Genome-based approaches to understanding phosphorus deprivation responses and PSR1 control in Chlamydomonas reinhardtii

Abstract

The Chlamydomonas reinhardtii transcription factor PSR1 is required for the control of activities involved in scavenging phosphate from the environment during periods of phosphorus limitation. Increased scavenging activity reflects the development of high-affinity phosphate transport and the expression of extracellular phosphatases that can cleave phosphate from organic compounds in the environment. A comparison of gene expression patterns using microarray analyses and quantitative PCRs with wild-type and psr1 mutant cells deprived of phosphorus has revealed that PSR1 also controls genes encoding proteins with potential "electron valve" functions--these proteins can serve as alternative electron acceptors that help prevent photodamage caused by overexcitation of the photosynthetic electron transport system. In accordance with this finding, phosphorus-starved psr1 mutants die when subjected to elevated light intensities; at these intensities, the wild-type cells still exhibit rapid growth. Acclimation to phosphorus deprivation also involves a reduction in the levels of transcripts encoding proteins involved in photosynthesis and both cytoplasmic and chloroplast translation as well as an increase in the levels of transcripts encoding stress-associated chaperones and proteases. Surprisingly, phosphorus-deficient psr1 cells (but not wild-type cells) also display expression patterns associated with specific responses to sulfur deprivation, suggesting a hitherto unsuspected link between the signal transduction pathways involved in controlling phosphorus and sulfur starvation responses. Together, these results demonstrate that PSR1 is critical for the survival of cells under conditions of suboptimal phosphorus availability and that it plays a key role in controlling both scavenging responses and the ability of the cell to manage excess absorbed excitation energy.

FIG. 1.

Genes that exhibit altered transcript abundance during P deprivation. Proportional Venn diagrams representing genes in microarray experiments with altered transcript levels during P starvation are shown. The areas of the circles and of the overlapping regions are directly proportional to the numbers of genes represented. The total number of genes in both the wild type and the psr1-1 strain that reached or surpassed the threshold ratio is shown above each overlapping diagram; the numbers below each diagram distinguish the genes altered in wild-type versus psr1-1 cells. (A) Transcript levels altered by ≥2-fold; (B) transcript levels altered by ≥2.5-fold; (C) transcript levels altered by ≥3-fold.

FIG. 2.

Amino acid alignment of V. carteri and C. reinhardtii PHOX polypeptides. The diagram shows a CLUSTALW alignment of V. carteri PHOX (accession no. CAA10030) and the predicted polypeptide encoded by gene model fgenesh1_pg.C_scaffold_86000013, corresponding to the potential PHOX homolog deduced from the C. reinhardtii draft genome sequence. Black shading denotes sequence identity, and conservative amino acid changes are shaded gray. Gaps introduced by the alignment program are represented by dots, while dashes indicate a gap in the C. reinhardtii PHOX polypeptide sequence that is due to a missing nucleotide sequence on the genomic scaffold.

FIG. 3.

P_i transporter type B paralogs. (A) Schematic representation of the arrangement of PTB genes in the C. reinhardtii genome. The direction of transcription is indicated by the direction of the arrows. Coding sequences are represented by light gray boxes or arrows, the connecting lines correspond to introns, and dark gray boxes or arrows correspond to the 5′ and 3′ untranslated regions of each gene. The gene models for PTB1 and PTB2 are based on BLASTN alignments with the cDNA sequences (accession no. AB074880 and AB074881, respectively); the other gene models were assembled using a combination of GreenGenie gene structure prediction, TBLASTN alignments with PTB1 and PTB2, and alignments of EST sequences. (B) CLUSTALW alignment of eight PTB proteins. A 1,107-amino-acid sequence between residues 252 and 1,358 which is not similar to any of the other PTB sequences was removed from the PTB1 sequence prior to construction of the alignment (represented by dashes). Black shading denotes 100% sequence identity or conservative replacement in all sequences, dark gray shading with white letters denotes sequence conservation in at least six of eight sequences, and light gray shading with black letters denotes sequence conservation in at least four of eight sequences.

FIG. 4.

P_i transporter type A paralogs. (A) Schematic representing the arrangement of PTA genes in the C. reinhardtii genome. The PTA1, PTA2, and PTA3 gene models are based on alignments with the cDNA sequences (accession no. AB074874, AB074875, and AB074876, respectively); the PTA4 model was constructed based on similarity to the other PTA genes and to matching EST sequences. (B) CLUSTALW alignment of four PTA proteins, as described in the legend to Fig. 3.

FIG. 5.

Immunodetection of PHOX. The immunoblot was created by using anti-PHOX antiserum (18) and whole-cell extracts from either P-replete (+) or P-deprived (24 h) (−) wild-type and psr1 mutant cells.

FIG. 6.

Light sensitivity of psr1 during P deprivation. (A) Comparison of growth of wild-type cells, psr1 mutant cells, and a psr1-complemented strain on solid TAP medium (left panels) or TA medium supplemented with 10 μM glucose-1-phosphate (right panels). Plates were grown for 6 days under the indicated light intensities. (B) Graph showing viability of wild-type (solid line, circles) and psr1-1 (dashed line, triangles) cells during growth in TA (P-free) medium at a constant light intensity of ∼700 μmol photons m⁻² s⁻¹.

FIG. 6.

Light sensitivity of psr1 during P deprivation. (A) Comparison of growth of wild-type cells, psr1 mutant cells, and a psr1-complemented strain on solid TAP medium (left panels) or TA medium supplemented with 10 μM glucose-1-phosphate (right panels). Plates were grown for 6 days under the indicated light intensities. (B) Graph showing viability of wild-type (solid line, circles) and psr1-1 (dashed line, triangles) cells during growth in TA (P-free) medium at a constant light intensity of ∼700 μmol photons m⁻² s⁻¹.