Pea Internodes Research Articles

Changes in the Pattern of Enzyme Development in Gibberellin-treated Pea Internodes

A study was made on the effect of gibberellic acid on amylase, cellulase, β-fructofuranosidase, pectinesterase, and starch phosphorylase activities in elongating dwarf-pea internodes. Hormonal stimulation of amylase and β-fructofuranosidase activities correlated closely with internode growth, the activity of starch phosphorylase less so, and gibberellic acid had no immediate effect on cellulase and pectinesterase activities. Injection of glucose (or glucose derivatives) into pea internodes mimicked the effect of gibberellic acid on fresh- and dry-weight accumulation, cell elongation, cell division, and cell-wall synthesis. It is proposed that the over-all effect of gibberellic acid on enzyme development is to provide more substrate (particularly glucose) for general cell metabolism and wall synthesis within elongating internodes.

Blockage by Gibberellic Acid of Phytochrome Effects on Growth, Auxin Responses, and Flavonoid Synthesis in Etiolated Pea Internodes

Red light inhibits the growth of etiolated pea internodes, causes a shift toward higher indoleacetic acid (IAA) concentrations in the IAA dose-response curve of excised sections, and promotes the synthesis in intact internodes of kaempferol-3-triglucoside. Gibberellic acid (GA(3)) prevents all 3 effects, the first effect substantially and the last 2 completely. This suggests GA(3) blockage of an early or basic event initiated by the active form of phytochrome. The red light-induced shift in the IAA dose-response curve of excised sections is consistent with a light-induced increase in the activity of an IAA destruction system, since the magnitude of the red light inhibition varied with IAA concentration. The red light and GA(3) effects on growth and on flavonoid synthesis are consistent with the view that phytochrome may control growth by regulating the synthesis of phenolic compounds which act as cofactors in an IAA-oxidase system. GA(3) reversal of the red light-induced shift in the IAA dose-response curve involves both growth promotion and inhibition by GA(3) at different IAA concentrations and this, together with the GA(3) reversal of light-induced flavonoid synthesis, supports the suggested regulatory role of phenolic compounds in growth.

Relations between DNA, RNA and Protein Synthesis, and the Cellular Basis of the Growth Response in Gibberellic acid-Treated Pea Internodes

Relations between DNA, RNA and Protein Synthesis, and the Cellular Basis of the Growth Response in Gibberellic acid-Treated Pea Internodes

Different Species, Sucrose, and Light in Plant Section Elongation Tests

Sections from both dark- and light-grown seedlings of 11 species were used to test responses to IAA (indoleacetic acid), sucrose, and an inhibitor prepared from cabbage seedlings. Variability among species was great; however, results indicate that many species, light-grown as well as dark-grown, could prove useful in bioassays and probably should be investigated. Although elongation of segments from high-intensity-light-grown cabbage and cucumber hypocotyls and oat coleoptiles had essentially stopped by the time of cutting, their growth and response to IAA as sections were considerable. Neither oat coleoptile nor pea internode sections can be considered representative because of differences in responses to sucrose, of dark-grown sections to light, and to an inhibitor prepared from cabbage. Sucrose generally did not stimulate and even inhibited response of most hypocotyls to IAA. Sucrose was absorbed by sections, increasing final dry weight while not affecting elongation. Sucrose reduced the rate of respiratory decay in cabbage and sunflower, but IAA did not affect respiration. Changes in length and fresh weight of cucumber hypocotyl sections were comparable.

DIFFERENT SPECIES, SUCROSE, AND LIGHT IN PLANT SECTION ELONGATION TESTS

Sections from both dark‐ and light‐grown seedlings of 11 species were used to test responses to IAA (indoleacetic acid), sucrose, and an inhibitor prepared from cabbage seedlings. Variability among species was great; however, results indicate that many species, light‐grown as well as dark‐grown, could prove useful in bioassays and probably should be investigated. Although elongation of segments from high‐intensity‐light‐grown cabbage and cucumber hypocotyls and oat coleoptiles had essentially stopped by the time of cutting, their growth and response to IAA as sections were considerable. Neither oat coleoptile nor pea internode sections can be considered representative because of differences in responses to sucrose, of dark‐grown sections to light, and to an inhibitor prepared from cabbage. Sucrose generally did not stimulate and even inhibited response of most hypocotyls to IAA. Sucrose was absorbed by sections, increasing final dry weight while not affecting elongation. Sucrose reduced the rate of respiratory decay in cabbage and sunflower, but IAA did not affect respiration. Changes in length and fresh weight of cucumber hypocotyl sections were comparable.

Influence of gibberellic acid on nucleic acid synthesis in dwarf pea internodes

Influence of gibberellic acid on nucleic acid synthesis in dwarf pea internodes

Inhibition of RNA synthesis and auxin-induced cell wall extensibility and growth by Actinomycin D.

A linear stress strain analyzer was used to determine the effects of inhibitors of RNA and protein synthesis on auxin-induced increases in cell wall extensibility. With etiolated soybean hypocotyl, maize mesocotyl and Avena coleoptile sections and light-grown pea internode sections, inhibition of RNA synthesis resulted in inhibition of auxin-induced extensibility changes and cell expansion. The results with both actinomycin D and cycloheximide support an earlier conclusion that unstable cell constituents, presumably enzymes, are essential for cell wall loosening induced by auxin as well as for cell elongation.

Growth, Turgor, Water Potential, and Young's Modulus in Pea Internodes

AbstractThe relations between longitudinal growth, Young's modulus, turgor, water potential, and tissue tensions have been studied on growing internodes of etiolated pea seedlings in an attempt to apply some physical concepts to the growth of a well‐known plant material. The modulus has been determined by the resonance frequency method and expressed as Etissue It increases nearly proportional to the turgor pressure and is at water saturation more than 50 times higher than at plasmolysis. Etissue is higher in the epidermis than in the ground parenchyma. Indoleacetic acid causes a decrease in Etissue Other properties have been studied on intact and split segments of internodes in solutions of graded mannitol additions. — The following tentative picture of the normal course of the growth has been obtained. Auxin induces growth both in the periphery (epidermis) and in the central core (parenchyma) under a decrease in Etissue This is followed by an increase of Etissue which is independent of auxin but depending upon the turgor pressure. It is assumed to involve internal structural changes of the cell walls of the type of creep. The rapid growth takes place in a dynamic system with a low water potential despite favourable water conditions. Epidermis and parenchyma grow equally rapid without tissue tensions. — Such can be produced artificially by splitting of segments and water uptake. The parenchyma thereby loses its sensitivity to auxin. This is the background of the split stem test for auxin. — Etissue increases when growth is slowing down, probably owing to both synthesis of wall substance and structural changes within the wall. The cells attain a more static condition with Etissue higher in epidermis than in parenchyma. This leads to the normal tissue tensions. — The result agrees with growth according to the multi‐net‐principle. The cause of the low water potential and low turgor is discussed with reference to the dynamic nature of both growth and water transport and a probably low matric potential of the streaming water. The decrease in Etissue following auxin addition is small but is the net difference between an auxin‐induced decrease and an increase through the assumed creep.

THE BASIC ACTION OF GIBBERELLIC ACID IN ELONGATION OF ‘METEOR’ PEA STEMS

SummaryThe published evidence on the histological action of gibberellins does not favour the widespread belief that the primary action of gibberellic acid (GA) in stem growth is on cell elongation. Histological evidence from Meteor pea internodes shows that the main effect of GA is to stimulate cell division; and even when applied to internodes which have passed into the vacuolation phase of growth GA often re‐activates cell division. Mitotic activity in the apical meristem of Meteor pea stems is also stimulated by GA. The experimental results suggest that the primary effect of GA on caulescent plants, as on rosette plants, is to increase mitotic activity in apical and sub‐apical meristems; the cell elongation effect of GA may well be a derivative of the increased mitotic activity, possibly through an increased production of auxin in the meristematic region.

Ricerche Sul Rigonfiamento di Piccola Ampiezza in Mitocondri Isolati di Pisello

Abstract Low amplitude swelling in isolated pea mitochondria. — The authors have studied the volume changes of isolated pea internode mitochondria occurring in a medium which allows the oxidative activity to proceed. With succinate as substrate respiratory control ratios as high as 9 have been obtained. They can be taken as an index of a tight coupling of mitochondria. The adding of succinate induces on the other hand a slight but continuous swelling which is strongly enhanced by ADP or ATP. Inhibitors of the respiratory chain like antimycin A and of the phosphorylation reactions like atractylate or oligomycin block completely the ADP or ATP-induced swelling. 2,4-dinitrophenol at 5×10−6 M concentration exerts a strong inhibitory action on succinate oxidation and on swelling. Both actions of 2,4-DNP can be reversed by ATP. It can be concluded from these findings that the substances which slow down or abolish the oxidative and phosphorylative reactions inhibit also mitochondrial swelling. This type of volum...

The mechanism of action of plant growth substances: growth substance stimulation of amino acid incorporation into nuclear protein.

Aluxin-induced stimulation of amino acid incorporation into protein of pea internodes is localised largely in the nucleus soon after auxin application. Low concentrations of auxin, kinetin and gibberellic acid also promote the incorporation of amino acids into the protein of free nuclei of coconut milk. The auxin-induced stimulation is abolished by actinomycin D but can be restored considerably by higher concentrations of the growth substances. Puromycin also retards the auxin-induced stimulation of amino acid incorporation into nuclear protein but the inhibition cannot be reversed by higher concentrations of auxin.

Ricerche Sui Fenomeni di Contrazione di Mitocondri Isolati di Internodio di Pisello

Abstract Study on the contraction phenomena of isolated mitochondria of pea internodes. — The action of the phosphorylative inhibitors atractylate and oligomycin on ATP-induced contraction of isolated pea mitochondria has been studied. Oligomycin has proved to be an efficient inhibitor of mitochondrial contraction; no effect is seen on the contrary upon addition of atractylate. Both atractylate and oligomycin inhibit completely oxygen consumption of mitochondria with succinate as substrate. On the base of these results it is postulated that the two inhibitors act upon different sites of the phosphorylative reaction sequence.

Studies on the Mechanism of Auxin Action: Auxin Regulation of Nucleic Acid Metabolism in Pea Internodes and Coconut Milk Nuclei

Studies on the Mechanism of Auxin Action: Auxin Regulation of Nucleic Acid Metabolism in Pea Internodes and Coconut Milk Nuclei

Osservazioni Sull'effetto Inibente di Elevate Pressioni Osmotiche su Diversi Tipi di Crescita

Abstract On the inhibition of seeds germination and of growth by cell enlargement by the osmotic pressure of the medium. — The mechanism of inhibition by osmotic pressure (O.P.) of the medium on growth and respiration of germinating wheat, castor bean and lettuce sèeds and of etiolated pea internode segments was investigated. The following results were obtained: 1 - External osmotic pressure (up to 0.3 M) of various substances such as mannitol, urea, glucose, NaCl, was shown to inhibit the germination and growth of lettuce, wheat and castor bean seeds. 2 - a) A remarkable decrease of the development of respiration during the first 48 h of germination was demonstrated in embryos of wheat seeds germinated and maintained in mannitol solutions at concentration from 0,2 to 0,3 M. b) A slight but reproducible inhibition of óxygen uptake by O.P. was also observed in embryos isolated from wheat seeds germinated in water for 24 and 34 h and transported respectively in water or into 0,2 M mannitol solutions. This is interpreted as indicating that high external O.P. inhibits both the respiratory metabolism and the development with time of enzyme systems supporting respiration. 3 - Mannitol solutions (0,2–0,3 M) inhibited completely growth by cell enlargement in pea internode sections, while they did not at all affect oxygen uptake and protein synthesis ( 14 C - leucine incorporation). The stimulatory effect of auxin on pea elongation was almost completely suppressed by mannitol, whereas the hormone stimulation of respiration remained unchanged. These data are interpreted as indicating that in tissues, presenting an advanced differentiation, high external O.P. inhibits growth by a direct physico-chemical mechanism; while the inhibitory effect in embrional tissues seems to comprehend, besides this direct effect, a complicated metabolic component, apparently influencing protein synthesis.

Effetto Del Dinitrofenolo Sulla Conversione Del Glicerolo A Glucidi in Fettine Di Carote E in Internodi Di Pisello

Abstract On the effect of dinitrophenol on carbohydrate activation in higher plant tissues. — Previous investigations on the effects of 2,4 dinitrophenol (DNP) on carbohydrate metabolism in isolated pea internodes and in yeast showed that the increased rate of glycolysis induced by the uncoupler corresponds to an increased rate of the conversion of free hexoses and polysaccarides to hexose phosphates. In yeast about 30% of the radioactivity supplied and taken up as 14C labelled glucose, and 20% of that supplied and taken up as glycerol is recovered as soluble sugar and glycogen; this phenomenon is almost completely suppressed by 10-4M DNP. This suggested that a mechanism involving kinase enzymes, on one hand, and phosphatases, on the other, is mediating the interconversion of phosphorylathed and free sugars, and that the apparent increase of hexose phosphorylation observed in the presence of DNP might depend on a decreased rate of phosphatase mediate reactions, consequent to the decrease of phosphorylated sugars level in the cell. The experiments here reported were planned to test the validity of this hypothesis in the case of higher plant tissues. Material used in these experiments were segments from the growing part of the third internode isolated from 7 day old, etiolated pea seedlings, and carrot root diks (0,7 mm thick, 7 mm diameter) preincubated for 24 hours in aerated distilled water. Both of these materials show an active, steady respiration and some growth activity, so that they may be taken as representing a condition close enough to that of the generally physiologically active higher plant tissues. The reversibility of the hexose phosphate-free sugar interconversion process was tested by feeding 10-3M 1-C14 labeled glycerol, and measuring after 150 minutes the amount of radioactivity incorporated into CO2, soluble sugars, organic acids and proteins. The results of these experiments are summarized in table I and II. Glycerol metabolism as well as its response to DNP appears very similar in the two material used. In both cases, glycerol uptake and incorporation into organic acids and amino acids is almost insensitive to DNP. In contrast large differences are observed for the free sugar fraction. In the absence of the uncoupler, a consistent amount of the radioactivity fed as glycerol is found in this fraction. It appears reasonable to assume that the glycerol-sugar interconversion comprehends, as intermediate steps, glycerol-P, fructose di-P (or sedoeptulose di-P) and hexose-6-P. If this is true, the observed data implicate that a continuous interconversion occurs, in the cell, between sugar phosphates and free sugars and vice-versa, one reaction direction involving the activity of phosphatases, and the other one that of kinases. The true rate of this interconversion process is probably much larger than indicated by the radioactivity found in free sugars: as a considerable part of the triose-P transormed into sugars must immediately re-enter the descending flux of glycolysis. This view finds some support in the fact that DNP almost completely inhibits the incorporation of radioactivity in the free sugar fraction. It has been previously observed that DNP very markedly decreases the level of hexose mono- and di-phosphates and of triose-phosphates in the pea stem tissues. If phosphatases acting on fructose di-phosphate and on hexose-6-P are not saturated by their substrates, a decrease of the rate of free hexose synthesis from sugar phosphates should be expected. The present results are thus consistent with the hypothesis that hexose phosphates and free sugars in the cell are continuously interconverted by the simultaneous action of phosphatases and kinases; and that the effect of DNP, and thus of any physiological conditions decreasing the ATP/ADP ratio in accelerating free hexose utilizations is at least in part due to a decreased rate of the reactions catalized by fructose diphosphate and hexose-6-P phosphatases. The reversibility of the kinase-phosphatase system would thus represent a crucial link in the mechanism by which the rate of carbohydrate activation and breackdown is controlled by the rate of utilization of high-energy phosphate bonds.

Sul Metabolismo Dell'Alcool Etilico in Tessuti Di Fusto Di « Pisum »

Abstract On the metabolism of ethanol in the Pea stem tissues. — The average concentration of ethanol in the growing part of the etiolated pea internodes is of the order of 10-3M. Previous work showed that auxin at growth promoting concentration markedly lowers this level in the excised internodes. This finding prompted a series of investigations on C14 labeled ethanol utilization in this material. The capacity of the segments to metabolize ethanol is remarkable: with an external ethanol concentration 5X10-3M the C14 labeled CO2 originated from 1-C14 ethanol accounted for about 10% of total CO2 produced during the first hour of treatment. Moreover, an amount of ethanol about 10 fold higher that that dissimilated to CO2 was metabolized to various yet unidentified compounds. The ratio between the contribution of ethanol to CO2 and that to other metabolites appeared maximal in the first period after feeding the labeled compound. This ratio was significantly higher then that found for 6-C14 glucose. These pre...

Azione Dell'auxina Sull'utilizzazione di Glicerolo-1-14C in Segmenti di Internodi di Pisello

Abstract The effect of auxin on the metabolism of 14C labelled glycerol in isolated pea internode segments. — The effect of auxin on the respiratory metabolism of isolated pea internode segments was investigated by measuring the auxin-induced modifications of glycerol-1-14C incorporation in some metabolites. The experiments showed that auxin induces: an increase of the rate of metabolized glycerol, that could not be interpreted as a consequence of an increased uptake. a strong increase of glycerol incorporation into monosaccharides. a significant fall of the radioactivity incorporated into fats. These result are discussed in relation with previous data on the auxin effects on oxidative metabolism.

Effect of indole-3-acetic acid (IAA) on phosphorus metabolism in the Avena coleptile.

IT is generally believed that energy-rich phosphate compounds act as a store-house of energy for different reactions during growth. The auxin-induced stimulation of respiration is presumed to be due to an increased availability of phosphate acceptors due to the rapid utilization of nucleotides during the growth process1–3. Rhodes and Ashworth4 have speculated on the mechanism by which growth substances may be metabolized for the generation of energy-rich phosphate bonds, but an unequivocal demonstration of the stimulation of nucleotide synthesis is not available. Ormrod and Williams5 and Key et al.6 have recently observed increased phosphorylating activities in plants sprayed with 2,4-dichlorophenoxyacetic acid. Marre and Forti7 had noted a rapid increase in high-energy phosphate level in auxin-treated pea internode segments during the first 30 min., followed by a decline. The method of estimation of adenosine triphosphate (ATP) adopted by them, which involves the adsorption of adenine nucleotides on charcoal followed by elution with hydrochloric acid, has several limitations when applied to tissue sections. Since numerous metabolic reactions go on in the cell at the same time, and a large number of them concern the participation of nucleotides, particularly reactions mediated by the kinase type of enzyme, and the phosphate residues are transferred to a variety of substrates, estimation of the level of adenine nucleotides at any given time is not the true measure of the total ATP formed during the experimental period. A more correct approach is to estimate the phosphate esterified to organic compounds including the nucleotides. Phosphate labelled with phosphorus-32 may be conveniently used for this purpose and the present communication summarizes the findings of an investigation directed towards this objective.

Vie di ossidazione della frazione extramitocondriale dei coenzimi piridinici: I. - Sull'EfficienzaIn Vivodel sistema ascorbato/deidroascorbato

Abstract OXIDATION PATHWAYS OF EXTRAMITOCHONDRIAL PIRIDINE COENZYMES. I. - ON THE « IN VIVO » EFFICIENCY OF THE ASCORBATE-DEYHDROASCORBATE SYSTEM. — An evaluation of the efficiency in vivo of the AA-DHA couple as an electron carrier system has been attempted, by measuring after short time of anaerobiosis the rate of the increase of AA and of the dicrease of DHA in etiolated pea internode segments and in potato tuber disks. The changes of reduced glutathione (GSH) contents as induced by anaerobiosis or by the addition of DHA to the incubation medium were also followed. In the pea segments anaerobiosis induced a significant increase of AA and a corresponding decrease of DHA. These changes were almost completed after 10 minutes from starting anaerobiosis. The value (extrapolated to 0 time) of the initial rate of DHA desappearance under anaerobiosis was taken as representing the rate of DHA reduction to AA « in vivo », under aerobic conditions. As this rate — in a steady state situation — corresponds to that of the inverse process of oxidation of AA to DHA, this value should give and indication on the « in vivo » efficiency of the AA-DHA system as an electron carrier in respiration. As some AA was probably reoxidized to DHA in the very short period required to kill the tissue, the value of the AA DHA turnover thus calculated is probably somewhat lower than the real one. According to the present work, the oxidative turnover of the AA-DHA system would results of 0,7 micromoles/g. fr. weght/h. for the pea internode tissues and of 0,9 micromoles/g. fr. weght−h for the potato tuber (aged disks). These values would account for 5% of total oxygen uptake, in the former, and for 3% in the latter material. The very high AA/DHA ratio usually prevailing in living cells suggests that the contents in DHA (and thus the activity of the AA oxidizing systems) is a limiting factor for the efficiency of the AA-DHA system as an electron carrier. This view is supported also by experiments in which DHA (at pH 5) was fed to pea internode segments and to potato tuber disks : as the presence of DHA into the medium induced — under anaerobiotic conditions — a rapid increase of the level of AA in both types of materials. In aerobiosis uptake and reduction of DHA to AA was evident in the potato tuber tissue, while it appeared very scarce in the pea internodes. As an interpretation of this behaviour it is suggested that, in aerobiosis, the very active and probably surface localized ascorbic acid oxidase of the pea tissue re-oxidises the AA formed from reduction of the DHA fed; an accumulation of DHA into the cells would follow, and this excess of DHA would inhibit the enzyme GSH-DHA reductase. This enzyme, in fact, appears, from « in vitro » experiments, to be strongly inhibited by DHA when the DHA/GSH ratio becomes higher than 1. On the other hand, the same hypothesis is also supported by the finding that the addition of DHA to the medium induces a significant drop in the GSH level (probably due to its oxidation to GSSG) only under those conditions in which DHA is absorbed and reduced to AA; that is, in the pea internodes, under anaerobiosis, and in the potato disks, under both anaerobiosis and aerobiosis. These results are also taken as confirming the indication from the enzymatic data that GSH is acting, in vivo as a reducing agent for DHA. The results of this investigation are thus interpred as showing that a comparatively small, but by no means negligeable fraction of respiration is mediated, in higher plant tissues such as those of the pea stem and the potato tuber, by and electron transfer system including glutathione and the ascorbate-dehydroascorbate couple. The efficiency of this system in the materials investigated appears to account for 3–5% of the total 02 uptake (minimum value). As enzyme systems transferring electrons from TPNH to ox. glutathione are widely distributed and generally very active in higher plant tissues, it is suggested that the sequence TPNH-GSH-AA/DHA - O2 is probably of considerable importance in mediating the reoxidation of extramitochondrial trophosphoridine nucleotide and thus in permitting the operation of the TPN requiring pentose phosphate pathway of respiration.

Effetti del trattamentoIn Vivocon auxlna di internodi di pisello sulla fosforilazione ossidativa dei mitocondriIn Vitro

Abstract EFFECT OF TREATMENT WITH IAA OF PEA INTERNODE SECTIONS ON THE OXIDATIVE PHOSPHORYLATION OF THEIR MITOCHONDRIA. — It was previously shown that auxin treatment raises the level of ATP in pea stem sections, under the conditions where the hormone stimulates growth (MARRe AND FORTI). It is also known that under the same conditions auxin stimulates oxygen uptake (MARRe, FORTI e ARRIGONI; MARRE and FORTI), and that the auxin induced respiration is most probably mediated by cytochrome oxidase (MARRE, FORTI and GAUR). However, auxin has no effect when added to isolated mitochondria. In this paper, the effect of auxin treatment of the tissue on the activity of mitochondria isolated after the hormone treatment has been studied. It has been found that oxidative phosphorylation of mitochondria from the auxin treated pea stem sections is 13% higher than that of controi sections. The auxin effect is significant at 96% probabilities. There is no effect of the hormone on the P/O ratio.