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. 2001 Apr 24;98(9):5341-6.
doi: 10.1073/pnas.101534498. Epub 2001 Apr 17.

The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii

Y Xiang  1 , J Zhang, D P Weeks
Affiliations

The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii

Y Xiang et al. Proc Natl Acad Sci U S A. .

Abstract

Wild-type Chlamydomonas reinhardtii cells shifted from high concentrations (5%) of CO2 to low, ambient levels (0.03%) rapidly increase transcription of mRNAs from several CO2-responsive genes. Simultaneously, they develop a functional carbon concentrating mechanism that allows the cells to greatly increase internal levels of CO2 and HCO3-. The cia5 mutant is defective in all of these phenotypes. A newly isolated gene, designated Cia5, restores transformed cia5 cells to the phenotype of wild-type cells. The 6,481-bp gene produces a 5.1-kb mRNA that is present constitutively in light in high and low CO2 both in wild-type cells and the cia5 mutant. It encodes a protein that has features of a putative transcription factor and that, likewise, is present constitutively in low and high CO2 conditions. Complementation of cia5 can be achieved with a truncated Cia5 gene that is missing the coding information for 54 C-terminal amino acids. Unlike wild-type cells or cia5 mutants transformed with an intact Cia5 gene, cia5 mutants complemented with the truncated gene exhibit constitutive synthesis of mRNAs from CO2-responsive genes in light under both high and low CO2 conditions. These discoveries suggest that posttranslational changes to the C-terminal domain control the ability of CIA5 to act as an inducer and directly or indirectly control transcription of CO2-responsive genes. Thus, CIA5 appears to be a master regulator of the carbon concentrating mechanism and is intimately involved in the signal transduction mechanism that senses and allows immediate responses to fluctuations in environmental CO2 and HCO3- concentrations.

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Figures

Figure 1
Figure 1
Cia5 gene, mRNA, and CIA5 protein. (A) Structure of the Cia5 gene and mRNA. The schematic diagram at the top shows the intron/exon configuration of the Cia5 gene along with a portion of the promoter region (from the SalI restriction site at −163 bp to the transcription initiation site at +1). The lower diagram illustrates the 2,094-nt coding region of the Cia5 mRNA along with its exceptionally long, 2,904-nt 3′ UTR. (B) Northern blot analysis of Cia5 mRNAs from wild-type cells (lanes 1, 5, 9, and 13), cia5 mutant cells (lanes 2, 6, 10, and 14), cia5 mutants complemented with an intact Cia5 gene (lanes 3, 7, 11, and 15), and cia5 mutants complemented with a slightly truncated Cia5 gene (lanes 4, 8, 12, and 16). Total RNA was extracted from cells maintained in high concentrations of CO2 (lanes 1–4) or from cells after they were switched from high CO2 levels to low levels of CO2 for 45 min (lanes 5–8), 90 min (lanes 9–12), or 180 min (lanes 13–16). The last lane of the Northern blot (M) contains labeled RNA markers of 1.5, 2.6, and 4.7 kb in size. (C) Deduced amino acid sequence of the CIA5 protein. The CIA5 protein contains a number of notable features including an abundance of glutamine residues (red) and repeats (residues 316–329), alanine residues (blue) and repeats (residues 352–363), and a region interspersed with glycines and alanines (italicized residues 550–627). The cysteine and histine residues contained in two potential, but noncanonical, zinc-finger motifs are underlined (residues C35 and C41, H54 and H69; C77 and C79, H89 or H90 and C93). The cia5 mutation is the result of a single nucleotide base pair change that converts the H54 histidine residue (noted above as a component of a potential zinc-finger motif) to a tyrosine residue. The underlined amino acid sequences at the C-terminal end of the protein represents a potential protein kinase C phosphorylation site. The italicized glutamine residue at position 642 represents the last complete codon encoded by the Cia5 gene truncated at its single NotI restriction enzyme cut site.
Figure 2
Figure 2
Cellular levels of CIA5 under low and high CO2 conditions. Western blot analysis was performed with total cell lysates of three separate cia5 mutants (lanes 4 and 5; lanes 6 and 7; lanes 8 and 9, respectively) complemented with an intact, HA-tagged Cia5 gene and maintained under low CO2 conditions (lanes 4, 6, and 8) or high CO2 conditions (lanes 5, 7, and 9). Epitope-tagged CIA5 was detected with antibodies to HA. Specificity of the antibody for epitope-tagged CIA5 was demonstrated by the lack of detection of proteins of the expected size in extracts from cia5 mutants (lane 1) or from wild-type cells maintained in high (lane 2) or low (lane 3) CO2 conditions.
Figure 3
Figure 3
Nuclear localization of the CIA5 protein. Onion epidermal cells were transformed by particle bombardment with a chimeric gene construct containing a truncated Cia5 gene fused in-frame with a β-GUS gene and driven by the promoter from the CaMV 35S promoter. Cells were fixed and prepared for detection of the GUS enzyme activity by reaction with 5-bromo-4-chloro-3-indoly-β-d-glucuronide. (A) Epidermal cells bombarded with the Cia5/GUS chimeric gene. (B) Cells in A stained with propidium iodide for DNA detection. (C) Epidermal cells bombarded with a CaMV 35S/GUS gene construct. (D) Cells in C stained with propidium iodide for DNA detection. (Magnification: ×100).
Figure 4
Figure 4
Northern blot analysis of the induction or derepression of synthesis of mRNAs encoded by four different CO2-responsive genes on shift of cells from high concentrations of CO2 to low, ambient concentrations of CO2. Total RNA was extracted from wild-type cells (lanes 1, 5, 9, and 13), cia5 mutant cells (lanes 2, 6, 10, and 14), cia5 mutants complemented with an intact Cia5 gene construct (lanes 3, 7, 11, and 15), and cia5 mutants complemented with a truncated Cia5 gene (lanes 4, 8, 12, and 16). Total RNA was extracted from cells maintained in high concentrations of CO2 (lanes 1–4) or from cells after they were switched from high CO2 levels to low levels of CO2 for 45 min (lanes 5–8), 90 min (lanes 9–12), or 180 min (lanes 13–16). (A) mRNAs detected by hybridization with probes produced from the cDNA encoding Ccp2 (LIP36), a chloroplast inner envelope protein. (B) mRNAs detected with hybridization probes to the CAH1 cDNA encoding the major C. reinhardtii periplasmic CA. (C) mRNAs detected with probes to Att1 cDNA sequences encoding an alanine:α-ketoglutarate aminotransferase. (D) mRNAs detected with probes produced from the mtCA1 cDNA encoding a mitochondrial CA. (E) Ethidium bromide-stained gel illustrating uniform loading of total RNAs in each lane. The same RNA preparations used for the Northern blot analyses depicted in Fig. 1B were used for the blots shown.
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