Chloroplast Ribosomes Research Articles

High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.

Chlamydomonas reinhardtii proteomics

Proteomics, based on the expanding genomic resources, has begun to reveal new details of Chlamydomonas reinhardtii biology. In particular, analyses focusing on subproteomes have already provided new insight into the dynamics and composition of the photosynthetic apparatus, the chloroplast ribosome, the oxidative phosphorylation machinery of the mitochondria, and the flagellum. It assisted to discovered putative new components of the circadian clockwork as well as shed a light on thioredoxin protein–protein interactions. In the future, quantitative techniques may allow large scale comparison of protein expression levels. Advances in software algorithms will likely improve the use of genomic databases for mass spectrometry (MS) based protein identification and validation of gene models that have been predicted from the genomic DNA sequences. Although proteomics has only been recently applied for exploring C. reinhardtii biology, it will likely be utilized extensively in the near future due to the already existing genetic, genomic, and biochemical tools.

Chloroplast Elongation Factor Ts Pro-Protein Is an Evolutionarily Conserved Fusion with the S1 Domain-Containing Plastid-Specific Ribosomal Protein-7

The components of chloroplast translation are similar to those of prokaryotic translation but contain some additional unique features. Proteomic analysis of the Chlamydomonas reinhardtii chloroplast ribosome identified an S1-like protein, plastid-specific ribosomal protein-7 (PSRP-7), as a stoichiometric component of the 30S subunit. Here, we report that PSRP-7 is part of a polyprotein that contains PSRP-7 on its amino end and two translation elongation factor Ts (EF-Ts) domains at the carboxy end. We named this polyprotein PETs (for polyprotein of EF-Ts). Pets is a single-copy gene containing the only chloroplast PSRP-7 and EF-Ts sequences found in the C. reinhardtii genome. The pets precursor transcript undergoes alternative splicing to generate three mRNAs with open reading frames (ORFs) of 1.68, 1.8, and 3 kb. A 110-kD pro-protein is translated from the 3-kb ORF, and the majority of this protein is likely posttranslationally processed into the 65-kD protein PSRP-7 and a 55-kD EF-Ts. PETs homologs are found in Arabidopsis thaliana and rice (Oryza sativa). The conservation of the 110-kD PETs polyprotein in the plant kingdom suggests that PSRP-7 and EF-Ts function together in some aspects of chloroplast translation and that the PETs pro-protein may have a novel function as a whole.

RNA editing in ribosome-less plastids of iojap maize.

In maize chloroplasts, 28 C-to-U editing events have been identified in the transcripts of 14 different genes. The iojap mutant of maize, which lacks chloroplast ribosomes, affords the opportunity to examine whether any chloroplast translation products are required for the editing of any of the 28 sites. Furthermore, the mode of action of the IOJAP protein itself is unknown, so we explored the possibility that homozygous ij1/ ij1 plants are defective in RNA editing. Current knowledge of RNA editing in chloroplasts indicates the existence of site-specific factors responsible for recognizing C targets of editing, but the factors have not yet been identified and their encoding genes are unknown. Our results indicate that all 28 editing sites can be recognized and processed in ribosome-less plastids. Transcripts of rpoB are more abundant and more highly edited in iojap mutants. The editing site in rpl2, which creates the mRNA start codon, is the most severely affected in homozygous ij/ ij plants, but nevertheless exhibits at least 10% editing in all mutant lines. Reduced editing of rpl2 may be an indirect effect of reduced splicing, rather than a defect caused by the iojap mutation. We conclude that neither the IOJAP protein nor chloroplast translation products are required for editing any of the 28 C targets of editing in maize chloroplast RNAs.

Proteomic Characterization of the Chlamydomonas reinhardtii Chloroplast Ribosome: IDENTIFICATION OF PROTEINS UNIQUE TO THE 70 S RIBOSOME

We have conducted a proteomic analysis of the 70 S ribosome from the Chlamydomonas reinhardtii chloroplast. Twenty-seven orthologs of Escherichia coli large subunit proteins were identified in the 50 S subunit, as well as an ortholog of the spinach plastid-specific ribosomal protein-6. Several of the large subunit proteins of C. reinhardtii have short extension or insertion sequences, but overall the large subunit proteins are very similar to those of spinach chloroplast and E. coli. Two proteins of 38 and 41 kDa, designated RAP38 and RAP41, were identified from the 70 S ribosome that were not found in either of the ribosomal subunits. Phylogenetic analysis identified RAP38 and RAP41 as paralogs of spinach CSP41, a chloroplast RNA-binding protein with endoribonuclease activity. Overall, the chloroplast ribosome of C. reinhardtii is similar to those of spinach chloroplast and E. coli, but the C. reinhardtii ribosome has proteins associated with the 70 S complex that are related to non-ribosomal proteins in other species. In addition, the 30 S subunit contains unusually large orthologs of E. coli S2, S3, and S5 and a novel S1-type protein (Yamaguchi, K. et al., (2002) Plant Cell 14, 2957-2974). These additional proteins and domains likely confer functions used to regulate chloroplast translation in C. reinhardtii.

Group II intron splicing factors derived by diversification of an ancient RNA-binding domain.

Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosome. Many group II introns have lost the ability to splice autonomously as the result of an evolutionary process in which the loss of self-splicing activity was compensated by the recruitment of host-encoded protein cofactors. Genetic screens previously identified CRS1 and CRS2 as host-encoded proteins required for the splicing of group II introns in maize chloroplasts. Here, we describe two additional host-encoded group II intron splicing factors, CRS2-associated factors 1 and 2 (CAF1 and CAF2). We show that CRS2 functions in the context of intron ribonucleoprotein particles that include either CAF1 or CAF2, and that CRS2-CAF1 and CRS2-CAF2 complexes have distinct intron specificities. CAF1, CAF2 and the previously described group II intron splicing factor CRS1 are characterized by similar repeated domains, which we name here the CRM (chloroplast RNA splicing and ribosome maturation) domains. We propose that the CRM domain is an ancient RNA-binding module that has diversified to mediate specific interactions with various highly structured RNAs.

Discovery and characterization of Acanthamoeba castellanii mitochondrial 5S rRNA.

Although 5S rRNA is a highly conserved and universal component of eubacterial, archaeal, chloroplast, and eukaryotic cytoplasmic ribosomes, a mitochondrial DNA-encoded 5S rRNA has so far been identified only in land plants and certain protists. This raises the question of whether 5S rRNA is actually required for and used in mitochondrial translation. In the protist Acanthamoeba castellanii, BLAST searches fail to reveal a 5S rRNA gene in the complete mitochondrial genome sequence, nor is a 5S-sized RNA species detectable in ethidium bromide-stained gels of highly purified mitochondrial RNA preparations. Here we show that an alternative visualization technique, UV shadowing, readily detects a novel, mitochondrion-specific small RNA in A. castellanii mitochondrial RNA preparations, and that this RNA species is, in fact, a 5S rRNA encoded by the A. castellanii mitochondrial genome. These results emphasize the need for caution when interpreting negative results that suggest the absence of 5S rRNA and/or a mitochondrial DNA-encoded 5S rRNA sequence in other (particularly protist) mitochondrial systems.

The Solution Structure of the Loop E Region of the 5 S rRNA from Spinach Chloroplasts

A structure has been obtained for the loop E region of the 5 S rRNA from Spinacia oleracia chloroplast ribosomes using residual dipolar coupling data as well as NOE, J coupling and chemical shift information. Even though the loop E sequence of this chloroplast 5 S rRNA differs from that of Escherichia coli loop E at ∼40% of its positions, its conformation is remarkably similar to that of E. coli loop E. Consistent with this conclusion, ribosomal protein L25 from E. coli , which binds to the loop E region of both intact E. coli 5 S rRNA and to oligonucleotides containing that sequence, also binds to the chloroplast-derived oligonucleotide discussed here.

Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.

Six ribosomal proteins are specific to higher plant chloroplast ribosomes [Subramanian, A.R. (1993) Trends Biochem. Sci.18, 177-180]. Three of them have been fully characterized [Yamaguchi, K., von Knoblauch, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465; Yamaguchi, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28466-28482]. The remaining three plastid-specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N-terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs). PSRP-3 exists in two forms (alpha/beta, N-terminus free and blocked by post-translational modification), whereas PSRP-2 and PSRP-4 appear, from MS data, to be unmodified. PSRP-2 contains two RNA-binding domains which occur in mRNA processing/stabilizing proteins (e.g. U1A snRNP, poly(A)-binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis. PSRP-3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga. The ycf65 gene is absent from the chloroplast genomes of higher plants. Therefore, we suggest that Psrp-3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle-to-nucleus gene transfer in chloroplast evolution. PSRP-4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis. We would therefore suggest that PSRP-4 is an example of gene capture (via horizontal gene transfer) during chloro-ribosome emergence. Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database. All six PSRPs are nucleus-encoded. The cytosolic precursors of PSRP-2, PSRP-3, and PSRP-4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively. Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.

Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5.

To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.

Modeling a Minimal Ribosome Based on Comparative Sequence Analysis

We have determined the three-dimensional organization of ribosomal RNAs and proteins essential for minimal ribosome function. Comparative sequence analysis identifies regions of the ribosome that have been evolutionarily conserved, and the spatial organization of conserved domains is determined by mapping these onto structures of the 30 S and 50 S subunits determined by X-ray crystallography. Several functional domains of the ribosome are conserved in their three-dimensional organization in the Archaea, Bacteria, Eucaryotic nuclear, mitochondria and chloroplast ribosomes. In contrast, other regions from both subunits have shifted their position in three-dimensional space during evolution, including the L11 binding domain and the α-sarcin–ricin loop (SRL). We examined conserved bridge interactions between the two ribosomal subunits, giving an indication of which contacts are more significant. The tRNA contacts that are conserved were also determined, highlighting functional interactions as the tRNA moves through the ribosome during protein synthesis. To augment these studies of a large collection of comparative structural models sampled from all major branches on the phylogenetic tree, Caenorhabditis elegans mitochondrial rRNA is considered individually because it is among the smallest rRNA sequences known. The C. elegans model supports the large collection of comparative structure models while providing insight into the evolution of mitochondrial ribosomes.

The Arabidopsis HUELLENLOS Gene, Which Is Essential for Normal Ovule Development, Encodes a Mitochondrial Ribosomal Protein

The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning.

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present.

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.

The Small Subunit of the Mammalian Mitochondrial Ribosome: IDENTIFICATION OF THE FULL COMPLEMENT OF RIBOSOMAL PROTEINS PRESENT

Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human mitochondrial ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster mitochondrial ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.

Discovery of a protein required for photosynthetic membrane assembly.

About one-half of the ≈57 proteins of the photosynthetic membranes in plant chloroplasts is encoded in the chloroplast genome, translated on chloroplast ribosomes and cotranslationally inserted into the lipid bilayer of the thylakoids (flattened sacs; ref. 1). The other half is translated on cytosolic ribosomes from messenger RNA of nuclear genes and posttranslationally imported across the outer and inner chloroplast envelope membranes. The import machinery consists of smart receptor proteins, translocation channels, and chaperon proteins (2, 3). The photosynthetic proteins are assembled into five multisubunit complexes. There, the pigments for light harvesting and the electron transport molecules for the primary light-dependent charge separation and for the generation of protons and oxygen in the water-splitting reaction are positioned at the optimal distances. The two photosystems cooperate via the cytochrome b6f complex in the transfer of electrons to provide reducing power in the form of NADPH and establishment of proton gradients from the stroma side to the lumen of the thylakoid. The electrochemical gradient formed by the accumulation of protons in the thylakoid lumen provides the driving force for the phosphorylation of ADP by the ATP synthase complex. In contrast to the profound knowledge of the organization of the photosynthetic membrane and the import of the protein components into the chloroplast and their targeting to the thylakoids, progress in learning how the lipid bilayer membranes are formed is less obvious. This situation may change with the discovery of a function of the vesicle-inducing protein in plastids (VIPP) by Kroll et al. and Westphal et al. as reported in this issue of PNAS (4, 5).

PLASTID FATTY ACID BIOSYNTHESIS IN THE DIATOMS NITZSCHIA ALBA AND NITZSCHIA LAEVIS

The role of the plastid in fatty acid biosynthesis in the non‐photosynthetic diatom Nitzschia alba was studied and compared to that in the photosynthetic, closely related Nitzschia laevis. Transmission electron microscopy was used to analyze structural features of the plastid that may be relevant to biochemical function. Typical of a photosynthetic diatom, N. laevis had a chloroplast envelope composed of four membranes, and had abundant chloroplast ribosomes. The leucoplast of N. alba also had a multi‐membrane envelope, chloroplast ribosomes, and a genome that encodes plastid specific proteins. This suggested that the plastid of N. alba may still possess the biochemical functions of the chloroplast, aside from photosynthesis. To determine whether plastidial fatty acid biosynthesis occurred in N. alba, the response of the two diatoms to the compound thiolactomycin was compared. Thiolactomycin has been shown to effect keto‐acyl‐ACP‐synthases, and specifically inhibits the plastidial fatty acid biosynthetic pathway. While growth of N. alba was not impacted by thiolactomycin as in N. laevis, neutral lipid accumulation and fatty acid composition was impacted by thiolactomycin in both organisms. These findings suggest that the plastidial fatty acid biosynthetic pathway does exist in the leucoplast of N. alba, although it lacks photosynthetic capacity.

TRANSFER OF PROTEINS FROM THE CHLOROPLAST TO VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): A PATHWAY FOR DEGRADATION

Several chloroplast proteins were detected by immunoelectron microscopy within dense granules in cytoplasmic vacuoles in the alga Chlamydomonas reinhardtii Dangeard. Transfer from chloroplast to vacuoles of two major, pulse‐labeled polypeptides, the large subunit of rubisco and the α subunit of ATPase, which are synthesized on chloroplast ribosomes, was demonstrated by the recovery of these polypeptides in vacuolar granules over a several‐hour time period. The ultrastructure of cryofixed algal cells was examined to search for structures that would provide insight into the transfer of chloroplast proteins to vacuoles. Micrographs showed that the two membranes of the envelope were appressed, with no detectable intermembrane space, over most of the chloroplast surface. Protrusions of the outer membrane of the envelope were occasionally found that enclosed stroma, with particles similar in size to chloroplast ribosomes, but generally not thylakoid membranes. These observations suggest that chloroplast material, especially the stromal phase, was extruded from the chloroplast in membrane‐bound structures, which then interacted with Golgi‐derived vesicles for degradation of the contents by typical lysosomal activities. A protein normally targeted to vacuoles through the endomembrane system for incorporation into the cell wall was detected in Golgi structures and vacuolar granules but not the chloroplast.

A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting.

A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO), was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein-protein interactions. These motifs include ankyrin repeats and chromodomains. Therefore, CAO encodes an SRP component that is unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes.

Cell ultrastructure of needles from saplings of Norway spruce, Picea abies (L) Karst., exposed to ozone and low phosphorus supply in open-top chambers

Two-year-old saplings of one clone of Norway spruce, Picea abies (L.) Karst., were exposed to ozone in open-top chambers. The treatments were; charcoal filtered air (CF); non-filtered air + 1.5 times ambient ozone (NF+); charcoal filtered air and low phosphorus supply (CF/PD); non-filtered air + ozone and low phosphorus supply (NF+/PD). Ozone exposure was started in 1992 and low phosphorus supply in 1993. Current (C) and 1-year-old (C+1) needles were sampled on 27 July 1994 for structural studies. Electron microscopy showed that the structure of the mesophyll cells in C and C+1 needles from CF treatment was intact. Statistically significant ozone-induced changes in ultrastructure were found only in the chloroplasts, all the other organelles were intact. In both ozone treatments the stroma was granulated. The granules were about 1.5 times the size of the chloroplast ribosomes. In the low phosphorus supply changes were seen in the chloroplasts, in the mitochondria, and in the cytoplasm. The mean chloroplast and starch grain areas were significantly increased. In both age classes about 12% and 20% of the mitochondria were injured in the CF/PD and NF+/PD treatments, respectively. Furthermore, the cytoplasm in the C needles from low phosphorus supply treatments was partly disintegrated. The symptoms induced by ozone and low phosphorus supply were clearly distinguishable from each other, also in combined treatments, which points out their diagnostic values.

Dr and spr/sr mutations of Chlamydomonas reinhardtii affecting D1 protein function and synthesis define two independent steps leading to chronic photoinhibition and confer differential fitness

ABSTRACTThe effects of introduced chloroplast gene mutations affecting D1 synthesis, turnover and function on photosynthesis, growth and competitive ability were examined in autotrophic cultures of Chlamydomonas reinhardtii (Chlorophyta) adapted to low or high irradiance. Few discernible effects were evident when the mutants were grown in low light (LL, 70 μmol m−2 s−1). The herbicide‐resistant psbA mutation Ser264→ Ala (dr) slowed electron transfer and accelerated D1 degradation in cells grown under high light (HL, 600 μmol m−2 s−1). The maximum rate of light‐and CO2‐saturated photosynthesis, cell growth rate and competitive ability in the dr mutant were reduced compared to wild type under HL. However, the wild‐type rate of D1 synthesis in dr was adequate to compensate for accelerated D1 degradation. 16S rRNA mutations conferring resistance to streptomycin and spectinomycin (spr/sr) that altered chloroplast ribosome structure and assembly were used to inhibit chloroplast protein synthesis. In spr/sr cells grown under HL, D1 synthesis was reduced by 40–60% compared to wild type and D1 degradation was accelerated, leading to a 4‐fold reduction in D1 pool size. The reduced D1 levels were accompanied by an elevation of Fo and a decline in Fv/Fm, quantum yield and maximum rate of CO2‐saturated photosynthesis. Chemostat experiments showed that the growth rate and competitive ability of spr/sr were reduced against both wild type and dr.