Molecular genetic study of acclimation mechanism to phosphate deprivation stress in photosynthetic euaryote.

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1. Seven phosphate transporter gene homologues (CrPTA1, CrPTA2, CrPTA3, CrPTA4, CrPTB1, CrPTB2, CrPTB3) and one phosphate repressible phosphatase gene (CrAP) were cloned in Chlamydomonas reinhardtii. Among these transporter homologue genes, at least CrPTA1 and CrPTA3 complemented the phosphate uptake phenotype of yeast phosphate transporter deficiency mutant. Southern blotting analysis revealed that there are no more CrPTA homologue gene in Chlamydomonas. Transcriptional regulation of these genes under phosphate limitation condition were examined by real-time RT-PCR. It was revealed that transcription of CrPTA1 and CrPTA3 were dramatically repressed under phosphate limitation condition. Meanwhile that of CrPTB2 and CrAP were drastically induced under the same condition.2. Extensive screening of mutant of Pi-uptake induction were performed by using the fact that relative density of the cells rapidly increase during reabsorption of Pi when Pi was resupplied to P-starved cells. The mutagenized cells were generated by insertional mutagenesis. The cells with different relative density were separated by isopycnic density gradient centrifugation using Percoll. Sixteen independent mutants of Pi-uptake induction were isolated from about one million of independent mutants. Genetic complementation test using PSR1 gene, which was previously characterized as a gene responsible for phosphate starvation acclimation, revealed that at least ten mutants out of sixteen were new class of mutants, and thus suggested to carry a mutation on a regulatory gene of Pi uptake induction previously unknown.