Leaf Polypeptides Research Articles

Analysis of Peanut Leaf Proteome

Peanut (Arachis hypogaea) is one of the most important sources of plant protein. Current selection of genotypes requires molecular characterization of available populations. Peanut genome database has several EST cDNAs which can be used to analyze gene expression. Analysis of proteins is a direct approach to define function of their associated genes. Proteome analysis linked to genome sequence information is critical for functional genomics. However, the available protein expression data is extremely inadequate. Proteome analysis of peanut leaf was conducted using two-dimensional gel electrophoresis in combination with sequence identification using MALDI/TOF to determine their identity and function related to growth, development and responses to stresses. Peanut leaf proteins were resolved into 300 polypeptides with pI values between 3.5 and 8.0 and relative molecular masses from 12 to 100 kDa. A master leaf polypeptide profile was generated based on the consistently expressed protein pattern. Proteins present in 205 spots were identified using GPS software and Viridiplantae database (NCBI). Identity of some of these proteins included RuBisCO, glutamine synthetase, glyoxisomal malate dehydrogenase, oxygen evolving enhancer protein and tubulin. Bioinformatical analyses showed that there are 133 unique protein identities. They were categorized into 10 and 8 groups according to their cellular compartmentalization and biological functionality, respectively. Enzymes necessary for carbohydrate metabolism and photosynthesis dominated in the set of identified proteins. The reference map derived from a drought-tolerant cv.Vemana should serve as the basis for further investigations of peanut physiology such as detection of expressed changes due to biotic and abiotic stresses, plant development. Furthermore, the leaf proteome map will lead to development of protein markers for cultivar identification at seedling stage of the plant. Overall, this study will contribute to improve our understanding of plant genetics and metabolism, and overall assist in the selection and breeding programs geared toward crop improvement.

Changes in the leaf polypeptide patterns of wheat flag leaves spraying with non-purine cytokinins

The changes in the polypeptide patterns of soluble proteins of wheat flag leaves after treatment with two synthetic cytokinin-like substances 3-CP-2-PU [1-(3-chlorophenyl)-3-(2-pyridyl)urea] and 3-CP-4-MPU [1-(3-chlorophenyl)-3-(4-methyl-2-pyridyl)urea] was studied. Both compounds increased differentially in quantity and composition most individual polypeptides identified compared to the non-treated leaves. While 3-CP-2-PU increased only the polypeptide quantity, 3-CP-4-MPU led to the appearance of new polypeptide with Mw of 51 kDa.

Improved phytoaccumulation of cadmium by genetically modified tobacco plants ( Nicotiana tabacum L.). Physiological and biochemical response of the transformants to cadmium toxicity

The response of tobacco plants ( Nicotiana tabacum L.)—non-transformed and transformed with a metallothionein gene MThis from Silene vulgaris L.—to increase cadmium supply in the nutrient solution was compared. The transgenic plants accumulated significantly more Cd both in the roots and the leaves. Visual toxicity symptoms and disturbance in water balance were correlated with Cd tissue content. Treatment with 300 μM CdCl 2 resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 μM CdCl 2 led to irreversible damage of photosynthesis and oxidative stress. An appearance of a new peroxidase isoform and changes in the leaf polypeptide pattern were observed at the highest Cd concentration. The level of non-protein thiols gradually increased following the Cd treatment both in transgenic and non-transformed plants.

Changes in the Leaf Polypeptide Patterns of Barley Plants Exposed to Soil Flooding

Exposure of barley plants (Hordeum vulgare L.) to soil flooding for 72 - 120 h led to decrease in the content of the both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The effect was more pronounced on the small subunit. Further, the changes in protein pattern were observed, mainly proteins with molecular masses 30 - 85 kD were down-regulated.

Polypeptides associated with the induction of direct somatic embryogenesis in Camellia japonica leaves I. Identification of embryo-specific polypeptides

Embryogenic and non-embryogenic induced leaves of Camellia japonica, with the same shoot origin and submitted to the same culture conditions, were used to study protein changes during the induction of direct embryogenesis. The analysis of protein changes in these samples, based on two-dimensional polyacrylamide gel electrophoresis, revealed that 91% and 66.2% of the polypeptides detected, respectively, in embryogenic and non-embryogenic induced leaves, were already present in the non-induced control leaves. The results of the differential expression of eight selected polypeptides detected in induced and non-induced leaves are presented. A spotlist report was obtained for the 9% (43) embryogenesis-associated leaf polypeptides. From these polypeptides, two polypeptides were identified which seem to be specifically associated with somatic embryogenesis in C. japonica: E1 (pl 5.6; mol. wt. 43.5) and E2 (pl 6.0; mol. wt. 25.7).

Cytochrome P-450-catalysed monoterpenoid oxidation in catmint ( Nepeta racemosa) and avocado ( Persea americana); evidence for related enzymes with different activities

A cytochrome P-450 present in ripening avocado ( Persea americana) fruit mesocarp (CYTP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.

Seasonal Variation in Polypeptide Profiles and Cellular Structure of Pecan Leaves

Polypeptides from pecan [Carya illinoensis (Wangenh.) C. .Koch] leaves were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Pecan leaf protein profiles were similar irrespective of cultivar (Desirable and Stuart), leaflet position, reproductive status of the allied shoot, or seasonal leaf age relative to fruit development. The large subunit of ribulose l,5-bisphosphate carboxylase and the majority of the other polypeptides were consistently present. However, the most striking change in the polypeptide composition was the seasonal decline of a polypeptide with an approximate molecular mass of 24.5 kDa. This leaf polypeptide was present in leaves collected in June and July, coinciding with the periods of initial fruit elongation and rapid increase in fruit volume. A detectable decrease occurred by mid-August, when kernel development was initiated. Changes in the abundance of this polypeptide relative to other polypeptides were observed over two growing seasons. Cells of young leaves collected early in the growing season contained more ribosomes and starch granules, but fewer vesicles and smaller electron-dense osmophilic granules than old leaves collected late in the growing season.

Drought‐stress‐induced polypeptide accumulation in tomato leaves

Abstract. Tomato (Lycopersicon esculentum Mill ev. Ailsa Craig) leaf polypeptides, radiolabelled with L‐[36S] methionine during drought stress, were separated by two‐dimensional polyacrylamide gel electrophoresis. Three sets of polypeptides were distinguished: those that decreased, increased or transiently increased in response to drought stress. Three sets of polypeptides also could be distinguished during recovery from drought stress: those that increased, decreased rapidly or decreased slowly and could still be detected after 6 h of recovery. The set of polypeptides that decreased during drought stress was the same set that increased during recovery and those that increased during stress, decreased during recovery. The leaf ABA concentration increased rapidly in response to drought stress and decreased rapidly during recovery from drought stress, returning to the nonstress level after 6 h of rehydration. There was a correlation between the accumulation of some of the drought‐induced polypeptides and the pattern of ABA accumulation during stress and recovery from stress.

Systemic accumulation of novel proteins in the apoplast of the leaves of potato plants following root invasion by the cyst-nematode Globodera rostochiensis

Systemic changes in gene expression have been analysed in potato plants following root infection with the cyst nematode Globodera rostochiensis. Root invasion by the pathogen induced major changes to occur in the composition of proteins extracted in intercellular fluid from the leaf apoplastic space. The changes involved the disappearance of innate leaf proteins and the accumulation of novel gene products. Analysis by SDS-PAGE indicated that the leaf polypeptides induced on nematode invasion are not cultivar or nematode pathotype-specific. Aspirin treatment of leaves induced a similar pattern to that induced systemically by nematodes, but several gene products were specific for the latter method of induction. In particular, two abundant [ 125I]-Con A binding polypeptides of high M r accumulated in intercellular fluid only on aspirin treatment. The results are discussed in the context of the role of the extracellular leaf proteins and their modulation by systemic signals.

Electrophoretic Analysis of Protoplast, Vacuole, and Tonoplast Vesicle Proteins in Crassulacean Acid Metabolism Plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.

Two-Dimensional Gel Electrophoresis of Polypeptides from Stems, Roots, Leaves, and Rhizophores of Selaginella kraussiana

Two-dimensional gel electrophoresis of polypeptides from stems, leaves, roots, and rhizophores of the vegetative sporophyte of Selaginella kraussiana shows unique patterns. Over 600 polypeptides can be resolved; 18% are common to the four organs. Stems yield the highest percentage (23%) of unique polypeptides, while only 3% of leaf polypeptides are unique. Pair comparisons of patterns of the four organs show the greatest similarity between stems and rhizophores: 58% common polypeptides. Of the total polypeptides of rhizophores and roots, 42% are similar. Stems and rhizophores have the largest number of polypeptides unique to a pair of organs, while rhizophores and roots have fewer unique polypeptides. These results indicate that, at the abundant gene-product level, stems and rhizophores are more similar than rhizophores and roots. These results contradict views that the rhizophore is essentially an aerial phase of the root system and that rhizophores and roots are homologous organs.

Purification and characterization of two leaf polypeptide inhibitors of leaf protease from alfalfa ( Medicago sativa)

Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40–50%) α-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.

Changes in the composition of in vitro translated leaf m‐RNA caused by photoperiodic flower induction ofHyoscyamus niger

Flowering of the long day plantHyoscyamus nigerL., which is strictly photoperiodically controlled, was induced by 58 h continuous white light. The RNA from the leaves was isolated from photoperiodically induced and non‐induced plants and the poly(A)‐rich RNA separated by affinity chromatography on oligo‐dT‐cellulose. The poly(A)‐rich RNA was translated in vitro in the presence of35S‐methionine using a rabbit reticulocyte lysate. Subsequent separation of the translation products by two‐dimensional polyacrylamide gel electrophoresis and fluorography allowed a comparison of the polypeptide pattern from induced and non‐induced leaf m‐RNA. The results indicate that induction of flowering is reflected by changes in the translation of several leaf polypeptides. These polypeptides were characterized by their isoelectric points and molecular masses.

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C4 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 vivo(35)S-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 (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-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 (35)S 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.