Excised Root Tips Research Articles

Rapid Respiratory Changes Due to Red Light or Acetylcholine during the Early Events of Phytochrome-mediated Photomorphogenesis

Two millimeter long secondary root tips of etiolated mung bean (Phaseolus aureus) plants were given 4 minute consecutive treatments of darkness, red light, far red light, and acetylcholine during darkness. We studied the effects of these treatments on exogenous (H(+)) changes, ATP utilization, O(2) uptake, P(1) levels, and ATPase activity. Red light and acetylcholine increased the level of P(1), O(2) uptake, and exogenous H(+), but decreased ATP concentrations. Darkness and far red light caused the amount of ATP to increase and decreased the O(2) uptake and P(1) level. O(2) uptake of both excised root tips and isolated mitochondria was promoted by acetylcholine levels of the same order of magnitude that promoted the other photomimetic phenomena. ADP-O ratios indicated that acetylcholine did not cause an appreciable decrease in ATP synthesis. The total ATPase activity remained constant throughout all treatments. Ouabain caused no adhesion to negatively charged glass in the dark, while the inhibitors valinomycin, atractyloside, digitoxin, gramicidin, and oligomycin caused immediate adhesion. All of the inhibitors prevented release from the glass. In red light ouabain increased adhesion, whereas the other inhibitors caused caused immediate and complete adhesion.These data seem to imply that one of the functions of the phytochrome-mediated response to red light in roots, regulated by acetylcholine, is to cause the rapid utilization of ATP pools; far red light appears to inhibit this utilization.

An Evaluation of the Excised Root Tip Method for Determining the Resistance of Pisum sativum to Aphanomyces euteiches1

Abstract Resistance in Pisum sativum to Aphanomyces euteiches was evaluated in the laboratory by the number of oospores formed in excised root tips inoculated with zoospores of the pathogen. Significantly lower mean numbers of oospores formed in root tips of 3 moderately resistant breeding lines than in those of 3 susceptible cultivars. Results were reproduceable if test procedures were adequately controlled. A sample size of 40 root tips gave 95% confidence in detecting a 40 oospore/root tip difference between means of resistant and susceptible genotypes. Lower mean numbers of oospores formed in excised root tips of genotypes which were resistant in greenhouse tests. Inoculum concentrations of 10 to 100 zoospores/plant caused severe disease in susceptible cv. New Era, while greater amounts of inoculum were needed to cause comparable disease in 2 resistant breeding lines.

Effects of Hydroxyurea and Deoxyadenosine on the Synthesis of Deoxycytidine Phosphate and on the Uptake of Nucleosides in Excised Root Tips of Vicia faba

AbstractThe effects of hydroxyurea and deoxyadenosine on the synthesis of deoxycytidine phosphate was studied by measuring the incorporation of [14C]‐cytidine into acid soluble deoxycytidine phosphate in root tips of Vicia faba. Hydroxyurea and deoxyadenosine both markedly depressed the incorporation of [14C]‐cytidine.Deoxyadenosine had the additional effect of inhibiting the uptake of [14C]‐cytidine. Furthermore, millimolar concentrations of deoxyadenosine inhibited the uptake of micromolar concentrations of adenosine, thymidine, and deoxycytidine.The incorporation of [14C]‐cytidine into RNA was only slightly affected by hydroxyurea. Deoxyadenosine inhibited the incorporation into RNA to about the same extent as the uptake of [14C]‐cytidine.It is suggested that hydroxyurea reduced the incorporation of radioactive cytidine into deoxycytidine phosphate mainly by interfering with ribonucleotide reduction. The depression of [14C]‐cytidine incorporation into deoxycytidine phosphate in the presence of deoxyadenosine is believed to be the result of an inhibition of both ribonucleotide reduction and [14C]‐cytidine uptake.

Studies on the Growth of Tobacco Roots : VIII. The effects of medium condition on the nicotine synthesis in root tips

In order to determine the effect on the nicotine synthesis, excised root tips were incubated in the media varying in concentration of nitrogen, sugar and gibberellin. The results obtained were as follows. 1. The contents of total nitrogen and total sugar in the big root tips of several varieties were much higher than those in the fine roots, while comparison of nicotine content and the ratio of nicotine-nitrogen to total-nitrogen between big roots and fine ones showed that the values in the former were lower than in the latter. 2. The amount per milligram of synthesized nicotine in the root tips was highest in the roots grown in rooting media with low nitrogen level and those grown in the elongation media with high leyel of nitrogen. 3. The sugar level in the medium seemed to give more effect than nitrogen level. Considerable amount of nicotine was decomposed in the medium of 0.5 per cent sugar level and optimum concentration of sugar for nicotine synthesis was found to be around 2 per cent. 4. With increasing gibberellin concentration from 0 ppm to 1000 ppm, the activities of nicotine synthesis decreased.

Regulation of Root Growth by Auxin-Ethylene Interaction

A large portion of indoleacetic acid (IAA)-induced inhibition of excised root tips and virtually all such inhibition of intact roots are the result of IAA-dependent ethylene production. Under certain conditions an additional effect of IAA accounts for a small portion of the inhibition of excised root tips. Ethylene production in response to applied IAA is governed by the level of applied auxin found inside the root. Evidence is presented to confirm the participation of ethylene in the geotropic response of roots.

AUXIN AND GROWTH OF EXCISED ROOTS OF Bryophyllum calycinum

Exogenous auxin (alpha-naphthalene acetic acid, indole acetic acid, or 2,4-dichlorophenoxyacetic acid) was essential for the growth of single excised root tips of Bryophyllum calycinum in 50 ml of a mineral salt-sucrose medium supplemented with vitamins. Large inocula with a dry weight of 2.0 mg or more grew with no auxin added to the medium. Evidence for the synthesis of auxin by the excised roots grown from the larger inocula is presented. Leaching of auxin from single root tips cultivated in 15 or 50 ml of basal medium is considered to account for their failure to grow.

Adenosine Triphosphatase Activity in Cell-Wall Preparations and Excised Roots of Barley

Cell-wall preparations from barley roots contain about 20 per cent of the ATP ase activity of the whole homogenate. This activity is maximal near pH 7, acti vated by calcium and magnesium ions and shows further activation when sodium and potassium chlorides are applied at alkaline pH values. High concentrations of sodium chloride and ammonium sulphate are needed to elute the activity from the walls which suggests an ionic binding with the wall fraction. Excised root tips release inorganic phosphate from ATP with no lag phase, and this activity shows a response to variation in substrate and magnesium concentration similar to that of the cell-wall preparations, suggesting a surface location of the enzyme. The properties of this hydrolytic activity are discussed in relation to those described in other plant systems and to animal transport ATP-ases.

Phytochrome-mediated bioelectric potentials in mung bean seedlings.

Excised root tips from dark-grown mung bean seedlings (Phaseolus aureus) that adhere to a negatively charged glass surface when irradiated with red light and release when irradiated by far-red light develop a positive bioelectric potential (about 1.0 millivolt) at the tip in red light and a negative bioelectric potential in far-red light. The sign of the bioelectric potential was repeatedly photo-reversible, and the adhesion and release kinetics were similar to those of the development of the bioelectric potentials. Photoconversion of the phytochrome holochrome perhaps changes permeability characteristics of the cell membrane, resulting in an induced localized electrochemical gradient manifested as a bioelectric potential. This supports the view that phytochrome in situ is membrane bound.

Localization and Kinetic Properties of β-Glycerophosphatase in Barley Roots

The study of /S-glycerophosphatase activity in cell-wall preparations and in excised root tips from barley seedlings supports the view that the former, which constitutes about 20 per cent of the activity of the whole homogenate, represents the fraction located at the surface of the roots in vivo. The activities of the cell wall suspension and intact roots are virtually identical, and further show identical relations to pH, substrate concentration (Km), and competitive inhibition by molybdate and inorganic phosphate (K{). The enzyme must therefore be freely exposed to the external solution without any permeability barrier separating it from either substrate or inhibitors. The absence of any lag phase in the hydrolysis in excised root tips suggests that the surface enzyme may be limited to the outer most layers of the root. The solubilization of some of the activity of the cell-wall preparation by treatment with sodium chloride and ammonium sulphate suggest that surface activity may have been lost from these preparations rather than adsorbed during homogenization and extraction. The Km and pH-activity curve of the supernatant activity remaining after centrifugation of the cell-wall frac tion indicate that only this enzyme and no other detectable glycerophosphatase exists in the roots.

On the Growth Ability of the Excised Seminal Roots of Rice Plant in vitro.

During the course of the previous studies on the nutrient medium for the culture of excised roots of rice, it was repeatedly observed that these excised roots showed a remarkable variation in their growth even with the same nutrient medium. Since this variation was considered as an obstacle for further studies, investigations were carried out in order to clarify this situation. The explants were obtained from the seedlings of rice, variety Noorin No. 29, germinated aseptically under well-controlled as well as semi-controlled temperature. Throughout the studies, the 1cm root tips used to initiate experiments were excised from seminal roots and were cultured with the "improved medium" reported previously. All the cultures were maint-ained at about 27°C in the dark for the periods from half to one month, until the root growth ceased. A series of cultures, from November 1963 to October 1964, were performed using the rice seeds harvested in the Autumn of 1963. The results of these cultures showed a seasonal fluctuation in root growth ; the roots cultured in winter months grew definitely longer than those cultured in spring and summer months. Maximum elongation of roots in each cuture was almost within the range of 13 to 22 cm in winter cultures and 8 to 13 cm in spring and summer cultuers. On the basis of the above data, it was of interest to study the factor (s) involved in this seasonal fluctuation of root growth. In order to elucidate this, the authors carried out the following experiments. In the first experiments on the effect of various controlled temperature, at the time of seed germination, upon the root growth in cultures, it was found that the growth of excised roots was hardly influenced by the germination temperature. In the second experiments on the relation between the length of seminal roots, at the time of excising root tips, and the growth of excised root tips (1cm), it was found that the growing stage of the seminal root at the time of excision affected the growth of excised root tips in culture. At last, it was thought that the age of seeds might affect the growth of seminal root tips in culture. Accordingly, a number of comparisons were made between the growth of excised roots derived from the seeds harvested in the autum of 1963 and those harvested in the autum of 1964. The results of these experiments showed that the growth of excised roots derived from "new" seeds exceeded distinctly that of excised roots derived from "old" seeds. Furthermore, a series of cultures, using the seeds harvested in the autumn of 1964, were performed. In this experiment, root tips were excised from 1.5 cm seminal roots (at the same growing stage) and were cultured as mentioned above. In these cultures, again, the root growth showed a remarkable seasonal fluctuation. This phenomenon might be due to the aging of seeds. From these experiments, it may be concluded that there are at least two factors responsible for the variation in the growth of excised seminal roots; the growing stage of seminal root at the time of excising the inoculum and the age of seeds used.

On the composition of the medium for the culture of excised seminal roots of rice.

The seminal root tips of the rice, var. Norin No. 29 were used as materials. Two root tips were grown in each flask containing 15 ml of liquid medium. Cultures were maintained at 26∼27°C in the dark for the periods from half to one month, until the root growth ceased. The following subjects were studied. First, the effects of yeast extract, peptone, casamino acid (casein acid hydrolysate), tryptophane and indole acetic acid on the growth of excised root tips have been investigated. These substances were added singly or in combination to the basic components of medium, i. e., modified White's inorganic salts (containing 0.4 mg per liter of Fe in the form of ferric citrate) and sucrose or dextrose. Among the substances tested, casamino acid were most stimulating to the growth of root tips. Other substances were without noticeably favorable effects. Basing upon these results, the "original medium" were obtained, which was consisted of modified White's inorganic salts, 4 percent sucrose and 0.2 percent casamino acid. Next, the several factors involved in the preparation of the "original medium" have been examined to study their respective effects on the growth of excised roots. The points which have emerged from these experiments were as follows: i. the optimum PH of the medium is . 4.0, ii. the concentration of Fe may be increased to 4.0 mg per liter, iii. the optimum concentration of sucrose is relatively high, about 6 percent, and iv. the autoclaving of the medium at 1.0 kg per cm2 air pressure for five minutes shows best results. In addition to these, it was found that the vitamins (thiamin, pyridoxine and nicotinic acid each in the concentration of 0.5 mg per liter) accelerated the growth of excised root tips distinctly unless they had been sterile-filtered. By all accounts, "improved medium" were constituted as follows: modified White's inorganic salts (containing 4.0 mg per liter of Fe in the form of ferric citrate), 6 percent sucrose, each 0.5 mg per liter of thiamin, pyridoxine and nicotinic acid and 0.2 percent casamino acid. Superiority of the "improved medium" to the "original medium" for the growth of excised root tips of rice were verified. Nevertheless, incapability of "improved medium" to sustain the unlimited growth of excised rice roots were the problem remaining for future work.

Experimental Control of Dna Synthesizing and Dividing Cells in Excised Root Tips of Pisum

Experimental Control of Dna Synthesizing and Dividing Cells in Excised Root Tips of Pisum

Transport of Amino Acids to the Maize Root

When 5-mm maize root tips were excised and placed in an inorganic salts solution for 6 hours, there was a loss of alcohol-insoluble nitrogen. The levels of threonine, proline, valine, isoleucine, leucine, tyrosine, phenylalanine, and lysine in the alcohol soluble fraction were severely reduced, whereas those of glutamate, aspartate, ornithine, and alanine were scarcely affected. There was a 4-fold increase in the level of gamma-aminobutyrate. Those amino acids whose synthesis appeared to be deficient in excised root tips also showed poor incorporation of acetate carbon. In addition, the results show that asparagine and the amino acids of the neutral and basic fraction were preferentially transported to the root tip region. The results therefore suggest that the synthesis of certain amino acids in the root tip region is restricted, and that this requirement for amino acids in the growing region could regulate the flow of amino acids to the root tip.

THE EFFECT OF ALUMINIUM ON THE UPTAKE AND INCORPORATION OF PHOSPHATE BY EXCISED SAINFOIN ROOTS

SummaryMovement of aluminium into excised root tips of sainfoin follows a normal curve which suggests little or no uptake into non‐free space. Washing in water removes very little aluminium from the roots but washing in dilute buffer solution removes a considerable exchangeable fraction. The pattern of phosphate incorporation in roots pretreated with aluminium is different from that obtained with untreated roots in several respects. Both percentage esterification and the proportions of the esterified fractions differ suggesting both a Donnan free space and a metabolic interference. The small amount of aluminium removed by aqueous washing suggests a minimum of precipitation with aluminium either external to the root, or in the water‐free space of the cortex. The importance of this aluminium‐phosphate effect as a component of aluminium toxicity, its specific sites and rates of reaction, require further experimental assessment.

Ribonucleic acid metabolism during growth of excised root tips from normal and X-irradiated corn seeds

Growth of excised corn root tips was found to involve a degradation and loss of protein and ribonucleic acid. During growth (mostly cell elongation) of the normal excised root tips there is a major redistribution of ribonucleic acid among the subcellular components. The microsomal and soluble ribonucleic acids rapidly decline with a concomitant increase in the nuclear and mitochondrial fractions. There was little redistribution of ribonucleic acid in the root tips from X-irradiated seeds, since the transformation had already occurred as a result of X-irradiation prior to incubation of the root tips. Incorporation of uracil into the subcellular ribonucleic acid fractions by excised root tips revealed that the greatest incorporation was in the nuclei, followed in order by mitochondria, microsomes and the soluble fraction. X-irradiation greatly enhanced the incorporation of uracil into ribonucleic acid. Growth of root tips from normal and X-irradiated seeds increased the ribonuclease activity as much as 2 to 4-fold. Root tips grown from X-irradiated seed had an initial ribonuclease activity 2 to 3-fold greater than normal tissue.

Ribonucleic acid metabolism during growth of excised root tips from normal and X-irradiated corn seeds

Growth of excised corn root tips was found to involve a degradation and loss of protein and ribonucleic acid. During growth (mostly cell elongation) of the normal excised root tips there is a major redistribution of ribonucleic acid among the subcellular components. The microsomal and soluble ribonucleic acids rapidly decline with a concomitant increase in the nuclear and mitochondrial fractions. There was little redistribution of ribonucleic acid in the root tips from X-irradiated seeds, since the transformation had already occurred as a result of X-irradiation prior to incubation of the root tips. Incorporation of uracil into the subcellular ribonucleic acid fractions by excised root tips revealed that the greatest incorporation was in the nuclei, followed in order by mitochondria, microsomes and the soluble fraction. X-irradiation greatly enhanced the incorporation of uracil into ribonucleic acid. Growth of root tips from normal and X-irradiated seeds increased the ribonuclease activity as much as 2 to 4-fold. Root tips grown from X-irradiated seed had an initial ribonuclease activity 2 to 3-fold greater than normal tissue.

Attractiveness of Plant Roots To Pratylenchus Penetrans (Cobb) 1)

Initial migration of P. penetrans from an inoculation point in a thin layer of agar or quartz sand was significantly greater in the direction of host seedlings. Tomato, carrot, and alfalfa seedlings were attractive in agar but only alfalfa seedlings were attractive in sand. Removal of seedling tops decreased attractiveness and areas where plants had been removed were less attractive than seedlings themselves. No attraction was found to excised root tips of mature plants and seedlings were not attractive when placed more than 12.5 mm from the inoculation site.

Mitotic Activity and Respiration in the Excised Pea Root Meristem

SUMMARYExperiments with 2 mm. excised root tips give the following results:1. Experiments in which various carbon sources were tested against the 1% glucose control, showed that fructose is utilized as well as glucose or better, both mitotically and in oxygen uptake. Ribose and DL-glyceraldehyde, on the other hand, did not give as great a response as the glucose control.2. Respiratory inhibitor studies demonstrated that potassium cyanide and 2, 4-dinitro-phenol inhibit both respiration and mitotic activity. Two concentration of potassium cyanide showed a differential effect on respiration but a common effect on mitosis.3. Mitotic inhibitors show a mitotic effect but no effect on respiration. Actidione inhibits the onset of prophase but does not inhibit oxygen uptake. Colchicine causes abnormal mitotic figures in that the spindle is effected. Colchicine does not inhibit onset of prophase or oxygen uptake.4. An experiment using C14-l-glucose and C14-6-glucose showed that there is fluctuation in pathways about six hours after glucose addition. It is speculated that during the first six hours dihydroxyacetone is shunted into glycerol synthesis. In the last six hours the hexose monophosphate shunt seems to be the primary metabolic pathway.5. In conclusion, our data seem to show that the meristem requires a certain level of oxygen uptake before mitosis can take place maximally. In addition, inhibition of mitosis does not effect oxygen uptake.

Studies on 3-Indoleacetic Acid Metabolism. V. Effect of Calcium Ions on 3-indoleacetic Acid Uptake and Metabolism by Pea Roots

It has previously been shown that indoleacetic acid (IAA) applied to pea roots is in part degraded and in part conjugated with aspartic acid to form indoleacetylaspartic acid (IAAspA) (2, 6). While other workers (9) have reported that IAA applied to pea roots is metabolically bound to tissue protein to give a Salkowski reactive derivative, we have found no evidence of such a complex, almost all the Salkowski material in the tissues being accountable for in our studies as IAAspA or IAA itself (3). During the present studies on IAA miietabolism by roots, it was found that whereas tips of intact roots accumulated IAAspA at a rate considerably greater than epicotyl segments, excised root tips almost entirely lost this ability. This decreased accumulation of IAAspA was paralleled by a decreased loss of IAA from solution. The addition of Ca+ + or certain divalent ions in combination with sucrose completely restored the ability of roots to accumulate IAAspA but had no effect on this process in epicotyls.

The Carbohydrate Nutrition of Tomato Roots

The respiratory responses of substrate-depleted excised root tips to a range of sugars, sugar phosphates, and sugar alcohols have been determined by measuring oxygen uptake by the direct method of Warburg. Sucrose, dextrose, and laevulose are the only sugars which promote a high level of oxygen uptake. The effects of azide and DNP on the oxygen uptake promoted by sucrose and by dextrose are described. Mannose is a strong inhibitor of respiration. This inhibition is reversed by the simultaneous addition of those sugars which also reverse the growth inhibition caused by mannose. Mannose inhibits the respiration of sucrose and of glycolytic intermediates. Galactose is slowly respired and does not, even at high concentration, inhibit the respiration of sucrose. The results are discussed in relation to the growth effects of the sugars tested.