Leaf Homogenates Research Articles

Multiplication of Pseudomonas syringae pv. phaseolicola“in planta”

AbstractIn the compatible combination of the halo blight disease of bean Pseudomonas phaseolicola was able to colonize large areas of the intercellular space of leaves, such that these confluent water congested areas became visible as water‐soaked spots. Most of the plant cell walls in the infected region maintained their normal shape, even when the cytoplasm had collapsed. Some inward bending of plant cell walls preceded their rather slow degradation and final replacement by bacterial masses. Neighbouring plant cells appeared to be metabolically active.In resistant leaves no indications of active bacterial attachment or encapsulation could be observed. However, bacteria appeared to be more densely packed in resistant leaves, and relatively more plant cells completely collapsed as compared with susceptible leaves.From 8—14 days after inoculation, the bacterial concentration did not change much in susceptible or resistant leaves, indicating the absence of bactericidal components. Even Pseudomonas pisi snowed some multiplication in bean leaves (immune reaction), but its growth stopped earlier than that of P. phaseolicola. in the resistant cultivars, probably due to a different mechanism of resistance. Although less bacteria were determined in the intercellular washing fluid (IF) compared with leaf homogenates, the high bacterial concentrations in the IF supported our observation that an effective encapsulation of bacteria in resistant leaves did not occur.

Isolation and Partial Characterization of a Cadmium-binding Protein from Pisum sativum

A Cd-binding protein was isolated from pea plants ( Pisum sativum L. c.v. Wunder von Kelvedon) grown in the presence of 0.5 mg Cd/1 nutrient solution. After Sephadex G-75 gel filtration of a Cd-containing leaf homogenate a high proportion of the metal was found in the 6,000–10,000 molecular weight fraction, which coincided with a 280/250 nm absorption ratio of < 1.0. DEAE-cellulose chromatography of the 6,000–10,000 molecular weight fraction resulted in one Cd-containing peak, which eluted at 0.67 M Tris/HCl and showed a low 280/250 nm absorption ratio. The ultraviolett absorption spectrum and its changes with pH, respectively, of this protein fraction were characteristic of metallothionein. Concomitant purification of a Cd-containing rat liver homogenate under nearly identical conditions resulted in two metallothioneins, MT-A and MT-B eluting from the DEAE-cellulose at 0.08–0.12 M Tris/ HCl. The metal contents (µg Cd/mg protein) of the proteins were 8.6 (pea plant) and 8.07 and 8.23 (MT-A and MT-B), respectively. Immunological comparison by Ouchterlony double diffusion did not reveal any cross-reactivity between the purified plant protein and antisera to the rat liver metallothioneins MT-A and MT-B. The properties of the Cd-binding protein from Pisum sativum are discussed.

A Role for manganese in the Regulation of Soybean Nitrate Reductase Activity?

The effect of manganese nutrient levels and light treatments on nitrate reductase activity (NRA) of soybean leaves was studied. Under dark conditions, soybean Mn-deficient leaves showed higher NRA than normal leaves, using the in vivo and in vitro assays. Under light, the differences in activities depended on the incubation media in the in vivo assay, whereas under the same conditions in the in vitro assay the differences observed between (- Mn)- and ( + Mn)-leaves were lower. In leaf homogenates, MV- and FMNH(2)-NR activities were also reduced in ( + Mn)-leaves in comparison to Mn-limiting leaves but DCPIP-NADH reductase was not affected. Apparently, differences in NRA could be localized on the final step of nitrate reduction, though reactivation by the addition of ferricyanide could not be obtained. Partial purification of NADH-NR-inhibitor from both, (- Mn)- and ( + Mn)-leaves, and activity assays showed lower inhibition rates of NRA by those fractions obtained from the Mn-deficient leaves. On the basis of the results obtained, an indirect role of manganese on the regulation of NRA is proposed.

Effect of pH on Proteolysis in Ensiled Legume Forage1

AbstractThe hydrolysis of proteins is extensive in ensiled forages but the production practices which may influence the extent of proteolysis remain to be characterized. An in vitro assay system was developed to estimate the relative rates of proteolysis and was compared to a laboratory silo system as a means of estimating the relative extent of proteolysis in three forage legumes, alfalfa (Medicago sativa L. cv. Iroquois), red clover (Trifolium pratense L. cv. Arlington) and birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Plants were harvested from field plots as first and second cuts at the same time and were in the early bud to 10% bloom stages, depending on species and sampling time. Leaf homogenates containing chloramphenicol as an antibiotic were used as in vitro systems to monitor changes in the level of free amino acids at pH values between 4.0 and 6.5 at incubation times up to 72 h. This method provided a more satisfactory estimate of the relative proteolysis in ensiled forage than previous attempts using azocasein as substrate. Proteolysis was more extensive in alfalfa than the other two species. The optimal pH of proteolysis was approximately 6.0 in alfalfa, but 6.5 or above in red clover and birdsfoot trefoil. Low pH inhibited proteolysis. In a second experiment, forage from the same field plots as the in vitro experiment was ensiled in laboratory silos with the addition of 1% Na2CO3, 1% formic acid, 0.7% HCl, 1% formaldehyde, or no treatment. After ensiling for 30 days, protein hydrolysis was consistently highest in the alfalfa silages. The acid additives reduced proteolysis in all species, but a significant (P≤0.01) additive ✕ species interaction was observed in the second cut. A close positive relationship between the initial pH of the forage at ensiling and proteolysis was established for each species. The amount of protein degraded to free amino acids during ensiling of these legumes appears to be dependent on the proteolytic activity in the leaves, the rate of pH decline during ensiling, and the interaction between these two factors.

Formula omitted] metabolism of deltamethrin by stem and leaf homogenates of tobacco plants

Stem and leaf homogenates from tobacco plants metabolised deltamethrin by oxidative and hydrolytic actions on the insecticide. Although a major portion of deltamethrin remained unchanged, metabolites such as 3-phenoxybenzaldehyde, 3-phenoxybenzoic acid and 3-(2,2-dibromonyl)-2,2-dimethylcyclopropanecarboxylic acid were formed. The data suggest a major portion of the insecticide would remain unchanged when absorbed by tobacco plants.

Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves ( Pisum sativum)

Enzymatic activity responsible for the conversion of fatty acids to alkanes catalyzed by pea leaf homogenate was found to be mainly in the microsomal fraction. This particulate preparation catalyzed alkane formation from n-C 18, n-C 22, and n-C 24 acids at rates comparable to that observed with n-C 32 acid with O 2 and ascorbate as required cofactors. In each case the major alkane contained two carbon atoms less than the precursor acid. Since the preparation also catalyzed α-oxidation, it was suspected that some α-oxidation intermediate, with one less carbon atom than the substrate acid, might lose another carbon to generate the alkane. Thin-layer and radio-gas-liquid chromatographic analysis of the products generated from [U- 14C]stearic acid by the particulate preparation after different periods of incubation showed that, at all time periods, α-hydroxy C 18 acid, C 17 aldehyde, and C 17 acid were the major products. Since C 16 alkane was the major product even after short periods of reaction, the C 17 aldehyde might have been the immediate precursor of the alkane. Exogenous labeled C 18 and C 24 aldehyde were converted to alkanes. The alkane-synthesizing activity was solubilized from the microsomal preparation using Triton X-100. The solubilized preparation was retarded in a Sepharose 6-B column, but the hydrocarbon-forming activity was not resolved from α-oxidation. The solubilized preparation produced alkane with two carbon atoms less than the parent acid in a time- and protein-dependent manner. The soluble preparation also required O 2 and ascorbate and, like the microsomal preparation, was inhibited by dithioerythritol and metal ion chelating agents.

Compartmentation of phenylacetic and cinnamic acid synthesis in spinach

Applying labelled phenylalanine or tyrosine to purified intact spinach chloroplasts, only the corresponding phenylacetic acids but not the cinnamic acids could be detected. The addition of mercaptoethanol or dl‐dithiothreitol and the variation of light conditions had only a slight effect. However, cinnamic acids could be found together with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine and/or tyrosine ammonia lyase outside the spinach chloroplasts. Similar results were obtained with barley leaf homogenates, where cinnamic acids were the main products. Reviewing recent findings on amino acid synthesis in spinach leaves, it may be concluded that the synthesis of aromatic amino acids is restricted to the chloroplast, whereas the metabolism of secondary aromatic compounds is predominantly localized outside the chloroplasts.

Subcellular localization of hexokinase in pea leaves. Evidence for the predominance of a mitochondrially bound form.

Hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) activity was determined in subcellular fractions prepared from pea (Pisum sativum) leaf homogenates. About 60% of the total detectable activity of hexokinase was found associated with a particulate fraction consisting essentially of mitochondria and chloroplasts and free of cytosol contamination. The hexokinase specific activity of the particulate fraction was 2-fold higher than that of the homogenate and about 4-fold higher than that of the cytosol. Using a specially designed isokinetic-isopycnic sucrose density gradient centrifugation method, the distribution of hexokinase activity correlated with that of the mitochondrial marker (cytochrome oxidase) and not with that of the chloroplast membrane marker ( chlorophyll ) or that of the cytosol marker (phosphoenolpyruvate carboxylase). Thus, the hexokinase/mitochondria ratio was close to 1.0 along the entire gradient, while the hexokinase/chloroplast ratio varied over a 10-fold range. The results strongly suggest that hexokinase is predominantly bound to mitochondria of pea leaves, and that pea leaf chloroplasts are essentially devoid of any specifically associated hexokinase activity. This work provides the first demonstration of a specific association of hexokinase with mitochondria from photosynthetic tissues of higher plants.

Activation State of Ribulose Bisphosphate Carboxylase in Soybean Leaves

Conditions for extraction and assay of ribulose-1,5-bisphophate carboxylase present in an in vivo active form (initial activity) and an inactive form able to be activated by Mg(2+) and CO(2) (total activity) were examined in leaves of soybean, Glycine max (L.) Merr. cv Will. Total activity was highest after extracts had preincubated in NaHCO(3) (5 millimolar saturating) and Mg(2+) (5 millimolar optimal) for 5 minutes at 25 degrees C or 30 minutes at 0 degrees C before assay. Initial activity was about 70% of total activity. K(act) (Mg(2+)) and K(act) (CO(2)) were approximately 0.3 millimolar and 36 micromolar, respectively. The carry-over of endogenous Mg(2+) in the leaf extract was sufficient to support considerable catalytic activity. While Mg(2+) was essential for both activation and catalysis, Mg(2+) levels greater than 5 millimolar were increasingly inhibitory of catalysis. Similar inhibition by high Mg(2+) was also observed in filtered, centrifuged, or desalted extracts and partially purified enzyme. Activities did not change upon storage of leaves for up to 4 hours in ice water or liquid nitrogen before homogenization, but were about 20% higher in the latter. Activities were also stable for up to 2 hours in leaf extracts stored at 0 degrees C. Initial activity quickly deactivated at 25 degrees C in the absence of high CO(2). Total activity slowly declined irreversibly upon storage of leaf homogenate at 25 degrees C.

The role of superoxide dismutase in the susceptibility of bean leaves to ozone injury

The primary leaves of bush bean plants pretreated daily with very low levels of ozone (0.02 or 0.05 ppm) pass through stages of varying susceptibility to a subsequent acute dose. This variation in response can only partly be accounted for by stomatal behaviour. Present studies indicate that the levels of the oxyradical scavenger, superoxide dismutase (SOD), assayed in leaf homogenates, appear to play no role in the phenomenon. No observed changes in the levels of extracted SOD following various low ozone pretreatment regimes could be related to changes in susceptibility to acute injury, in comparisons with control plants maintained in charcoal-filtered air prior to acute fumigation. The only significant increases in SOD levels which appeared to be directly related to ozone exposure occurred simultaneously with the appearance of visible symptoms of injury from either the cumulative chronic or acute ozone exposures.

An Association Between Acid Invertase Activity and Cell Growth during Leaf Expansion inPhaseolus vulgarisL.

Changes in lamina area, dimensions of epidermal and palisade cells, acid invertase activity and content of sucrose and hexose in the primary leaves of Phaseolus vulgaris L. were determined between emergence of the hypocotyl hook and the completion of leaf expansion. Growth in area and thickness of the primary leaf after emergence was attributable to the expansion of cells already present in the lamina at emergence. The major invertase in the expanding leaf was a readily soluble acid invertase; little insoluble invertase activity was detected. Soluble and insoluble fractions of leaf homogenates contained little neutral invertase activity. The specific activity of the soluble acid invertase increased rapidly during the early stages of leaf expansion, reaching a peak at the time of most rapid cell enlargement (5 d after emergence) and then declining as the leaf matured. Highly significant positive correlations were found between enzyme specific activity and the rates of cell and leaf enlargement. The early, rapid phase of lamina expansion was characterized by high concentrations of hexose sugar and low concentrations of sucrose. As the rates of leaf cell enlargement declined the concentration of hexose fell and that of sucrose increased. Between 5 d and 11 d after hypocotyl emergence, the hexose/sucrose ratio in the primary leaf decreased approximately 10-fold as the specific activity of acid invertase decreased. The results are discussed with reference to sources of carbon substrates for cell growth and to the sink/source transition during leaf development.

Isolation and photosynthetic characteristics of mesophyll cells from developing leaves of soybean

Mesophyll cells were isolated from developing sink leaves (25 to 30 mm in length) of soybean, Glycine max (L.) Merr. cv. Will. Leaf strips were incubated for two h in a buffered medium containing osmoticum and 0.2% Pectolyase Y‐23. Gently stirring the leaf strips released from 7 to 16% of the total leaf mesophyll cells. Other pectinase enzymes, effective in releasing cells from mature source leaves (70 to 75 mm in length), did not release cells from sink leaves. Sink and source cell preparations were about 50 and 95% intact, respectively, based on the exclusion of Evans Blue dye. Intact cells could not be separated from broken cells on Ficoll or metrizamide density gradients. Total protein and catalase, glyceraldehyde‐3‐phosphate dehydrogenase, glycolate oxidase, phosphoenolpyruvate carboxylase, and ribulose 1,5‐bisphosphate carboxylase activities on a chlorophyll basis were about 50% lower in sink mesophyll cells than in sink leaf homogenates indicating that broken sink cells lost soluble protein to the medium. Source cells and source leaf homogenates had comparable amounts of protein and enzymatic activities. Enzymatic activities on a chlorophyll basis were similar in source and sink leaves with the exception of phosphenolpyruvate carboxylase, which was two times higher in sink leaves. This enzyme was also exceptionally low in source and sink cells being only 61 and 23%, respectively, of whole leaf activities. Sink cell rates of 14CO2 fixation were only 7% of source cell rates and sink cells did not show light‐dependent O2 evolution. Both cell preparations had photosystem II activiteis which were comparable to rates of 14CO2 fixation at satuarating light and CO2 concentration. It was concluded that the reduced photosynthetic rate of sink cells was limited by the low photochemical capacity rather than a limitation of Calvin cycle enzymes.

Catalase activity of C 3 and C 4 species and its relationship to mercury vapor uptake

Catalase activity of seven gramineous species was measured. Significant differences of catalase activity were observed between C 3 and C 4 species; the average activity of C 3 plants was approximately 4-fold greater than that of C 4 plants. Positive correlations were observed between the catalase activity and in vivo uptake of mercury by leaves or in vitro uptake by leaf homogenates. Sodium azide and sodium cyanide completely inhibited in vitro mercury uptake by the leaf homogenates. 3-Amino-1,2,4-triazole (ATA), a specific catalase inhibitor, gave 95–100% inhibition on mercury uptake by a wheat homogenate and only 40–60% inhibition by a corn homogenate. Treatment of intact plants with various amounts of ATA caused various degrees of inhibition in both mercury vapor uptake and catalase activity. For comparable amount of ATA, a greater inhibition of mercury uptake was observed in C 3 plants as compared to that in C 4 plants. However, there were no differences on inhibition of catalase activity between the C 3 and C 4 species. Observations from the results of both in vivo and in vitro experiment suggest that catalase plays a key role in mercury vapor uptake by gramineous C 3 species and a lessor role in C 4 species, particularly sorghum.

Asparagine Synthesis in Pea Leaves, and the Occurrence of an Asparagine Synthetase Inhibitor

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [(14)C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [(14)C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.

Plant Transketolase: Subcellular Distribution, Search for Multiple Forms, Site of Synthesis

Summary Sucrose density gradient analysis of pea and rye leaf homogenates showed that transketolase activity was only present in the supernatant and in the intact chloroplast fraction. It was estimated that in the intact tissue transketolase was predominantly (at least 90%) associated with the chloroplasts. Transketolase was also contained in the proplastids of dark-grown pea roots. By chromatography on DEAE-Sepharose Cl-6B, ammonium sulfate-gradient solubilization, polyacrylamide gel electrophoresis, or isoelectric focusing it was not possible to separate any multiple forms of transketolase from cell-free leaf extracts of rye, wheat, pea, or spinach. Transketolase was also present in the plastids of 70S ribosome-deficient heat-bleached rye leaves, indicating that the chloroplast-localized enzyme was synthesized on cytoplasmic 80S ribosomes.

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.

Subcellular localization of saponins in green and etiolated leaves and green protoplasts of oat (Avena sativa L.)

Investigations on the subcellular localization of the two steroidal saponins avenacoside A and B of oat by fractionation of leaf homogenates indicate that these saponins are located in the soluble cell fraction of etiolated as well as green leaves. The analysis of isolated green mesophyll protoplasts reveals that the mesophyll of green oat leaves contains only 50% of the leaf saponins. This reduction is nearly exclusively caused by a reduction of avenacoside B in mesophyll protoplasts in contrast to the epidermis which shows high concentrations of avenacoside B and only traces of avenacoside A. Investigations of isolated vacuoles in comparison to isolated mesophyll protoplasts showed that the steroidal saponins are predominantely localized in the vacuole. Furthermore, results are presented which indicate that the avenacosides A and B may be used as markers for the integrity of isolated protoplasts and vacuoles.

Selective Adsorption of Nitrate and Nitrite from Tobacco Leaf Homogenates

AbstractA procedure has been developed for the reduction of nitrate / nitrite in tobacco during the homogenized leaf curing process. This procedure involves the precipitation of these anions with a polymer composed of the nitron 1,4-diphenyl-3,5-endoanilino-4,5-dihydro-1,2,4-triazole linked to poly(vinylbenzyl chloride). This poly(vinylbenzyl-nitron chloride) complex was coated onto several inert supporting materials and tested for anion binding capacity using nitrate/nitrite standards and tobacco leaf extracts. When coated onto the surface of acid-washed sand, the nitron polymer was capable of repeated regeneration with no detectable loss of nitrate / nitrite binding capacity. The nitron polymer had a slightly greater affinity for nitrite than for nitrate; however, due to the high ratio of nitrate to nitrite in tobacco leaf tissues, nitrate was the principle anion adsorbed from leaf homogenates. Large scale application of this selective nitrate adsorbing polymer during homogenized leaf curing of tobacco could significantly reduce these potentially harmful components in smoking products.

Synthesis and composition of vitamin D-3 metabolites in Solanum malacoxylon

The synthesis of vitamin D-3 hydroxylated metabolites in Solanum malacoxylon was investigated. When crude leaf homogenates and subcellular fractions were incubated with [ 3H]vitamin D-3 and [ 3H]25-hydroxy-vitamin D-3 under conditions described for animal vitamin D-3-25-hydroxylase and 25-hydroxy-vitamin D-3-1α-hydroxylase, respectively, labelled metabolites identified on the basis of their chromatographic properties as 25-hydroxy-vitamin D-3 and 1,25-dyhydroxy-vitamin D-3 were formed. Other unidentified product metabolites were also detected. Vitamin D-3-25-hydroxylase activity was localized in microsomes and 25-hydroxy-vitamin D-3-1α-hydroxylase in mitochondria and microsomes. Chromatography of sterols isolated from leaf extracts preincubated with β-glucosidase on Sephadex LH-20 columns permitted the isolation of three biologically active fractions with elution properties similar to vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3, respectively. Ultraviolet spectra characteristic of vitamin D-3 and its metabolites were obtained after purification of the fractions by TLC. Co-chromatography of individual fractions with authentic metabolites on TLC provided further evidence that the plant contains vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3 as glucoside derivatives. These results suggest that a similar pathway of vitamin D-3 hydroxylation as in animals may be operative in S. malacoxylon.

Two-dimensional analysis of enhanced synthesis of proteins in oat leaves responding to the crown rust infection

Changes in protein composition of primary leaves of oat (Avena sativa L.) cultivar Shokan 1 were studied in relation to resistance expression at the early stage of infection (16 to 20 h after inoculation) with incompatible race 226 of Puccinia coronata avenae. The incorporation ratio of [14C]-leucine into the acid-insoluble fraction was increased by inoculation with race 226 in both the leaf homogenate and the cytoplasmic fraction, while in the microsomal fraction it was unchanged. The pre-treatment of inoculated leaves with blasticidin S, a protein synthesis inhibitor, largely inhibited this incorporation into the cytoplasmic fraction, but only slightly into the microsomal fraction. Two-dimensional polyacrylamide gels stained with Coomassie blue revealed no changes in protein composition in leaves inoculated with either race 226 or race 203. However, the tracer experiments made with [14C]-leucine revealed that the number of protein spots detectable by fluorography were 87, 77 and 67 for cytoplasmic fractions from leaves inoculated with races 226 and 203 and uninoculated leaves respectively. Eleven spots were found to be specific for the incompatible interaction but were not detected in leaves treated with blasticidin S. Sixteen spots labeled with [14C]-leucine were detected in microsomal fractions from leaves inoculated with race 226 and from the uninoculated control but no difference was observed between them. It is suggested that enhanced synthesis of cytoplasmic proteins is involved in the resistance expression of oat leaves against the incompatible race of the crown rust fungus.