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

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.

Figure 1

Experimental strategy for targeted replacement of the ycf3 reading frame. (A) Map of the plastid DNA region containing ycf3. Genes above the line are transcribed from left to right; genes below the line are transcribed in the opposite direction. Restriction sites relevant for vector construction, RFLP analysis, or generation of hybridization probes are marked. Introns are shown as open boxes. (B) Map of the plastid DNA fragment in the final transformation vector pSR2. A chimeric spectinomycin resistance gene (aadA) replaces ycf3. Restriction sites eliminated by ligation with different half-sites are shown in parentheses. Note that the aadA gene is transcribed in the same direction as ycf3 in the cognate sequence of the plastid genome.

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.

Figure 3

Phenotype of homoplasmic Δycf3 plants. (A) A mutant plant kept under standard light conditions (3.5–4 W/m²). Massive photooxidative damage in mutant chloroplasts results in completely white plants. (B) A mutant plant grown under low light conditions (0.4–0.5 W/m²). Bars, 1 cm.

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.

Figure 5

Fluorescence measurements as test for PSII activity in Δycf3 plants. Wild-type and mutant plants grown under low light conditions were dark adapted, and leaf samples were illuminated with white actinic light. As a control, a wild-type sample treated with the plastoquinone-reducing herbicide 3-(3,4-dichlorphenyl)- 1,1-dimethylurea (DCMU) was included. PSII activity is clearly detectable in Δycf3 plants. However, comparison of the variable fluorescent yields indicates that the mutant accumulates fewer functional PSII reaction centers than the wild type, which is most likely the result of photooxidative damage as caused by the lack of functional electron acceptors downstream of PSII. The course of the fluorescence curve recorded for the mutant is virtually identical with the one of the wild-type sample treated with DCMU demonstrating that in both cases electrons accumulate in PSII and are not transferred to downstream components of the photosynthetic electron transport chain.

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).

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.