Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.     

Induction and segregation of chloroplast mutations in vegetative cell cultures of chlamydomonas reinhardtii

The single chloroplast of the alga Chlamydomonas reinhardtii contains at least 100 copies of the chloroplast chromosome. It is not known how the chloroplast (or cell) becomes homoplasmic for a mutation that arises in one of these copies. Under suitable selection conditions, clones with chloroplast mutations for streptomycin resistance induced by methyl methanesulfonate can be recovered with direct plating after mutagenesis. Using an adaptation of the Luria-Delbrück fluctuation test, mutagenized cultures grown on nonselective liquid medium for seven to nine doublings show negligible proliferation of cells capable of forming such mutant colonies. In contrast, cells among the same cultures with reduced nuclear mutations conferring streptomycin resistance reveal considerable clonal propagation prior to plating on selection medium. Reconstruction growth-rate experiments show no reduced growth of cells with chloroplast mutations relative to either wild-type cells or to those with nuclear mutations. We propose that newly arising chloroplast mutations and their copies are usually transmitted to only one daughter cell for several cell generations by reductional divisions of the chloroplast genome. In the absence of recombination and mixing, such a reductional partition of chloroplast alleles would readily permit the formation of homoplasmic lines without the need for selection.     

Zeolin is a recombinant storage protein with different solubility and stability properties according to its localization in the endoplasmic reticulum or in the chloroplast

Several strategies have been exploited to maximize heterologous protein accumulation in the plant cell. Recently, it has been shown that a portion of a maize prolamin storage protein, gamma-zein, can be used for the high accumulation of a recombinant protein in novel endoplasmic reticulum (ER)-derived protein bodies of vegetative tissues. In this study, we investigate whether this protein can be expressed in the chloroplast. Our long-term purpose is to use zeolin to produce value-added proteins by fusing these polypeptides with its gamma-zein portion and targeting the recombinant proteins to the ER or to the chloroplast. We show here that zeolin accumulates in the chloroplast to lower levels than in the ER and its stability is compromised by chloroplast proteolytic activity. Co-localization of zeolin and the ER chaperone BiP in the chloroplast does not have a beneficial effect on zeolin accumulation. In this organelle, zeolin is not stored in protein bodies, nor do zeolin polypeptides seem to be linked by inter-chain disulfide bonds, which are usually formed by the six cysteine of the gamma-zein portion, indicating abnormal folding of the recombinant protein. Therefore, it is concluded that to accumulate zeolin in the chloroplast it is necessary to facilitate inter-chain disulfide bond formation.     

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.     

Mutant phenotypes support a trans-splicing mechanism for the expression of the tripartite psaA gene in the C. reinhardtii chloroplast

The chloroplast psaA gene of the green unicellular alga Chlamydomonas reinhardtii consists of three exons that are transcribed from different strands. Analysis of numerous nuclear and chloroplast mutants that are deficient in photosystem I activity reveals that roughly one-quarter of them are specifically affected in psaA mRNA maturation. These mutants can be grouped into three phenotypic classes, based on their inability to perform either one or both splicing reactions. The data indicate that the three exons are transcribed independently as precursors which are normally assembled in trans and that the splicing reactions can occur in either order. While some chloroplast mutations could act in cis, the nuclear mutations that fall into several complementation groups probably affect factors specifically required for assembling psaA mRNA.     

Differential Synthesis of Photosystem Cores and Light-Harvesting Antenna during Proplastid to Chloroplast Development in Spirodela oligorrhiza

Proplastids and etioplasts are common starting points for monitoring chloroplast development in higher plants. Although proplastids are the primary precursor of chloroplasts, most proplastid to chloroplast systems are cumbersome to study temporally. Conversely, the etioplast to chloroplast transition is initiated by light and is readily examined as a function of time. Etioplasts, however, are found mostly in plants germinated in the dark and are not an obligatory step in chloroplast development. We have chosen to study chloroplast ontogeny in Spirodela oligorrhiza (Kurtz) Hegelm (a C₃-monocot) because of its unique ability to grow indefinitely in the dark. Ultrastructural, physiological, and molecular evidence is presented in support of a temporal, light-triggered proplastid to chloroplast transition in Spirodela. The dark-grown plants are devoid of chlorophyll, and upon illumination synchronously green over a 3- to 5-day period. Synthesis of chloroplast proteins involved in photosynthesis is coincident with thylakoid assembly, chlorophyll accumulation, and appearance of CO₂ fixation activity. Interestingly, the developmental sequence in Spirodela was slow enough to reveal that biosynthesis of the D1 photosystem II reaction center protein precedes biosynthesis of the major light-harvesting antenna proteins. This, coupled with the high chlorophyll a/b ratio observed early in development, indicated that reaction center assembly occurred prior to accumulation of the light-harvesting complexes. Thus, with Spirodela one can study proplastid to chloroplast conversions temporally in higher plants and follow the process on a time scale that enables a detailed dissection of plastid maturation processes.     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive gamma-Irradiation of the Seed

Spinach seeds (Spinacia oleracea L.) given massive doses of γ-irradiation (500 krad) germinate and form a seedling with two green cotyledons and a radicle, but develop no further. Irradiated cotyledons show no increase in cell number or total DNA over a 7-day period in the light, while in control cotyledons there is a small increase in cell number and large increases in total DNA and chloroplast number. The chloroplasts of irradiated cotyledons are delayed in their division, become greatly enlarged and contain large amounts of starch. The whole population of chloroplasts subsequently undergoes a wave of division. The daughter chloroplasts show normal thylakoid development, but have some abnormal structural features caused by the radiation stress. Information on the effect of X-irradiation, ultraviolet irradiation, and 5-fluorodeoxyuridine on chloroplast replication and on chloroplast and nuclear DNA synthesis was obtained from cultured spinach leaf discs. It appears that chloroplast replication is more resistant to ionizing radiation than cell division and can proceed in the absence of nuclear DNA synthesis and greatly reduced chloroplast DNA synthesis.     

The Effect of Light and Inhibitors on Chloroplast and Cytoplasmic RNA Synthesis

Chloroplast RNA is synthesized in dark-grown radish cotyledons at about one-third the rate of that in the light. The synthesis, however, continues for longer in the dark and the percentage of chloroplast RNA can approach that in light-grown tissue. Light stimulates the synthesis and accumulation of both cytoplasmic and chloroplast RNA, but shows a 4-fold greater stimulation of the chloroplast RNA. Chloramphenicol, streptomycin and cycloheximide inhibit the synthesis of chloroplast RNA with little effect on cytoplasmic RNA. 5-Fluorouracil inhibits the synthesis of cytoplasmic more than chloroplast RNA. Synthesis of the 0.56 x 10(6) mol wt chloroplast RNA is inhibited much less than the other ribosomal RNA components by actinomycin D.     

Integration of light and plastid signals

Light and plastid signals promote chloroplast biogenesis and are among the most potent inducers and repressors of photosynthesis-related gene expression, respectively. These signals can be likened to a 'gas and brake system' that promotes efficient chloroplast biogenesis and function. Recent findings indicate that a particular plastid signal can 'rewire' a light signaling network, converting it from an inducer into a repressor of particular photosynthesis-related genes. Therefore, a plastid signal appears to be an endogenous regulator of light signaling rather than a signal acting independently from light. This integration of light and plastid signals may allow plants to proactively manage chloroplast dysfunction when performing chloroplast biogenesis and maintenance in adverse light conditions.     

Plastid division and morphology in the genus Peperomia

We have investigated several factors determining plastid size and number in Peperomia, a genus in the Piperaceae family whose species naturally display great interspecific variation in chloroplast size and number per cell. Using microscopic techniques, we show that chloroplast size and number are differently regulated in the palisade parenchyma and the spongy parenchyma, suggesting that chloroplast division in these cell types is controlled in different ways. Microscopic studies of iodine-stained root cells revealed a correlation between amyloplast size in root cells and chloroplast size in palisade parenchyma cells. However, despite substantial variation in chloroplast number in leaf mesophyll cells, amyloplast number in root cells was very similar in all species. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. We also demonstrate that plastid size determines the size but not the number of starch grains in root amyloplasts.     

Effect of High Temperature on Chloroplast Ribosomes and Biosynthesis of Chloroplast Proteins in Wheat

The chloroplasts of wheat have chanced greatly at high temperature condition(34℃). When wheat grown at 34℃ for 10 days, its chlorophyll content was 6 times less than that under the normal condition(22℃). The ribosomes were isolated from the leaves by sucrose density gradient centrifugation. It is found that only 80 S ribosomes existed in wheat leaves grown at the high temperature and the formation of 70 S ribosomes is specifically prevented. Since the absence of 70 S ribosomes in chloroplast, proteins synthesis can no longer proceed. Analysis of SDS-polyacrylamide gel electrophoresis indicates that the bands of chloroplast proteins from the leaves of wheat at the high temperature are less than those under normal condition. One of the poly- peptides the large subunit(MW=57000 daltons) of ribulose bisphosphate carboxylase, which is coded for by chloroplast genome and synthesized on 70 S ribosome in chloroplast, was lost. The photosynthetic intensity is decreased due to the blocking synthesis in chloroplast of some polypeptides which play the important role in photosynthesis.     

Evolution of the chloroplast genome and polymorphic ITS regions in Allium subg. Melanocrommyum.

Relationships based on PCR-RFLPs of non-coding regions of cpDNA indicate that some of the largest subgenera of the genus Allium and five of the largest sections of the Central Asian subg. Melanocrommyum are artificial. Internested synapomorphic mutations without homoplasy were found only in the chloroplast genomes of plants of subg. Melanocrommyum that occur in the border region of Tajikistan, Uzbekistan, Afghanistan, and Kyrgyzstan. Eighteen of 49 plants surveyed were polymorphic for their ITS regions. Even plants that had identical chloroplast genomes were polymorphic for nuclear ribosomal regions. These individuals had markedly different frequencies of ITS variants that were detected with various restriction enzymes. The geographic partitioning of chloroplast haplotypes and the fact that the ITS variants could not be ordered hierarchically can readily be envisioned to result from gene flow. Processes such as concerted evolution and parallel morphological evolution may also be partly responsible for the disconcordance of mutations in the chloroplast and nuclear genome. However, the chimeric nature of the nuclear ribosomal regions indicates that concerted evolution is not the dominating process in Allium subg. Melanocrommyum.     

玉米叶绿体表达载体的构建及AsRED基因原核表达

从玉米幼嫩叶片中提取玉米叶绿体基因DNA,通过PCR克隆出叶绿体同源重组片段trnA和trnI、叶绿体特异性启动子Prrn以及终止子psbA.构建玉米叶绿体表达载体pBAIRTARED,含有一个人工操纵子,其中,筛选标记基因aadA和红色荧光蛋白报告基因AsRED处于Prrn启动子和psbA终止子控制.将构建的载体转化大肠杆菌BL21（DE3）,观测到重组细胞呈现红色,表明构建的载体可以用于玉米叶绿体转化以及表达报告基因.