions within the respective proteins.

Fig 4. Repression of NIA1 and NRT2.1 in the 42.49 mutant is partially insensitive to treatments with NO donors, A23187 and IBMX.

NIA1 and NRT2.1 transcript levels were quantified by qRT-PCR in the 704 parental and in the 42.49 mutant after treatment with (A) and (B) DEA NONOate, (C) and (D) SNP and FeCN chemical control, (E) A23187, and (F) IBMX. The strains were originally grown in medium containing NH₄⁺ 8 mM as the sole nitrogen source until the cultures reached the exponential growth phase. Cells were then washed and transferred to the induction medium containing NO₃^- 100 μM plus different chemicals at the indicated concentration. The NO₃^- concentration used in this experiment was lower compared to previous experiments because, among other reasons, the chemicals are less potent repressors of gene expression than ammonium. These technical issues are discussed in the Material and Methods section. Samples were harvested 1 hour after treatment. Results are expressed in % relative to the untreated control.

Fig 5. AMT1.2 is highly insensitive to DEA NONOate in 42.49.

Experiments were performed and results treated as in Fig 4. DEA NONOate was applied at the indicated concentrations.