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.

The timing of GIGANTEA expression during day/night cycles varies with the geographical origin of Arabidopsis accessions.

Latitudinal clines in circadian rhythms have consistently been described in various plant species, with the most recent examples appearing in soybean cultivars and in monkey flower natural populations. These latitudinal clines provide evidence that natural variation in circadian rhythms is adaptive, but it is still unclear what adaptive benefits this variation confers, particularly because circadian rhythms are not usually measured in day/night conditions that reflect those experienced by organisms in nature. Here, we report that daily rhythms of GIGANTEA expression respond to day length in a way that depends on the latitude of origin of Arabidopsis accessions. We additionally extend previous findings by confirming that natural variation in GI expression affects growth related traits, and alters the expression of different target genes. The results support the idea that natural variation in daily rhythms of expression have broad effects on plant development and are of potential adaptive value.

The Root Growth-Regulating Brevicompanine Natural Products Modulate the Plant Circadian Clock.

Plant growth regulating properties of brevicompanines (Brvs), natural products of the fungus Penicillium brevicompactum, have been known for several years, but further investigations into the molecular mechanism of their bioactivity have not been performed. Following chemical synthesis of brevicompanine derivatives, we studied their activity in the model plant Arabidopsis by a combination of plant growth assays, transcriptional profiling, and numerous additional bioassays. These studies demonstrated that brevicompanines cause transcriptional misregulation of core components of the circadian clock, whereas other biological read-outs were not affected. Brevicompanines thus represent promising chemical tools for investigating the regulation of the plant circadian clock. In addition, our study also illustrates the potential of an unbiased -omics-based characterization of bioactive compounds for identifying the often cryptic modes of action of small molecules.

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.

A Luciferase-Based Assay to Test Whether Gene Expression Responses to Environmental Inputs Are Temporally Restricted by the Circadian Clock.

Gating is the mechanism by which the influence of an environmental signal on a particular output is temporally restricted by the circadian clock, so that the maximum response of the output to the signal occurs at a specific time. Gated regulation mechanisms have been described for several genes whose expression is strongly induced by light or temperature at certain times but repressed by the circadian clock at others. To reveal a gated pattern of expression in response to light, light pulses are applied in the dark at different times of the 24 h cycle and the transcriptional response of the gene of interest is then monitored with an appropriate technique. Luciferase (LUC) reporters have been the method of choice to study circadian rhythms in the past decades, but this methodology also provides an ideal platform for performing a gating assay. In this chapter, we describe a LUC imaging based protocol designed to test whether the influence of light on the expression of a gene of interest is gated by the circadian clock.

Evolution of CONSTANS Regulation and Function after Gene Duplication Produced a Photoperiodic Flowering Switch in the Brassicaceae.

Environmental control of flowering allows plant reproduction to occur under optimal conditions and facilitates adaptation to different locations. At high latitude, flowering of many plants is controlled by seasonal changes in day length. The photoperiodic flowering pathway confers this response in the Brassicaceae, which colonized temperate latitudes after divergence from the Cleomaceae, their subtropical sister family. The CONSTANS (CO) transcription factor of Arabidopsis thaliana, a member of the Brassicaceae, is central to the photoperiodic flowering response and shows characteristic patterns of transcription required for day-length sensing. CO is believed to be widely conserved among flowering plants; however, we show that it arose after gene duplication at the root of the Brassicaceae followed by divergence of transcriptional regulation and protein function. CO has two close homologs, CONSTANS-LIKE1 (COL1) and COL2, which are related to CO by tandem duplication and whole-genome duplication, respectively. The single CO homolog present in the Cleomaceae shows transcriptional and functional features similar to those of COL1 and COL2, suggesting that these were ancestral. We detect cis-regulatory and codon changes characteristic of CO and use transgenic assays to demonstrate their significance in the day-length-dependent activation of the CO target gene FLOWERING LOCUS T. Thus, the function of CO as a potent photoperiodic flowering switch evolved in the Brassicaceae after gene duplication. The origin of CO may have contributed to the range expansion of the Brassicaceae and suggests that in other families CO genes involved in photoperiodic flowering arose by convergent evolution.

The GI-CDF module of Arabidopsis affects freezing tolerance and growth as well as flowering.

Plants monitor and integrate temperature, photoperiod and light quality signals to respond to continuous changes in their environment. The GIGANTEA (GI) protein is central in diverse signaling pathways, including photoperiodic, sugar and light signaling pathways, stress responses and circadian clock regulation. Previously, GI was shown to activate expression of the key floral regulators CONSTANS (CO) and FLOWERING LOCUS T (FT) by facilitating degradation of a family of CYCLING DOF FACTOR (CDF) transcriptional repressors. However, whether CDFs are implicated in other processes affected by GI remains unclear. We investigated the contribution of the GI-CDF module to traits that depend on GI. Transcriptome profiling indicated that mutations in GI and the CDF genes have antagonistic effects on expression of a wider set of genes than CO and FT, whilst other genes are regulated by GI independently of the CDFs. Detailed expression studies followed by phenotypic assays showed that the CDFs function downstream of GI, influencing responses to freezing temperatures and growth, but are not necessary for proper clock function. Thus GI-mediated regulation of CDFs contributes to several processes in addition to flowering, but is not implicated in all of the traits influenced by GI.

Natural diversity in daily rhythms of gene expression contributes to phenotypic variation.

Daily rhythms of gene expression provide a benefit to most organisms by ensuring that biological processes are activated at the optimal time of day. Although temporal patterns of expression control plant traits of agricultural importance, how natural genetic variation modifies these patterns during the day and how precisely these patterns influence phenotypes is poorly understood. The circadian clock regulates the timing of gene expression, and natural variation in circadian rhythms has been described, but circadian rhythms are measured in artificial continuous conditions that do not reflect the complexity of biologically relevant day/night cycles. By studying transcriptional rhythms of the evening-expressed gene gigantea (GI) at high temporal resolution and during day/night cycles, we show that natural variation in the timing of GI expression occurs mostly under long days in 77 Arabidopsis accessions. This variation is explained by natural alleles that alter light sensitivity of GI, specifically in the evening, and that act at least partly independent of circadian rhythms. Natural alleles induce precise changes in the temporal waveform of GI expression, and these changes have detectable effects on phytochrome interacting factor 4 expression and growth. Our findings provide a paradigm for how natural alleles act within day/night cycles to precisely modify temporal gene expression waveforms and cause phenotypic diversity. Such alleles could confer an advantage by adjusting the activity of temporally regulated processes without severely disrupting the circadian system.

THB1, a truncated hemoglobin, modulates nitric oxide levels and nitrate reductase activity.

Hemoglobins are ubiquitous proteins that sense, store and transport oxygen, but the physiological processes in which they are implicated is currently expanding. Recent examples of previously unknown hemoglobin functions, which include scavenging of the signaling molecule nitric oxide (NO), illustrate how the implication of hemoglobins in different cell signaling processes is only starting to be unraveled. The extent and diversity of the hemoglobin protein family suggest that hemoglobins have diverged and have potentially evolved specialized functions in certain organisms. A unique model organism to study this functional diversity at the cellular level is the green alga Chlamydomonas reinhardtii because, among other reasons, it contains an unusually high number of a particular type of hemoglobins known as truncated hemoglobins (THB1-THB12). Here, we reveal a cell signaling function for a truncated hemoglobin of Chlamydomonas that affects the nitrogen assimilation pathway by simultaneously modulating NO levels and nitrate reductase (NR) activity. First, we found that THB1 and THB2 expression is modulated by the nitrogen source and depends on NIT2, a transcription factor required for nitrate assimilation genes expression. Furthermore, THB1 is highly expressed in the presence of NO and is able to convert NO into nitrate in vitro. Finally, THB1 is maintained on its active and reduced form by NR, and in vivo lower expression of THB1 results in increased NR activity. Thus, THB1 plays a dual role in NO detoxification and in the modulation of NR activity. This mechanism can partly explain how NO inhibits NR post-translationally.

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.

Transcriptional regulation of CDP1 and CYG56 is required for proper NH4+ sensing in Chlamydomonas.

The assimilation of inorganic nitrogen is an essential process for all plant-like organisms. In the presence of ammonium and nitrate as nitrogen sources, Chlamydomonas reinhardtii preferentially assimilates ammonium and represses the nitrate assimilation pathway through an unknown mechanism that in part involves the guanylate cyclase CYG56. It is demonstrated that cells not only respond quantitatively to the NH(4)(+) signal but are also able to sense a balance between both nitrogen sources. This quantitative response was altered in a collection of mutants that were partially insensitive to NH(4)(+). In one of these mutants, reduced function of a gene named CDP1 encoding a cysteine domain-containing protein was genetically linked to NH(4)(+) insensitivity. Alteration of CYG56 or CDP1 transcription was detected in several mutants, and combined down-regulation of both genes seemed to enhance the incapacity to sense NH(4)(+) properly. These results suggest that transcriptional regulation of CYG56 and CDP1 are central and independent steps of the NH(4)(+) signalling pathway.

Plant development goes like clockwork.

The plant circadian clock promotes daily rhythms in the activity of many processes. These rhythms are synchronized to the diurnal day/night cycle by environmental cues such as light and temperature. Output pathways link the clock to particular biological processes, ensuring that they peak in activity at the appropriate times of day or night. Recently, significant progress was made in defining the mechanisms by which output pathways are activated at specific times. Here these issues are emphasized by describing how the clock regulates growth and development throughout the life cycle of Arabidopsis thaliana, including seed germination, seedling growth, stress responses and the transition to flowering. This wide impact of the clock on growth and development appears to provide an advantage by enhancing growth and seed production in different environments.

A soluble guanylate cyclase mediates negative signaling by ammonium on expression of nitrate reductase in Chlamydomonas.

Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in plants.

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA.

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.

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.