A nuclear-encoded function essential for translation of the chloroplast psaB mRNA in chlamydomonas.

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, that contains a mutation at the TAB1 (for translation of psaB mRNA) nuclear locus. Pulse labeling of chloroplast proteins revealed that the synthesis of the two photosystem I reaction center polypeptides PSAA and PSAB was undetectable in this mutant. The mRNA levels of these proteins were only moderately reduced, suggesting that the primary defect occurs at a step during or after translation. We constructed chimeric genes consisting of the psaA and psaB 5' untranslated region (5' UTR) fused to the aminoglycoside adenyltransferase (aadA) coding sequence, which confers spectinomycin resistance. Insertion of these genes into the chloroplast genome through biolistic transformation and analysis of their expression in the TAB1 mutant nuclear background revealed that the psaB (but not the psaA) 5' UTR is the target of the wild-type TAB1 function. This suggests that TAB1 is required for the initiation of psaB mRNA translation. The dependence of PSAA synthesis or accumulation on PSAB synthesis is strongly suggested by the identification of a suppressor mutation within the psaB 5' UTR. The suppressor specifically restores the synthesis of both proteins in the presence of the tab1-F15 mutation. The location of the suppressor mutation within a putative base-paired region near the psaB initiation codon suggests a role for TAB1 in the activation of translation of the psaB mRNA.     

Missense Mutation in the Chlamydomonas Chloroplast Gene that Encodes the Rubisco Large Subunit

The 69-12Q mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase activity, but retains holoenzyme protein. It results from a mutation in the chloroplast large-subunit gene that causes an isoleucine-for-threonine substitution at amino-acid residue 173. Considering that lysine-175 is involved in catalysis, it appears that mutations cluster at the active site.     

A mutation in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that reduces the rate of its incorporation into holoenzyme

A mutant of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), in which Arg53 is replaced by Glu, was synthesized and imported into isolated chloroplasts. The mutant protein was efficiently imported into the chloroplast and correctly processed to the mature size. Like the wild type protein, it was stable over a period of at least 2 h. Unlike the wilk-type protein however, most of the mutant protein was not assembled with holo-Rubisco at the end of a 10-min import reaction. It migrated instead as a diffused band on a non-denaturing gel, slower than the precursor protein, but faster than the holoenzyme. The level of the unassembled mutant protein in the stroma decreased with time, while its level in the assembled fraction has increased, indicating that this protein is a slowly-assembled, rather than a non-assembled, mutant of the small suubunit of Rubisco. Accumulation of the mutant protein in the holoenzyme fraction was dependent on ATP and light. The transient species, migrating faster than the holoenzyme but slower than the precursor protein, may represent an intermediate in the assembly process of the small subunit of RubiscoAbbreviations LSU large subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of Rubisco     

Molecular basis of the ribulose-1,5-bisphosphate carboxylase/oxygenase activase mutation in Arabidopsis thaliana is a guanine-to-adenine transition at the 5'-splice junction of intron 3.

Analysis of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase gene and gene products from Arabidopsis thaliana wild-type plants and the Rubisco activase-deficient mutant strain showed that the rca mutation caused GT to be changed to AT at the 5'-splice junction of intron 3 in the six-intron pre-mRNA. Northern blot analysis, genomic and cDNA sequencing, and primer extension analysis indicated that the mutation causes inefficient and incomplete splicing of the pre-mRNA, resulting in the accumulation of three aberrant mRNAs. One mutant mRNA was identical with wild-type mRNA except that it included intron 3, a second mRNA comprised intron 3 and exons 4 through 7, and the third mRNA contained exons 1 through 3. The G-to-A transition is consistent with the known mechanism of mutagenesis by ethyl methanesulfonate, the mutagen used to create the Rubisco activase-deficient strain.     

A Dominant Mutation in the Chlamydomonas Reinhardtii Nuclear Gene Sim30 Suppresses Translational Defects Caused by Initiation Codon Mutations in Chloroplast Genes

A suppressor of a translation initiation defect caused by an AUG to AUU mutation in the Chlamydomonas reinhardtii chloroplast petD gene was isolated, defining a nuclear locus that we have named SIM30. A dominant mutant allele at this locus, sim30-1d, was found to increase the translation initiation rate of the mutant petD mRNA. sim30-1d was also able to suppress the translational defect caused by an AUG to AUC mutation in the petD gene, and an AUG to AUU mutation in the chloroplast petA gene. We therefore suggest that the SIM30 gene may encode a general chloroplast translation factor. The ability of sim30-1d to suppress the petD AUG to AUU mutation is diminished in the presence of one or more antibiotic resistance markers located within the 16S and 23S rRNAs, suggesting that the activity of the sim30-1d gene product in translation initiation may involve interaction with ribosomal subunits.     

Pseudoreversion substitution at large-subunit residue 54 influences the CO2/O2 specificity of chloroplast ribulose-bisphosphate carboxylase/oxygenase.

Chlamydomonas reinhardtii mutant 31-4E lacks ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) holoenzyme due to a mutation in the chloroplast rbcL gene. This mutation causes a glycine54-to-aspartate substitution within the N-terminal domain of the Rubisco large subunit. In the present study, photosynthesis-competent revertants were selected to determine whether other amino acid substitutions might complement the primary defect. Revertants were found to arise from only true reversion or either of two forms of pseudoreversion affecting residue 54. One pseudorevertant has a glycine54-to-alanine substitution that decreases the accumulation of holoenzyme, but the purified Rubisco has near-normal kinetic properties. The other pseudorevertant has a glycine54-to-valine substitution that causes an even greater decrease in holoenzyme accumulation. Rubisco purified from this strain was found to have an 83% decrease in the Vmax of carboxylation and an 18% decrease in the CO2/O2 specificity factor. These results indicate that small increases in the size of amino acid side chains can influence Rubisco assembly or stability. Even though such changes occur far from the active site, they also play a significant role in determining Rubisco catalytic efficiency.     

拟南芥叶绿体分裂突变体pdl37的基因鉴定与分析

pd137是经甲基磺酸乙脂（ethyl methane sulphonate, EMS）诱变并通过筛选得到的一个拟南芥叶绿体分裂突变体。该突变体的叶绿体表型与野生型相比有很大差异： 叶绿体面积显著增大, 细胞中叶绿体数量明显减少。遗传分析显示pd137的突变表型受隐性单基因控制。本研究通过遗传作图将该突变基因粗定位于拟南芥2号染色体的分子标记CH2-13.70和CH2-16.0区间内。该区间内已知的与叶绿体分裂相关的基因只有FtsZ2-1。对FtsZ2-1基因的测序结果显示pd137突变体的FtsZ2-1基因第505位碱基发生了无义突变, 使蛋白质翻译提前终止。该突变还严重影响了FtsZ2-1基因的mRNA水平。转基因互补实验进一步验证了该突变体表型是由于FtsZ2-1基因突变引起。本项工作为研究叶绿体分裂的机制提供了新材料和一些有用的线索。     

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast.

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.     

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.     

A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis

Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.     

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO₂ fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.     

Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii.

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N-terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser-Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild-type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5' untranslated region of psbC where the RNA can be folded into a stem-loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem-loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.     

Function of 3′ non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3 untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3 flanking sequences of psaB or extended the open reading frame into the 3 inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3 stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3 inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.