Leaf Tissue Extracts Research Articles

Cellular distribution of 3-hydroxy-3-methylglutaryl coenzyme a reductase and mevalonate kinase in leaves of Nepeta cataria

In Nepeta cataria leaf tissue there are two separate activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and mevalonate (MVA) kinase respectively as determined by the use of a 20–45% discontinuous sucrose density gradient. Cell-free extracts of leaf and callus tissue were prepared and HMG-CoA reductase and MVA kinase activities were compared to activities in extracts from porcine livers and yeast autolysates. Callus tissue from N. cataria has only one peak of HMG-CoA reductase and MVA kinase activity located at the top of the sucrose density gradient. Isolated chloroplast from N. cataria leaves have one peak of HMG-CoA reductase and MVA kinase activity, located near the bottom of a sucrose density gradient. MVA kinase activities in porcine livers and yeast autolysate also showed only one activity profile, located at the top of the sucrose gradient. Partial purification of the leaf extract through the use of differential centrifugation, 30–70% ammonium sulfate precipitation and Bio-Gel P-100 column chromatography shows that MVA kinase, 5-phosphomevalonate (MVAP) kinase and 5-pyrophosphomevalonate (MVAPP) decarboxylase activities remain in the same fractions. The extra-chloroplastidic HMG-CoA reductase activity may be separated from MVA kinase activity by differential centrifugation. These results suggest the presence of two HMG-CoA reductase and MVA kinase enzymes in N. cataria leaf tissue—one located in the chloroplast and a second being extra-chloroplastidic.

Sunflowers (Helianthus annuus) are Allelopathic to Weeds

Laboratory, greenhouse, and field studies were conducted to determine the allelopathic potential of sunflower (Helianthus annuusL.) cultivars to suppress weed germination and growth. Germination of wild mustard [Brassica kaber(DC.) L.C. Wheeler var.pinnatifida(Stokes) L.C. Wheeler] seeds at 25 C in undiluted aqueous extracts of sunflower leaf tissue was inhibited 75%, but was stimulated by up to 150% at 10- and 100-fold dilutions. Stem-tissue extracts at all concentrations stimulated wild mustard seed germination. The germination response of other weed species varied with the sunflower cultivar and concentration of tissue extract. In sand culture, leachates of dried sunflower leaf and stem tissue inhibited broadleaf - weed seedling growth, but had little or no effect on the growth of grass weeds. Sunflower root exudates inhibited seedling growth, but were less effective than leaf and stem tissue leachates. Germination of weed seeds was unaffected by root exudates. Over a 5 -yr period, weed density and percent ground cover increased less in field plots of sunflower than in control plots.

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba

The cytoplasmic male sterility (CMS) trait of at least one line of Vicia faba plants is always associated with the presence of high molecular weight double-stranded RNA in the leaf tissue extracts. Subcellular fractions of leaf tissue from CMS and fertile maintainer plants were initially analyzed in an attempt to locate, identify, and characterize the genetic material involved with the sterility trait. This CMS-associated high molecular weight RNA was found only in the cytosol of the "447" male sterile line of V.faba plants and could not be isolated from the recurrent parent (maintainer), from lines that had been fertility-restored, or from lines that had reverted from the sterile condition. We have been able to move the CMS-associated RNA from donor to fertile host plants through a dodder bridge. These hots not only contain the RNA but now exhibit a male sterile phenotype, as detected by visual examination of the flower, the pollen, morphological characteristics, and pollen staining ability.

Effect of Ethephon and UV Irradiation on Auxin Destruction in «Little Marvel» Dwarf Pea (Pisum sativum)

Summary Treatment of «Little Marvel» dwarf pea (Pisum sativum) seeds with 3 ppm ethephon was followed by subsequent exposure to: 1. complete darkness; 2. visible light; and 3. ultraviolet irradiation (UV). Cell free extracts of distal root, root tip, proximal stem, and leaf tissues (20-day-old) were assayed for IAA-1-14C destruction. Distal root and root tip tissue extracts of ethephon-treated seeds and subsequent exposure of the seedlings to UV showed marked IAA-1-14C destruction compared to proximal stem and leaf cell-free extracts. The present study suggests that IAA-1-14C destruction may be enzymic in nature and different in all four tissues (root tip, dorsal root, proximal stem, and leaf) depending upon the exposure treatment (dark, visible light and UV), with or without ethephonpretreatment.

Changes in Enzyme Regulation during Growth of Maize

Extracts of leaf tissue of Zea mays L. seedlings were fractionated on nonlinear sucrose gradients to separate subcellular organelles. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact chloroplasts, as judged by the presence of chlorophyll and triosephosphate isomerase activity. Neither enzyme activity was detected in fractions containing ruptured chloroplasts, mitochondria, or microbodies. Quantitative measurements of enzyme activity and chlorophyll, and electron microscopic analysis of plastid preparations support the conclusion that maize mesophyll chloroplasts contain a significant fraction of the total cellular content of homoserine dehydrogenase.A survey of representative kinetic, regulatory, and physical properties did not reveal any significant differences between enzyme released from isolated, undamaged chloroplasts and that obtained from soluble cellular fractions.Examination of enzyme prepared from chloroplasts of different age seedlings indicated that the sensitivity of homoserine dehydrogenase to inhibition by the feedback modifier l-threonine was progressively diminished during growth of the plants. This systematic change in regulatory properties of the enzyme occurred to the same extent for the enzymes obtained from chloroplasts and soluble fractions.

Accumulation of antibacterial isoflavonoids in hypersensitively responding bean leaf tissues inoculated with Pseudomonas phaseolicola

Red Kidney bean ( Phaseolus vulgaris L.) plants inoculated with Pseudomonas phaseolicola (isolate HB-36) showed water-soaked infection centers on the inoculated leaf surfaces about 48 h after inoculation. In contrast, resistant bean plants (GN Nebraska 27) inoculated with the same isolate responded by developing a hypersensitive reaction (HR) after roughly the same period after inoculation. High concentrations of phenolic compounds were detected in the ethanolic extracts of leaf tissues showing HR. Among these were identified the isoflavonoid phytoalexins phaseollin (pterocarpan), phaseollinisoflavan (isoflavan), coumestrol (coumestan) and kievitone (isoflavanone). These isoflavonoids were detected in substantial quantities 24 h after inoculation and their concentrations increased further up to 72 h, after which time they began to decrease. In susceptible Red Kidney leaves, isolate HB-36 induced much lower concentrations of these compounds at all stages of disease development. In repeated in vitro bioassays the above four compounds consistently restricted colony formation by P. phaseolicola isolates. However, the race 2 isolates, HB-36 and G-50, were found to be comparatively more tolerant to these compounds than the race 1 isolates, HB-33 and HB-20. Colony formation in race 1 isolates was completely inhibited by phaseollin and phaseollinisoflavan at 42 and 21 μg/ml; colony formation of race 2 isolates was retarded (90 to 98%) but not completely suppressed at 52·5 μg/ml of phaseollin and 52 μg/ml of phaseollinisoflavan. Coumestrol and kievitone at 21 and 33 μg/ml respectively were totally toxic to race 1 isolates but they were relatively less toxic to race 2 isolates even at higher test concentrations. All four phytoalexins also restricted the growth of P. glycinea isolates, R-2 and 724 (Leben). Isolate 724, however, exhibited a greater degree of tolerance than isolate R-2. P. fluorescens, a saprophyte, was relatively insensitive to the phytoalexins and showed only 26 to 30% retardation in colony formation at the maximum test concentrations used. These studies show that the virulent isolates of P. phaseolicola are not as sensitive to bean phytoalexins as the relatively less virulent isolates of P. phaseolicola.

The effect of fruit-removal on cytokinins and gibberellin-like substances in grape leaves

Removal of fruit from potted cuttings of Vitis vinifera L. increased the concentration of a cytokininglucoside in leaf tissue extracts and decreased the level of extractable gibberellin-like substances. The glucoside (of zeatin riboside) is not present in xylem exudate of V. vinifera L., and appears to be synthesized in the leaves. Berry extracts contain zeatin-riboside and smaller amounts of cytokinin-glucoside. The changes in the level of these hormones are discussed in relation to previous results on abscisic acid and phaseic acid levels in grape leaves.

A messenger RNA for capsid protein isolated from tobacco mosaic virus-infected tissue

A low molecular weight virus-related RNA component (LMC) was isolated from extracts of TMV-infected leaf tissue by employing a combination of the techniques of sucrose density gradient sedimentation and polyacrylamide gel electrophoresis. It was found that LMC has an estimated molecular weight of 2.5 × 10 5 and acts as a template for the synthesis of TMV capsid protein in a wheat germ-derived cell-free protein synthesizing system. Evidence is presented for the possible existence of other unique virus-related RNA species in extracts of infected tissue.

The detection of phytoalexins in jute leaves after infection by Colletotrichum corchorum and their possible rôle in lesion development

Colletotrichum corchorum readily produces lesions on the leaves of different cultivars of jute ( Corchorus capsularis L.) but the percentage of spreading lesions on cv. JRC 412 is significantly higher than on cv. JRC 321. This difference is less apparent when 1% sorbitol or orange juice is added to the inocula. The rate of germ tube growth on glass and leaf surfaces indicates that growth is inhibited on leaf surfaces of all the six cultivars of jute tested, but the greatest inhibition occurs on JRC 321. The germ tubes are markedly shorter over lesions than apparently healthy areas. Inhibition of growth also occurs in the underlying cells even if the cuticle is damaged prior to inoculation. Drops of water left for 48 h on healthy leaf surfaces became stimulatory, while similar drops containing spores of C. corchorum became highly inhibitory. The diffusates collected from cv. JRC 321 were more inhibitory than those from JRC 412. The amount of inhibitor in the diffusates gradually increased with incubation time up to 48 h. The bacterial population was always found to be much greater in diffusates than in exudates. Control of bacterial growth did not significantly reduce the antifungal activity of diffusates. Again, a highly concentrated suspension of bacteria isolated from the diffusate was much less inhibitory to C. corchorum than diffusates with a low concentration of bacteria. The extraction of leaf tissue beneath the infection droplets as well as of water droplets by organic solvents and chromatographic separation of inhibitor from the diseased tissue suggest that an antifungal substance or phytoalexin is formed post-infectionally in host tissue and presumably this is involved in lesion development in jute leaves.

Mevalonate-activating enzymes in callus culture cells from Nepeta cataria

The 30000 g supernatants from cell-free extracts of Nepeta cataria leaf tissue and leaf callus tissue have mevalonic acid kinase, mevalonic acid phosphate kinase and mevalonic acid pyrophosphate decarboxylase activities. The callus tissue cell-free extract produced mevalonic acid pyrophosphate and isopentenyl pyrophosphate; however, very little mevalonic acid phosphate was observed. The leaf cell-free extracts incubated with [ 14C]-mevalonic acid produced higher amounts of mevalonic acid phosphate. When both the leaf cell-free extract and the callus cell-free extract were incubated with [ 14C]-mevalonic acid in the presence of iodoacetamide, the ion exchange column elution profile was cleaner, which was confirmed by PC. Apparently the callus tissue 30000 g supernatant contains mevalonic acid phosphorylating enzymes even though there is no production of the methyl cyclopentane monoterpenes.

Adenosine diphosphate sulphurylase activity in leaf tissue.

1. A new method is described for the assay of ADP sulphurylase. The method involves sulphate-dependent [(32)P]P(i)-ADP exchange; the method is simpler, more sensitive and more direct than the method involving adenosine 5'-sulphatophosphate-dependent uptake of P(i). 2. ADP sulphurylase activity was demonstrated in crude extracts of leaf tissue from a range of plants. Crude spinach extract catalysed the sulphate-dependent synthesis of [(32)P]ADP from [(32)P]P(i); spinach extracts did not catalyse sulphate-dependent AMP-P(i), ADP-PP(i) or ATP-P(i) exchange under standard assay conditions. ADP sulphurylase activity in spinach leaf tissue was associated with chloroplasts and was liberated by sonication. 3. Some elementary kinetics of crude spinach leaf and purified yeast ADP sulphurylases in the standard assay are described; addition of Ba(2+) was necessary to minimize endogenous P(i)-ADP exchange of the yeast enzyme and crude extracts of winter-grown spinach. 4. Spinach leaf ADP sulphurylase was activated by Ba(2+) and Ca(2+); Mg(2+) was ineffective. The yeast enzyme was also activated by Ba(2+). The activity of both enzymes decreased with increasing ionic strength. 5. Purified yeast and spinach leaf ADP sulphurylases were sensitive to thiol-group reagents and fluoride. The pH optimum was 8. ATP inhibited sulphate-dependent P(i)-ADP exchange. Neither selenate nor molybdate inhibited sulphate-dependent P(i)-ADP exchange and crude spinach extracts did not catalyse selenate-dependent P(i)-ADP exchange. 6. The presence of ADP sulphurylase activity jeopardizes the enzymic synthesis of adenosine 5'-sulphatophosphate from ATP and sulphate with purified ATP sulphurylase and pyrophosphatase.

Replication of tobacco mosaic virus: IV. Further characterization of viral related RNAs

Experiments were performed to characterize the viral related RNA species which appear in extracts of tobacco mosaic virus (TMV)-infected tissue and, in particular, the low molecular weight (ca. 350,000 daltons) component, LMC. It was determined that LMC is probably not a component of the virus rod but is a fragment of unincapsidated TMV RNA. Synthesis of LMC in diseased tissue is not inhibited by the presence of actinomycin D. Because LMC would not reconstitute into a rod with TMV protein, it was considered not to contain a detectable amount of the 5′ terminus of TMV RNA. Polyadenylic acid sequences could not be detected by three analytical methods in any RNA component. In addition to LMC, which is homogeneous in size, TMV RNA fragments polydisperse in size are also present in leaf tissue extracts. In contrast, RNA complementary to TMV RNA was present in extracts only as a component of the double-stranded TMV replicative form and was not found free in the infected cell. Neither fragments of replicative form nor single-stranded TMV complementary RNA could be detected. In addition, the only RNA fraction of uniform size found to contain both an RNA species and its complement was the TMV replicative form.

Chitinase activity in Lycopersicon esculentum and its relationship to the in vivo lysis of Verticillium albo-atrum mycelium

Healthy and Verticillium albo-atrum-infected tomato plants were shown to possess a constitutive enzyme capable of hydrolysing chitin of N-acetylglucosamine. It was present in leaf, stem and root tissue extracts, vacuum extracted xylem sap and to a lesser extent in the bleeding exudate. The partially purified enzyme showed optimal activity between pH 5·4 and 5·8 and at a temperature of 28°C. The K m and V max values for chitin were 0·408 mg/ml and 3·7 μg N-acetylglucosamine N/h ml respectively. No free hexosamines were detected in healthy or diseased plants. Authentic lysozyme and β-glucosidase showed slight chitinase activity. Only trace amounts of lysozyme activity could be detected in tissue extracts. Crude extracts possessed high β-glucosidase and low chitinase activities; after partial purification the β-glucosidase activity was negligible while chitinase activity increased tenfold. Chitinases from Streptomyces and V. albo-atrum and the tomato enzyme responded identically to selected inhibitors. Inoculation of tomato cultivars with isolates of V. albo-atrum from hop and tomato gave a range of disease expression from susceptible to resistant. Infection resulted in a significant increase in chitinase activity which was greater in the susceptible reaction. The apparent pH of xylem exudate from diseased plants (6·8 to 7·1) was too high for appreciable enzyme activity. The activity of host polysaccharases is discussed in relation to the well-documented in vivo lysis of plant pathogens.

THE ACCUMULATION AND BINDING OF COPPER IN LEAF TISSUE OF BECIUM HOMBLEI (DE WILD.) DUVIGN. & PLANCKE

SummaryThe flowering plant Beciutn homblei is able to accumulate copper and other heavy metals to a relatively high level in its leaves. Approximately 17% of the total copper is concentrated in cell walls where it is bound as an organic complex. Copper in water extracts of leaf tissue is not in free ionic form but is complexed with amino acids or peptides. In vitro experiments indicate the strong complexing powers with copper of certain substances in leaf tissue.

Effect of Variety, Seed Lot, Soil pH, Soil Fertility, and Light on Cotyledon and Plant Color in Alfalfa1

Environmental conditions such as light intensity, temperature, soil pH, and fertility significantly affected the expression of alfalfa plant and cotyledon color. Soil pH and soil fertility had only minor effects on cotyledon color. Plant color was affected to a greater extent, plants grown on limed and fertilized soil being about 20% darker, i.e., acetone extracts of leaf tissue averaged about 20% less transmission than those grown on a relatively acid (pH 5.6) and infertile soil. Plants grown in growth chamber at alternating 18 and 9 C with light for 14 hours at the higher temperature were significantly darker than those grown in a warm greenhouse (25 to 20 C) and 24‐hour photoperiods. Differences in cotyledon and plant color associated with seed lot were relatively minor. When grown and sampled under standardized conditions, varieties derived in part from Medicago falcata such as ‘Vernal’ and ‘Narragansett’ were generally darker green than varieties derived from Medicago sativa. Measurements of cotyledon and plant color in 10 common varieties of alfalfa indicated the presence of significant varietal differences. Results of these studies indicate that colorimetric measurements of cotyledon and plant color can be used to separate alfalfa varieties into groups. Additional tests can then be applied to further characterize varieties within each group. Cotyledon color was of lesser value for this purpose than plant color.

The Relationship Between Calcium, Magnesium, and Potassium Accumulation and Titratable Acidity in the Leaves of a Selected Mcintosh Apple Clone1

Abstract Water extracts of dry leaf tissues from a selected McIntosh apple clone were analyzed by titration with sodium hydroxide and by atomic absorption spectrophotometry. It was shown that titratable acidity decreases during the period July 4 - August 1 and increases as the season advances beyond August 1. The data suggest that calcium and magnesium which accumulate may be the principal cations associated with bases involved in the buffer system to maintain a relatively constant pH in the leaf. It is theorized that changes in leaf pH may be a major factor in the damage characteristic of certain mineral deficiencies in apples.

Activation of the Latent Tyrosinase of Broad Bean

EXTRACTS of leaf tissue of broad bean (Vicia faba, L.) contain an enzyme which shows little tyrosinase (E.C. 1.10.3.1)1 activity unless treated with a denaturing agent2–5 when the activity toward both orthodiphenols and mono-phenols normally increases 50-fold. Conditions under which the denaturing agents are used for activation (Table 1) are normally considered to be insufficient to break covalent bonds associated with the protein molecules but are known to alter the tertiary structure and to cause some proteins to dissociate into sub-units6. Like other tyrosinases7 the isolated latent enzyme contains copper and exists in several forms separable by column chromatography or by electrophoresis on starch gel. Inhibition investigations have shown that the latent enzyme is less sensitive toward copper complexing agents, such as cyanide and diethyldithiocarbamate, than the active form, indicating that activation is probably accompanied by an unmasking of the prosthetic group.

An electrophoretic analysis of tobacco mosaic virus biosynthesis

1. 1. A detailed electrophoretic analysis of the time-course of tobacco mosaic virus biosynthesis in nutrient-cultured tobacco leaf blade tissue is described. The mobility of the TMV formed (av.: −9.7 × 10 −5 cm 2/v./sec.) is fairly constant during the time-course, and agrees with the mobility of the inoculum. 2. 2. Calculations of the concentration of TMV from the areas of scanning peaks of the electrophoretic patterns give results comparable with independent determination of TMV concentration by microanalysis. Tobacco mosaic virus appears at 72 hr. after inoculation, increases in concentration until 200 hr., and remains constant thereafter. 3. 3. A new electrophoretic component, B, appears in infected tissue when the TMV content is about one-third maximum. It increases in concentration thereafter in parallel with TMV. The properties of component B are consistent with those of a protein. It has an average mobility of −3.4 × 10 −5 cm. 2/v./sec., is soluble between pH 7.0 and 4.6, and does not appear to have a high molecular weight. 4. 4. Both uninfected and infected leaf tissue contain a heterogeneous electrophoretic component, ` A, which appears to be variable in composition, and has an average mobility of −6.9 × 10 −5 cm. 2/v./sec. These proteins precipitate between pH 5.5 and 4.6 and appear to account for the major part of extractable nonvirus protein. The concentration of group A calculated from the scanning peak areas is variable with time, indicating that the proteins, in contrast with TMV and component B, are readily metabolized. TMV biosynthesis in infected leaf tissue is associated with the development of a transitory excess in the concentration of group A as compared with uninfected controls. 5. 5. Most of the group A proteins are of low molecular weight, but in all samples this group also includes a high-molecular-weight component of S 20 = 19–23. The concentration of the high-molecular-weight component is proportional to that of group A and to that of the total protein content of leaf extracts. 6. 6. Low concentrations of thiouracil block TMV biosynthesis. The TMV formed in partially inhibitory concentrations of thiouracil has identical mobility with TMV formed normally. In 0.0001 M thiouracil, leaf tissue forms 40% of the TMV concurrently present in untreated tissue, but component B does not appear. The excess in group A associated with TMV formation is greater in thiouracil-inhibited tissue than in untreated tissue. In 0.001 M thiouracil, no TMV is formed, and the group A concentration is at a minimum. In higher concentrations of thiouracil the group A concentration in infected tissue approaches that of the uninfected controls. 7. 7. Thiouracil alters the concentration of group A in uninfected tissue. The concentration is at a minimum in about 0.001 M thiouracil, but in higher concentrations of the inhibitor is above that of untreated tissue. It is likely that the inhibitory effect of thiouracil on TMV biosynthesis is associated with its effect on the group A proteins. 8. 8. The effect of thiouracil on group A was confirmed by pH fractionation of tobacco leaf tissue extracts. 9. 9. The possibility that component B represents a TMV precursor, and that some part of the group A complex is involved in TMV biosynthesis, is discussed. The data available at present do not permit final conclusions on these questions.

Beiträge zu Sterilitätsfragen: unter besonderer Berücksichtigung einiger „guter Arten“, wie Secale montanum Gussone und verschiedener Iris.

The behavior of the flavonoid diglycosides, relevant constituents of parsley (Petroselinum crispum) fruit (PFr) and leaf (PLe) samples was characterized upon their enzymatic hydrolyses applying complementary liquid chromatography-ultraviolet (LC-UV) and gas chromatography mass selective (GC–MS) detections. Analyses were performed in quantitative manner, from the same extracts as a function of hydrolysis times. Both in fruit and leaf tissue extracts, in intact and in enzyme hydrolyzed ones, apigenin, chrysoeriol, their glycosides, sugars, sugar alcohols, carboxylic acids and phytosterols, in total 17 constituents were identified and quantified. Based primarily on the selective mass fragmentation properties of the trimethylsilyl (oxime) ether/ester derivatives of constituents, we confirmed several novelties to the field. (i) It was shown for the first time that in parsley tissues different types of glycosidase enzyme are active. In PFr samples, both the stepwise and disaccharide specific endogenous mechanisms were certified, quantifying simultaneously the continuous release of apigenin, chrysoeriol, 2-O-apiosyl-apiose, apiose and glucose. (ii) 2-O-Apiosyl-glucose was demonstrated as disaccharide due to its formation under derivatization conditions from parsley glycosides. (iii) Both in PFr and in PLe samples even the invertase enzyme activity was attainable: sucrose decomposition in both tissues was going on with the same intensity. Three different types of enzymatic glycosidase processes were followed with their specific hydrolysis products by means of HPLC-UV and GC–MS, simultaneously.