Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi

Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.     

The Rubisco complex protein: A protein induced by fruit removal that forms a complex with ribulose-1,5-bisphosphate carboxylase/oxygenase

Continuous removal of fruits from soybean plants (Glycine max [L.] Merr.) causes a redistribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) from the soluble to the insoluble phase of leaf extracts. The extent of this redistribution is genotype-dependent. We previously reported that insoluble Rubisco occurs in a high-molecular-mass complex together with a protein composed of 30-kDa subunits (S.J. Crafts-Brander et al., Planta, 183, 300–306). In the present study, the Rubisco Complex Protein (RCP), was isolated from the Rubisco-RCP complex by gel-filtration chromatography in 4 M urea. Under these conditions, RCP migrated with an apparent molecular mass of 120 kDa, indicating that the protein maintains a tetrameric structure even in 4 M urea. Once freed of urea, purified RCP was soluble, but formed insoluble complexes with Rubisco from soybean, tobacco and spinach when RCP and Rubisco were incubated in a ratio of 11 by weight. Purified Rubisco and RCP also associated into a high-molecular-mass complex when either component was in several-fold excess, but in this case the complex was soluble. Similarly, the amount of Rubisco sequestered as an insoluble Rubisco:RCP complex in leaf extracts of different soybean genotypes was related to the relative amounts of Rubisco and RCP present in the extracts. Thus, with both purified components and in leaf extracts, formation of an insoluble complex between Rubisco and RCP required a precise stoichiometry. Antibodies directed against purified RCP detected an accumulation of RCP in soybean leaves around the time of flowering. The RCP was also detected in petioles, stems, and pod walls of soybean, but not seeds. Fruit removal caused a marked increase in the amount of RCP in the leaves to levels as high as 15% of the total soluble protein. The accumulation of RCP in response to source:sink manipulations was similar to soybean vegetative storage proteins (VSPs). However, immunogold-localization showed that RCP was located in the cytosol of leaves, compartmentalized separate from both Rubisco and the VSPs. Thus, the physiological relevance of the specific association between RCP and Rubisco is obscure.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - RCP rubisco complex protein - VSPs vegetative storage proteins Kentucky Agricultural Experiment Station Journal Article No. 93-3-162We wish to acknowledge L.F. Staples and J.C. Anderson for their expert technical assistance. Electron microscopy was performed by the Nano-Probe Laboratory, Lucille Parker Markey Cancer Center, University of Kentucky. We thank Dr. K.C. Vaughn, USDA-ARS, for providing guidance pertaining to immunogold-localization procedures.     

Detection of an immunoglobulin-like serum protein possessing a high affinity for collagen

Fibronectin isolated from bovine serum by affinity chromatography on collagen-Sepharose was found to contain a great number of concomitant proteins. Polyacrylamide gel electrophoresis of experimental samples pretreated with beta-mercaptoethanol under denaturation conditions resulted in the polypeptide fractions with Mr of 25, 54 and 82 KD, while the non-treated samples contained only one protein of non-fibronectin type (Mr = = 180-190 KD). This protein was isolated from the total preparations of collagen-binding proteins by the procedures generally employed for the isolation of purified preparations of immunoglobulins G; this protein was also isolated from purified immunoglobulins G using affinity chromatography on collagen-Sepharose. In terms of its molecular weight, subunit composition and immunological and chromatographical behaviour this protein can be related to immunoglobulins. The immunoglobulin-like protein isolated together with fibronectin revealed an affinity for denatured collagen, but not for fibronectin or Sepharose. The content of immunoglobulin with an affinity for denatured collagen in the total fraction of immunoglobulins G is 0.3-0.5%.     

Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.     

烟草Rubisco活化酶的纯化及其特性

利用35％饱和硫酸铵分部、DEAE－Sephacel和FPIC－MonoQ柱层析等步骤从烟草叶片中纯化了Rubisco活化酶,并制备了其专一性抗体。此法不仅快速,而且比活力高。以往认为菠菜和拟南芥Rubisco活化酶由两种亚基组成。通过快速制备的粗提液分析．发现烟草Rubisco活化酶由一种42kD的亚基组成。即使在有多种蛋白酶抑制剂存在的情况下,此亚基仍很易降解为39kD的亚基。ATP不仅对酶的活性所必需,而且也有利于维持酶的稳定性。该酶的热稳定性远比Rubisco差。     

Relative association of Rubisco with manganese and magnesium as a regulatory mechanism in plants

Rubisco, the enzyme that constitutes as much as half of the protein in a leaf, initiates either the photorespiratory pathway that supplies reductant for the assimilation of nitrate into amino acids or the C3 carbon fixation pathway that generates carbohydrates. The relative rates of these two pathways depend both on the relative extent to which O₂ and CO₂ occupies the active site of Rubisco and on whether manganese or magnesium is bound to the enzyme. This study quantified the activities of manganese and magnesium in isolated tobacco chloroplasts and the thermodynamics of binding of these metals to Rubisco purified from tobacco or a bacterium. In tobacco chloroplasts, manganese was less active than magnesium, but Rubisco purified from tobacco had a higher affinity for manganese. The activity of each metal in the chloroplast was similar in magnitude to the affinity of tobacco Rubisco for each. This indicates that, in tobacco chloroplasts, Rubisco associates almost equally with both metals and rapidly exchanges one metal for the other. Binding of magnesium was similar in Rubisco from tobacco and a bacterium, whereas binding of manganese differed greatly between the Rubisco from these species. Moreover, the ratio of leaf manganese to magnesium in C3 plants increased as atmospheric CO₂ increased. These results suggest that Rubisco has evolved to improve the energy transfers between photorespiration and nitrate assimilation and that plants regulate manganese and magnesium activities in the chloroplast to mitigate detrimental changes in their nitrogen/carbon balance as atmospheric CO₂ varies.     

Glycine 176 affects catalytic properties and stability of the Synechococcus sp. strain PCC6301 ribulose-1,5-bisphosphate carboxylase/oxygenase

A previously described system for biological selection of randomly mutagenized ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) employing the phototrophic bacterium Rhodobacter capsulatus was used to select a catalytically altered form of a cyanobacterial (Synechococcus sp. strain PCC6301) enzyme. This mutant Rubisco, in which conserved glycine 176 was replaced with an aspartate residue, was not able to support CO(2)-dependent growth of the host strain. Site-directed mutant proteins were also constructed, e.g. asparagine and alanine residues replaced the native glycine with the result that these mutant proteins either greatly reduced the ability of R. capsulatus to support growth or had little effect, respectively. Growth phenotypes were consistent with the Rubisco activity levels associated with these proteins, and this was also borne out with purified recombinant proteins. Despite being catalytically challenged, the G176D and G176N mutant proteins were found to exhibit a more favorable interaction with CO(2) than the wild type protein but exhibited a reduced affinity for the substrate ribulose 1,5-bisphosphate. The G176A enzyme differed little from the wild type protein in these properties. None of the mutants had CO(2)/O(2) specificities that differed markedly from the wild type. Further studies taken from the known structure of the Synechococcus Rubisco indicated that substitutions at Gly-176 affected associations between large subunits. Supporting experimental data included an unusual protein concentration-dependent effect on in vitro activity, differences in thermal stability relative to the wild type protein, and aberrant migration on nondenaturing polyacrylamide gels. From these results, it is apparent that residues not directly located within the active site but near large subunit interfaces can affect key kinetic properties of Rubisco. These results suggest that further bioselection protocols (using these proteins as starting material) might yield novel mutant forms of Rubisco that relate to key functional properties.     

Structure and functional annotation of hypothetical proteins having putative Rubisco activase function from Vitis vinifera

Rubisco is a very large, complex and one of the most abundant proteins in the world and comprises up to 50% of all soluble protein in plants. The activity of Rubisco, the enzyme that catalyzes CO₂ assimilation in photosynthesis, is regulated by Rubisco activase (Rca). In the present study, we searched for hypothetical protein of Vitis vinifera which has putative Rubisco activase function. The Arabidopsis and tobacco Rubisco activase protein sequences were used as seed sequences to search against Vitis vinifera in UniprotKB database. The selected hypothetical proteins of Vitis vinifera were subjected to sequence, structural and functional annotation. Subcellular localization predictions suggested it to be cytoplasmic protein. Homology modelling was used to define the three-dimensional (3D) structure of selected hypothetical proteins of Vitis vinifera. Template search revealed that all the hypothetical proteins share more than 80% sequence identity with structure of green-type Rubisco activase from tobacco, indicating proteins are evolutionary conserved. The homology modelling was generated using SWISS-MODEL. Several quality assessment and validation parameters computed indicated that homology models are reliable. Further, functional annotation through PFAM, CATH, SUPERFAMILY, CDART suggested that selected hypothetical proteins of Vitis vinifera contain ATPase family associated with various cellular activities (AAA) and belong to the AAA+ super family of ring-shaped P-loop containing nucleoside triphosphate hydrolases. This study will lead to research in the optimization of the functionality of Rubisco which has large implication in the improvement of plant productivity and resource use efficiency.     

Occurrence of beauvericin and enniatins in wheat affected by Fusarium avenaceum head blight.

We evaluated Fusarium contamination and the levels of hexadepsipeptide mycotoxins in 13 wheat samples affected by head blight in Finland. Fusarium avenaceum was the dominant species (91%) isolated from all samples, but isolates of F. culmorum (4%), F. tricinctum (3%), and F. poae (2%) also were recovered. Beauvericin (0.64 to 3.5 microg/g) was detected in all 13 samples. Enniatin B (trace to 4.8 microg/g) was detected in 12 samples, enniatin B(1) (trace to 1.9 microg/g) was detected in 8 samples, and enniatin A(1) (trace to 6.9 microg/g) was detected in 10 samples. Ten of 13 strains of F. avenaceum and 2 strains of F. poae and F. tricinctum produced beauvericin in culture on rice (trace to 70, 9.4, and 33 microg/g, respectively). All strains also produced enniatins (trace to 2,700 microg/g). This is the first report on the natural co-occurence of beauvericin and enniatins in wheat infected predominantly by F. avenaceum.     

Natural contamination with mycotoxins in forage maize and green coffee in Nayarit State (Mexico)

The presence of mycotoxins in forage maize (zearalenone, fumonisin B1, T-2 toxin and diacetoxyscirpenol) and green coffee (ochratoxin A) from Nayarit State (Mexico) has been studied. All maize samples analyzed showed fumonisin B1 contamination, with an average concentration of 2,541 microg/kg. Fifteen percent of the samples contained zearalenone, with an average concentration of 1,610 microg/kg. Only one sample showed T-2 toxin contamination (7 microg/kg), and no diacetoxyscirpenol was detected. Sixty-seven per cent of green coffee samples were contaminated with ochratoxin A, with an average concentration of 30.1 microg/kg. This is the first study about mycotoxins developed in Nayarit and it has shown that mycotoxin contamination is a real problem in both foodstuffs studied.     

Antibacterial effect of various mycotoxins and fungal metabolites against Bacillus thuringiensis (Berliner) strains sensitive or resistant to aflatoxin B1]

Antimicrobial activity of pure preparations of mycotoxins and fungal metabolites was studied against strains of Bacillus thuringiensis (Berliner). Two resistant strains, called stable-variant, were isolated after treatment with high concentrations of aflatoxin B1. These strains were then resistant also towards compounds with a double furan system (aflatoxins B1, B2, G1, G2, and sterigmatocystin).     

Tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 microg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.     

Prolonged stability of the plantibody HB-01 directed against the hepatitis B surface antigen in cryo-preserved tobacco leaves

Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at ?20 °C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at ?20 °C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.     

Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver

Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol. The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates. The purified recombinant protein was also used to raise polyclonal antibodies in rabbits. Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus. The identity of the cleavage boundary was verified by protein sequence analysis. Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations. An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site. Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta-hydroxylation as markers for P450 2B6.     

Alterations in Photosystem II electron transport as revealed by thermoluminescence of Cu-poisoned chloroplasts

The Rubisco activase amino acid sequences of spinach and tobacco are 79% identical, yet the tobacco protein does not facilitate the activation of the uncarbamylated, ribulose bisphosphate bound form of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and vice versa. In contrast, combinations of the spinach Rubisco activase with Rubisco from non-Solanaceae species and combinations of tobacco Rubisco activase with Rubisco from other Solanaceae species are almost as effective as the analogous combination. To examine the basis of the preference of an activase protein for either Solanaceae or non-Solanaceae Rubisco, several recombinant chimeric proteins were obtained by combining regions from the cDNAs of spinach and tobacco activase and expression in Escherichia coli. The chimeric proteins were analyzed for ATP hydrolysis and ability to activate spinach and tobacco Rubisco. Comparisons of Rubisco preference with composition of the various activase chimeras indicate that the major determinants of Rubisco preference seem to be localized in the carboxyl-terminal region.     

Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in <Emphasis Type="Italic">E. coli</Emphasis> and isolated by metal affinity chromatography

Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni²⁺-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni²⁺ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.     

Comparative studies on fraction 1 protein from spinach and tobacco leaves

Fraction 1 protein of spinach and tobacco leaves was purifiedby Sephadex G-200 and DEAE-cellulose column chromatography.Immunological comparison of purified preparations of these proteinswas carried out with precipitin analysis using an antiserumof tobacco fraction 1 protein. Two different antigenic activitieswere found in tobacco fraction 1 protein, of which one was commonlyfound in the spinach protein and the other was specific to tobaccofraction 1 protein. The immunological results suggest that fraction 1 protein iscomposed of at least two different structures, one of whichis common to both spinach and tobacco proteins and the otherof which is specific to each of these proteins. This was confirmedfrom a chemical experiment. Fraction 1 protein was divided intolarge and small polypeptide components by sodium dodecyl sulfatetreatment and subsequent Sephadex G-100 column chromatography,then the amino acid compositions of each polypeptide of theseproteins were compared. The amino acid composition of the smallpolypeptide of spinach was different from that of tobacco, whileamino acid compositions of the large polypeptides of those proteinswere similar to each other. (Received July 1, 1968; )     

Photosynthetic Acclimation to Elevated CO2 Occurs in Transformed Tobacco with Decreased Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content

Inhibition of net carbon assimilation rates during growth at elevated CO2 was studied in transgenic tobacco (Nicotiana tabacum L.) plants containing zero to two copies of antisense DNA sequences to the small subunit polypeptide (rbcS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). High- and low-Rubisco tobacco plants were obtained from the selfed progeny of the original line 3 transformant (S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). Assimilation rates of high- and low-Rubisco tobacco plants increased 22 and 71%, respectively, when transferred from 35- to 70-Pa CO2 chamber air at 900 [mu]mol m-2 s-1 photon flux density. However, CO2-dependent increases of net carbon assimilation rates of high- and low-Rubisco plants virtually disappeared after 9 d of growth in elevated CO2 chamber air. Total above-ground dry matter production of high- and low-Rubisco plants was 28 and 53% greater, respectively, after 9 d of growth at 70 Pa compared with 35 Pa CO2. Most of this dry weight gain was due to increased specific leaf weight. Rubisco activity, Rubisco protein, and total chlorophyll were lower in both high- and low-Rubisco plants grown in enriched compared with ambient CO2 chamber air. Soluble leaf protein also decreased in response to CO2 enrichment in high- but not in low-Rubisco tobacco plants. Decreased Rubisco activities in CO2-adapted high- and low-Rubisco plants were not attributable to changes in activation state of the enzyme. Carbonic anhydrase activities and subunit levels measured with specific antibodies were similar in high- and low-Rubisco tobacco plants and were unchanged by CO2 enrichment. Collectively, these findings suggested that photosynthetic acclimation to enriched CO2 occurred in tobacco plants either with or without transgenically decreased Rubisco levels and also indicated that the down-regulation of Rubisco in CO2-adapted tobacco plants was related to decreased specific activity of this enzyme.     

The DNA-binding protease,CND41, and the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase in senescent leaves of tobacco

Plastids bear their own genome, organized into DNA–protein complexes (nucleoids). Recently, we identified a DNA-binding protease (CND41) in the chloroplast nucleoids of cultured tobacco (Nicotiana tabacum L.) cells. In this study, we examine the biochemical function of this novel DNA-binding protease, particularly in senescent leaves, because antisense tobacco with a reduced amount of CND41 showed retarded senescence. Nitrogen-depletion experiments clearly showed that CND41 antisense tobacco maintained green leaves and constant protein levels, especially ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), throughout the whole plant, whereas wild-type tobacco showed marked senescence and the reduction of protein levels in the lower leaves. In vitro analyses confirmed that CND41 showed proteolytic activity at physiological pH when denatured Rubisco was used as the substrate. These results suggest that CND41 is involved in Rubisco degradation and the translocation of nitrogen during senescence. The possible regulation of protease activity of CND41 through DNA-binding is discussed.Abbreviations CABP 2-Carboxyarabinitol-1,5-bisphosphate - CBB Coomassie Brilliant Blue - GS Glutamine synthetase - OEC33 The extrinsic 33-kDa protein in the oxygen-evolving complex - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase     

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.