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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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Test for association  of PSI gene transcripts with  polysomes in Δycf3 plants.  (A) RNAs extracted from  fractions 2–5 of analytical  polysome isolation gradients  were separated on 1% formaldehyde-containing agarose  gels, transferred to nylon  membranes, and hybridized  to a psaA-specific probe  mainly detecting the dicistronic psaA/B transcripts.  Comparison of EDTA-free  with EDTA-containing gradient fractions identifies fractions 2 and 3 as mainly  monosome containing, and  fractions 4 and 5 as polysome  containing. The psaA/B transcripts in Δycf3 plastids are  as efficiently associated with  polysomes as in wild-type  plastids (Wt). Note the prominent band for the read-through transcript initiating  upstream of the aadA  marker gene in mutant plastids (compare with Fig. 6 B),  which is translated with extraordinarily high efficiency (most probably owing to the strong  [rbcL-derived] Shine-Dalgarno sequence of the chimeric aadA).  Transcript sizes are given at the right. The direction of polysome  sedimentation is marked by horizontal arrows below the blot. (B)  Analysis of polysome association for psaC transcripts. As psaA/B  mRNAs, psaC transcripts are loaded with ribosomes with comparable efficiencies in wild-type and mutant plastids. The polysome-associated monocistronic psaC transcript (horizontal  arrow) is predominantly present in fractions 3 and 4 for both  wild-type and mutant plastids, but nearly exclusively in fraction 2  of the EDTA-containing gradient. (C) Ribosome content of the  fractions collected. RNA aliquots of the fractions were separated  under nondenaturing conditions on 2% agarose gels stained with  ethidium bromide. The ribosome-containing fractions show  prominent bands representing the ribosomal RNA species.
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Figure 7: Test for association of PSI gene transcripts with polysomes in Δycf3 plants. (A) RNAs extracted from fractions 2–5 of analytical polysome isolation gradients were separated on 1% formaldehyde-containing agarose gels, transferred to nylon membranes, and hybridized to a psaA-specific probe mainly detecting the dicistronic psaA/B transcripts. Comparison of EDTA-free with EDTA-containing gradient fractions identifies fractions 2 and 3 as mainly monosome containing, and fractions 4 and 5 as polysome containing. The psaA/B transcripts in Δycf3 plastids are as efficiently associated with polysomes as in wild-type plastids (Wt). Note the prominent band for the read-through transcript initiating upstream of the aadA marker gene in mutant plastids (compare with Fig. 6 B), which is translated with extraordinarily high efficiency (most probably owing to the strong [rbcL-derived] Shine-Dalgarno sequence of the chimeric aadA). Transcript sizes are given at the right. The direction of polysome sedimentation is marked by horizontal arrows below the blot. (B) Analysis of polysome association for psaC transcripts. As psaA/B mRNAs, psaC transcripts are loaded with ribosomes with comparable efficiencies in wild-type and mutant plastids. The polysome-associated monocistronic psaC transcript (horizontal arrow) is predominantly present in fractions 3 and 4 for both wild-type and mutant plastids, but nearly exclusively in fraction 2 of the EDTA-containing gradient. (C) Ribosome content of the fractions collected. RNA aliquots of the fractions were separated under nondenaturing conditions on 2% agarose gels stained with ethidium bromide. The ribosome-containing fractions show prominent bands representing the ribosomal RNA species.

Mentions: It has frequently been observed that unassembled subunits of PSI complexes are highly unstable (10, 25, 30). The rather lengthy pulse-labeling experiments may thus prevent the detection of PSI translation products in Δycf3 plastids by in organello translation assays. Analysis of the polysomal association of PSI mRNAs is therefore the method of choice to test for faithful translation initiation on PSI transcripts in Δycf3 plants. Wild-type and mutant leaf samples were lysed under conditions maintaining the integrity of polysomes (3). The lysates were then fractionated in sucrose gradients, and the distribution of chloroplast transcripts was analyzed by performing Northern hybridization experiments with RNA purified from gradient fractions. As a control, EDTA was added to a gradient containing lysate from mutant plants. EDTA treatment releases ribosomes from mRNAs. Comparison of EDTA-containing with EDTA-free gradient fractions thus allows for the identification of monosome- versus polysome-containing fractions (Fig. 7).


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)

Test for association  of PSI gene transcripts with  polysomes in Δycf3 plants.  (A) RNAs extracted from  fractions 2–5 of analytical  polysome isolation gradients  were separated on 1% formaldehyde-containing agarose  gels, transferred to nylon  membranes, and hybridized  to a psaA-specific probe  mainly detecting the dicistronic psaA/B transcripts.  Comparison of EDTA-free  with EDTA-containing gradient fractions identifies fractions 2 and 3 as mainly  monosome containing, and  fractions 4 and 5 as polysome  containing. The psaA/B transcripts in Δycf3 plastids are  as efficiently associated with  polysomes as in wild-type  plastids (Wt). Note the prominent band for the read-through transcript initiating  upstream of the aadA  marker gene in mutant plastids (compare with Fig. 6 B),  which is translated with extraordinarily high efficiency (most probably owing to the strong  [rbcL-derived] Shine-Dalgarno sequence of the chimeric aadA).  Transcript sizes are given at the right. The direction of polysome  sedimentation is marked by horizontal arrows below the blot. (B)  Analysis of polysome association for psaC transcripts. As psaA/B  mRNAs, psaC transcripts are loaded with ribosomes with comparable efficiencies in wild-type and mutant plastids. The polysome-associated monocistronic psaC transcript (horizontal  arrow) is predominantly present in fractions 3 and 4 for both  wild-type and mutant plastids, but nearly exclusively in fraction 2  of the EDTA-containing gradient. (C) Ribosome content of the  fractions collected. RNA aliquots of the fractions were separated  under nondenaturing conditions on 2% agarose gels stained with  ethidium bromide. The ribosome-containing fractions show  prominent bands representing the ribosomal RNA species.
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Figure 7: Test for association of PSI gene transcripts with polysomes in Δycf3 plants. (A) RNAs extracted from fractions 2–5 of analytical polysome isolation gradients were separated on 1% formaldehyde-containing agarose gels, transferred to nylon membranes, and hybridized to a psaA-specific probe mainly detecting the dicistronic psaA/B transcripts. Comparison of EDTA-free with EDTA-containing gradient fractions identifies fractions 2 and 3 as mainly monosome containing, and fractions 4 and 5 as polysome containing. The psaA/B transcripts in Δycf3 plastids are as efficiently associated with polysomes as in wild-type plastids (Wt). Note the prominent band for the read-through transcript initiating upstream of the aadA marker gene in mutant plastids (compare with Fig. 6 B), which is translated with extraordinarily high efficiency (most probably owing to the strong [rbcL-derived] Shine-Dalgarno sequence of the chimeric aadA). Transcript sizes are given at the right. The direction of polysome sedimentation is marked by horizontal arrows below the blot. (B) Analysis of polysome association for psaC transcripts. As psaA/B mRNAs, psaC transcripts are loaded with ribosomes with comparable efficiencies in wild-type and mutant plastids. The polysome-associated monocistronic psaC transcript (horizontal arrow) is predominantly present in fractions 3 and 4 for both wild-type and mutant plastids, but nearly exclusively in fraction 2 of the EDTA-containing gradient. (C) Ribosome content of the fractions collected. RNA aliquots of the fractions were separated under nondenaturing conditions on 2% agarose gels stained with ethidium bromide. The ribosome-containing fractions show prominent bands representing the ribosomal RNA species.
Mentions: It has frequently been observed that unassembled subunits of PSI complexes are highly unstable (10, 25, 30). The rather lengthy pulse-labeling experiments may thus prevent the detection of PSI translation products in Δycf3 plastids by in organello translation assays. Analysis of the polysomal association of PSI mRNAs is therefore the method of choice to test for faithful translation initiation on PSI transcripts in Δycf3 plants. Wild-type and mutant leaf samples were lysed under conditions maintaining the integrity of polysomes (3). The lysates were then fractionated in sucrose gradients, and the distribution of chloroplast transcripts was analyzed by performing Northern hybridization experiments with RNA purified from gradient fractions. As a control, EDTA was added to a gradient containing lysate from mutant plants. EDTA treatment releases ribosomes from mRNAs. Comparison of EDTA-containing with EDTA-free gradient fractions thus allows for the identification of monosome- versus polysome-containing fractions (Fig. 7).

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