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

Meinecke L, Alawady A, Schroda M, Willows R, Kobayashi MC, Niyogi KK, Grimm B, Beck CF - Plant Mol. Biol. (2010)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Fakultaet fuer Biologie, Institut fuer Biologie III, Universitaet Freiburg, Schaenzlestrasse 1, 79104, Freiburg, Germany.

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.

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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
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Fig4: 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

Mentions: We next investigated whether in mutants chlM-1 and chlM-2 the steady-state and light-inducible expression of CHLM at the RNA and protein levels were affected. Accumulation of CHLM mRNA was induced by a shift of wild-type and mutant cultures from dark to light. Due to the light sensitivity of the mutants only a fluence rate of 15 μmol m−2 s−1 was employed (Fig. 4a). In the two mutants, CHLM mRNA accumulated to higher levels than in wild type (in the case of chlM-2 the RNA concentration at time point 0 already was elevated as shown in Fig. 2). This mRNA accumulation was maintained for a longer time period.Fig. 4


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

Meinecke L, Alawady A, Schroda M, Willows R, Kobayashi MC, Niyogi KK, Grimm B, Beck CF - Plant Mol. Biol. (2010)

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
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Related In: Results  -  Collection

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Fig4: 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
Mentions: We next investigated whether in mutants chlM-1 and chlM-2 the steady-state and light-inducible expression of CHLM at the RNA and protein levels were affected. Accumulation of CHLM mRNA was induced by a shift of wild-type and mutant cultures from dark to light. Due to the light sensitivity of the mutants only a fluence rate of 15 μmol m−2 s−1 was employed (Fig. 4a). In the two mutants, CHLM mRNA accumulated to higher levels than in wild type (in the case of chlM-2 the RNA concentration at time point 0 already was elevated as shown in Fig. 2). This mRNA accumulation was maintained for a longer time period.Fig. 4

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Fakultaet fuer Biologie, Institut fuer Biologie III, Universitaet Freiburg, Schaenzlestrasse 1, 79104, Freiburg, Germany.

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.

Show MeSH
Related in: MedlinePlus