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Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene.

Ruf S, Kössel H, Bock R - J. Cell Biol. (1997)

Bottom Line: Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Deltaycf3 plants suggest a posttranslational cause of the PSI-defective phenotype.We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units.This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie III, Universität Freiburg, Germany.

ABSTRACT
The chloroplast genome of all higher plants encodes, in its large single-copy region, a conserved open reading frame of unknown function (ycf3), which is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the function of ycf3 we have deleted the reading frame from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic Deltaycf3 plants display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not due to a defect in any of the general functions of the plastid genetic apparatus. Instead, the mutant plants specifically lack detectable amounts of all photosystem I (PSI) subunits analyzed. In contrast, at least under low light conditions, photosystem II subunits are still present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Deltaycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units. This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

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Accumulation of thylakoid proteins in Δycf3 plants.  Immunoblots probed with antisera against the ATPase subunit  AtpB, the PSII proteins PsbA,  PsbD, PsbO, PsbP, and Lhcb6,  the cytochrome bf complex subunit PetA, and the PSI proteins  PsaC, PsaD, and PsaF are shown  for wild-type plants and two  or three independently transformed Δycf3 lines. For comparison, a dilution series of the  wild-type extract is shown. Chlorophyll concentrations were wild  type (higher concentration; first  lane)/wild type (lower concentration; second lane)/mutant (1: 0.2:1.) Note that PSI proteins  are undetectable in mutant  plants whereas all the other protein complexes of the thylakoid  membrane appear to be not primarily affected by the absence  of the ycf3 gene product.
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Figure 4: Accumulation of thylakoid proteins in Δycf3 plants. Immunoblots probed with antisera against the ATPase subunit AtpB, the PSII proteins PsbA, PsbD, PsbO, PsbP, and Lhcb6, the cytochrome bf complex subunit PetA, and the PSI proteins PsaC, PsaD, and PsaF are shown for wild-type plants and two or three independently transformed Δycf3 lines. For comparison, a dilution series of the wild-type extract is shown. Chlorophyll concentrations were wild type (higher concentration; first lane)/wild type (lower concentration; second lane)/mutant (1: 0.2:1.) Note that PSI proteins are undetectable in mutant plants whereas all the other protein complexes of the thylakoid membrane appear to be not primarily affected by the absence of the ycf3 gene product.

Mentions: The phenotype of the homoplasmic transformants suggests that the ycf3 gene product is directly or indirectly involved in photosynthetic electron transfer. To test whether the photosynthetic deficiency of the Δycf3 plants can be attributed to a specific complex in the thylakoid membrane we performed immunoblot analyses using various antibodies raised against proteins of PSII, PSI, the cytochrome bf complex, and the plastid ATP synthase complex (Table I). Whereas PSII proteins as well as cytochrome bf complex and ATPase subunits are readily detected in thylakoid membrane protein preparations from Δycf3 plants, PSI proteins appear to be absent or accumulate to levels falling below the sensitivity of our Western blots (Fig. 4). PSI subunits are also undetectable in the soluble protein fraction excluding the possibility that the proteins are stable in the stroma but cannot be incorporated into the thylakoid membrane.


Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene.

Ruf S, Kössel H, Bock R - J. Cell Biol. (1997)

Accumulation of thylakoid proteins in Δycf3 plants.  Immunoblots probed with antisera against the ATPase subunit  AtpB, the PSII proteins PsbA,  PsbD, PsbO, PsbP, and Lhcb6,  the cytochrome bf complex subunit PetA, and the PSI proteins  PsaC, PsaD, and PsaF are shown  for wild-type plants and two  or three independently transformed Δycf3 lines. For comparison, a dilution series of the  wild-type extract is shown. Chlorophyll concentrations were wild  type (higher concentration; first  lane)/wild type (lower concentration; second lane)/mutant (1: 0.2:1.) Note that PSI proteins  are undetectable in mutant  plants whereas all the other protein complexes of the thylakoid  membrane appear to be not primarily affected by the absence  of the ycf3 gene product.
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Related In: Results  -  Collection

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Figure 4: Accumulation of thylakoid proteins in Δycf3 plants. Immunoblots probed with antisera against the ATPase subunit AtpB, the PSII proteins PsbA, PsbD, PsbO, PsbP, and Lhcb6, the cytochrome bf complex subunit PetA, and the PSI proteins PsaC, PsaD, and PsaF are shown for wild-type plants and two or three independently transformed Δycf3 lines. For comparison, a dilution series of the wild-type extract is shown. Chlorophyll concentrations were wild type (higher concentration; first lane)/wild type (lower concentration; second lane)/mutant (1: 0.2:1.) Note that PSI proteins are undetectable in mutant plants whereas all the other protein complexes of the thylakoid membrane appear to be not primarily affected by the absence of the ycf3 gene product.
Mentions: The phenotype of the homoplasmic transformants suggests that the ycf3 gene product is directly or indirectly involved in photosynthetic electron transfer. To test whether the photosynthetic deficiency of the Δycf3 plants can be attributed to a specific complex in the thylakoid membrane we performed immunoblot analyses using various antibodies raised against proteins of PSII, PSI, the cytochrome bf complex, and the plastid ATP synthase complex (Table I). Whereas PSII proteins as well as cytochrome bf complex and ATPase subunits are readily detected in thylakoid membrane protein preparations from Δycf3 plants, PSI proteins appear to be absent or accumulate to levels falling below the sensitivity of our Western blots (Fig. 4). PSI subunits are also undetectable in the soluble protein fraction excluding the possibility that the proteins are stable in the stroma but cannot be incorporated into the thylakoid membrane.

Bottom Line: Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Deltaycf3 plants suggest a posttranslational cause of the PSI-defective phenotype.We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units.This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie III, Universität Freiburg, Germany.

ABSTRACT
The chloroplast genome of all higher plants encodes, in its large single-copy region, a conserved open reading frame of unknown function (ycf3), which is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the function of ycf3 we have deleted the reading frame from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic Deltaycf3 plants display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not due to a defect in any of the general functions of the plastid genetic apparatus. Instead, the mutant plants specifically lack detectable amounts of all photosystem I (PSI) subunits analyzed. In contrast, at least under low light conditions, photosystem II subunits are still present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Deltaycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units. This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

Show MeSH
Related in: MedlinePlus