Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

Subunit polypeptide composition of rubisco and the origin of allopolyploid Arabidopsis suecica (Brassicaceae)

The polypeptide composition of the large and small subunits of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) from Arabidopsis thaliana, A. suecica and Cardaminopsis arenosa have been studied by IEF (isoelectric focusing) analysis. The putative recent alopolyploid origin of A. suecica is supported. The chloroplast encoded large subunits served to identify solely A. thaliana as the maternal parent whereas the nuclear encoded small subunits indicate C. arenosa as the paternal species.     

Phytochrome-mediated plastid development in etiolated pea stem apices

The effects of red and far-red light on growth and plastid development in the stem apices of etiolated pea seedlings have been examined. Changes were determined in various growth parameters (DNA, soluble protein and fresh weight) and also in the activities of the plastid-localized enzymes ribulose-1,5-bisphosphate carboxylase, NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline-1,6-bisphosphatase and the non-photosynthetic (cytoplasmic) enzymes NADP-isocitrate dehydrogenase, enolase and NAD-malate dehydrogenase. Changes in the amounts of Fraction I protein were also measured. Brief daily irradiation with low intensity red light increased growth 5·1–7·6-fold which was correlated with increases of about 3·5-fold in activities of the non-photosynthetic enzymes. The chloroplast enzymes, however, showed much greater increases in activity ranging from 15- to 91-fold. Fraction I protein increased 11·7-fold. These increases approached the levels attained in fully green leaves. All these responses were largely prevented by far-red light indicating that they were mediated by phytochrome. In experiments with red light given at daily intervals there was a lag of 24 hr before the initially very low activity of ribulose-1,5-bisphosphate carboxylase increased. Fraction I protein which was initially present in significant amounts showed a similar lag in its synthesis. However, for 3 days after the initial irradiation, the rate of increase of the enzymic activity was much greater than the rate of net synthesis of Fraction I protein. A single initial red irradiation was as effective as 3 daily irradiations in increasing the activity of ribulose-1,5-bisphosphate carboxylase. A fourth irradiation, however, gave an additional response which exceeded that of the single initial irradiation. It was shown that there was a rapid activation of ribulose-1,5-bisphosphate carboxylase by either continuous white or 3 min of red light. The red light response was slowly reversed in the dark. These results are discussed with particular emphasis on the relation between growth and plastid development in a phytochrome-mediated system.     

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.     

Arabidopsis rbcS genes are differentially regulated by light.

Individual members of the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene family are differentially regulated by light of different qualities. In 10-d-old etiolated seedlings, the expression of only three of the four genes is under inductive phytochrome control. rbcS mRNA levels reach a maximum (3- to 5-fold higher than the dark level) about 6 h after a red light pulse, but the rate of decay differs among the genes. Moreover, rbcS 2B requires a higher fluence for induction. At early stages of development, rbcS 1A, 2B, and 3B are highly expressed in the dark and cannot be further induced by red light, indicating a developmental component in the overall regulatory mechanism. Continuous light experiments indicate that high-irradiance responses may play a role in the induction of at least three of the four rbcS genes. Under conditions of phytochrome saturation, rbcS 1A is insensitive to blue light pulses, whereas among the three B locus genes, at least rbcS 3B appears to respond to a blue-light photoreceptor. These results add to the data suggesting that individual members of rbcS gene families in higher plants may be subject to a variety of differing regulatory mechanisms.     

Recent developments in chloroplast protein transport

Most proteins located in chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and transported into the organelle. The study of protein uptake by chloroplasts has greatly expanded over the past few years. The increased activity in this field is due, in part, to the application of recombinant DNA methodology to the analysis of protein translocation. Added interest has also been gained by the realization that the transport mechanisms that mediate protein uptake by chloroplasts, mitochondria and the endoplasmic reticulum display certain characteristics in common. These include amino terminal sequences that target proteins to particular organelles, a transport process that is mechanistically independent from the events of translation, and an ATP-requiring transport step that is thought to involve partial unfolding of the protein to be translocated. In this review we examine recent studies on the binding of precursors to the chloroplast surface, the energy-dependent uptake of proteins into the stroma, and the targeting of proteins to the thylakoid lumen. These aspects of protein transport into chloroplasts are discussed in the context of recent studies on protein uptake by mitochondria.Abbrevlations CAT chloramphenicol acetyl transferase - CCCP carbonylcyanide m-chlorophenylhydrazone - DHFR dihydrofolate reductase - EPSP 5-enol-pyruvylshikimate-3-phosphate - ER endoplasmic reticulum - LHCP light harvesting chlorophyll a/b apoprotein - NPT neomycin phosphotransferase - oATP adenosine-2,3-dialdehyde-5-triphosphate - P-inorganic phosphate Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - SRP signal recognition particle     

Differential Involvement of the Circadian Clock in the Expression of Genes Required for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Synthesis,Assembly, and Activation in Arabidopsis thaliana

We have investigated the role of the circadian clock in the regulation of expression of genes required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) synthesis, assembly, and activation. Circadian oscillations in RCA (the gene encoding Rubisco activase) and RBCS (the gene encoding Rubisco small subunit) mRNA accumulation, with peak abundance occurring soon after dawn, occur in Arabidopsis thaliana grown in a light-dark (LD) photoperiod. These oscillations persist in plants that have been transferred from LD to either continuous darkness (DD) or continuous light (LL). In contrast, CPN60[alpha] (the gene encoding [alpha]-chaperonin) and CPN60[beta] (the gene encoding [beta]-chaperonin) mRNA abundance oscillates in a diurnal, but not in a circadian, fashion. Although rapid damping of the circadian oscillation in RCA mRNA abundance is observed in Arabidopsis that have been grown in LD and then transferred to DD for 2 d, the circadian oscillations in RCA and RBCS mRNA abundance persist for at least five continuous cycles in LL, demonstrating the robustness of the circadian oscillator.     

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 X g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2+-dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.     

Missense mutation in the amino terminus of phytochrome A disrupts the nuclear import of the photoreceptor

Phytochromes are the red/far-red photoreceptors in higher plants. Among them, phytochrome A (PHYA) is responsible for the far-red high-irradiance response and for the perception of very low amounts of light, initiating the very-low-fluence response. Here, we report a detailed physiological and molecular characterization of the phyA-5 mutant of Arabidopsis (Arabidopsis thaliana), which displays hyposensitivity to continuous low-intensity far-red light and shows reduced very-low-fluence response and high-irradiance response. Red light-induced degradation of the mutant phyA-5 protein appears to be normal, yet higher residual amounts of phyA-5 are detected in seedlings grown under low-intensity far-red light. We show that (1) the phyA-5 mutant harbors a new missense mutation in the PHYA amino-terminal extension domain and that (2) the complex phenotype of the mutant is caused by reduced nuclear import of phyA-5 under low fluences of far-red light. We also demonstrate that impaired nuclear import of phyA-5 is brought about by weakened binding affinity of the mutant photoreceptor to nuclear import facilitators FHY1 (for FAR-RED ELONGATED HYPOCOTYL1) and FHL (for FHY1-LIKE). Finally, we provide evidence that the signaling and degradation kinetics of constitutively nuclear-localized phyA-5 and phyA are identical. Taken together, our data show that aberrant nucleo/cytoplasmic distribution impairs light-induced degradation of this photoreceptor and that the amino-terminal extension domain mediates the formation of the FHY1/FHL/PHYA far-red-absorbing form complex, whereby it plays a role in regulating the nuclear import of phyA.     

Nicotiana chloroplast genes for components of the photosynthetic apparatus

In order to understand more fully chloroplast genetic systems, we have determined the complete nucleotide sequence (155, 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA. It contains two copies of an identical 25,339 bp inverted repeat, which are separated by 86, 684 bp and 18,482 bp single-copy regions. The genes for 4 different rRNAs, 30 different tRNAs, 44 different proteins and 9 other predicted protein-coding genes have been located. Fifteen different genes contain introns.Twenty-two genes for components of the photosynthetic apparatus have so far been identified. Most of the genes (except the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) code for thylakoid membrane proteins. Twenty of them are located in the large single-copy region and one gene for a 9-kd polypeptide of photosystem I is located in the small single-copy region. The gene for the 32-kd protein of photosystem II as well as the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase have strong promoters and are transcribed monocistronically while the other genes are transcribed polycistronically. We have found that the predicted amino acid sequences of six DNA sequences resemble those of components of the respiratory-chain NADH dehydrogenase from human mitochondria. As these six sequences are highly transcribed in tobacco chloroplasts, they are probably genes for components of a chloroplast NADH dehydrogenase. These observations suggest the existence of a respiratory-chain in the chloroplast of higher plants.     

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.     

The Expression of Light-Regulated Genes in the High-Pigment-1 Mutant of Tomato

Three light-regulated genes, chlorophyll a/b-binding protein (CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, and chalcone synthase (CHS), are demonstrated to be up-regulated in the high-pigment-1 (hp-1) mutant of tomato (Lycopersicon esculentum Mill.) compared with wild type (WT). However, the pattern of up-regulation of the three genes depends on the light conditions, stage of development, and tissue studied. Compared with WT, the hp-1 mutant showed higher CAB gene expression in the dark after a single red-light pulse and in the pericarp of immature fruits. However, in vegetative tissues of light-grown seedlings and adult plants, CAB mRNA accumulation did not differ between WT and the hp-1 mutant. The ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA accumulated to a higher level in the hp-1 mutant than WT under all light conditions and tissues studied, whereas CHS gene expression was up-regulated in de-etiolated vegetative hp-1-mutant tissues only. The CAB and CHS genes were shown to be phytochrome regulated and both phytochrome A and B1 play a role in CAB gene expression. These observations support the hypothesis that the HP-1 protein plays a general repressive role in phytochrome signal transduction.     

Photooxidative destruction of chloroplasts and its consequences for expression of nuclear genes

Expression of nuclear genes involved in plastidogenesis is known to be controlled by light via phytochrome. Examples are the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b binding protein of photosystem II (LHCP). In the present study we show that, beside phytochrome, the integrity of the plastid is essential for the expression of the pertinent nuclear genes as measured at the level of translatable mRNA. When the plastids are severely damaged by photooxidation in virtually carotenoid-free mustard (Sinapis alba L.) seedling cotyledons (made carotenoid-free by the application of Norflurazon, NF), almost no SSU, no SSU precursor, LHCP and LHCP precursor can be detected by immunological assays, and almost no translatable mRNA of SSU and LHCP can be found, although the levels and rates of phytochrome-mediated syntheses of representative cytoplasmic, mitochondrial and glyoxisomal enzymes are not adversely affected and morphogenesis of the mustard seedling proceeds normally (Reiß et al. 1983; Planta 159, 518–528). Norflurazon per se has no effect on the amount of translatable mRNA of SSU and LHCP as shown by irradiation of NF-treated seedlings with far-red light (FR) which strongly activates phytochrome but does not cause photooxidation in the plastids. It is concluded that a signal from the plastid is required to allow the phytochrome-mediated appearance of translatable mRNA for SSU and LHCP. Seedlings not treated with NF show a higher level of translatable mRNA_LHCP in red light (RL) compared to FR, whereas the mRNA_SSU levels are the same in RL and FR. These facts indicate that the level of translatable mRNA_LHCP is adversely affected if the apoprotein is not incorporated into the thylakoid membrane.Abbreviations FR far-red light (3.5 W m^-2) - LHCP light harvesting chlorophyll a/b binding protein of photosystem II - LSU large subunit of RuBPCase - NF Norflurazon - RL red light (6.8 W m^-2) - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - SSU small subunit of RuBPCase - WL white light (28 W m^-2)     

叶绿体超氧化物歧化酶（ChlSOD）基因的构建及序列分析

构建叶绿体超氧化物歧化酶基因（ChlSOD),采用RT-PCR方法分离豌豆RUBP羧化酶小亚基导肽基因（TP）,定向克隆至pUC19测序,定向克隆烟草MnSOD成熟蛋白基因（SODm)至pUC19;采用平粘端连接法将二者在pUC19中构成嵌合基因ChlSOD,并对此基因进行序列分析,序列分析表明：TPcDNATP,12bp的Linker及615bp SODm。TP与ChlSOD基因的序列分析与国外报道序列完全吻合。