Excised Pea Roots Research Articles

Some effects of analogues of uracil on cell elongation and wall metabolism in excised pea root segments

Excised root segments of Pisum sativum (L.) cut from the region 2-4 mm behind the root tip were cultured in a 2% sucrose medium containing analogues of uracil and proline. Of several uracil analogues tested only those containing a thiol group (2-thiouracil and 2-thio-6-azauracil) markedly stimulated the growth rate and prolonged the duration of growth of the segments, whereas other uracil analogues which enhanced growth affected only its duration. Uracil had no effect on cell elongation and did not completely prevent the effects of 2-thiouracil and 2-thio-6-azauracil; it did, however, prevent the stimulation by those analogues without a thiol group. Two analogues of proline, thioproline and hydroxyproline, also enhanced cell elongation but whereas the effect of hydroxyproline was completely prevented by proline, the stimulation produced by thioproline was not.During cell elongation, [(14)C]thiouracil was incorporated into RNA, where it replaced uracil, and into a non-nucleotide fraction of the cell wall from which it could not be removed with perchloric acid, sodium hydroxide, ribonuclease or pronase. Experiments using labelled thiouracil, orotic acid, leucine, proline and hydroxyproline strongly suggest that 2-thiouracil stimulates the growth rate of segments by becoming attached to cysteine in cell-wall proteins and delaying an increase in wall rigidity caused by the formation of disulphide bridges between proteins.

Effects of some chelating and phenolic substances on the growth of excised pea root segments

Several chelating substances, such as 2,2'-dipyridyl, 8-hydroxyquinoline and desferal enhanced the growth of elongating segments of excised pea root tips when cultured in the presence of 2% sucrose, but their non-chelating analogues such as 4,4'-dipyridyl and 2-hydroxyquinoline were without effect. Some phenolic substances, such as cinnamic, ferulic, chlorogenic and caffeic acids, also enhanced the growth of excised segments. In general the substances which enhanced cell elongation also enhanced the development of invertase, but generally they did not enhance the increase in peroxidase activity or have any effect on the decrease in RNA content.[(14)C]cinnamic acid was continuously incorporated into the cell walls of elongating segments from which it could only be partially removed with NaOH, pronase, or HClO4. There was some evidence that the radioactivity was transferred from the cytoplasm and not incorporated directly into the cell walls. The implications of these results are discussed.

A possible mechanism for humic acid action on cell elongation in root segments of Pisum sativum under aseptic conditions

At low concentrations (up to 50 mg/l) humic acid enhances cell elongation in excised pea root segments, but at higher concentrations it is inhibitory. At concentrations stimulating cell elongation humic acid has no effect on protein metabolism but does inhibit the formation of cell-wall bound hydroxyproline from proline. Humic acid does not stimulate cell elongation or inhibit the formation of cell-wall bound hydroxyproline in the presence of ferrous iron. Results similar to those of humic acid were obtained using the iron chelator αα'-dipyridyl, but γγ'-dipyridyl. which does not chelate iron. was without effect on cell elongation. The cessation of cell elongation corresponds to the time at which there is a substantial increase in cellwall bound hydroxyproline, this latter process requiring ferrous iron. Thus humic acid may enhance growth by virtue of its ability to complex ferrous iron within the plant tissues.

Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips

Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 mum and 10 mum). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 mum) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 mum) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production.

The regulation of glutamate dehydrogenase, nitrite reductase, and nitrate reductase in excised pea roots by nitrite

Both nitrite reductase and nitrate reductase were induced by nitrite, but there were differences in the time course of induction and in the response to different NO2 - concentrations between these enzymes. NH4 + depressed the induction of nitrite reductase. NADH2 dependent glutamate dehydrogenase activity was enhanced by those NO2-concentrations in the medium at which unmetabolized NO2 - occurred in the roots. NADPH2 and NAD+ dependent GDh activities were not affected. In vivo modification and (or) in vivo activation were probably responsible for the increase in NADH2 dependent GDH activity.

EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE RADIATIONS ON CELL CYCLE KINETICS IN EXCISED ROOT MERISTEMS OF PISUM SATIVUM

Near‐ultraviolet and visible radiations increased the duration of the mitotic cycle in excised pea root meristems primarily by lengthening the duration of the pre‐DNA synthetic period (G1). All radiations tested shortened the duration of the post‐DNA synthetic period (G2). The most pronounced effects were exhibited by green radiation, which lengthened the duration of the cell cycle, G1, DNA synthesis (S), and mitosis (M), and shortened the duration of G2. Progression of cells arrested by starvation in G1 and G2 into DNA synthesis and mitosis was also affected by light treatments. Green radiation appeared to arrest a group of cells in DNA synthesis as well as in G1 and G2. Meristems receiving green and near‐ultraviolet radiations exhibited the most rapid progression of G1 cells through S and G2.

The Regulation of the Synthesis of Glutamate Dehydrogenase in Excised Pea Roots by ExogenousL- aspartic andL- glutamic Acids

Exogenous L-aspartic and L-glutamic acids enhance glutamate dehydrogenase activity in isolated pea roots. The results obtained indicate that both ammo acids induce increased GDH synthesis.

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase, Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO3R) activity, nitrite reductase (NO2R) activity and NADH2 dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10−5; 3 × 10−5; 5 × 10−5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1−1, that is 4.65 × 10−7;4.65 × 10−6 and 2.3 × 10−5 M). NO3R activity was not influenced by IAA, NO2R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO3R activity significantly both after 9 h and 24 h incubation, slightly increased NO2R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO2R activity. Nevertheless the depressing effect of kinetin on NO3R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.

Soil physical conditions affecting seedling root growth

The lengths, fresh weights, and dry weights of pea seedling roots grown for 48 hours in loose-soil columns were progressively less as oxygen partial pressures of the soil atmosphere varied from 0.16 to 0 atm. The roots were thicker at intermediate oxygen levels than in either 0 or 0.21 atm oxygen. The number of vacuolated cells per root were the same in 0.21 and 0.08 atm oxygen indicating that rate of cell division was the same. Therefore over this range decrease in cell elongation and increased cell breadth were responsible for the shorter, thicker roots. Rate of cell division was reduced at 0.03 atm oxygen. The utilisation of the seedling metabolite decreased from 0.10 to 0.03 atm oxygen and the hyperbolic relationship for oxygen uptake by excised pea roots suggests a causal relationship between growth, cell development, and oxygen availability. On this assumption pea root elongation was calibrated at different mechanical impedance and oxygen levels in order to predict the oxygen concentration at the surface of pea roots growing in poorly aerated soils.

The Regulation of Nitrate Reductase, Nitrite Reductase, and Glutamate Dehydrogenase in Excised Pea Roots by Some Exogenous Amino Acids

The effect of growth retarding amino acids (L-aspartic acid,L-leucine,L-methionine, andL-threonine) on nitrate reductase (NO3R), nitrite reductase (NO2R), and NADH2 dependent glutamate dehydrogenase (GDH) in excised pea roots was followed,L-methionine andL-threonine slightly depressed NO3R activity,L-aspartic acid enhanced NO2R and GDH activities.L-methionine andL-threonine also slightly decreased nitrate uptake. The results obtained are discussed in connection with the growth effects of the amino acids investigated.

Nuclear DNA contents of Pisum genotypes grown in vivo and in vitro

The nuclear DNA content of prophase nuclei in root tips of two cultivars and two primitive lines of Pisum sativum and of Pisum fulvum have been determined, using a scanning microdensitometer. The nuclear DNA contents differed significantly between the genotypes investigated but there was no correlation with their supposed phylogenetic positions.A loss of 73% of the DNA from cells of aseptically cultured excised pea roots has been recently reported (Abbott, 1971). In marked contrast to this claim, our measurements of the nuclear 4C DNA content of root tip meristematic cells have shown that there is no significant loss in excised roots compared with attached roots.

On the mechanism of nitrate uptake

A study on the time effect on the influx and outflux of nitrogen by excised pea roots grown under specified conditions was carried out. The results indicate the close relationship between the two directional movement of nitrogen compounds. The outflux mechanism seems to depend on the influx or ion uptake. In distilled water no appreciable outflux took place. This calls for further work on the nitrogen balance between the roots and outside medium.

In vitro loss of nucleic acids from cells of aseptically cultured excised pea roots

Analysis of pea root tips taken from attached seedling roots and excised roots cultured in vitro has revealed major differences in cell constituents. The cells of cultured roots have only 40% and 13% of the protein and amino acid content of attached root cells. The nucleic acid content of cultured root cells was shown to be only 20% and 27% of the RNA and DNA respectively found in attached roots. It is suggested that there is excess nucleic acid in whole plant tissues above that required for transfer of genetic information necessary for normal growth and differentiation of root cells.

Metabolism and the prolonged retention of cells in the G1 and S period of the mitotic cycle of cultured pea roots

Stationary phase (SP) meristems were established by the culture of excised pea root tips in medium without carbohydrate and the kinetics of the resumption of cell progression in the cycle measured when carbohydrate was provided. The initiation of DNA synthesis was increasingly delayed with extended time in the SP. However, once cells began to enter S, they did so at a relatively constant rate of 2%/h in all meristems. The length of the SP affected neither the rate of entry into S, once it began, nor the subsequent transit time through S and G2. The initial delay before the onset of DNA synthesis was interpreted as time needed to fulfill necessary molecular prerequisites and hence constituted a principal control point for cell progression in the cycle. Also demonstrated was the ability first to position and then to retain cells in the S period for up to 24 h. Positioning was accomplished by an 8 h incubation of 48 h SP meristems in medium with sucrose and retention was achieved with hypoxia. The results showed that cells could be held in S for the duration of hypoxia and that insufficient oxygen for up to 24 h did not impair DNA synthesis nor the ability of cells to undergo mitosis. The combined data support the idea that the level of cellular metabolism involved in the initiation of DNA synthesis may be higher than that of mid- or late S.

Does Ethylene Mediate Root Growth Inhibition by Indole-3-Acetic Acid?

The effects of ethylene and of indole-3-acetic acid (IAA) on growth of excised pea root sections have been compared under a variety of conditions. After 16 hours treatment the inhibitory action of IAA is fully reversible on transfer of the root sections to IAA-free solutions. In contrast, inhibition by ethylene is almost totally irreversible. IAA inhibits growth from zero time; ethylene is generally without effect during the first 3 to 6 hours. The inhibitory action of ethylene is dependent on factors such as tissue age and solution composition which have no major effect on IAA inhibition. Ethylene production is enhanced by 100 mum IAA, but conditions which reduce the rate of ethylene evolution 2 to 3-fold at the same IAA concentration fail to affect the inhibitory action of IAA on elongation. It seems unlikely that ethylene can play more than a minor role in mediating inhibition of pea root growth by IAA.

Some Kinetic Aspects of Recovery of G 1 and G 2 Cells in Stationary-Phase Excised Pea Roots

Recovery after 300 R of γ-rays by G1 and G2 stationary-phase meristematic cells of excised pea root tips was measured as a function of the duration of an induced postirradiation stationary phase. The recovery indices measured were the initiation of DNA synthesis by G1 cells (3 H-thymidine incorporation) and the entrance of G2 cells into mitosis. Results showed that cells in each phase required 24 to 26 hours for full recovery. Recovery rate of G1 cells was relatively constant throughout the 24-hour period, while that of G2 cells varied, being higher in the first half and lower during the last half of the recovery period. Recovery of G1 and G2 cells exposed to a total of 300 R at a rate of 300 R/min or 0.19 R/min was the same, provided the stationary phase was not continued after γ-ray exposure.

Control of cell progression through the mitotic cycle by carbohydrate provision. I. Regulation of cell division in excised plant tissue.

A stationary phase in the root meristem of excised pea roots was established by prolonged carbohydrate deprivation in sterile culture medium. When the stationary phase had been established, cells that had collected in the G1 period of the mitotic cycle were induced to enter the S stage by subjection to relatively short intervals of carbohydrate provision (sucrose spurts). Progression and cycle location of the G1 cells induced to enter S were measured with tritiated thymidine and radioautography. The results indicated that the number of G1 cells induced to enter S increased directly with the spurt duration and that cells could be positioned and retained in the S and/or G2 periods by varying the duration of the spurt. The data support the hypothesis that S and maybe M stages have a relatively larger dependence on carbohydrate availability, and presumably a greater energy requirement, than G1 and G2.

Recovery Enhancement of G 1 and G 2 Meristematic Cells in Excised Pea Roots during an Induced Postirradiation Stationary Phase

Prolonged carbohydrate starvation of sterile excised pea roots induces a stationary phase characterized by the accumulation of meristematic cells in either the G1 (90%) or G2 (10%) periods of the mitotic cycle which is relieved by carbohydrate supply. With the presence of carbohydrate, DNA synthesis and mitosis are resumed and the cells return to a normal mitotic cycle. In these experiments three groups of roots were irradiated with 300 R or 600 R of X-rays after: (1) 24 hours in the stationary phase, (2) 48 hours in the stationary phase, or (3) 24 hours in the stationary phase followed by another 24 hours in this phase before carbohydrate provision. Three cytological measurements were made: the increase with time in the number of ${}^{3}{\rm H}\text{-thymidine-labeled}$ cells, the frequency of mitotic figures, and the appearance of ${}^{3}{\rm H}\text{-thymidine-labeled}$ cells in mitosis. Cells in the first and second groups of roots displayed a delay in the increase of ${}^{3}{\rm H}\text{-thymidine-la...

DNA synthesis in relation to polyploid mitoses in excised pea root segments cultured in vitro

Diploid and polyploid mitoses were observed in pea root segments cultured on a medium containing auxins and kinetin for 72 to 74 hr. Initial diploid mitoses were observed as early as 24 hr after the beginning of culture; initial polyploid mitoses were observed only after about 60 hr. The presence of kinetin was not required for the first 24 hr in culture for the appearance of polyploid mitoses at 74 hr. However, kinetin must be present in the culture medium after 24 hr. Auxin was required during the first 24 hr of the culture and its continuous presence may be required for polyploid mitoses. The question whether DNA synthesis must occur preceding the initial diploid and polyploid mitoses in cultured segments was studied using 5-fluoro-deoxyruridine and tritiated thymidine. The results, while not definitive, support the hypothesis that DNA synthesis is required for most of the mitoses, both diploid and polyploid. The same methods were used to study the time of DNA synthesis. In those cells in which DNA synthesis took place it seemed to occur between about 24 and 10 hr before mitosis for both polyploid and diploid mitoses. The results on the timing of the hormone requirements and the time of DNA synthesis make it unlikely that auxin or kinetin acts in this system primarily by directly triggering DNA synthesis.

THE EFFECTS OF INDOLEACETIC ACID AND 2,4-DICHLOROPHENOXYACETIC ACID ON THE UPTAKE AND METABOLISM OF 14C-GLUCOSE AND ON THE INCORPORATION OF 14C-LABELLED AMINO ACIDS INTO PROTEIN IN PEA ROOT TIPS

The effect of indoleacetic acid and 2,4-dichlorophenoxyacetic acid on the uptake and metabolism of 14C-labelled glucose and amino acids by excised pea root tips was studied. The intention was to determine whether the observed reduction of root growth by growth hormones was caused by interference in the uptake or in the metabolism of compounds by roots. The results indicate that the main effect of auxins on sugar metabolism in root tips is not on uptake, but on the subsequent metabolism of glucose. Auxins also had several specific rather than general effects on the synthesis of proteins. The production of certain amino acids from glucose was prevented, and the entry of others into protein was affected. This indicates that effects of auxin on protein metabolism were specific, and not necessarily merely consequences of decreased rates of growth and metabolism.