Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.

Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo³⁵S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major ³⁵S-labeled proteins. The major incorporation of ³⁵S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major ³⁵S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the ³⁵S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

     

Antigenic patterns of seed proteins in Opuntioideae (Cactaceae)

The antigenic patterns recognised by Western blotting in seed proteins of species of Opuntioideae (Cactaceae) were analysed in an attempt to evaluate their usefulness in systematics. Total protein profiles were also analysed by SDS–PAGE. The resulting similarity and distance matrices were further used to carry out Cluster Analysis (UPGMA) and Principal Coordinates Analysis. Populations of Opuntia cardiosperma were found to exhibit a prominent morphological uniformity, a unique electrophoretic pattern and a uniform antigenic pattern. The latter was obtained using anti-O. cardiosperma as antiserum. Results from the qualitative and quantitative interspecific analyses of antigenic profiles helped to characterise all the species studied. Tephrocactus articulatus and Cylindropuntia imbricata evidenced lower affinity with O. cardiosperma than the species of Opuntia s.s. Our results demonstrate that in Cactaceae, Western blotting analysis broadens the usefulness range of immunological techniques at the specific level and complements the information collected from electrophoretic profiles.     

The effects of copper nutrition and developmental state on the biosynthesis of diamine oxidase in clover leaves

The biosynthesis of diamine oxidase (DAO; EC 1.4.3.6) in leaf blades of subterranean clover (Trifolium subterraneum L. cv Seaton Park) was followed by labeling whole plants with ¹⁴CO₂. A pulse-chase experiment where DAO was immunoprecipitated with anti-DAO antibodies showed that only leaf primordia and the youngest emerged leaves were able to synthesize the enzyme. The amount of DAO in young leaves of clover grown with a range of Cu treatments was determined by its enzymic activity and by single radial immunodiffusion against anti-DAO antibodies; both parameters were highly correlated with the Cu concentration of the leaf. Further, anti-DAO antibodies reacted against apo-DAO prepared in vitro indicating that apo-DAO was absent from Cu-deficient leaves. These results suggest that the biosynthesis of DAO in young clover leaves is controlled by the Cu concentrations of the leaves. Poly(A) mRNA purified from leaf primordia and young emerging leaves of plants with either a high Cu or low Cu supply was translated in wheat germ and rabbit reticulocyte cell-free systems. No differences between the two Cu treatments could be seen in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the translation products after fluorography. However, anti-DAO antibodies did not detect any DAO synthesized in vitro from either treatment.     

Environmentally controlled changes of phosphoenolpyruvate carboxylases in Mesembryanthemum

NaCl treated Mesembryanthemum crystallinum plants exhibit a Crassulacean acid metabolism. The activity of phosphoenolpyruvate (PEP) carboxylase, the enzyme responsible for CO₂ dark fixation, depends on leaf age showing maximum activity in mature leaves. Electrophoresis revealed that the young leaves possess only two protein bands with PEP carboxylase activity, while older leaves have 3 bands. The removal of NaCl from the soil resulted in the disappearance of the 3rd band obtained after electrophoresis and a decline in the total activity of the PEP carboxylase. The reintroduction of NaCl at the same concentration as before did not restore the activity of the PEP carboxylase nor did it restore the initial electrophoretic band pattern.     

Effect of head removal on leaf senescence of sunflower

Greenhouse and field studies examined the effect of flower or seedhead removal on leaf senescence and associated changes in sunflower (Helianthus annuus L.) plants. At intervals during seed development, selected leaves (leaves 6 through 8 from the top in the greenhouse and leaf 7 from the top in the field) were harvested and analyzed for chlorophyll, specific leaf weight, N, P, soluble protein, and electrophoretic gel profiles of soluble polypeptides. In both the greenhouse and the field, the leaves of headless plants retained or accumulated more N, P, soluble protein, and dry weight than leaves of plants with heads. Obviously, head removal affected the partitioning of these metabolites during seed development. None of the treatments resulted in the formation of new polypeptides (electrophoretic gel profiles). Comparisons of the rates and extent of loss of chlorophyll, soluble protein, and polypeptide bands (especially ribulose 1,5-bisphosphate carboxylase) from the leaves of headed and deheaded plants showed that head removal delayed the rate of development of leaf senescence for the greenhouse-grown but had much less effect on field-grown plants. These findings illustrate the variability in different parameters commonly associated with the leaf senescence processes of headed and deheaded sunflower plants grown under different environments.     

Purification of plant complex protein extracts in non-denaturing conditions by in-solution isoelectric focusing

An alternative approach for plant complex protein extracts pre-purification by in-solution isoelectric focusing in non-denaturing conditions is presented. The separation of biologically active proteins, in narrow ranges of isoelectric point (pI) was obtained by a modified OFFGEL electrophoresis. Two different water-soluble protein extracts from Phragmites leaves were fractionated into 24 fractions within a 3–10 pI range at 10 °C in the absence of denaturing/reducing agents. One-dimensional electrophoretic analysis revealed different protein distribution patterns and the effective fractionation of both protein extracts. Peroxidase activity of each fraction confirmed that proteins remained active and pre-purification occurred. Biological triplicates assured the needed reproducibility.     

Juvenoid effects on Rhodnius prolixus embryogenesis

Protein electrophoretic techniques demonstrate that the normal patterns of protein synthesis accompanying embryogenesis of Rhodnius prolixus are altered by the juvenoid compound Ro 13.5223 (fenoxycarb). Electrophoretic analysis reveals that embryonic perturbation by fenoxycarb is due in part to alterations in the normal molecular events accompanying development. Using high-resolution 2-D gel electrophoresis, major differences in protein patterns were observed. Three proteins normally absent, were expressed in fenoxycarb perturbed embryos (two on day 3 and one on day 7).     

Schistosoma mansoni: resolution and molecular weight estimates of tegument glycoproteins by polyacrylamide gel electrophoresis

Tegument samples of Schistosoma mansoni, extracted either by freezing and thawing or saponin, were fractionated in sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The electrophoretic patterns varied if different extraction techniques were used. Lighter backgrounds and sharper bands were always observed with frozen and thawed preparations, although one of the major glycoprotein components and a PAS-positive diffuse material which migrates very rapidly were not extracted by this technique. The number of bands which could be identified by slab gel electrophoresis was approximately 25. Electrophoretic differences could be detected when teguments from male and female as well as young and adult parasites were compared. On the other hand, tegument preparations of 30-day parasites obtained from mice, rats, guinea pigs, and hamsters showed a remarkable similarity to each other.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Lipopolysaccharide Profiles from Nodules as Markers of Bradyrhizobium Strains Nodulating Wild Legumes

To develop the use of electrophoretic lipopolysaccharide profiles for Bradyrhizobium strain identification, we studied the feasibility of using electrophoresis of whole legume nodule homogenates to obtain distinctive lipopolysaccharide profiles. The electrophoretic patterns were the same whether we used nodule extracts, bacteroids, or cultured bacteria as samples, and there was no evidence of changes in the ladder-like pattern during the nodulation process. To assess the reliability of using lipopolysaccharide profiling performed with individual nodules for studying the diversity and microdistribution of the rhizobia nodulating wild shrub legumes, we used a population of Adenocarpus foliolosus seedlings. We obtained 75 different profiles from the 147 nodules studied. There was no dominant profile in the sample, and a plant with different nodules generally produced different profiles. Electrophoresis of legume root nodules proved to be a fast and discriminating technique for determining the diversity of a bradyrhizobial population, although it did not allow the genetic relationships among the nodulating strains to be studied.     

COMPARATIVE ELECTROPHORESIS AND ISOZYME ANALYSIS OF SEED PROTEINS FROM CULTIVATED RACES OF SORGHUM

Comparative disc electrophoresis of acidic proteins, basic proteins, and isozymes of esterase, MDH, and peroxidase were performed with aqueous extracts of seeds from seven cultivars belonging to five races of Sorghum bicolor ssp. bicolor: bicolor, caudatum, durra, guinea, and kafir. Two disc electrophoretic systems were employed. Acidic proteins were electrophoresed in an anionic system (tris-glycine buffer, pH 8.3). Basic proteins were electrophoresed in a cationic system (β-alanin-acetate buffer, pH 4.5). Soluble proteins were stained with Coomassie brilliant blue. Isozyme activity was detected by using specific enzyme stains and substrates. Each cultivar yielded reproducible, characteristic patterns of distinct acidic and basic proteins. Cultivars belonging to the same race produced identical protein and isozyme patterns. The degree of electrophoretic similarity among races was estimated by calculating similarity index values for each of the 10 possible pairs of races. Bicolor, caudatum, durra, and guinea produced very similar acidic and basic protein patterns and esterase, MDH, and peroxidase isozyme patterns. Differences, however, were observed among all races. All of kafir patterns were significantly different from the patterns of other races. Comparative electrophoresis may provide a new source of taxonomic characters for investigating phenetic and phylogenetic relationships in Sorghum.     

Synthesis of early heat shock proteins in young leaves of barley and sorghum

The in vivo synthesis of early heat-shock proteins in young leaves of barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) was studied by one- and two-dimensional electrophoresis. Analysis of whole leaf protein patterns demonstrated clearly the enhanced resolution of heat-shock proteins, especially those of low molecular weight, when separated by two-dimensional electrophoresis. Comparison between the two cereals showed that a greater number and diversity of heat-shock proteins were induced in the subtropical C₄ (sorghum) species compared to the temperate C₃ (barley) species. Fractionation of whole leaf proteins into soluble and membrane fractions showed the majority of heat-shock proteins to be associated with the soluble fraction in both sorghum and barley. However, several low molecular mass (17-24 kilodalton) heat-shock proteins were clearly identified in the membrane fractions, indicating a likely association with thylakoid membranes in vivo during the early stages of a heat-shock response in both species.     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Regulation of Corn Leaf Nitrate Reductase : I. Immunochemical Methods for Analysis of the Enzyme's Protein Component

NADH:nitrate reductase was extracted from corn leaves (Zea mays L. W64A × W182E) and purified on blue Sepharose. After the nitrate reductase was further purified by polyacrylamide gel electrophoresis, it was used to immunize mice and a rabbit. Western blots of crude leaf extracts were used to demonstrate monospecificity of the mouse ascitic fluids and the rabbit antiserum. The electrophoretic properties of purified corn and squash NADH:nitrate reductases in both native and denatured states were shown to be similar using western blotting with mouse ascitic fluid. The corn leaf enzyme has a 115,000 polypeptide subunit like that of squash. Western blots could detect 3 to 10 nanograms of nitrate reductase protein. But the detection of proteolytic degradation products using western blotting was inconsistent and remains to be established. An enzyme-linked immunosorbent assay (ELISA) was developed for quantifying nitrate reductase protein in the crude extracts of corn leaves. Using a standard curve based on nitrate reductase activity, the ELISA for corn nitrate reductase could detect 0.5 to 10 nanograms of nitrate reductase protein and was adequately sensitive for quantitative analysis of nitrate reductase in crude extracts of leaves even when activity levels were very low. When the ELISA was used to compare the nitrate reductase protein content of corn roots and leaves, these tissues were estimated to contain 0.24 to 0.5 and 4 to 5 micrograms nitrate reductase protein/gram root and leaf, respectively.     

Variation of the polypeptide composition of mitochondria isolated from different potato tissues

The protein contents of mitochondria from different potato (Solanum tuberosum L.) tissues (tubers, dark-grown shoots, and green leaves) grown in a greenhouse or in vitro were compared by two-dimensional polyacrylamide gel electrophoresis. Two different methods were used: using the method that gave the highest resolution, an average number of 360 polypeptides was revealed on the mitochondrial patterns after silver staining. The mitochondrial protein patterns of etiolated tissues (tubers, dark-grown shoots) are roughly similar but distinct from those of green leaves. The four subunits of the glycine decarboxylase complex (involved in photorespiration) and a few other polypeptides are very abundant in green tissues, compared with nonphotosynthetic tissues. Conversely, some other polypeptides that are abundant in tubers and dark-grown shoots are hardly detectable in green leaf mitochondria. A rabbit antiserum was raised against a 40 kilodalton polypeptide that is among the most characteristic of these nonphotosynthetic tissue-specific polypeptides, and the N-terminal sequence of this polypeptide was determined. No effect of in vitro culture was observed on the protein composition of mitochondria isolated from differentiated tissues. However, the protein patterns of callus and cell suspension mitochondria are distinct from those of any differentiated tissues, although their basic pattern is clearly mitochondrial.     

Malate Dehydrogenases of Pisum sativum: Tissue Distribution and Properties of the Particulate Forms

Mitochondria and leaf microbodies isolated from leaves of pea (Pisum sativum) by sucrose density gradient centrifugation were each shown to have a unique form (isoenzyme) of malate dehydrogenase (EC 1.1.1.37) based on chromatographic and kinetic properties. Root organelle preparations were shown to contain only a mitochondrial malate dehydrogenase with physical and kinetic properties similar to the leaf form. The absence of a detectable root microbody malate dehydrogenase similar to the leaf enzyme, which is intermediate in electrophoretic and chromatographic properties between the mitochondrial and soluble isoenzymes, was confirmed by diethylaminoethyl cellulose column chromatography and starch-gel electrophoresis of total homogenates from leaf and root tissue. These findings tend to support the role of the leaf microbody isoenzyme in a pathway unique to photosynthetic tissue.     

Degradation of cytoskeletal proteins by the human immunodeficiency virus type 1 protease.

Triton X-100-extracted human skin fibroblasts were exposed to human immunodeficiency virus type 1 protease and analysed by 2D-gel electrophoresis and immunofluorescence microscopy. Vimentin, two of the tropomyosin isoforms, a protein with M_r ∼ 90,000 and a protein with M_r ∼ 200,000 were found to be degraded. Structurally, this was accompanied by the disintegration of the vimentin filament network and the disappearance of the microfilament network. In contrast to our in vivo observations (Höner et al., 1991), prominent stress fibers and chromatin structure seemed to be rather resistant to the action of this protease.     

Disc-Electrophoretic Studies of Soluble Proteins and Enzymes of Meloidogyne incognita and M. arenaria

Soluble-protein and eight enzyme profiles obtained by polyacrylamide-gel electrophoresis were compared between Meloidogyne incognita and M. arenaria. Esterase, malate dehydrogenase, and α-glycerophosphate dehydrogenase patterns were distinctly characteristic for each species. Peroxidase and α-glycerophosphate dehydrogenase isoenzyme patterns varied when nematodes were propagated on different host plants. Similar profiles were obtained for two populations within each species. Antigenic proteins of these two species were compared following separation by electrophoresis.