Responses of Chlamydomonas reinhardtii during the transition from P-deficient to P-sufficient growth (the P-overplus response): The roles of the vacuolar transport chaperones and polyphosphate synthesis.

Phosphorus (P) assimilation and polyphosphate (polyP) synthesis were investigated in Chlamydomonas reinhardtii by supplying phosphate (PO ; 10 mg P·L ) to P-depleted cultures of wildtypes, mutants with defects in genes involved in the vacuolar transporter chaperone (VTC) complex, and VTC-complemented strains. Wildtype C. reinhardtii assimilated PO and stored polyP within minutes of adding PO to cultures that were P-deprived, demonstrating that these cells were metabolically primed to assimilate and store PO . In contrast, vtc1 and vtc4 mutant lines assayed under the same conditions never accumulated polyP, and PO assimilation was considerably decreased in comparison with the wildtypes. In addition, to confirm the bioinformatics inferences and previous experimental work that the VTC complex of C. reinhardtii has a polyP polymerase function, these results evidence the influence of polyP synthesis on PO assimilation in C. reinhardtii. RNA-sequencing was carried out on C. reinhardtii cells that were either P-depleted (control) or supplied with PO following P depletion (treatment) in order to identify changes in the levels of mRNAs correlated with the P status of the cells. This analysis showed that the levels of VTC1 and VTC4 transcripts were strongly reduced at 5 and 24 h after the addition of PO to the cells, although polyP granules were continuously synthesized during this 24 h period. These results suggest that the VTC complex remains active for at least 24 h after supplying the cells with PO . Further bioassays and sequence analyses suggest that inositol phosphates may control polyP synthesis via binding to the VTC SPX domain.