Understanding the ways in which phosphorus metabolism is regulated in photosynthetic eukaryotes is critical for optimizing crop productivity and managing aquatic ecosystems in which phosphorus can be a major source of pollution. Here we describe a gene encoding a regulator of phosphorus metabolism, designated Psr1 (phosphorus starvation response), from a photosynthetic eukaryote. The Psr1 protein is critical for acclimation of the unicellular green alga Chlamydomonas reinhardtii to phosphorus starvation. The N-terminal half of Psr1 contains a region similar to myb DNA-binding domains and the C-terminal half possesses glutamine-rich sequences characteristic of transcriptional activators. The level of Psr1 increases at least 10-fold upon phosphate starvation, and immunocytochemical studies demonstrate that this protein is nuclear-localized under both nutrientreplete and phosphorus-starvation conditions. Finally, Psr1 and angiosperm proteins have domains that are similar, suggesting a possible role for Psr1 homologs in the control of phosphorus metabolism in vascular plants. With the identification of regulators such as Psr1 it may become possible to engineer photosynthetic organisms for more efficient utilization of phosphorus and to establish better practices for the management of agricultural lands and natural ecosystems. P hosphorus (P) is a major component of nucleic acids and phospholipids and is present in the biosphere as the oxidized anion, phosphate (P i ). P i is not easily accessible to most plants and microbes because it forms insoluble precipitates with common cations or is covalently bound to organic molecules (1-3). Crop yields are limited by P availability, and, consequently, P, in the form of P i , is an important component of commercial fertilizers. A considerable proportion of this ''supplementary'' P i is leached from agricultural fields and deposited into aquatic ecosystems, triggering rapid algal proliferation (algal blooms), which leads to eutrophication and fish kills (2). The sustainability of agricultural yields and quality of aquatic ecosystems would benefit from more efficient acquisition and utilization of P. Efficient P utilization by crop plants would decrease our dependence on rock P i reserves, the mining of which has serious economic and ecological consequences (3). Thus, a more complete understanding of P utilization in plants has significant implications with respect to both the environment and world agriculture.P limitation triggers a suite of ''starvation responses'' in most organisms. These responses can be divided into two categories, the P-specific responses and the general responses (4-6). The P-specific responses promote efficient mobilization and acquisition of P from extracellular and intracellular stores (e.g., synthesis and secretion of phosphatases with broad substrate specificity, accumulation of high affinity P i transporters) (5, 7). The general responses allow for long-term survival by coordinating the metabolism of the cell to nutrient availability and growth po...

The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants, but not in eukaryotic algae. To establish a new model for molecular analysis of the circadian clock, here we identified a large number of components of the circadian system in the eukaryotic unicellular alga Chlamydomonas reinhardtii by a systematic forward genetic approach. We isolated 105 insertional mutants that exhibited defects in period, phase angle, and/or amplitude of circadian rhythms in bioluminescence derived from a luciferase reporter gene in their chloroplast genome. Simultaneous measurement of circadian rhythms in bioluminescence and growth rate revealed that some of these mutants had defects in the circadian clock itself, whereas one mutant had a defect in a specific process for the chloroplast bioluminescence rhythm. We identified 30 genes (or gene loci) that would be responsible for rhythm defects in 37 mutants. Classification of these genes revealed that various biological processes are involved in regulation of the chloroplast rhythmicity. Amino acid sequences of six genes that would have crucial roles in the circadian clock revealed features of the Chlamydomonas clock that have both partially plant-like and original components. The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants (Dunlap 1999;Harmer et al. 2001). Despite the striking biochemical features of circadian clocks (e.g., oscillation with long periodicity [∼24 h] and its temperature compensation) (Bünning 1973), their central components are not conserved between these kingdoms (Dunlap 1999;Harmer et al. 2001). To understand the nature of oscillation mechanisms and the evolutionary history of clock components, it is important to understand circadian clock systems of a wide range of organisms.Circadian rhythms of unicellular algae have been studied extensively (Mittag 2001), but no clock component of eukaryotic algae has yet been identified. Chlamydomonas reinhardtii is one of the best-studied algae in circadian rhythm research. A forward genetic approach to identify circadian clock components of Chlamydomonas was started more than three decades ago (Bruce 1970). Although several clock mutants have been isolated (Bruce 1972(Bruce , 1974Mergenhagen 1984), the genes responsible could not be identified because of limitations of tools for genetic analyses. However, since Chlamydomonas is now one of the most attractive model organisms in molecular genetics (Harris 2001), it is possible to re-establish it as a model for studying the molecular mechanism of the circadian clock. For this purpose, we previously developed bioluminescence reporter strains with a codon-optimized luciferase gene in their chloroplast genomes to enable real-time monitoring of circadian rhythms (Breton and Kay 2006;Matsuo et al. 2006).In this study, we screened ∼16,000 insertional mutants for defects in circadian rhythmicity of the chloroplast bioluminescence reporter, isolated 105 mutants, and identified 30 genes (or gene loci...

P-starved plants scavenge inorganic phosphate (Pi) by developing elevated rates of Pi uptake, synthesizing extracellular phosphatases, and secreting organic acids. To elucidate mechanisms controlling these acclimation responses in photosynthetic organisms, we characterized the responses of the green alga Chlamydomonas reinhardtii to P starvation and developed screens for isolating mutants (designated psr [phosphorus-stress response]) abnormal in their responses to environmental levels of Pi. The psr1-1 mutant was identified in a selection for cells that survived exposure to high concentrations of radioactive Pi. psr1-2 and psr2 were isolated as strains with aberrant levels of extracellular phosphatase activity during P-deficient or nutrient-replete growth. The psr1-1 and psr1-2 mutants were phenotypically similar, and the lesions in these strains were recessive and allelic. They exhibited no increase in extracellular phosphatase activity or Pi uptake upon starvation. Furthermore, when placed in medium devoid of P, the psr1 strains lost photosynthetic O 2 evolution and stopped growing more rapidly than wild-type cells; they may not be as efficient as wild-type cells at scavenging/accessing P stores. In contrast, psr2 showed elevated extracellular phosphatase activity during growth in nutrient-replete medium, and the mutation was dominant. The mutant phenotypes and the roles of Psr1 and Psr2 in P-limitation responses are discussed.