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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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Northern blot analysis to test transcript patterns and  mRNA accumulation in wild-type and homoplasmic transformed  Δycf3 plants. Total plant RNA was hybridized to probes specific  for psaC (A), psaA (B), psaI (C), and psaJ (D). The major transcripts of ∼0.5 kb for psaC (18), 5.2 kb for psaA and psaB (17),  0.6 kb for psaI, and 0.5 kb for psaJ, respectively, are marked by  arrows. No significant differences in mRNA accumulation between wild-type and mutant plants could be detected, thus excluding a pretranslational cause of the PSI-deficient phenotype.  Note a difference in the size of a minor RNA species detected by  the psaA-specific probe (asterisks; 6.9-kb transcript in mutant  plants). This RNA species represents a polycistronic transcript  initiating far upstream of psaA. The polymorphism thus reflects  the size difference of ycf3 in wild type versus the chimeric aadA  gene in mutant plastids. (The diffuse signal in the wild-type lane  (wt) is due to the presence of splicing intermediates of the intron-containing ycf3 gene, which give rise to multiple bands.) Read-through transcription, as the cause of the appearance of these  high molecular weight mRNA species, was verified by hybridizing the blot with an aadA-specific probe (B, right panel). This  probe detects the same 6.9-kb transcript as the psaA-specific  probe in Δycf3 plants and, in addition, the 1.0-kb monocistronic  aadA transcript (and a 1.4-kb aadA transcript stabilized by the  downstream 3′-UTR of the deleted ycf3 gene).
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Figure 6: Northern blot analysis to test transcript patterns and mRNA accumulation in wild-type and homoplasmic transformed Δycf3 plants. Total plant RNA was hybridized to probes specific for psaC (A), psaA (B), psaI (C), and psaJ (D). The major transcripts of ∼0.5 kb for psaC (18), 5.2 kb for psaA and psaB (17), 0.6 kb for psaI, and 0.5 kb for psaJ, respectively, are marked by arrows. No significant differences in mRNA accumulation between wild-type and mutant plants could be detected, thus excluding a pretranslational cause of the PSI-deficient phenotype. Note a difference in the size of a minor RNA species detected by the psaA-specific probe (asterisks; 6.9-kb transcript in mutant plants). This RNA species represents a polycistronic transcript initiating far upstream of psaA. The polymorphism thus reflects the size difference of ycf3 in wild type versus the chimeric aadA gene in mutant plastids. (The diffuse signal in the wild-type lane (wt) is due to the presence of splicing intermediates of the intron-containing ycf3 gene, which give rise to multiple bands.) Read-through transcription, as the cause of the appearance of these high molecular weight mRNA species, was verified by hybridizing the blot with an aadA-specific probe (B, right panel). This probe detects the same 6.9-kb transcript as the psaA-specific probe in Δycf3 plants and, in addition, the 1.0-kb monocistronic aadA transcript (and a 1.4-kb aadA transcript stabilized by the downstream 3′-UTR of the deleted ycf3 gene).

Mentions: psaC is located in the small, single-copy region of higher plant plastid genomes. It is cotranscribed with six genes homologous to NADPH dehydrogenase subunits as part of the plastid ndhH operon (18). Hybridization with a psaC-specific probe detects a complex transcript pattern (Fig. 6 A), most likely resulting from cleavage of the polycistronic precursor transcript into numerous processing intermediates and from splicing of the intron-containing ndhA gene. The major transcript of ∼0.5 kb represents the monocistronic psaC mRNA being one of the final maturation products (18). No differences between wild-type and mutant plants could be detected in mRNA accumulation or transcript pattern (Fig. 6 A) thus excluding a pretranslational defect as the reason for the lack of PsaC protein accumulation in Δycf3 plants.


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)

Northern blot analysis to test transcript patterns and  mRNA accumulation in wild-type and homoplasmic transformed  Δycf3 plants. Total plant RNA was hybridized to probes specific  for psaC (A), psaA (B), psaI (C), and psaJ (D). The major transcripts of ∼0.5 kb for psaC (18), 5.2 kb for psaA and psaB (17),  0.6 kb for psaI, and 0.5 kb for psaJ, respectively, are marked by  arrows. No significant differences in mRNA accumulation between wild-type and mutant plants could be detected, thus excluding a pretranslational cause of the PSI-deficient phenotype.  Note a difference in the size of a minor RNA species detected by  the psaA-specific probe (asterisks; 6.9-kb transcript in mutant  plants). This RNA species represents a polycistronic transcript  initiating far upstream of psaA. The polymorphism thus reflects  the size difference of ycf3 in wild type versus the chimeric aadA  gene in mutant plastids. (The diffuse signal in the wild-type lane  (wt) is due to the presence of splicing intermediates of the intron-containing ycf3 gene, which give rise to multiple bands.) Read-through transcription, as the cause of the appearance of these  high molecular weight mRNA species, was verified by hybridizing the blot with an aadA-specific probe (B, right panel). This  probe detects the same 6.9-kb transcript as the psaA-specific  probe in Δycf3 plants and, in addition, the 1.0-kb monocistronic  aadA transcript (and a 1.4-kb aadA transcript stabilized by the  downstream 3′-UTR of the deleted ycf3 gene).
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Figure 6: Northern blot analysis to test transcript patterns and mRNA accumulation in wild-type and homoplasmic transformed Δycf3 plants. Total plant RNA was hybridized to probes specific for psaC (A), psaA (B), psaI (C), and psaJ (D). The major transcripts of ∼0.5 kb for psaC (18), 5.2 kb for psaA and psaB (17), 0.6 kb for psaI, and 0.5 kb for psaJ, respectively, are marked by arrows. No significant differences in mRNA accumulation between wild-type and mutant plants could be detected, thus excluding a pretranslational cause of the PSI-deficient phenotype. Note a difference in the size of a minor RNA species detected by the psaA-specific probe (asterisks; 6.9-kb transcript in mutant plants). This RNA species represents a polycistronic transcript initiating far upstream of psaA. The polymorphism thus reflects the size difference of ycf3 in wild type versus the chimeric aadA gene in mutant plastids. (The diffuse signal in the wild-type lane (wt) is due to the presence of splicing intermediates of the intron-containing ycf3 gene, which give rise to multiple bands.) Read-through transcription, as the cause of the appearance of these high molecular weight mRNA species, was verified by hybridizing the blot with an aadA-specific probe (B, right panel). This probe detects the same 6.9-kb transcript as the psaA-specific probe in Δycf3 plants and, in addition, the 1.0-kb monocistronic aadA transcript (and a 1.4-kb aadA transcript stabilized by the downstream 3′-UTR of the deleted ycf3 gene).
Mentions: psaC is located in the small, single-copy region of higher plant plastid genomes. It is cotranscribed with six genes homologous to NADPH dehydrogenase subunits as part of the plastid ndhH operon (18). Hybridization with a psaC-specific probe detects a complex transcript pattern (Fig. 6 A), most likely resulting from cleavage of the polycistronic precursor transcript into numerous processing intermediates and from splicing of the intron-containing ndhA gene. The major transcript of ∼0.5 kb represents the monocistronic psaC mRNA being one of the final maturation products (18). No differences between wild-type and mutant plants could be detected in mRNA accumulation or transcript pattern (Fig. 6 A) thus excluding a pretranslational defect as the reason for the lack of PsaC protein accumulation in Δycf3 plants.

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