Overcoming Poor Transgene Expression in the Wild-Type Chlamydomonas Chloroplast: Creation of Highly Mosquitocidal Strains of Chlamydomonas reinhardtii.

High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g., the cry toxin genes from Bacillus thuringiensis israelensis). The bottleneck is presumed to be post-transcriptional and mediated by the 5' element and the coding region. Using 5' elements from highly expressed photosynthesis genes such as atpA did not improve the outcome with cry11A regardless of the promoter. However, when we employed the 5' UTR from mature rps4 mRNA with clean fusions to promoters, production of the rCry11A protein became largely promoter-dependent. The best results were obtained with the native 16S rrn promoter (−91 to −1). When it was fused to the mature 5' rps4 UTR, rCry11A protein levels were ~50% higher than was obtained with the inducible system, or ~0.6% of total protein. This level was sufficient to visualize the 73-kDa rCry11A protein on Coomassie-stained gels of total algal protein. In addition, analysis of the expression of these transgenes by RT-PCR indicated that RNA levels roughly correlated with protein production. Live cell bioassays using the best strains as food for 3rd instar Aedes aegypti larvae showed that most larvae were killed even when the cell concentration was as low as 2 × 104 cells/mL. Finally, the results indicate that these highly toxic strains are also quite stable, and thus represent a key milestone in using C. reinhardtii for mosquito control.

Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa) of Bacillus thuringiensis subsp. israelensis in the Chloroplast of Wild-Type Chlamydomonas.

Chlamydomonas reinhardtii (Chlamydomonas) strains that are toxic to mosquito larvae because they express chloroplast transgenes that are based on the mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis (Bti) could be very useful in mosquito control. Chlamydomonas has several advantages for this approach, including genetic controls not generally available with industrial algae. The Bti toxin is produced by sporulating bacteria and has been used for mosquito control for >30 years without creating highly resistant mosquito populations. The suite of toxins is four main proteins: three Cry proteins and the cytotoxic Cyt1Aa (27 kDa). Cyt1Aa is not very toxic to mosquitoes by itself, but it prevents the development of resistance. The production of Cyt1Aa in other microbes, however, has been challenging due to its affinity for certain membrane phospholipids. Here we report on the production of recombinant Cyt1Aa (rCyt1A) in the chloroplast of photosynthetic Chlamydomonas at levels of at least 0.3% total protein. Live cell bioassays demonstrated toxicity of the rCyt1Aa Chlamydomonas to larvae of Aedes aegypti. We also expressed the chloroplast cyt1Aa gene in a wild-type Chlamydomonas strain (21 gr) that can grow on nitrate. These results have implications for developing a Chlamydomonas strain that will be toxic to mosquito larvae but will not induce strongly resistant populations.

Toward mosquito control with a green alga: Expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas.

We are developing Chlamydomonas strains that can be used for safe and sustainable control of mosquitoes, because they produce proteins from Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast. Chlamydomonas has a number of advantages for this approach, including genetic controls that are not generally available with industrial algae. The Bti toxin has been used for mosquito control for > 30 years and does not engender resistance; it contains three Cry proteins, Cry4Aa (135 kDa), Cry4Ba (128 kDa) and Cry11Aa (72 kDa), and Cyt1Aa (25 kDa). To express the Cry proteins in the chloroplast, the three genes were resynthesized and cry4Aa was truncated to the first 700 amino acids (cry4Aa700 ); also, since they can be toxic to host cells, the inducible Cyc6:Nac2-psbD expression system was used. Western blots of total protein from the chloroplast transformants showed accumulation of the intact polypeptides, and the relative expression level was Cry11Aa > Cry4Aa700 > Cry4Ba. Quantitative western blots with purified Cry11Aa as a standard showed that Cry11Aa accumulated to 0.35% of total cell protein. Live cell bioassays in dH20 demonstrated toxicity of the cry4Aa700 and cry11Aa transformants to larvae of Aedes aegypti and Culex quinquefasciatus. These results demonstrate that the Cry proteins that are most toxic to Aedes and Culex mosquitoes, Cry4Aa and Cry11Aa, can be successfully expressed in the chloroplast of Chlamydomonas.

PCR analysis of chloroplast double-strand break (DSB) repair products induced by I-CreII in Chlamydomonas and Arabidopsis.

Homing endonuclease I-CreII has been used to study the consequences and repair of a double-strand break (DSB) in the chloroplast genome of Chlamydomonas and Arabidopsis. Since I-CreII is from a mobile psbA intron of Chlamydomonas, it cleaves the psbA gene of an intronless-psbA strain of Chlamydomonas. And it cleaves specifically in the psbA gene of Arabidopsis, which is naturally intronless. We have shown further that most of the repair products of an I-CreII-induced break in chloroplast DNA can be defined by PCR analysis with total nucleic acids and the appropriate primers. Here, we provide protocols for small-scale preparation of nucleic acids from Chlamydomonas and Arabidopsis, as well as guidelines for the subsequent PCR analysis.

Reverse transcription of spliced psbA mRNA in Chlamydomonas spp. and its possible role in evolutionary intron loss.

Reverse transcription of mRNA is thought to be an important first step in a model that explains certain evolutionary changes within genes, such as the loss of introns or RNA editing sites. In this model, reverse transcription of mRNA produces cDNA molecules that replace part of the parental gene by homologous recombination. In vivo evidence of reverse transcription of physiologically relevant mRNAs is generally lacking, however, except in genetically engineered cells. Here, we provide in vivo evidence for reverse transcription of the chloroplast psbA mRNA in two naturally occurring species of Chlamydomonas (raudensis and subcaudata) that is based on the presence of spliced cDNAs in both organisms. The psbA cDNAs, which lack the group II intron of the genomic gene, are nearly full length, and the majority of them--though not all--are in the form of RNA-cDNA hybrids. Moreover, the presence in these species of psbA cDNAs is correlated with the loss of an early group I intron from the same psbA gene. The group II intron that interrupts psbA in C. raudensis and C. subcaudata potentially encodes a protein with a reverse transcriptase domain, and the C. raudensis protein was shown to have reverse transcriptase activity in vitro. These results provide strong evidence for reverse transcription of a physiologically important mRNA (psbA) in two species of Chlamydomonas that have also lost an intron from the same gene, possibly through recombination with the cDNA.

Circadian regulation of chloroplast transcription in Chlamydomonas is accompanied by little or no fluctuation in RPOD levels or core RNAP activity.

In Chlamydomonas growing under 24 h light-dark cycles, chloroplast transcription is under circadian clock control, and peaks early in the morning. The peak (but not trough) requires ongoing cytoplasmic translation, as it is sensitive to cycloheximide (CH). The chloroplast transcriptional apparatus in Chlamydomonas is simpler than in land plants, with only one type of RNA polymerase (RNAP, bacterial) and apparently only one sigma factor (RPOD). Core RNAP can be assayed in vitro with a non-sigma factor dependent template, and is sensitive to rifampicin. We developed a membrane-based assay for RNAP activity, and used it to determine that core activity is only weakly affected by pre-treating cells with CH. Moreover, core chloroplast RNAP activity was steady during a 24 h light-dark cycle. Levels of the sigma factor (RPOD) were examined using western blots, and found to fluctuate less than 25 % during light-dark cycles. These data indicate that circadian regulation of chloroplast transcription is distinct from regulation by sulfur availability, which involves significant changes in RPOD levels. The implications of this data for hypotheses that purport to explain the circadian control mechanism are discussed.

Mapping of the css (chloroplast splicing suppressor) gene(s) to a recombinationally suppressed region of chromosome III in Chlamydomonas reinhardtii.

In previous work, three suppressors of defective group I introns (7151, 71N1, 7120) were isolated from a mutant of Chlamydomonas reinhardtii that had a splicing-deficient chloroplast large subunit (LSU) rRNA intron. Genetic analysis indicated that the 7151 and 71N1 suppressor mutations each involved single nuclear loci, and that the 7151 mutation was dominant. Here we present genetic evidence that the 7120 suppressor also involves a single nuclear locus and that the mutation is dominant in vegetative diploids. Moreover, we have employed crosses with the S1D2 strain and molecular markers to map the 7120 and 71N1 suppressors. Based on an analysis of 800 progeny from 7120 × S1D2, the 7120 suppressor is located in a region of ~400 kb on chromosome III that is devoid of recombination. The ~400-kb region contains at least 72 genes, about one-third of which (i.e., 22) are predicted to be organelle targeted. Similar analysis of 71N1 × S1D2 using 400 progeny also pointed to the recombination-deficient region of chromosome III, raising the possibility that these mutations could affect the same gene. These efforts lay the foundation for identifying the css (chloroplast splicing suppressor) gene(s), which promotes splicing of multiple chloroplast group I introns.

A novel rhodanese is required to maintain chloroplast translation in Chlamydomonas.

Rhodanese-domain proteins (RDPs) are widespread in plants and other organisms, but their biological roles are mostly unknown. Here we report on a novel RDP from Chlamydomonas that has a single rhodanese domain, and a predicted chloroplast transit peptide. The protein was produced in Escherichia coli with a His-tag, but lacking most of the N-terminal transit peptide, and after purification was found to have rhodanese activity in vitro. It was also used to elicit antibodies for western blot analysis, which showed that the native Chlamydomonas protein migrated slower on SDS gels (apparent M(r) =34 kDa) than its predicted size (27 kDa), and co-fractionated with chloroplasts. To assess function in vivo, the tandem-RNAi approach was used to generate Chlamydomonas strains that had reductions of 30-70% for the mRNA and ~20-40% for the 34-kDa protein. These strains showed reduced growth under all trophic conditions, and were sensitive to even moderate light; properties reminiscent of chloroplast translation mutants. Pulse-labeling in the presence of cycloheximide indicated that chloroplast protein synthesis was broadly reduced in the RNAi strains, and transcript analysis (by RT-PCR and northern blotting) indicated the effect was mainly translational. These results identify a novel rhodanese-like protein that we have named CRLT, because it is required to maintain chloroplast translation.

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis.

Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomology-mediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle's ability to repair broken chromosomes.

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs.

Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG. Previously, we showed that cleavage by I-CreII leaves ends (2-nt 3' overhangs) that are characteristic of GIY-YIG endonucleases, yet it has a relaxed metal requirement like H-N-H enzymes. Here we show that I-CreII can bind DNA without an added metal ion, and that it binds as a monomer, akin to GIY-YIG enzymes. Moreover, cleavage of supercoiled DNA, and estimates of strand-specific cleavage rates, suggest that I-CreII uses a sequential cleavage mechanism. Alanine substitution of a number of residues in the GIY-YIG motif, however, did not block cleavage activity, although DNA binding was substantially reduced in several variants. Substitution of conserved histidines in the H-N-H motif resulted in variants that did not promote DNA cleavage, but retained high-affinity DNA binding-thus identifying it as the catalytic motif. Unlike the non-specific H-N-H colicins, however; substitution of the conserved asparagine substantially reduced DNA binding (though not the ability to promote cleavage). These results indicate that, in I-CreII, two catalytic motifs have evolved to play important roles in specific DNA binding. The data also indicate that only the H-N-H motif has retained catalytic ability.

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

Distinct roles for the 5' and 3' untranslated regions in the degradation and accumulation of chloroplast tufA mRNA: identification of an early intermediate in the in vivo degradation pathway.

Elongation factor Tu in Chlamydomonas reinhardtii is a chloroplast-encoded gene (tufA) whose 1.7-kb mRNA has a relatively short half-life. In the presence of chloramphenicol (CAP), which freezes translating chloroplast ribosomes, a 1.5-kb tufA RNA becomes prominent. Rifampicin-chase analysis indicates that the 1.5-kb RNA is a degradation intermediate, and mapping studies show that it is missing 176-180 nucleotides from the 5' end of tufA. The 5' terminus of the intermediate maps to a section of the untranslated region (UTR) predicted to be highly structured and to encode a small ORF. The intermediate could be detected in older cultures in the absence of CAP, indicating that it is not an artifact of drug treatment. Also, it did not overaccumulate in the chloroplast ribosome-deficient mutant, ac20 cr1, indicating its stabilization is specific to elongation-arrested ribosomes. To determine if the 5' UTR of tufA is destabilizing, the corresponding region of the atpA-aadA-rbcL gene was replaced with the tufA sequence, and introduced into the chloroplast genome; the 3' UTR was also substituted for comparison. Analysis of these transformants showed that the transcripts containing the tufA 3'-UTR accumulate to significantly lower levels. Data from constructs based on the vital reporter, Renilla luciferase, confirmed the importance of the tufA 3'-UTR in determining RNA levels, and suggested that the 5' UTR of tufA affects translation efficiency. These data indicate that the in vivo degradation of tufA mRNA begins in the 5' UTR, and is promoted by translation. The data also suggest, however, that the level of the mature RNA is determined more by the 3' UTR than the 5' UTR.

Unusual metal specificity and structure of the group I ribozyme from Chlamydomonas reinhardtii 23S rRNA.

Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg2+ and Mn2+ support both processes. However, some group I introns are active only with Mg2+, e.g. three of the five group I introns in Chlamydomonas reinhardtii. We have investigated one of these ribozymes, an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii (Cr.LSU), by determining if the inhibition by Mn2+ involves catalysis, folding, or both. Kinetic analysis of guanosine-dependent cleavage by a Cr.LSU ribozyme, 23S.5 Delta Gb, that lacks the 3' exon and intron-terminal G shows that Mn2+ does not affect guanosine binding or catalysis, but instead promotes misfolding of the ribozyme. Surprisingly, ribozyme misfolding induced by Mn2+ is highly cooperative, with a Hill coefficient larger than that of native folding induced by Mg2+. At lower Mn2+ concentrations, metal inhibition is largely alleviated by the guanosine cosubstrate (GMP). The concentration dependence of guanosine cosubstrate-induced folding suggests that it functions by interacting with the G binding site, perhaps by displacing an inhibitory Mn2+. Because of these and other properties of Cr.LSU, the tertiary structure of the intron from 23S.5 Delta Gb was examined using Fe2+-EDTA cleavage. The ground-state structure shows evidence of an unusually open ribozyme core: the catalytic P3-P7 domain and the nucleotides that connect it to the P4-P5-P6 domain are exposed to solvent. The implications of this structure for the in vitro and in vivo properties of this intron ribozyme are discussed.

Mg2+ mimicry in the promotion of group I ribozyme activities by aminoglycoside antibiotics.

Aminoglycoside antibiotics inhibit several types of ribozymes, including group I introns, by displacing critical Mg2+ ions. However, they stimulate activity of the small hairpin ribozyme. We show here that aminoglycosides promote self-splicing of the Cr.psbA2 group I intron at subthreshold Mg2+ concentrations. Neomycin is the most effective of the aminoglycosides tested; it stimulates splicing of Cr.psbA2 at micromolar concentrations, and, in this respect, is >100-fold more effective than spermidine. At optimal Mg2+ for Cr.psbA2 splicing, these drugs, especially kanamycin B and tobramycin, promote GTP attack at the 3' splice-site. Kinetic analysis suggests that this is due to an alternatively folded state of the ribozyme that is induced, or stabilized, by aminoglycosides. A similar effect is observed at high Mg2+ concentrations. Comparing the effects of structurally related aminoglycosides indicates that splicing promotion is more sensitive to drug structure than misfolding and occurs at lower drug concentrations. These data show that aminoglycosides can promote biochemical activities of a large ribozyme by acting as a Mg2+ mimic. The results also underscore the functional diversity of group I introns in nature.

Expression, purification, and biochemical characterization of the intron-encoded endonuclease, I-CreII.

The ORF of the Cr.psbA4 intron of Chlamydomonas reinhardtii mediates efficient intron homing, and contains an H-N-H and possibly a GIY-YIG motif. The ORF was over-expressed in Escherichia coli without non-native amino acids, but was mostly insoluble. However, co-over-expression of E. coli chaperonins GroEL/GroES solubilized approximately 50% of the protein, which was purified by ion-exchange and heparin-affinity chromatography. Biochemical characterization showed that the protein is a double-strand-specific endonuclease that cleaves fused psbA exon 4-exon 5 DNA, and was named I-CreII. I-CreII has a relatively relaxed divalent metal ion requirement (Mg(2+), Mn(2+), Ca(2+), and Fe(2+) supported cleavage), is insensitive to salt <350 mM, and is stabilized by DNA. Cleavage of target DNA occurs close (4 nt on the top strand) to the intron-insertion site, and leaves 2-nt 3'-OH overhangs, similar to GIY-YIG endonucleases. The boundaries of the recognition sequence span approximately 30 bp, and encompass the cleavage and intron-insertion sites. Cleavage of heterologous psbA DNAs indicates the enzyme can tolerate multiple, but not all, substitutions in the recognition site. This work will facilitate further study of this novel endonuclease, which may also find use in site-specific manipulation of chloroplast DNA.

A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self-splicing and genetic evidence for maturase activity.

The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp. from Antarctica that self-splices accurately in vitro. Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E. myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria. In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg(2+) (>12 mM) and NH(4)(+). Self-splicing was improved by deleting most of the ORF and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription. Self-splicing of Chs.psbA1 pre-RNAs showed temperature optima of ~44 degrees C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50 degrees C, indicative of thermolability. Splicing of wild-type Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work provides the first description of a ribozyme from a psychrophilic organism. It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts.

Chloroplast RNA processing and stability.

Primary chloroplast transcripts are processed in a number of ways, including intron splicing, internal cleavage of polycistronic RNAs, and endonucleolytic or exonucleolytic cleavages at the transcript termini. All chloroplast RNAs are also subject to degradation, although a curious feature of many chloroplast mRNAs is their relative longevity. Some of these processes, e.g., psbA splicing and stability of a number of chloroplast mRNAs, are regulated in response to light-dark cycles or nutrient availability. This review highlights recent advances in our understanding of these processes in the model organism Chlamydomonas reinhardtii, focusing on results since the extensive reviews published in 1998 [Herrin DL et al. 1998 (pp. 183-195), Nickelsen Y 1998 (pp. 151-163), Stern DB and Drager RG 1998 (pp. 164-182), in Rochaix JD et al. (eds) The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands]. We also allude to studies with other organisms, and to the potential impact of the Chlamydomonas genome project where appropriate.

Structure, circadian regulation and bioinformatic analysis of the unique sigma factor gene in Chlamydomonas reinhardtii.

In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded bacterial RNAP in which promoter specificity is conferred by nuclear-encoded sigma factors, and a nuclear-encoded phage-like RNAP. Green algae, however, appear to possess only the bacterial enzyme. Since transcription of much, if not most, of the chloroplast genome in Chlamydomonas reinhardtii is regulated by the circadian clock and the nucleus, we sought to identify sigma factor genes that might be responsible for this regulation. We describe a nuclear gene (RPOD) that is predicted to encode an 80 kDa protein that, in addition to a predicted chloroplast transit peptide at the N-terminus, has the conserved motifs (2.1- 4.2) diagnostic of bacterial sigma-70 factors. We also identified two motifs not previously recognized for sigma factors, adjacent PEST sequences and a leucine zipper, both suggested to be involved in protein-protein interactions. PEST sequences were also found in approximately 40% of sigma factors examined, indicating they may be of general significance. Southern blot hybridization and BLAST searches of the genome and EST databases suggest that RPODmay be the only sigma factor gene in C. reinhardtii. The levels of RPODmRNA increased 2- 3-fold in the mid-to-late dark period of light-dark cycling cells, just prior to, or coincident with, the peak in chloroplast transcription. Also, the dark-period peak in RPOD mRNA persisted in cells shifted to continuous light or continuous dark for at least one cycle, indicating that RPODis under circadian clock control. These results suggest that regulation of RPODexpression contributes to the circadian clock's control of chloroplast transcription.

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth.

The chloroplast-encoded psbA gene encodes the D1 polypeptide of the photosystem II reaction center, which is synthesized at high rates in the light. In Chlamydomonas reinhardtii, the psbA gene contains four self-splicing group I introns whose rates of splicing in vivo are increased at least 6-10-fold by light. However, because psbA is an abundant mRNA, and some chloroplast mRNAs appear to be in great excess of what is needed to sustain translation rates, the developmental significance of light-promoted splicing has not been clear. To address this and other questions, potentially destabilizing substitutions were made in several predicted helices of the fourth psbA intron, Cr.psbA4, and their effects on in vitro and in vivo splicing assessed. Two-nucleotide substitutions in P4 and P7 were necessary to substantially reduce splicing of this intron in vivo, although most mutations reduced self-splicing in vitro. The P7-4,5 mutant, whose splicing was completely blocked, showed no photoautotrophic growth and synthesis of a truncated D1 (exons 1-4) polypeptide from the unspliced mRNA. Most informative was the P4'-3,4 mutant, which exhibited a 45% reduction in spliced psbA mRNA, a 28% reduction in synthesis of full-length D1, and an 18% reduction in photoautotrophic growth. These results indicate that psbA mRNA is not in great excess, and that highly efficient splicing of psbA introns, which is afforded by light conditions, is necessary for optimal photosynthetic growth.