Integration of Chloroplast Nucleic Acid Metabolism into the Phosphate Deprivation Response in Chlamydomonas reinhardtii

Abstract

Cell survival depends on the cell's ability to acclimate to phosphorus (P) limitation. We studied the chloroplast ribonuclease polynucleotide phosphorylase (PNPase), which consumes and generates phosphate, by comparing wild-type Chlamydomonas reinhardtii cells with strains with reduced PNPase expression. In the wild type, chloroplast RNA (cpRNA) accumulates under P limitation, correlating with reduced PNPase expression. PNPase-deficient strains do not exhibit cpRNA variation under these conditions, suggesting that in the wild type PNPase limits cpRNA accumulation under P stress. PNPase levels appear to be mediated by the P response regulator PHOSPHORUS STARVATION RESPONSE1 (PSR1), because in psr1 mutant cells, cpRNA declines under P limitation and PNPase expression is not reduced. PNPase-deficient cells begin to lose viability after 24 h of P depletion, suggesting that PNPase is important for cellular acclimation. PNPasedeficient strains do not have enhanced sensitivity to other physiological or nutrient stresses, and their RNA and cell growth phenotypes are not observed under P stress with phosphite, a phosphate analog that blocks the stress signal. In contrast with RNA metabolism, chloroplast DNA (cpDNA) levels declined under P deprivation, suggesting that P mobilization occurs from DNA rather than RNA. This unusual phenomenon, which is phosphite-and PSR1-insensitive, may have evolved as a result of the polyploid nature of cpDNA and the requirement of P for cpRNA degradation by PNPase.

Polyadenylation in Arabidopsis and Chlamydomonas organelles: the input of nucleotidyltransferases, poly(A) polymerases and polynucleotide phosphorylase

“…However, subsequent isolation and analysis of longer tails revealed that although rich in adenosines, most of the tails contained other nucleotides, suggesting that the tails in Chlamydomonas chloroplasts are often heteropolymeric (see below and Figure S3). As mentioned earlier, Chlamydomonas appears to encode a single, chloroplast PNPase (Yehudai-Resheff et al, 2007), although a dual targeting to mitochondria has not been excluded. In order to analyze the possible involvement of PNPase and CrPAP4 in the polyadenylation of chloroplast or mitochondrial transcripts, we used RNAi to reduce expression of these enzymes and analyzed the tail composition in the resultant strains.…”

“…In order to analyze the possible involvement of PNPase and CrPAP4 in the polyadenylation of chloroplast or mitochondrial transcripts, we used RNAi to reduce expression of these enzymes and analyzed the tail composition in the resultant strains. The generation of a stable PNPasedepleted strain PNPi3 (also called ip3; with a reduction in the protein amount of approximately 90%) has been previously described (Yehudai-Resheff et al, 2007), and an analogous strain, PAP4i, was obtained for CrPAP4 ( Figures S1 and S2).…”

“…Chlamydomonas transformants of the recipient strain CC-849, generated by electroporation (Shimogawara et al, 1998), were selected on TAP medium containing 12 lM 5-fluoroindole and 1.5 mM tryptophan (Yehudai-Resheff et al, 2007).…”

“…In addition, genes encoding several members of the Ntr family were identified, and the localizations and activities of the proteins, either polyadenylation or tRNA 3¢ end addition/correction, were predicted (Martin and Keller, 2004). There is, however, only one PNPase-encoding gene in Chlamydomonas, and available evidence suggests that the protein is localized to the chloroplast (Yehudai-Resheff et al, 2007). There are also several Ntr proteins encoded in Chlamydomonas, the functions of which remain to be determined (Zimmer et al, 2008).…”

Tissue-specific organelle DNA degradation mediated by DPD1 exonuclease

“…6,7 Under phosphate-limiting conditions, chloroplast DNA was reportedly degraded in Chlamydomonas, possibly as a compensation mechanism for survival. 8 Furthermore, the characterization of plastidial uracil phosphoribosyltransferases showed that plastid functions as the…”