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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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RFLP analysis to  verify chloroplast transformation and homoplasmy of the  Δycf3 plants. Total cellular  DNA from wild-type plants  and from three independently  transformed lines (Nt-pSR2-1,  Nt-pSR2-2, and Nt-pSR2-5, subsequently referred to as 2-1, 2-2,  and 2-5) was digested with XhoI and hybridized to the radiolabeled SacI/XhoI fragment covering the region downstream of the  ycf3 reading frame (i. e., the psaA gene and the 5′ portion of  psaB; Fig. 1). The probe detects a 5.6-kb fragment in wild-type  plants (corresponding to nucleotide positions 40,883 to 46,524;  29; Fig. 1) and a 4.9-kb fragment in the transplastomic lines. Absence of the 5.6-kb signal in the lanes representing the Δycf3  plants indicates a uniformly transformed population of plastid  DNA molecules.
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Figure 2: RFLP analysis to verify chloroplast transformation and homoplasmy of the Δycf3 plants. Total cellular DNA from wild-type plants and from three independently transformed lines (Nt-pSR2-1, Nt-pSR2-2, and Nt-pSR2-5, subsequently referred to as 2-1, 2-2, and 2-5) was digested with XhoI and hybridized to the radiolabeled SacI/XhoI fragment covering the region downstream of the ycf3 reading frame (i. e., the psaA gene and the 5′ portion of psaB; Fig. 1). The probe detects a 5.6-kb fragment in wild-type plants (corresponding to nucleotide positions 40,883 to 46,524; 29; Fig. 1) and a 4.9-kb fragment in the transplastomic lines. Absence of the 5.6-kb signal in the lanes representing the Δycf3 plants indicates a uniformly transformed population of plastid DNA molecules.

Mentions: Young leaves from sterile tobacco plants were bombarded with plasmid pSR2–coated tungsten particles using the DuPont biolistic gun (PDS1000He; BioRad, Hercules, CA) (12, 36). Primary spectinomycin-resistant lines were selected on RMOP regeneration medium containing 500 mg/liter spectinomycin dihydrochloride (37). Plastid transformants were identified by PCR amplification according to standard protocols using the primer pair P10 (complementary to the psbA 3′-untranslated region of the chimeric aadA gene) and P11 (derived from the 3′ portion of the aadA coding region). Three independent transplastomic lines were subjected to four additional rounds of regeneration on RMOP/spectinomycin to obtain homoplasmic tissue. Homoplasmy was verified by DNA gel blot analysis (see Fig. 2).


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)

RFLP analysis to  verify chloroplast transformation and homoplasmy of the  Δycf3 plants. Total cellular  DNA from wild-type plants  and from three independently  transformed lines (Nt-pSR2-1,  Nt-pSR2-2, and Nt-pSR2-5, subsequently referred to as 2-1, 2-2,  and 2-5) was digested with XhoI and hybridized to the radiolabeled SacI/XhoI fragment covering the region downstream of the  ycf3 reading frame (i. e., the psaA gene and the 5′ portion of  psaB; Fig. 1). The probe detects a 5.6-kb fragment in wild-type  plants (corresponding to nucleotide positions 40,883 to 46,524;  29; Fig. 1) and a 4.9-kb fragment in the transplastomic lines. Absence of the 5.6-kb signal in the lanes representing the Δycf3  plants indicates a uniformly transformed population of plastid  DNA molecules.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139824&req=5

Figure 2: RFLP analysis to verify chloroplast transformation and homoplasmy of the Δycf3 plants. Total cellular DNA from wild-type plants and from three independently transformed lines (Nt-pSR2-1, Nt-pSR2-2, and Nt-pSR2-5, subsequently referred to as 2-1, 2-2, and 2-5) was digested with XhoI and hybridized to the radiolabeled SacI/XhoI fragment covering the region downstream of the ycf3 reading frame (i. e., the psaA gene and the 5′ portion of psaB; Fig. 1). The probe detects a 5.6-kb fragment in wild-type plants (corresponding to nucleotide positions 40,883 to 46,524; 29; Fig. 1) and a 4.9-kb fragment in the transplastomic lines. Absence of the 5.6-kb signal in the lanes representing the Δycf3 plants indicates a uniformly transformed population of plastid DNA molecules.
Mentions: Young leaves from sterile tobacco plants were bombarded with plasmid pSR2–coated tungsten particles using the DuPont biolistic gun (PDS1000He; BioRad, Hercules, CA) (12, 36). Primary spectinomycin-resistant lines were selected on RMOP regeneration medium containing 500 mg/liter spectinomycin dihydrochloride (37). Plastid transformants were identified by PCR amplification according to standard protocols using the primer pair P10 (complementary to the psbA 3′-untranslated region of the chimeric aadA gene) and P11 (derived from the 3′ portion of the aadA coding region). Three independent transplastomic lines were subjected to four additional rounds of regeneration on RMOP/spectinomycin to obtain homoplasmic tissue. Homoplasmy was verified by DNA gel blot analysis (see Fig. 2).

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