Acid phosphatase fractions from various growth zones of broad bean root revealed by disc electrophoresis

Fractions of acid phosphate (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) were studied in extracts of segments from three growth zones of broad bean roots by means of electrophoresis in acrylamide gel. The azocoupling reaction with α-naphtyl phosphate was used for detection. The phosphatase activity was investigated in the range of pH 3·6–7·2. Altogether nine fractions moving towards the anode were revealed. Some fractions differed slightly in their pH optimum. The presence of Mg⁺⁺ in the incubation medium resulted in the activation of two fractions, Mn⁺⁺ showed activation of three fractions and inhibition of the rest of the fractions; the presence of Zn⁺⁺ resulted in a slight inhibition of all fractions. Between electrophoreograms of extracts of segments from the division zone and electrophoreograms of extracts of segments from the enlargement zone and from the maturation zone considerable quantitative differences were found with one fraction; proportions of the other fractions were approximately identical in electrophoreograms of all three growth zones. The response to the presence of Mg⁺⁺, Mn⁺⁺ and Zn⁺⁺ in the incubation medium as well as the pH optima of the individual fractions were identical for all three growth zones.     

Electrophoretic investigation of ribonuclease in roots and leaves ofVicia faba L.

Isozyme patterns and specific activity of ribonuclease (ribonucleate pyridinenucleotido-2′-transferase, E. C. 2.7.7.16) were followed in the extracts of segments from three growth zones of the root and in extracts of young and senescent leaves ofVicia faba L. Electrophoreograms of extracts from all three investigated root zones were identical, in the electrophoreograms of extracts from senescent leaves however one new ribonuclease occurred which could not be detected in the electrophoreograms of extracts from young leaves. Extracts from senescent leaves had higher specific activity of ribonuclease than extracts from young leaves. Extracts from the enlargement zone of the root and those from the maturation zone had a three times higher specific activity of RNase than extracts from the division zone.     

Effect of host-plant manipulation by a gall-inducing insect on abundance of herbivores on chestnut trees

We investigated plant-mediated effects of the stem gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), on other herbivores on the chestnut tree Castanea crenata. In the early season, leaves emerged earlier and in greater numbers on galled shoots than on ungalled shoots. On galled shoots the leaf to shoot biomass ratio was lower and the leaves were physically different. In May and June the concentration of nitrogen in leaves was higher on galled shoots than on ungalled shoots. In July, the water content of leaves was lower on galled shoots. In May and June, the number of aphids, Myzocallis kuricola Matsumura (Homoptera: Aphidoidea), on leaves was higher on galled shoots than on ungalled shoots, but the opposite was true at the end of July. Laboratory experiments showed that aphid fecundity and body weight decrease were higher in May and June when aphids fed on leaves on a galled shoot than when they fed on those on ungalled shoots. In contrast, aphid performance in July was greater on ungalled leaves than on galled leaves. Our findings suggest that gall initiation in a chestnut tree affected aphid performance by affecting host plant quality.     

Contribution to the study of seasonal dynamics of endogenous stimulators and inhibitors in peach trees

For three consecutive years the content of natural stimulators and inhibitors was observed in leaves and shoots of peach trees. Research was directed to the question of development of flower buds. The substances under study were isolated from the acidic fraction of ether extracts by means of paper chromatography and their concentration was determined by biological test. The activities of substances which either stimulate or inhibit the growth of oat coleoptiles, were added up. The curve expressing the content of stimulators in shoots in relation to the fresh weight showed its maximum in the period of full growth of shoots (15 June–15 July according to the fluctuating vegetative conditions) and it showed a decreasing tendency at the end of the season. The decline of the curve showing the content of inhibitors is of similar direction but less steep. The trend of substances under study is the same in leaves as in shoots, but their quantity is lower. Stimulation and inhibition effects in individual sampling intervals were added up to make a common curve which seems to express the combined action of stimulators and inhibitors in the plant, which determines its growth pattern.     

Contribution to the study of seasonal dynamics of endogenous stimulators and inhlbitors in peach trees

For three consecutive years the content of natural stimulators and inhibitors was observed in leaves and shoots of peach trees. Research was directed to the question of development of flower buds. The substances under study were isolated from the acidic fraction of ether extracts by means of paper chromatography and their concentration was determined by biological test. The activities of substances which either stimulate or inhibit the growth of oat coleoptiles, were added up. The curve expressing the content of stimulators in shoots in relation to the fresh weight showed its maximum in the period of full growth of shoots (15 June–15 July according to the fluctuating vegetative conditions) and it showed a decreasing tendency at the end of the season. The decline of the curve showing the content of inhibitors is of similar direction but less steep. The trend of substances under study is the same in leaves as in shoots, but their quantity is lower. Stimulation and inhibition effects in individual sampling intervals were added up to make a common curve which seems to express the combined action of stimulators and inhibitors in the plant, which determines its growth pattern.     

Fractions of non-specific esterase in root tip ofVicia faba L. revealed by disc electrophoresis in acrylamide gel

Fractions of non-specific esterase were studied in homogenates ofVicia faba L. root tips using disc electrophoresis in acrylamide gel. With α-naphthyl acetate, 7 bands were revealed in electrophoreograms, whereas only 5 bands appeared with naphthol AS acetate; the position of bands detected with naphthol AS acetate corresponds to 5 of 7 bands which appear when using α-naphthyl acetate. If incubated with α-naphthyl acetate, 1 band is totally blocked by the inhibitor E 600, whereas the other bands are weakened slightly and—more or less— proportionally, similarly, as if incubated with naphthol AS acetate. No bands appear after detection with α-naphthyl caprylate and with α-naphthyl myristate. Electrophoreograms developed with α-naphthyl propionate differ both from those treated with α-naphthyl acetate and from those treated with α-naphthyl butyrate. Remarkable, but only quantitative differences were revealed when comparing electrophoreograms from division, enlargement and maturation zones, detected with either α-naphthyl acetate, or α-naphthyl propionate. The non-specific esterase fractions revealed by disc electrophoresis are correlated with protein fractions distinguished in the same material by the same technique using xylene brilliant cyanine G staining and with fractions of the same enzyme found in sections of the same object treated histochemically.     

Electrophoretic study on peroxidase,indoleacetic acid oxidase,and o-diphenol oxidase fractions in extracts from different growth zones ofvicia faba L. roots

Using electrophoresis in acrylamide gel, fractions of peroxidase, indoleacetic acid oxidase, and o-diphenol oxidase were investigated in extracts from three growth zones ofVicia faba L. roots. Three peroxidase fractions (zones) moving towards the anode were revealed as well as four peroxidase fractions (zones) migrating towards the cathode. Three peroxidase fractions showed detectable indoleacetic acid oxidase activity. The o-diphenol oxidase activity was revealed in all peroxidase fractions moving towards the anode, in those moving towards the cathode the o-diphenol oxidase activity differred according to the substrate used. One fraction with both peroxidase and o-diphenol oxidase activity occurred only in electrophoreograms of extracts from the maturation zone; in this fraction no indoleacetic acid oxidase activity was demonstrable.     

N-glycans in liver-secreted and immunoglogulin-derived protein fractions

N-glycosylation of proteins provides a rich source of information on liver disease progression because majority of serum glycoproteins, with the exception of immunoglobulins, are secreted by the liver. In this report, we present results of an optimized workflow for MALDI-TOF analysis of permethylated N-glycans detached from serum proteins and separated into liver secreted and immunoglobulin fractions. We have compared relative intensities of N-glycans in 23 healthy controls and 23 cirrhosis patients. We were able to detect 82 N-glycans associated primarily with liver secreted glycoproteins, 54 N-glycans in the protein G bound fraction and 52 N-glycans in the fraction bound to protein A. The N-glycan composition of the fractions differed substantially, independent of liver disease. The relative abundance of approximately 53% N-glycans in all fractions was significantly altered in the cirrhotic liver. The removal of immunoglobulins allowed detection of an increase in a series of high mannose and hybrid N-glycans associated with the liver secreted protein fraction.     

Differential Induction of Endoproteinases during Senescence of Attached and Detached Barley Leaves

Endoproteinase activities and species were compared during dark-induced senescence of attached and detached primary barley leaves by isoelectric focusing and polyacrylamide gel electrophoresis of cell-free extracts. Neither of the two major endoproteinases (EP1 and EP2) changed in amounts during senescence of attached leaves, nor did new endoproteinases appear. In contrast, during senescence of detached leaves, both EP1 and EP2 activities increased and four new species of endoproteinases appeared. Proteolytic activity was evenly distributed throughout attached leaves, but activity in the detached leaf increased sharply from the tip to the base with the four new higher molecular weight species of proteinases present only in the bottom half of the leaf nearest the cut end. Thus a wound response may be superimposed on the processes of senescence in detached leaves. Cycloheximide and kinetin both inhibited the increase of EP1, EP2, and the induction of the four new endoproteinases; chloramphenicol had no effect. Indications are that both the increases in activity and the induction of new species of proteinases were the result of activity of cytoplasmic ribosomes.

Hydrolysis of total protein and ribulose-1,5-bisphosphate carboxylase protein in vivo was somewhat faster in detached than attached leaves. The difference, however, was much less than would be expected from the great increase in proteolytic activity in detached leaves.

     

In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.Abbreviation SDS sodium dodecyl sulfate     

The Changes in Soluble Protein of Excised Barley Leaves during Senescence and Kinetin Treatment

Sterile detached barley leaves were floated on water or kinetin (10 mg/1)and supernatant extracts (30,000 x g for 30 min) were prepared from these leaves over an 8 day incubation period. Changes in selected total enzyme levels and in individual soluble protein components wore compared. Ribonuclease, deoxyribonuclease and peptidase activities rose in senescing leaves, even as total protein levels fell. Kinetin to some extent depressed these activities. Evidence of considerable loss of ribonuclease and to a lesser extent of deoxyribonuclease into the surrounding medium was obtained. Soluble supernatant ant extracts were resolved on DEAE-Sephadex (A-50) columns into about 15 components. While most components were degraded during senescence they did so at different rates. Kinetin lowered the rate of degradation of all components. Since no conclusive evidence of a new protein(s) was obtained in water and kinetin treated leaves, it was considered that any protein changes may have been of a quantitative rather than qualitative nature.     

Comparison of soluble starch synthases and branching enzymes from leaves and kernels of normal andamylose-extender maize

Soluble starch synthases (SS) and branching enzymes (BE) from 20-day-old maize leaves and 22-day-old seeds of normal and amylose-extender (ae) were purified by DEAE-cellulose chromatography. Elution profiles of leaf extracts showed one major SS and two BE fractions from both genotypes. The SS fractions from normal and ae leaf extracts were capable of citrate-stimulated starch synthesis and had different reaction rates with various primers. The two BE fractions from normal leaf extracts differed significantly from each other but not when compared to the same BE from ae. Comparison of BE fractions from ae and normal leaves showed no differences based on chromatographic, kinetic, and immunological properties. Comparison of the leaf enzymes with endosperm enzymes showed major differences. Leaf extracts did not contain SSII or BEIIb observed in endosperm extracts. Developing ae endosperm lacks BEIIb activity and ae is the structural gene for BEIIb. The tissue specific expression of BEIIb in the endosperm provides the basis for explaining the tissue-specific expression of ae. We propose that as BEIIb is expressed in the endosperm, but not leaves, allelic substitution at the ae locus modifies only endosperm starch synthesis.     

Photosynthetic productivity of an immature maize crop: changes in quantum yield of CO2 assimilation, conversion efficiency and thylakoid proteins

Abstract. The effect of growth temperatures on quantum yield (φ) was examined for leaves at different stages of development within the immature canopies of two crops of field grown maize ( Zea mays cv. LG11) sown on 3 May and 20 June 1990. During the period of 23 to 49d after sowing, the crop sown on the 3 May experienced temperatures below 10°C on 19 occasions compared with only two for the crop sown on 20 June. A period of severe chilling at the end of May and the beginning of June was associated with a marked reduction in φ for all leaves in the early-sown crop. This chill-induced depression in φ was greater in recently emerged than more mature leaves in the canopy and was found to be accompanied by modifications in the polypeptide profiles of thylakoids isolated from the leaves. During the chilling period, decreases in some polypeptides, notably in the range of 41–42 and 20kDa apparent molecular size, and increases of polypeptides of c. 15–16kDa were observed compared with leaves developing at warmer temperatures in July. The efficiency of converting intercepted radiation into dry matter (conversion efficiency) was 42% lower in the early- than late-sown crop, but no significant relationship between conversion efficiency and quantum yield was found in either treatment.     

Comparison of growth and gibberellin concentrations in shoots from orchard-grown standard and thermosensitive dwarf apple trees

Apple (Malus domestica Borkh.) trees were propagated by budding from selected fully grown hybrids that ranged in height from 1.5 to 8 m. The growth and development of the selected budded trees after 7 years in the orchard was similar to that of the parent trees. Additional grafting studies showed that the dwarfism was not associated with the roots. Differences in photosynthetic activity and associated processes were not related to the size difference between tissue culture-propagated orchard-grown standard cv. Golden Delicious and dwarf hybrid trees. Applications of GA₃ did not stimulate elongation of shoots of dwarf trees. Shoots of both standard and dwarf trees started to develop in mid-April when they contained nearly the same amounts of GA₁, GA₃ and GA₈, but standard shoots contained higher concentrations of GA₁₉, GA₂₀ and GA₂₉. On 2 June standard shoots were almost three times the length of dwarf shoots, but the number of leaves and area per leaf were nearly the same. The relative amounts of GAs on 12 May and 2 June for both plant types were similar to those on 20 April, except that GA₁₉, GA₂₀, GA₁ and GA₂₉ levels had declined. Gibberellin levels in standard shoots declined further between 2 and 22 June, after which there was no further shoot elongation or production of new leaves. Between 2 June and the end of the growing season, when summer temperatures were high, dwarf shoots continued to elongate slowly and to develop new leaves, which expanded little. During this time, the GA₁₉ content of dwarf shoots nearly doubled, whereas the amounts of GA₂₀, GA₁, GA₂₉ and GA₈ declined. By the end of the season, standard shoots were 40 cm in length with 20 leaves and dwarf shoots were 28 cm in length, but with 36 leaves. High summer temperatures appear to induce loss of GA-responsiveness in orchard-grown dwarf trees and to cause a reduced rate of conversion of GA₁₉ to GA₂₀ in these genotypes.     

Transport of14C-IAA from leaves and shoots to different fruit parts

Transport of¹⁴C-IAA was studied in apple spurs of a 20-year-old McIntosh with one fruit and one shoot. Water solutions of IAA were applied to intact, pricked or scratched leaf blades, to decapitated shoots or to petioles (leaf-blade removed) at the end of June, July and August.¹⁴C-IAA (in an unknown form) was transported from intact leaves and shoots to pedicel, pericarp and seeds. Radioactivity of the pedicels increased every month while that of seeds reached maximum at the end of July and then markedly decreased in August. Total radioactivity of whole fruit doubled, at least, with every month due to enlargement of the pericarp. Pedicels deprived of fruits had their retention prolonged on spurs with leaves or shoots treated with 1% IAA in lanoline. It is assumed that auxin delivered from shoots or still growing leaves at the time of its deficiency in seeds, restrains fruits from premature dropping. At the same time seeds seem to be protected by a regulatory system in pedicel against too massive flow of auxin from outside.     

Physicochemical studies on the proteins of the peanut cotyledon and embryonic axis

Subcellular fractions from the cotyledon obtained by differential and density gradient centrifugation, and extracts of total proteins from both cotyledon and axial tissues were analyzed by diethylaminoethyl cellulose chromatography, zone electrophoresis, ultracentrifugation, immunodiffusion, and immunoelectrophoresis. Fractionation and characterization of proteins in subcellular organelles of the peanut reaffirm that α-arachin is located in the protein bodies of the cells. Results obtained by diethylaminoethyl cellulose chromatography of subcellular fractions suggest that some of the conarachin proteins are cytoplasmic. α₁-Conarachin is cytoplasmic, and α₂-conarachin is particle-bound. α-Arachin and α₂-conarachin predominate in the cotyledon. Quantitative differences for other proteins were also observed. Although qualitative similarities are apparent by immunoelectrophoresis, major differences were observed in the sedimentation patterns, zone electrophoreograms, and in the diethylaminoethyl cellulose chromatograms of total protein extracts from the cotyledon and the axis.     

The appearance of a new molecular species of phosphoenolpyruvate carboxylase (PEPC) and the rapid induction of CAM in Sedum telephium L.

Abstract. When detached leaves of Sedum telephium are incubated in the absence of water, a rapid switch from C₃ photosynthesis to CAM (as indicated by the onset of day-to-night fluctuations in titratable acidity. ΔH⁺) occurs within the first dark period. The C₃-CAM switch in intact plants occurs within 3 5d. Extractable activity of phospho enol pyruvate carboxylase (PEPC) increases five-fold in intact plants during CAM induction; however, during rapid CAM induction in detached leaves, there is only a very small increase in PEPC activity. Fractionation by anion exchange chromatography of crude extracts from leaves of intact plants subjected to water deficit shows that CAM induction is associated with the appearance of a molecular species of PEPC termed PEPC I. PEPC I is barely detectable in well-watered plants which are not performing CAM. The major form in these plants is termed PEPC II. In leaves from intact plants, there is a significant positive correlation between PEPC I activity and ΔH⁺ during a period of increasing water deficit. PEPC I exhibits day to night fluctuations in malate sensitivity, being less sensitive during the dark period. In contrast, PEPC II is more sensitive to inhibition by malate and has no day to night fluctuation in sensitivity. In detached leaves deprived of water, a small increase in PEPC I capacity is detected at the end of the first dark period (20 h after the start of treatment). The results suggest that PEPC I is required for attainment of maximum nocturnal malic acid synthesis. There is a significant correlation between leaf water status (relative water content), ΔH⁺, total PEPC and PEPC I activity suggesting that the internal water status of the plant may be a trigger for CAM induction. Abscisic acid applied to detached leaves does not cause nocturnal acidification.     

The timing of sprays for the protection of terminal buds on apple shoots from powdery mildew

Post-blossom sprays of fungicides, repeated at 10-day intervals until leader (syn. extension) shoots had stopped producing new leaves, provided the best protection of terminal buds against Podosphaera leucotricha on the apple cv. Lane's Prince Albert. Spraying was most effective in early summer, although many of these buds were not invaded until later, when the rate of shoot growth declined; applications from July to September did not compensate for the enhanced infection which followed interruptions of the post-blossom programme between late May and early July. This early period was critical because most leaf infections occurred then, and because this phase of the epidemic on foliage determined the eventual intensity of mildew on terminal leaves, and hence the inoculum available for infecting terminal buds. Also, many lateral shoots ceased growth early and their apices were directly protected by sprays applied in June. Applications after early June were too late to protect newly formed fruit buds on spur branches.     

Comparative electrophoretic investigation of some enzymes in extracts from different growth zones ofVicia faba L. Root

Activities of five enzymes were followed in electrophoreograms of extracts from three growth zones of broad bean root. With ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, E.C. 1.10.3.3) one new fraction was found in the electrophoreograms of extracts from the maturation zone, electrophoreograms of α-glucan phosphorylase (α-l,4-glucan: orthophosphate glucosyltransferase, E.C. 2.4.1.1), amylase (α-l,4-glucan 4-glucanohydrolase, E.C. 3.2.1.1), sucrose glucosyltransferase (disaccharide glucosyltransferase, E.C. 2.4.1.7) and leucine aminopeptidase (L-leucyl-peptide hydrolase, E.C. 3.4.1.1) were identical for all three investigated growth zones. Ascorbate oxidase activity occurred in the same regions of electrophoreograms as peroxidase activity.     

The Use of the Pressure Chamber Technique for Measurement of the Water Potential of Transpiring Plant Organs

The existence of water potential gradients in flowering shoots and leaves of roses (Rosa sp., cv. Baccara) and along flag leaves of wheat (Triticum aestivum L.) were studied by means of the Scholander pressure chamber. In roses grown in greenhouse, the water potential measured in transpiring shoots was higher than in leaves detached from these shoots, whereas the potential differences between leaf and shoot after equilibration in the dark were small or negligible. A progressive decrease in water potential was found upon repeated measurement on the same organ; this decline was steeper in leaves than in shoots. Extrapolating this decline to excision time resulted in water potential values which, in transpiring shoots, were 3 to 5 bars higher than in leaves. Detopping the flower bud did not alter this pattern, indicating that the highest water potential in the shoot was in the stem. In field-grown wheat, the water potential measured in a whole flag leaf was about 6 bars higher than that measured in the apical one-third of the leaf, and this difference disappeared after equilibrating the detached leaf for 1 h in the dark. These potential differences indicate the presence of resistances along the water path in the organ. The results obtained by the pressure chamber represent the highest water potential in the organ, rather than the average water potential.