Electrophoretic investigation of proteins in different root zones ofvicia faba L.

The differentiation of tissues is closely connected with the proteosynthesis. One can therfore assume that tissues with different types of cell growth (meristematic or elongation growth) and with different degrees of differentiation are different in their protein composition. In order to compare the protein composition of different plant organs, the method of disc electrophoresis on a polyacrylamide gel has been used by some authors. As compared with other methods used up to now, e. g. isolating proteins on DEAE cellulose or in Sephadex, this method does not need so much material and its resolution ability seems to be higher. It is also quicker and enables the study of several samples simultaneously. Its disadvantage is that proteins can be identified mainly by means of Rf and their quantity, measured from the intensity of staining of individual fractions in the gel, which may be misleading due to different sorption capacity of different proteins (Fri?Fri?ová 1967). None the less, it is good for comparison of protein composition of individual parts of the plant body. Different methods have been used to compare protein composition of individual growth zones in roots.Barsky,Ivanov andPushakova (1965) used luminiscence microscopy and found that in maize roots it is not possible to find substantial differences by this method.Morgan andReith (1954) arrived at similar conclusions. On the other hand,Steward et al. (1965) andMorris (1966) found qualitative differences in protein composition of different parts of pea roots using acrylamide electrophoresis. The results of the last named authors show considerable discrepancies in details, due perhaps to a different method of extraction (buffer, pH, purifying method). We have used acrylamide gel electrophoresis for investigating proteins in precisely defined growth zones of theVicia faba root.     

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.     

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.     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

The effect of kinetin on the indoleacetic acid level and indoleacetic acid oxidase activity in roots of young plants

Kinetin treatment increased the extractable IAA content in roots of young plants of Phaseolus vulgaris L., Zea mays L. and Avena sativa L. The highest increase was obtained with roots of beans and the lowest with oat roots. Maize was intermediate between these two species. Kinetin treatment decreased the activity of IAA-oxidase but the correlation between the decrease of the activity of this enzyme and the increase in the level of IAA was not good. The decrease of the oxidase activity was greatest in oat roots where kinetin had very little effect on the IAA level, and was rather small in bean roots, where kinetin treatment significantly increased the IAA level.     

Electrophoretic assay of protein fractions,non-specific esterase fractions and acid phosphatase fractions in extracts from leaves and apple blossoms

Electrophoreograms of protein extracts from leaves of the variety Lord Lambourne, detached at the flowering period toward the end of April, differed quantitatively from electrophoreograms of protein extracts from leaves detached from shoots at the end of May and June. Electrophoreograms of protein extracts of leaves from juvenile seedlings, detached at the end of May and June, did not differ from electrophoreograms of protein extracts of leaves from shoots of the variety Lord Lambourne detached at the same period. Electrophoretic pattern of protein fractions and the fractions of non-specific esterase from leaves differed from the corresponding electrophoretic pattern of flowers quantitatively and in some areas even qualitatively; the pattern of acid phosphatase differed quantitatively. The results obtained are discussed in relation to organogenesis.     

Electrophoretic studies of the relationship of peroxidases,polyphenol oxidase,and indoleacetic acid oxidase to cold tolerance of alfalfa

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to investigate the relationship of soluble proteins and enzyme activity and solubility characteristics to cold tolerance.Evaluations of cold-tolerance levels developed in crown and root samples were compared with results of soluble protein analyses and were in agreement with previously reported observations. Soluble protein content was associated with increases in cold tolerance and related to the environment from which samples were obtained; however, the degree of protein differences within samples of the same cultivar as well as between the two cultivars seemed to be influenced by the type of extractant used.Polyacrylamide disc gel electrophoresis of the extracted soluble proteins was performed on the basis of equal dry weights and equal quantities of protein. Amido black straining of gels indicated mainly quantitative changes and slight qualitative differences in component bands influenced by environment and extractant.Gels assayed for peroxidase, polyphenol oxidase, and indoleacetic acid oxidase enzymes exhibited mainly quantitative differences in constitutive isoenzyme components of both cultivars which were associated with environmental changes. Enzyme activities generally increased in winter, as cold tolerance and soluble protein content increased, and decreased during dehardening. The few qualitative differences in isoenzyme bands that were detected, appeared to be influenced by cultivar, environment, extractant, or substrate specificity differences.Variation in isoenzyme components between cultivars was maximum in summer samples, and minimum in winter samples, suggesting that overall reaction rates or activities of individual isoenzymes, preceding or during hardening, could be a limiting factor in cold-tolerance development.     

Isoenzymes of lactate dehydrogenase in the root growth zones ofVicia faba L.

The present studies of the LDH isoenzymes have been made as a part of our studies on the respiration systems of the roots ofVicia faba. All the root zones had 5 LDH isoenzymes and 2 antibands, but there were differences in their relative quantity. Cathodic isoenzymes (LDH₅, LDH₄) prevailed in the meristematic zone, whereas the anodic ones (LDH₁) prevailed in the others. The different enzyme activity in individual zones is probably connected with the transition of the anaerobic metabolism in the meristem to the aerobic one in the differentiating parts of the root.     

Effect of indoleacetic acid,abscisic acid,root tips and coleoptile tips on growth and curvature of maize roots

The effect of externally applied indoleacetic acid (IAA) and abscisic acid (ABA) on the growth of roots of Zea mays L. was measured. Donor blocks of agar with IAA or ABA were placed laterally on the roots and root curvature was measured. When IAA was applied to vertical roots, a curvature directed toward the donor block was observed. This curvature corresponded to a growth inhibition at the side of the root where the donor was applied. When IAA was applied to horizontal roots from the upper side, normal geotropic downward bending was delayed or totally inhibited. The extent of retardation and the inhibition of curvature were found to depend on the concentration of IAA in the donor block. ABA neither induced curvature in vertical roots nor inhibited geotropic curvature in horizontal roots; thus the growth of roots was not inhibited by ABA. However, when, instead of donor blocks, root tips or coleoptile tips were placed onto vertical roots, a curvature of the roots was observed.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

Studies on induced changes in growth patterns and polarity in roots of Vicia faba L.

Summary Roots of Vicia faba were treated with colchicine (0.025%), or IAA (4.7×10^-6 M), or both, for 3 hours and fixed at various intervals over the following 11 days. The axis of spindle orientation and the distribution of mitotic figures, lateral root primordia and xylem vessel elements was examined in the apical 10 mm of median longitudinal sections of these roots.No effect of IAA was found on the orientation of the spindle. However, evidence was obtained indicating that the systems controlling the polarity of cell division and cell expansion differ in some way.The number of lateral root primordia formed was greater in roots treated with IAA or colchicine than in control roots. These primordia were always initiated adjacent to a xylem vessel. Thus, no primordium was closer to the apex than the most apical xylem vessel, suggesting that an endogenous factor involved in primordia initiation is transported in the xylem. The primordia which develop after colchicine treatment grow out as lateral roots; this is in contrast with those which form after IAA treatment and which do not undergo elongation. These results, which it must be emphasized apply only to the apical 1 cm of treated roots, indicate that lateral root primordia become sensitive to IAA at a certain stage in their development. Exogenous IAA acts as an inhibitor.The new meristem, which forms in the primary root apex after colchicine treatment, contains both diploid and polyploid cells, i.e. it was formed from cells that were unaffected and from cells that were affected by colchicine. Following colchicine treatment the size of the meristem shrinks and this can be prevented by treatment with IAA. This and other evidence presented here, suggests that IAA is a factor involved in the control of the size of the apical meristem in normal roots.     

Effects of cadmium and copper on peroxidase, NADH oxidase and IAA oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots

Twelve-day-old seedlings of pea (Pisum sativum L.) that were treated for 4 days by 20 and 100 micromol/l Cd(NO3)2 or CuSO4 showed a growth reduction in all organs. From root protein extracts, the activities of guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11), coniferyl alcohol peroxidase (CAPX), NADH oxidase, and indole-3-acetic acid (IAA) oxidase were measured in covalently--and ionically--[symbol: see text] bound cell wall, soluble, and microsomal membrane fractions. With the exception of 20 micromol/l Cu, metal treatments enhanced GPX activity in all fractions. Only IAA oxidase activity was metal-elevated in the covalently bound cell wall fraction, while the ionic one showed Cd stimulation for all assayed enzymic activities. These effects were not entirely observed in Cu-treated plants, since APX and IAA oxidase activities were only enhanced in this fraction. However, soluble extract showed stimulation of APX activity, while in the microsomal fraction metal exposure also increased the activities of CAPX and NADH oxidase. Differential responses of root cell fractions to the presence of cadmium and copper ions are discussed in regard to the contribution of their enzymic capacities in antioxidant, lignification, and auxin degradation pathways. Comparisons between metals and dose effects are also underlined.     

Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L

To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-gamma-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis.     

Initial phases of indoleacetic acid induced growth in coleoptile segments of Avena sativa L.

The intial phases of auxin-induced growth in coleoptile segments of Avena sativa L. were investigated using a high resolution growth recording technique, based on an angular position sensing transducer. The first response to the hormone is a slight, transient reduction of the growth rate lasting about 5 min. After this phase growth rate increases to a maximum. The duration of the increase and the maximum clearly depend on the concentration of the hormone. With increasing auxin concentration the duration of the growth rate increase is reduced from about 80 min in 10^-9 M indoleacetic acid (IAA) to about 14 min in 10^-4 M IAA. After the maximum the growth rate declines. Looking at the maximum of the growth rate, we obtained a dose-response curve with a sharp increase between 10^-9 M and 10^-6 M IAA and a slight decline between 10^-6 M and 10^-4 M IAA. This result is confirmed by growth rates measured one and two hours after the application of the hormone.Abbreviations IAA indoleacetic acid     

Auxin, transport and growth in intact roots of Vicia faba

Transport of IAA applied to the intact root of Vicia seedlingswas investigated in relation to root growth. The root was treatedat 3–4, 4–5 or 7–8 mm from the tip with anarrow ring of lanolin paste containing IAA or IAA-2-¹⁴C ingrowth or transport experiments, respectively. The growth processalong the root axis was examined in every 1-mm part from thetip at 30 min, 1 or 4 hr intervals. The elongation zone of thecontrol root was 1–9 mm from the tip. IAA treatment broughtabout no significant change in the growth of the region apicalto the treated site, whereas distinct inhibition of growth occurredin the region basal to the treated site within 1 hr. The transportof radioactivity was observed in both acropetal and basipetaldirections within 1 hr, but the latter predominated for 8 hror more; the nearer to the tip the treatment site, the longerthe predominance lasted. The velocities of acropetal and basipetaltransport were estimated at about 4 and 8 mm/hr, respectively.Autoradiographs of transverse section of roots showed that basipetaltransport occurred mainly through the outer part of the root,whereas acropetal transport occurred mainly through the innerpart, the central cylinder. It may be concluded that the basipetallytransported IAA which passed through the outer part of the rootinhibited the elongation of the intact root. (Received November 25, 1975; )     

Contribution to the estimation of malic dehydrogenase isoenzymes in the root growth zones ofVicia faba L.

Highly active NAD-MDH (E.C.1.1.1.37) and low activity of NADP-MDH (E.C.1.1.1.40) were found inVicia faba roots. The NAD-MDH activity is associated with 6 to 12 isoenzymes. The number of isoenzymes is dependent on the extraction (use of Triton X-100etc.) and detection procudures (presence of KCN, phenazine methosulphate). The meristematic zone does not contain one isoenzyme (X) which is present in the other two zones. The meristematic zone, elongation zone and zone with the beginning differentiation differ in their activity of individual isoenzymes.     

Efficiency of indoleacetic acid,gibberellic acid and ethylene synthesized in vitro by Fusarium culmorum strains with different effects on cereal growth

Three different Fusarium culmorum strains having a pathogenic, a deleterious (deleterious rhizosphere microorganism), or a promoting (plant growth promoting fungus) effect on plant growth were studied for their ability to synthesize in vitro the phytohormones indoleacetic acid (IAA), gibberellic acid (GA), and ethylene. All the phytohormones tested were synthesized in cultures supplemented with wide concentration ranges of glucose and tryptophan or methionine (precursors of phytohormone synthesis). The amounts of these secondary metabolites synthesized by the particular strains were found to be significantly different. The non-pathogenic PGPF strain (DEMFc2) synthesized the highest amounts of IAA and GA, a fact that could be responsible for the growth-promoting properties of this strain. A pathogenic strain synthesized the highest amount of ethylene, which could be responsible for the negative effect of this strain on plant growth. F. culmorum isolates with a high capacity for IAA synthesis also have a high capacity for GA synthesis and irrespective of the growth conditions, a high positive correlation (R > 0.9) between the concentrations of synthesized IAA and GA in F. culmorum cultures was found. It is worth mentioning that the optimal conditions for the growth of F. culmorum isolates and the synthesis of the individual phytohormones differed from one another. The optimal growth conditions were 1.0% of glucose and 9.9 mM of methionine or 6.0 mM of tryptophan. The optimal conditions for ethylene synthesis were 0.5% of glucose and 6.6 mM of methionine, whereas 1.0% of glucose and 9.0 mM of tryptophan were optimal for IAA and GA synthesis.     

Effect of crude seaweed extracts on seed germination, seedling growth and some metabolic processes of Vicia faba L

Crude extracts from three green seaweeds (Cladophora dalmatica, Enteromorpha intestinalis, Ulva lactuca) and the three red algae (Corallina mediterranea, Jania rubens, Pterocladia pinnate) were prepared. Their effects on germination, growth of seedlings, chlorophyll content and other metabolic activities of Vicia faba were investigated. The crude extract of C. dalmatica showed maximal activity, and it increased seed germination, length of main root and shoot systems and the number of lateral roots. All the crude extracts of seaweed increased protein content in both root and shoot systems, total soluble sugars and chlorophyll content in leaves. The cytokinin content of the green algae was higher than that in red algae. Growth of seedlings of V. faba was stimulated but to different degrees.