Immuno-gold localization of cytochrome f,light-harvesting complex,ATP synthase and ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteins ribulose 1,5-bisphosphate carboxylase/oxygenase, ATP synthase, light-harvesting chlorophyll a/b protein, and cytochrome f, have been localized in mesophyll chloroplasts of barley (Hordeum vulgare L.) by electron microscopy of immunogold-labelled sections. The light-harvesting chlorophyll a/b protein and cytochrome f are shown to be present in the grana, both within the stacks and at the margins, and in the stromal membranes. Although the absolute amount of labelling for these proteins is greater in the grana than in the stromal membranes, when expressed as label/membrane length the partitioning appears approximately equal between appressed and non-appressed membranes for both the light-harvesting chlorophyll a/b protein and cytochrome f. ATP synthase is restricted to the non-appressed thylakoid membranes, and ribulose 1,5-bisphosphate carboxylase/oxygenase is uniformly distributed through the stromal contents.Abbreviations CF₁ ATP synthase - LHCPII light-harvesting chlorophyll a/b protein - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Effect of polyamines on stabilization of molecular complexes in thylakoid membranes of osmotically stressed oat leaves

Monocotyledonous leaves subjected to osmotica used for protoplast isolation accumulate a massive amount of putrescine (Put), lose chlorophyll and senesce rapidly. Treatment with spermidine (Spd) or spermine (Spm) prevents the loss of chlorophyll, indicating preservation of the thylakoid membranes at the site of the chlorophyll-protein complexes. Using several recently produced antibody probes, the effects on the stabilization of thylakoid membranes of applying either difluoromethylarginine (DFMA), a specific inhibitor of putrescine synthesis via arginine decarboxylase, or the polyamines Spd, Spm, or diaminopropane (Dap) to osmotically shocked oat leaves (Avena sativa L.) have been investigated. High protein levels were maintained in thylakoid membranes of leaf tissue incubated in the dark in the presence of 0.6 M sorbitol when pretreated with DFMA. After 48 h incubation, the level of the thylakoid protein D1, at the core of photosystem II, was higher in the DFMA-pretreated leaves as was the stromal protein ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; as indicated by the level of large subunits). Applications of Spd, Spm or Dap were effective in retarding the loss of D1, D2 and cytochrome f from the thylakoid membranes as well as Rubisco large subunits and chlorophyll from the leaf tissue. The effects of polyamine applications may be mediated through Dap since most of the added Spd or Spm was converted to Dap within 6 h. The possible mechanisms of action of polyamine applications and DFMA-pretreatment on stabilizing the composition of the thylakoid membrane are also discussed.Abbreviations Cyt cytochrome - Dap diaminopropane - DFMA DL--difluoromethylarginine - LSU large subunit (of Rubisco) - Put putrescine - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Spd spermidine - Spm spermine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This research was supported by the Agricultural and Food Research Council and by the British-Spanish joint research programme Acción Integrade HB-079 (R.T.B. and A.F.T.), British Council SPN/BAR/991 (R.T.B.) and Comision Interministerial de Cienica y Tecnologia 90-130 (A.F.T.). We thank Merrell Dow Research Center (Cincinnati, Ohio) for the gift of DFMA and Teresa Capell and Xavier Figueras (Univ. Barcelona) for help and suggestions.     

Identification of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase as a Substrate for Transglutaminase in Medicago sativa L. (Alfalfa)

Extracts prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [¹⁴C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [¹⁴C]putrescine incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts. Analysis of the primary structure of plant large subunit has revealed numerous potential glutaminyl and lysyl sites for transglutaminase-directed modifications of ribulose 1,5-bisphosphate carboxylase/oxygenase.     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

A<Emphasis Type="Italic"> Zea mays</Emphasis> 39-kDa thylakoid transglutaminase catalyses the modification by polyamines of light-harvesting complex II in a light-dependent way

A transglutaminase (TGase; EC 2.3.2.13) activity, which shared many properties with the TGase activity of the Helianthus tuberosus chloroplast, was observed in the Zea mays L. chloroplast and in its fractions. This activity was found to be prevalent in thylakoids; bis-(glutamyl) spermidine and bis-(glutamyl) putrescine were the main polyamine conjugates formed. Light stimulated the endogenous thylakoid activity. Putrescine, spermidine and spermine were conjugated to the isolated light-harvesting complex of photosystem II (LHCII) with different degrees of efficiency, spermine being the polyamine most efficiently conjugated. A TGase with a light-sensitive activity was identified in the photosystem II-enriched fraction. Its partial purification on a sucrose gradient allowed the separation of a 39-kDa band, which was immunorecognised by two anti-TGase antibodies (Ab-3 and rat prostatic gland-TGase). Both a colorimetric and a radiometric assay for TGase activity, the former carried out in the presence of biotinylated cadaverine and the latter in the presence of polyamines labelled with radioactive isotopes and resulting in the isolation of glutamyl-polyamines, further confirmed that the thylakoid enzyme is indeed a calcium-dependent transglutaminase (Thyl-TGase). At variance with guinea pig liver and erythrocyte TGases, which are insensitive to light, the activity of the thylakoid transglutaminase is affected by light. Moreover, this enzyme, when tested with purified LHCII as substrate, catalysed the production of mono- and bis-glutamyl-polyamines in equal amounts, whereas the animal enzymes produced mainly mono-derivatives. Herein, it is discussed whether this light sensitivity is due to the enzyme or the substrate.Abbreviations bis-PU Bis-(-glutamyl) putrescine - bis-SD Bis-(-glutamyl) spermidine - bis-SM Bis-(-glutamyl) spermine - Er-TGase Erythrocyte transglutaminase - Gpl-TGase Guinea pig liver transglutaminase - LHCII Light-harvesting complex II - mono-PU Mono-(-glutamyl) putrescine - mono-SD Mono-(-glutamyl) spermidine - mono-SM Mono-(-glutamyl) spermine - PA Polyamine - PU Putrescine - SD Spermidine - SM Spermine - Rpg-TGase Rat prostatic gland transglutaminase - Thyl-TGase Thylakoid transglutaminase     

Light-independent degradation of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations

Intact chloroplasts were isolated from mature pea (Pisum sativum L.) leaves in order to study the degradation of several stromal proteins in organello. Changes in the abundances of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), glutamine synthetase (EC 6.3.1.2) and ferredoxin-dependent glutamine:α-ketoglutarate aminotransferase (glutamate synthase; EC 1.4.7.1) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie-staining of the gels or immunoblotting using specific antibodies for the different enzymes. Degradation of several stromal proteins was strongly stimulated when intact chloroplasts were incubated in the light in the presence of dithiothreitol. Since free radicals may artificially accumulate in the chloroplast under such conditions and interfere with the stability of stromal proteins, the general relevance of these processes remains questionable. In the absence of light, proteolysis proceeded slowly in isolated chloroplasts and was not stimulated by dithiothreitol. Inhibition by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or excess zinc ions as well as the requirement for divalent cations suggested that a zinc-containing metalloprotease participated in this process. Furthermore, light-independent degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase was enhanced in chloroplasts isolated from leaves in which senescence was accelerated by nitrogen starvation. Our results indicate that light-independent stromal protein degradation in intact chloroplasts may be analogous to proteolysis that occurs in intact leaves during senescence. Received: 3 July 1997 / Accepted: 15 October 1997     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery

The electron-microscopic technique for immunogold labelling of thawed cryosectioned material (K.T. Tokuyasu, 1989, Histochem J 21: 163–171) has been adapted for use with isolated chloroplasts. Percoll-purified pea (Pisum Sativum L. cv Feltham First) chloroplasts were fixed in a buffered glutaraldehyde solution and then infiltrated with a buffered solution of 10% polyvinylpyrrolidone in 2.07 M sucrose prior to freezing in liquid nitrogen and sectioning in an ultracryomicrotome. Sections were thawed, immunolabelled, and stained with ammonium molybdate in methyl cellulose on Formvar/carbon-coated Cu or Cu/Pd electron-microscope grids. Cryosectioning gave excellent structural preservation and retained antigenicity. The effectiveness of this technique in localizing proteins to their specific chloroplast compartment was assayed using antibodies raised against: (i) the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a stromal protein, (ii) the chloroplast ATP synthase (CF₁), a peripheral thylakoid protein, and (iii) different envelope membrane proteins. Antibodies raised against three members of the chloroplasticouterenvelopeprotein (OEP) import machinery, a 34-kDa protein (OEP34 or IAP34), the channel-forming 75-kDa protein (OEP75 or IAP75), and the 86-kDa precursor protein receptor (OEP86 or IAP86) were tested for their localization. The previous localization of OEP86, OEP75 and OEP34 to the outer envelope by biochemical methods was confirmed by our immuno electronmicroscopic analysis. Additionally, a constituent of the chloroplastic inner envelope protein (IEP) import machinery IEP 110 (IAP 100) was clearly localized to this membrane. Therefore, cryosectioning and immunogold labelling of intact chloroplasts provides a method for studying the localization of chloroplast proteins, especially those residing in the inner and outer envelope membranes.Abbreviations FCS fetal calf serum - IAP import intermediate associated protein - IEP inner envelope protein - OEP outer envelope protein (numbers signifying the relative molecular mass in kilodaltons) - PBS phosphate buffered saline - PVP polyvinyl pyrrolidone - Rubisco ribulose-1,5-biophosphate carboxylase/oxygenase     

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase     

Phycoerythrin is absent from the pyrenoid of Porphyridium cruentum: photosynthetic implications

The thylakoid lamellae which traverse the pyrenoid of the unicellular red alga Porphyridium cruentum (Agardh) Nägeli appear to lack phycobilisomes. We have confirmed by immuno-electron microscopy that phycoerythrin (PE), an important structural component of the phycobilisomes of red algae, is absent from the pyrenoid. To characterize pyrenoid thylakoids further, electron-microscopic cytochemical methods were employed to detect photosystem activity. Photosystem (PS) I activity was demonstrated in both stromal and pyrenoid thylakoids by the photooxidation of 3,3-diaminobenzidine. In contrast, the localization of photoreduced distyryl nitroblue tetrazolium demonstrated that PSII activity was restricted to stromal thylakoids. The observed partitioning of PE and PSII activity within the plastid may be related to another observation, that being the localization of nearly all ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) within the pyrenoid of this alga. It is possible that the pyrenoid of P. cruentum functions as a specific metabolic compartment where CO₂ fixation is enhanced by the absence of photosynthetic O₂ evolution.Abbreviations DAB 3,3-diaminobenzidine-4HCl - DS-NBT distyryl nitroblue tetrazolium chloride - EF exoplasmic face - LSU large subunit of RuBisCO - PE phycoerythrin - PS photosystem - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Drs. Jacqueline Fleck (CNRS, Strasbourg) and Robert MacColl (New York State Department of Health, Albany) for providing us with the antibodies used in this study. We also thank Dr. C.E. Smith for use of the Zeiss MOP-3 digital analyser and Dr. Geneviève Bricheux for kindly providing Lowicryl-embedded samples of P. cruentum. Aatrex^® was kindly donated by Ciba-Geigy. This research was supported by the Natural Sciences and Engineering Research Council of Canada (grant No. A-2921).     

Stromal protein phosphorylation in spinach (Spinacia oleracea) chloroplasts.

When intact spinach chloroplasts were supplied with [32P]Pi, stromal protein phosphorylation was found to occur in the dark. On illumination the thylakoid protein kinase was activated and the amount of label found in thylakoid proteins quickly exceeded that incorporated into stromal protein, such that the latter was found to account for only 10-15% of the total radioactivity bound to chloroplast proteins after 5 min illumination. The rate of phosphorylation of stromal polypeptides was unchanged by light. After SDS/polyacrylamide-gel electrophoresis, more than 15 labelled polypeptides of stromal origin were observed. A polypeptide with an Mr of approx. 70 000 had the highest specific activity of labelling. Both the large and small subunits of the ribulose-1,5-bisphosphate carboxylase were phosphorylated. The level of phosphorylation of stromal protein was increased by CO2 fixation in intact chloroplasts. This increase was not observed in the absence of NaHCO3 or in the presence of the phosphoribulokinase inhibitor DL-glyceraldehyde. These effects appeared to be largely due to changes in the phosphorylation state of the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Studies with the reconstituted chloroplast system showed that the thylakoid protein kinase(s) played no part in the phosphorylation of stromal protein. The rate and level of phosphorylation of stromal protein was unaffected by the activation state of the thylakoid protein kinase and was unchanged when thylakoids were omitted from the reaction medium. The phosphorylation of stromal proteins is therefore catalysed by a discrete soluble protein kinase.     

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。     

Leaf senescence in a non-yellowing mutant of Festuca pratensis

Soluble and thylakoid membrane polypeptides from senescing leaf tissue of Rossa, a normal yellowing Festuca pratensis genotype, were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and compared with those of the non-yellowing mutant Bf 993. Subunits of ribulose-1,5-bisphosphate carboxylase were the major soluble polypeptides and declined to low levels in senescing leaves of both genotypes. The major thylakoid polypeptides were those associated with the chlorophyllprotein complexes CPI and CPII. The levels of all thylakoid polypeptide species fell during senescence of Rossa leaf tissue but Bf993 lamellae retained CPI, CPII and a number of other hydrophobic low molecular weight polypeptides. The increasing hydrophobicity and decreasing protein complement of Bf 993 thylakoids were reflected in a fall in membrane density from 1.16 to 1.13 g cm^-3 over 8 d of senescence and a decline in the extractability of chlorophyll-containing membranes in the same period. In Bf993 the molar ratio of chlorophyll to hydrophobic membrane protein increased from 92 at day 0 to 296 at day 8. In the same time the ratio for Rossa increased from 88 to 722 and 8 d-senesced Rossa tissue yielded less than 2% of the solvent-soluble protein it contained at day 0 as compared with 24% for the protein of Bf993. These results are discussed in relation to the nature of the non-yellowing lesion.Abbreviations RuBPC ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - EDTA ethylenediaminetetraacetate - SDS sodium dodecyl sulphate - CP chlorophyll-protein complex     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.Abbreviations ELIP early light-inducible protein - LHCP light-harvesting chlorophyll alb protein; poly(A)RNA=polyadenylated RNA - (ss)RuBPCase (small subunit) ribulose-1,5-bisphosphate carboxylase     

Precursor of the nuclear-encoded extrinsic 30 kDa protein in photosystem II of Euglena gracilis Z is not a polyprotein

Polyprotein-type precursors have been reported for the nuclear-encoded proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the apoproteins of light-harvesting chlorophyll-protein (LHC) in Euglena. We report here that the precursor of the extrinsic 30 kDa protein of photosystem II (PS II) encoded by nuclear DNA is not a polyprotein. The precursor was identified as a 45 kDa protein by immunoprecipitation of in vitro translation products of mRNA and by a pulse-chase experiment. It is probable that the structure of the precursor of the nuclear-encoded protein in Euglena chloroplast is closely related to the feature of assembly, as well as of transport, of the protein in chloroplast.     

Variation in cellular ribulose-1,5-bisphosphate-carboxylase content in leaves of Triticum genotypes at three levels of ploidy

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 1.1.39) (RuBPCase) was quantified using polyacrylamide-gel electrophoresis in whole 9-d-old first leaves of 14 genotypes of Triticum, and cellular RuBPCase levels calculated. Diploids, tetraploids and hexaploids were analysed and it was confirmed that the RuBPCase level per cell is closely related to ploidy in wheat. Inter-genotypic variation in RuBPCase levels per cell and per leaf were surveyed. It was found that the interactions between leaf size, cell size and RuBPCase levels result in small variations in RuBPCase levels per unit leaf area between genotypes.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase