Excised Pea Roots Research Articles

Mode of Growth Inhibitory Activity and Metabolism of Imazosulfuron in Excised Roots of Plants

Growth inhibition activity of imazosulfuron, 1-(2-chloroimidazo[1, 2-a] pyridin-3-ylsulfonyl)-3-(4, 6-dimethoxypyrimidin-2-yl)urea, against excised pea and soybean roots was investigated. Fifty percents growth inhibition concentrations of imazosulfuron (I 50 ) against pea and soybean roots were 17.6 and 54.2 ppb, respectively. When the excised pea roots were incubated in the culture medium containing 25 ppb of imazosulfuron and 1 mM of either branched-chain amino acids or intermediates of their biosynthetic pathways, the growth inhibition activity of excised roots was alleviated by the combination of isoleucine and valine or α-ketoisovalerate and α-keto-β-methyl-n-valerate. These findings suggest that imazosulfuron exhibits inhibition of acetolactate synthase which catalyses the biosynthetic pathways of branched-chain amino acids. Metabolic fate of imazosulfuron in excised pea roots was studied using the 14 C-labeled compounds. Imazosulfuron was predominantly demethylated to afford a monodemethyl derivative (HMS) in the roots, and hydrolytically cleaved at the sulfonylurea bond to give a sulfonamide (IPSN) and an aminopyrimidine (ADPM) in the culture medium. Since HMS did not inhibit the growth of excised roots, metabolic fate of imazosulfuron is involved in a selectivity between plants.

Is there a relationship between branched amino acid pool size and cell cycle inhibition in roots treated with imidazolinone herbicides?

Excised pea root tips were cultured in White's medium for 24 h and then treated for 12 h with one of the imidazolinone herbicides at 0.2, 2, 20, or 200 μM. Pursuit and Assert were almost ineffective in inhibiting the mitotic index (MI), except at the highest concentrations. Arsenal (ARS) and Scepter both showed good inhibition with 20 μM by 8 h. Adding all three branched amino acids (BAA) (VAL, ILE, and LEU) at 0.1 mM blocked herbicide action. Treatment with the BAAs singly had no protective effect. Experiments were performed to determine the BAA pool size and MI after an 8-h treatment with ARS at 2 and 200 μM and Chlorsulfuron (CS), a sulfonylurea herbicide, at 28 nM. Both CS and ARS at 200 μM inhibited the MI to almost 0 by 8 h. ARS at 2 μM inhibited the MI by about 40%. The BAA pool size in all three treatments was reduced by approximately 50%, whether the MI was totally blocked or not. The 1-mm root tips had a greater amount of VAL than did the mature portions of the roots, whereas ILE and LEU were slightly less in the root tip. Other soluble amino acids did not show consistent differences between herbicide-treated roots and controls. The implications of the pool size reduction, in instances where the MI was not totally inhibited, is discussed in light of new data from other laboratories on the mode of action of the imidazolinone herbicides.

Physiological Responses to Sethoxydim in Tissues of Corn (Zea mays) and Pea (Pisum sativum)

The physiological responses of corn (Zea maysL. ‘Goldencrossbantam′) and pea (Pisum sativumL. ‘Alaska′) to sethoxydim {2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio) propyl]-3-hydroxy-2-cyclohexen-1-one} were investigated. Sethoxydim did not affect excised pea root growth at 1 × 10−4M but inhibited excised corn root growth at concentrations of 1 × 10−8M and above. Treating corn impeded root growth with 1 × 10−7M sethoxydim within 4 h after treatment; however, little histological change of the roots was observed at 48 h. At 1 × 10−6M, growth nearly stopped within 4 h after treatment, and clear cytological changes of the roots were observed at 24 and 48 h. Sethoxydim inhibited both mitosis and DNA synthesis of excised corn root tips between 4 and 48 h after treatment. Respiration of corn roots measured by oxygen uptake and the TTC (2,3,5-triphenyltetrazolium chloride) test was not affected by the herbicide directly. Sethoxydim (1 × 10−4M) inhibited IAA-induced cell elongation of corn coleoptile and pea epicotyl by 37 and 12%, respectively. Sethoxydim selectively inhibited the growth of excised root tips of susceptible corn by affecting cell division rather than cell enlargement, and the inhibition mechanism of cell division (inhibition of mitosis) by the herbicide was not by direct inhibition of DNA synthesis or by effects on respiration.

Uptake and Accumulation of the Herbicides Chlorsulfuron and Clopyralid in Excised Pea Root Tissue

The herbicides chlorsulfuron and clopyralid were taken up rapidly by excised pea root tissue and accumulated in the tissue to concentrations ten and four times those in the external medium, respectively. Uptake was related linearly to external herbicide concentration over a wide concentration range, implying that transport across the membrane is by nonfacilitated diffusion. Uptake of both compounds was influenced by pH, with greatest uptake at low pH. The pH dependence of uptake suggests that the herbicides (both of which are weak acids) are transported across the plasma membrane in the undissociated form, and accumulate in the cytoplasm by an ion trap mechanism. Most of the absorbed herbicide effluxed from the tissue when it was transferred to herbicide-free buffer, indicating that the accumulation was not due to irreversible binding. Consequently, both herbicides remain available for transfer to the phloem. These results can explain the high reported phloem mobility of clopyralid in intact plants. The low phloem mobility of chlorsulfuron must be accounted for by factors that override its ability to accumulate in the symplast.

Sucrose regulation of protein synthesis in pea root meristem cells

The enhanced synthesis of a polypeptide of apparent molecular weight of 93,000 daltons in apical meristem cells of excised pea roots appears to be a specific response to low sugar levels in these cells. Following excision of roots, the levels of soluble sugars in the meristems drop precipitously, this drop preceding the switch to selective incorporation of 3H-leucine, detected by SDS-PAGE fluorography, into the polypeptide; removal of cotyledons from otherwise intact seedlings also results in an increase, in the meristem, in the relative amount of a polypeptide of the same apparent molecular weight. When exogenous sucrose is provided to starved, excised roots, the preferential incorporation of 3H-leucine into the 93,000 dalton polypeptide is greatly reduced, indicating an immediate and specific repression of its synthesis by sucrose. Finally, exposure of starved meristems to elevated temperature results in a switch to incorporation of labeled leucine into typical heat-shock proteins, rather than into the sucrose-regulated polypeptide.

Salt-induced Changes in the Distribution of Amyloplasts in the Root Cap of Excised Pea Roots in Culture

Salt-induced Changes in the Distribution of Amyloplasts in the Root Cap of Excised Pea Roots in Culture

Ion Absorption and Allocation of Carbon Resources in Excised Pea Roots Grown in Liquid Medium in Absence or Presence of NaCl

Ion Absorption and Allocation of Carbon Resources in Excised Pea Roots Grown in Liquid Medium in Absence or Presence of NaCl

Changes Induced by Salinity to the Anatomy and Morphology of Excised Pea Roots in Culture

Ann. Bot. 57, 811–818, 1986

Changes Induced by Salinity to the Anatomy and Morphology of Excised Pea Roots in Culture

Changes induced by salinity to the anatomy and morphology of exiised pea roots in culture , Changes induced by salinity to the anatomy and morphology of exiised pea roots in culture , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Site of Action of Chlorsulfuron

The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I(50) values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.

Interaction of root‐lesion nematodes, Pratylenchus thornei and P. crenatus, with the fungus Thielaviopsis basicola and a grey sterile fungus on roots of pea (Pisum sativum)

Pratylenchus thornei invaded excised pea roots in agar in greater numbers and penetrated the cortex more deeply than P. crenatus. Both species fed on the roots ectoparasitically and displaced root cells into the surrounding medium. The cytoplasm of cortical cells near cither nematode became granulated, with enlarged vacuoles and nuclei. P. thornei also caused these responses in the endodermis. Infection of the root surface with a grey sterile fungus inhibited invasion by P. crenatus and P. thornei. Infection by Thielaviopsis basicola inhibited P. thornei invasion but encouraged penetration by P. crenatus and the hyphae were found deeper in the cortex when P. crenatus was present.

Differences in protein content of sister nuclei: evidence from binucleate and mononucleate cells.

DNA and protein contents of pairs of sister nuclei were determined using a combined Feulgen-dinitrofluorobenzene technique. Sister nuclei were studied in binucleate cells, induced by treatment with 0.1% caffeine, and in sister mononucleate cells of untreated roots. Excised pea roots, grown in culture, were treated with 5-aminouracil to induce mitotic synchrony and with caffeine at the time of peak mitotic index, to provide the maximum number of binucleate cells. The induced binucleate cells form a marked population which was followed through a cell cycle; sister nuclei showed a correlation of volume and protein content, r = 0.79. Protein contents of sister nuclei were rarely identical and at 1 + 2 and 1 + 6 h the difference in protein contents of sister nuclei was significant (p = 0.05). Mean nuclear protein content decreased from 1 + 2 to 1 + 6 h; then, as nuclei entered S phase, their protein content increased. From 1 + 2 to 1 + 14 h the increase in protein content, in absolute amount, was identical in both sister nuclei. This suggests that there was a biphasic pattern of protein uptake; it is differential, in sister nuclei, in the first part of G1 but is identical throughout the rest of interphase. Analysis of sister nuclei from sister mononucleate cells showed a similar pattern of change; this is further evidence, from untreated cells, of a biophasic pattern of protein uptake. Caffeine-treated nuclei had lower protein contents than untreated nuclei, yet they completed a cell cycle and entered mitosis; this suggests that nonessential proteins were no longer present. It is proposed that mitosis is asymmetrical for molecules that regulate rates of macromolecular synthesis, cell growth, and progress through a cell cycle and that once the initial asymmetry has been established, it is maintained throughout interphase, even in binucleate cells in which the two nuclei share a common cytoplasm.

Effect of some disaccharides, hexoses and pentoses on nitrate reductase, glutamine synthetase and glutamate dehydrogenase in excised pea roots

AbstractThe effects of exogenous sucrose, lactose, d‐glucose, d(‐)fructose, d‐galactose, d‐mannose, l‐sorbose, l‐arabinose and d‐xylose on nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) levels, on anaerobic nitrite production and on respiratory O2 consumption were studied in excised roots of pea (Pisum sativum L. cv. Raman). Sucrose, glucose and fructose increase NR and GS levels and decrease GDH level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Precultivation of roots with mannose and galactose results in an increase in anaerobic nitrite production in a medium consisting of phosphate buffer and KNO3. GS reaches its maximum at lower sugar concentrations, this fact being especially clear‐cut with galactose. The decrease in GS level observed in roots cultured without sucrose is enhanced by higher sorbose concentrations. The increase in GDH level occurring in roots cultured without sucrose is depressed by low galactose and mannose concentrations but enhanced by high galactose, mannose, xylose and a wide range of sorbose concentrations. Lactose exerts only slight influence on the enzymes. The effects of sugars are in no case consistent with their effect on respiratory O2 consumption which is most pronounced with NR.The above results show that the effects of sugars on NR, GS and GDH are not mediated by one universal mechanism.

The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised pisum sativum roots

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.

The effect of chloride on nitrate reductase level, on anaerobic nitrite production, and on nitrate content in excisedPisum sativum L. roots

The effect was studied of chloride ions, added in the form of different salts, on nitrate reductase (NR) level in excised pea roots, on anaerobic nitrite production in an assay medium lacking both nitrate and n-propanol, on nitrate content in the roots, and on in vivo NR activity determined in an assay medium containing 5% n-propanol. The presence of Cl in nitrate containing nutrient solutions resulted in lower NR levels, however counterions supplied together with Cl tended to modify slightly this general trend. The negative effect of Cl ions was also apparent, when Cl ions were applied before nitrate ions. Anaerobic nitrite production in the medium lacking both nitrate and n-propanol was not influenced by chloride ions. Nitrate content in the roots was reduced in the presence of chloride both at 3 mM and 15 mM NO3 in nutrient solutions; however, at 16 mM NO3, nitrate content in the roots exoeeded even in the presence of 15 mM Cl nitrate content in those root segments which were cultivated in a nutrient solution with 6 mM nitrate, which is the concentration at which NR reaches the level of saturation in excised pea roots. The results obtained suggest that a special induction nitrate pool exists in plant cells besides the storage and metabolic nitrate pools.

The effect of some ammonium salts on nitrate reductase level, onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO3, Ca(NO3)2 and NaNO3 at 15 mM NO3 - concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH4 + at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH4H2PO4, (NH4)2SO4 and NH4NO3). However their positive effect is independent of the presence of NH4 + ions, and it is obviously the result of an increased concentration of H+ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH4NO3 solution where NH4 + is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH3). (H2O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence.Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO3 - pool (i.e. NO2 - production under anaerobic conditions), nor between NR level and the total nitrate content in the roots.One-salt solutions of NaNO3, Ca(NO3)2 and KNO3 exert different effects on the level of nitrate reductase and on the content of NO3 - in the roots, but the in vivo NO3 - reduction shows the same trend as NR level in the roots influenced by these salts. Cl- ions, supplied in NH4C1, depress both NR level and NO3 - content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.

Enhancement of Glutamate Dehydrogenase Activity in Excised Pea Roots by Exogenously Supplied Acids

Summary The addition of acids to the nutrient medium and the increase in the concentration of hydrogen ions in the external medium and within the roots therein obviously the determining factor causes an increase in GDH activity in excised pea roots. This increase is dependent on a proteosynthesis indicated by the complete inhibition of the increase by actidione; it is not, however, the result of generally enhanced proteosynthesis. Nitrate reductase activity was not induced by the increase in the concentration of H + ions; the activity of aspartate aminotransferase was slightly decreased. The increase in GDH activity caused by the addition of the acids was abolished by the addition of bases (incl. NH 4 OH), if the pH of the medium was shifted by this treatment nearer to neutral region (pH from 5 to 7). The shifting of pH into the alkaline region resulted in the inhibition of that increase in GDH level caused by the omission of sugar from the medium. The GDH level was not influenced significantly by this treatment in roots cultured with sugar. Among ammonium salts, NH 4 Cl (10 meq NH 4 + ) caused a statistically significant increase in GDH activity and simultaneously a statistically significant increase in the concentration of H + ions in roots cultured with sugar, whereas (NH 4 ) 2 HPO 4 (10 meq NH 4 + ) tendend to bring about an opposite, even though statistically insignificant, effect. These results indicate that the effect of higher concentrations of ammonium salts on GDH activity observed in the roots of intact plants by other authors does not reside in the direct influence of NH 4 + ions on GDH synthesis, but in the combination of (1) indirect influence of physiologically acid ammonium salts mediated by the increase in the concentration of H + ions inside the roots and of (2) another indirect influence mediated by the mechanism controlled by sugars (which was described and discussed in our previous paper - Sahulka et al., 1975). The GDH level could be increased up to four times during a 22 h incubation even in excised pea roots by the combined effects of the addition of acids and of the omission of sugar from the medium.

Regulation of Glutamate Dehydrogenase and Nitrate Reductase Levels in Excised Pea Roots by Exogeneously Supplied Sugar

The activities of NADH2 dependent glutamate dehydrogenase (GDH) and nitrate reductase were followed in excised pea roots cultured in liquid medium with or without sucrose. Whilst the persistance of nitrate reductase activity in excised pea roots depends directly on the presence of sugar in the medium and whilst in its absence nitrate reductase activity drops to the initial value before the induction with nitrate, both NADH9 dependent GDH and NADPH2 dependent GDH activities rise in excised pea roots in the absence of sugar in the medium, and vice versa, their activities are maintained at approximately the same level as in corresponding parts of roots on intact seedlings in the presence of sugar in the medium. The increase in NADH2 dependent GDH activity is inhibited by actidione which indicates that de novo synthesis of the enzyme is probably responsible for the increase. Glucose and fructose are in their effects equivalent to sucrose. The omission of sugar from the medium causes also a change in the ratio of the activities of individual isoenzymes of NAD+ dependent GDH. Physiological concentrations of ammonium and nitrate ions neither influence GDH level in the presence of sugar nor in the absence of it. The above mentioned sugars are apparently involved in the regulation of these enzymes directly and not indirectly by means of their metabolites formed during respiration. This is indicated by the results obtained in the experiments with exogenously supplied metabolites of glycolysis, pentose phosphate cycle and Krebs cycle, which did not show any significant effect on GDH and nitrate reductase levels. The increase in GDH level observed after the addition of sublethal concentrations of some respiratory inhibitors to the medium containing sugar was probably caused by decreased sugar uptake and transport. The regulation of GDH level controlled by sugars is in isolated pea roots independent of some other regulatory mechanisms (controlled by acids, nitrite and hydroxylamine), and vice versa. On the other hand, the results of our experiments indicate that the increase in the level of NADH2 dependent GDH observed by other authors in the roots of whole intact plants after their exposure to high concentrations of ammonium salts, may be, at least partly, caused indirectly by changes in the level of sugars in these roots.

Auxin and the Response of Pea Roots to Auxin Transport Inhibitors: Morphactin

The auxin transport inhibitor methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (CFM), a morphactin, inhibits negative geotropism, causes cellular swelling, and induces root hair formation in roots of intact Pisum sativum L. seedlings. In excised pea root tips, CFM inhibits elongation more than increase in fresh weight (swell ratio = 1.3 at 20 mum CFM). CFM growth inhibition was expressed in the presence of ethylene. Indoleacetic acid (IAA) prevented the expression of CFM growth inhibition possibly because IAA inhibited the accumulation of CFM into the tissue sections. CFM inhibited the accumulation of IAA and 2,4-dichlorophenoxyacetic acid into excised root tips. Applying Leopold's (1963. Brookhaven Symp. Biol. 16: 218-234) model for polar auxin transport, this result suggests a possible explanation for CFM inhibition of geotropism in pea roots, i.e. disruption of auxin transport by interfering with auxin binding.

Regulation of glutamate dehydrogenase level in excised pea roots by some exogenously supplied compounds with auxin activity

Besides IAA, other compounds with auxin activity (of both indole and non-indole nature) can also cause an increase in glutamate dehydrogenase level in excised pea roots. The effect of these substances is probably not mediated by ethylene, depends on proteosynthesis, is independent of the regulatory effect of sugars and need not be influenced by the antagonism between auxins and cytokinins.