Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX

Abstract

Two Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.

Fig. 1

Scheme of the tetrapyrrole biosynthetic pathway in Chlamydomonas reinhardtii and vascular plants. Shown are the major intermediates and the genes mentioned in the text. Dashed lines indicate multiple steps

Fig. 2

Structure of the CHLM gene, alterations caused by the mutations chlM-1 and chlM-2, and test of the mutants for CHLM encoded RNA and protein. a Gene CHLM with 7 exons. Open boxes indicate deduced protein coding exons, black boxes indicate 5′ and 3′ untranslated regions, and lines indicate introns. The positions and sequence alterations of mutations chlM-1 and chlM-2 are indicated. b Genomic and RT–PCR sequences in the vicinity of the splice site between exon 4 and intron 4. The vertical arrows indicate the end of exon 4. The bases of intron 4 are indicated in italics. The altered base of intron 4 in mutant chlM-2 is shown in bold. The asterisk indicates a stop codon. c For the determination of CHLM mRNA levels, samples were taken from wild type and mutant cultures grown in the dark. RNA gel blot hybridizations were performed using specific probes for CHLM and CBLP, the latter serving as a loading control. The size of CHLM mRNAs was deduced by comparison with an RNA marker. The average levels of CHLM mRNA in the mutants relative to the wild type (corrected for differences in loading) ±SEM are given below the RNA blot. For details see Materials and methods. Cultures for protein extraction were grown with low intensity irradiation (15 μmol m⁻² s⁻¹). After SDS–PAGE separation of 60 μg of total soluble protein per slot and blotting to nitrocellulose membranes, MgPMT was detected by immunodecoration with MgPMT-specific antibodies. For a loading control, the same membrane was incubated with antibodies directed against the 1β subunit of the chloroplast ATPase (CF₁β)

Fig. 3

Growth and pigmentation of the two chlM mutants. The wild- type strain as well as a yellow-in-the-dark mutant (y7) are shown for control. After inoculation the plates were incubated for 15 days either in the dark, or irradiated with the fluence rates indicated

Fig. 4

Light induction of CHLM in wild type and mutants. a mRNA accumulation in cultures that after growth in the dark were shifted into dim light (15 μmol m⁻² s⁻¹). Samples were taken at the time points indicated (hours). RNA gel blots were performed as described in Materials and methods using specific probes for CHLM and CBLP, the latter serving as a loading control. The average fold induction of mRNA relative to the dark control (corrected for differences in loading) from 9 independent experiments ±SEM were determined. b Changes in MgPMT upon shift of cultures from dark to light. For the assay of MgPMT, total soluble protein was extracted from cultures grown in the dark and after exposure to light (15 μmol m⁻² s⁻¹) for one or 2 h. After blotting, the proteins were detected by immunodecoration with MgPMT-specific antibodies. The weak signal seen in chlM-2 mutant extracts may reflect an unspecific reaction since it was not seen in Fig. 1c and also does not increase upon light incubation. For a loading control, the same membrane was decorated with an antiserum directed against the chloroplast ATPase subunit CF₁β

Fig. 5

Immuno and RNA blot analyses of genes coding for components of the photosynthetic apparatus in mutants defective in CHLM. a Detection of proteins of the light harvesting complex. Cultures of the strains prior to harvest were grown in darkness for at least 3 days. Each lane of the SDS-gel received the same amount of protein. We used an antiserum that specifically detected the LHCII protein and one that reacted with 3 different light harvesting proteins. Antisera that detected the CF₁β protein of plastid ATPase served as a loading control. b mRNA abundance of three genes encoding light harvesting chlorophyll a/b binding proteins. Cultures were grown in the dark and RNA was isolated, processed, and hybridized with the gene-specific probes indicated. The quantitative evaluation of the RNA gel blots comprised at least 4 independent experiments. The average mRNA levels relative to those of the wild type (set as 1) and corrected for differences in loading ±SEM are given. CBLP served as a loading control. c Detection of selected proteins of PSI, PSII, and the cytb ₆ /f complex. Besides extracts of the chlM mutants and wild type also those from mutants with defined lesions were employed for control: mutant F15 is deficient in the synthesis of PsaB (62), mutant F35 does not produce protein D1 (Yohn et al. 1996), and mutant ΔpetA has an inactivated petA (cytochrome f) gene (Kuras and Wollman 1994)

Fig. 6

Immuno and RNA blot analyses of genes coding for enzymes of chlorophyll synthesis. a Levels of proteins involved in chlorophyll biosynthesis of wild type and chlM mutants cultivated in the dark. Each slot of the SDS-gel received the same amount of protein. Immunodecoration was done using antisera against CHLH and CRD1, the latter detecting subunits of the cyclase (Moseley et al. 2002). Antisera that detect the CF₁β protein of plastid ATPase served as a loading control. b Comparison between chlM mutants and wild type in expression levels of genes involved in tetrapyrrole biosynthesis. mRNA steady state levels of CHLH, CHLI, CHLD, CRD-1, HEMA (encoding glutamyl-tRNA reductase), ALAD (encoding ALA dehydratase) and HEM15 (encoding Fe-chelatase) in wild type and mutant cultures. c Steady state levels of mRNA of HSP70 genes encoding a cytosolic (HSP70A) and a plastidic (HSP70B) chaperone. Cultures were grown in the dark. RNA was isolated, processed, and hybridized with the gene-specific probes indicated. The quantitative evaluation of the RNA gel blots comprised at least three independent experiments. The average mRNA levels relative to those of the wild type (set as 1) and corrected for differences in loading ±SEM are given. CBLP served as a loading control

Fig. 7

Activities of selected enzymes for heme and chlorophyll biosynthesis. The specific activities in dark-grown cells of wild type and two chlM mutants were determined for MgPMT, Mg-chelatase, ferrochelatase, and for the 5-aminolevulinic acid (ALA) synthesizing enzymes. The activities were determined as described in Materials and methods. The average of 3 independent experiments ±SEM are given. In the case of ALA synthesis the average of 2 independent experiments which did not differ by more than 10% is presented

Fig. 8

Effect of mutation chlM-2 on gene expression after a shift from dark to light. Cultures of wild type and the chlM-2 mutant grown in the dark at time 0 were shifted into dim light (15 μmol m⁻² s⁻¹) and samples for RNA isolation were taken at the time points indicated (hours). The average levels of mRNA accumulation relative to the dark control and corrected for differences in loading ±SEM from at least 3 independent experiments are indicated. CBLP served as a loading control. a Test for light induction of Mg-chelatase genes. b Test for light induction of genes encoding light harvesting chlorophyll a/b binding proteins. c Test for light induction of HSP70 genes