Etiolated Mung Bean Hypocotyl Segments Research Articles

Rapid repression of vacuolar invertase in mungbean hypocotyl segments and regulation by sucrose, auxin and light

Plant vacuolar invertase is implicated in the rapid regulation ofcellular energy demand by environmental changes. In etiolated mungbeanhypocotylsegments, a source-limited system, the soluble acid invertase activity andtranscript level of a vacuolar invertase gene, Vr-AI1 wasrapidly down regulated when hypocotyls were excised from the intact seedlings.Kinetic data show that the enzyme activity dropped to 50% of the initial levelwithin 3 h and the steady state level ofVr-AI1 mRNA was sharply decreased as well. Exogenoussupplyof sucrose suppressed the decrease in both enzyme activity and the transcriptlevel. Treatment with indole-3-acetic acid (IAA) and/or light also retarded thespeed of down-regulation. Studies with α-amanitin, an inhibitor of eukaryoticRNA polymerase II showed that the Vr-AI1 mRNA was morestable in the presence of IAA. Cycloheximide treatment also increased thetranscript level, raising the possibility that the Vr-AI1gene is controlled by mRNA stability. There was a discrepancy that theVr-AI1 mRNA disappeared much earlier than the enzymeactivity. In the light condition, moreover, the decay kinetics and IAAdose-response curve for the enzyme activity was markedly different from thosefor the Vr-AI1 mRNA level. Our results imply that multipledistinct mechanisms are involved in the regulation of soluble acid invertaseactivity by auxin and light

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments.

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.

The Inhibition of Brassinosteroid-Induced Ethylene Biosynthesis in Etiolated Mung Bean Hypocotyl Segments by 2,3,5-Triiodobenzoic Acid and 2-(p-Chlorophenoxy)-2-methylpropionic Acid

The effect of two auxin antagonists, 2,3,5-triiodobenzoic acid (TIBA) and 2-(p-chlorophenoxy)-2-methyl propionic acid (CMPA) on brassinosteroid (BR)-induced ethylene biosynthesis in etiolated mung bean hypocotyl ( Vigna radiata L. Rwilcz cv. Berken) segments was studied. The maximum inhibition of both ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) biosynthesis by TIBA and CMPA was achieved at concentrations of 50 μM and 10 μM, respectively. With increasing concentrations of BR, there were significant increases in both ethylene and ACC production. When varying concentrations of BR were used in combination with 50 μM TIBA or 10 μM CMPA, there was a decrease in ethylene and ACC production at all BR concentrations tested, with the exception of the lowest. After 18 h, mung bean hypocotyl segments pre-incubated for 8 h with either 50 μM TIBA or 10 μM CMPA showed a maximum inhibition of BR-induced ethylene and ACC production. The results of treatment sequences showed that both TIBA and CMP A caused the same inhibitory effect whether applied before or after BR-treatment.

Characterization of 1-Aminocyclopropane-1-carboxylate synthase purified from mung bean hypocotyls

Summary Analysis of the properties of the key enzyme for ethylene production, 1-aminocyclopropane-1-carboxylate (ACC) synthase, isolated in pure form from hormonally induced etiolated mung bean hypocotyl segments, has revealed that the optimum pH for catalytic activity under normal assay conditions was pH 8.0 although maximal stability was noted at pH 6 to 7. The purified enzyme has an isoelectric point of 5.1 when analyzed by isoelectric focusing and the enzyme's content of pyridoxal phosphate was determined by two separate procedures to be one per subunit of molecular weight 65,000. Edman degradation provided the sequence of 26 residues from the N-terminal portion of the protein: V-A-H-A-K-D-D-A-Y-L-Q-A-A-I-P-K-R-I-K-L-F-E-T-I-Q-A-. Routine assays conducted for 5 minutes with the hydrogen sulfate salt of S-adenosyl methionine (AdoMet) revealed no apparent inhibition. Longer incubation time with substrate, however, indicated that AdoMet was irreversibly inactivating the enzyme and suggested the possibility that this might have important consequences for enzyme turnover. Aminooxyacetic acid was a potent competitive inhibitor of enzyme activity with a Ki of 2 µM. No inhibition was noted with L-homoserine, methylthioadenosine, 5'-AMP, 5'-ADP, 5'-ATP or fusicoccin. An investigation of the methods required to stabilize ACC synthase from mung beans, tomato and apple found that high concentration (>0.5M) of potassium phosphate buffer were effective. Activity remained unchanged during 48-hour storage at 4 °C, and after 10 days in storage, 50 % of the original activity remained for enzyme extracted from mung bean and tomato, while activity from apple tissue was lost after 7 days. Such findings should facilitate the comparative analyses of these important ACC synhases when all have been purified.

Effects of Indole-3-acetic Acid and Brassinosteroid on Ethylene Biosynthesis in Etiolated Mung Bean Hypocotyl Segments

Summary Brassinosteroid (BR) and indole-3-acetic acid (IAA) alone and in combination were evaluated for their effects on ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC-synthase production in etiolated mung bean hypocotyl segments. Eighteen hours following treatment with BR (0.25 to 2.0 μM) or IAA (0.5 to 10.0 μM) alone promoted an increase in ethylene and ACC production. When BR and IAA were used in combination there was a synergistic increase in ethylene and ACC production at all concentrations tested 18 h following treatment. Aminooxyacetic acid (AOA), cycloheximide (CHI), and cobalt chloride (COCl2) treatments after 18 h inhibited BR, IAA, and IAA + BR-induced ethylene and ACC production at 2 mM, 100 μM and 10 mM concentrations respectively. Segments treated with 10 μM IAA showed an increase in ACC and ACC-synthase production 3 h after treatment initiation with little change between 3 to 6 h. This plateau was followed by a sharp decline in both ACC and ACC-synthase from 6 to 12 h following treatment. BR treated segments showed a sharp increase in both ACC and ACC-synthase between 6 to 24 h following treatment. When IAA was used in combination with BR there was a sharp increase in ACC and ACC-synthase for the first 12 h following treatment, followed by a plateau. Cobalt chloride dramatically inhibited BR-induced ACC and ACCsynthase over a 24 h period whereas it had a stimulatory effect on both IAA and BR + IAA-induced ACC and ACC-synthase production shortly following treatment. AOA stimulated IAA-induced ACCsynthase activity but not BR-induced. Both IAA and BR induced ethylene and ACC production were both inhibited by AOA.

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyls

1-Aminocyclopropane-l-carboxylate (ACC) synthase, EC 4.4.1.14, was purified to homogeneity from etiolated mung bean hypocotyl segments. This was made possible by the ability to elevate the enzyme level markedly through hormone treatments and by stabilization of the enzyme with high phosphate concentrations. The four-step procedure resulted in 1050-fold purification with 25% yield, and consisted of stepwise elution from hydroxylapatite, chromatography on phenyl-Sepharose CL-4B, gradient elution from hydroxylapatite, and fast protein liquid chromatography (FPLC) on a MonoQ anion-exchange column. FPLC-purified ACC synthase migrated as a single band of M r 65,000 on denaturing polyacrylamide gel electrophoresis. The molecular weight of native enzyme by Bio-Gel A-0.5 m chromatography was 125,000, indicating that the enzyme probably exists as a dimer of identical 65,000 M r subunits. The mung bean ACC synthase exhibited a pH optimum of 8.0 for activity and a K m for S-adenosylmethionine (AdoMet) of 55 μ m at 30 °C. It exhibited an Arrhenius activation energy of 12 kcal mol −1 degree −1 and was inactivated at temperatures in excess of 40 °C. The specific activity for pure ACC synthase was 21 μmol of ACC formed/mg protein/h when determined under optimal conditions with 400 μ m AdoMet.

Inactivation of 1-Aminocyclopropane-1-carboxylic Acid Synthase of Etiolated Mung Bean Hypocotyl Segments by its Substrate, S-Adenosyl-L-methionine

Inactivation of 1-Aminocyclopropane-1-carboxylic Acid Synthase of Etiolated Mung Bean Hypocotyl Segments by its Substrate, S-Adenosyl-L-methionine

Inhibition of IAA‐induced ethylene production in etiolated mung bean hypocotyl segments by 2,3,5‐triiodobenzoic acid and 2‐(p‐chlorophenoxy)‐2‐methyl propionic acid

The effect of two auxin antagonists, 2,3,5‐triiodobenzoic acid (TIBA) and 2‐(p‐chlorophenoxy)‐2‐methyl propionic acid (CMPA) on IAA‐induced ethylene production in etiolated mung bean hypocotyl (Vigna radiata L. Rwilcz cv. Berken) segments was studied. Both TIBA and CMPA inhibited IAA‐induced ethylene production and CO2 production at concentrations from 0.001 mM to 0.1 mM and 0.01 mM to 1.0 mM, respectively. The optimum concentration for inhibition of ethylene production by TIBA was 0.05 mM and CMPA was 0.5 mM. At the optimum concentration of TIBA and CMPA, there was a significant decrease in IAA‐induced ethylene production without a decrease in respiration rates below control levels. After 18 h, mung bean hypocotyl segments treated with 0.05 mM TIBA for 6 h or 0.5 mM CMPA for 8 h showed a maximum inhibition of IAA‐induced ethylene production. Treatments longer than 8 h caused no further inhibition. The uptake of [14C]‐naphthaleneacetic acid by mung bean segments was greatly reduced by the addition of either TIBA (0.05mM) or CMPA (0.5 mM) to the incubation media. The results of treatment sequences showed that TIBA needed to be applied prior to IAA in order to inhibit IAA‐induced ethylene production, but CMPA caused the same inhibitory effect whether applied before or after IAA treatment. These findings provide evidence that TIBA inhibits auxin‐induced ethylene production in etiolated mung bean hypocotyl segments by blocking auxin movement into the tissue whereas CMPA may work on both auxin transport and action.

Ca2+ acts synergistically with brassinosteroid and indole‐3‐acetic acid in stimulating ethylene production in etiolated mung bean hypocotyl segments

Brassinosteroid (BR) and indole‐3‐acetic acid (IAA) were used in combination with Ca2+ in order to determine if there was a synergistic effect in the stimulation of ethylene production in etiolated mung bean (Vigna radiata L. Rwilez ev. Berken) hypocotyl segments. Ca2+ was found to act synergistically with BR. IAA or a combination of the two in promoting a stimulation in ethylene production. EDTA, which chelates Ca2+, greatly reduced the effectiveness of calcium salts in promoting ethylene production in the presene of either BR, IAA or a combination of the two. Neither K+, Mg2+ nor Mn24 (chloride salts) acted synergistically with BR and IAA.

Effect of the Defoliant Thidiazuron on Ethylene Evolution from Mung Bean Hypocotyl Segments

The effect of the defoliant thidiazuron (N-phenyl-N'1,2,3-thiadiazol-5-ylurea) on ethylene evolution from etiolated mung bean hypocotyl segments was examined. Treatment of hypocotyl segments with concentrations of thidiazuron equal to or greater than 30 nanomolar stimulated ethylene evolution. Increased rates of ethylene evolution from thidiazuron-treated tissues could be detected within 90 minutes of treatment and persisted up to 30 hours after treatment. Radioactive methionine was readily taken up by thidiazuron-treated tissues and was converted to ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and an acidic conjugate of ACC. Aminoethoxyvinylglycine, aminooxyacetic acid, cobalt chloride, and alpha-aminoisobutyric acid reduced ethylene evolution from treated tissues. An increase in the endogenous content of free ACC coincided with the increase in ethylene evolution following thidiazuron treatment. Uptake and conversion of exogenous ACC to ethylene were not affected by thidiazuron treatment. No increases in the extractable activities of ACC synthase were detected following thidiazuron treatment.

(+)-5-Hydroxy-dioxindole-3-acetic acid, a synergist from rice bran of auxin-induced ethylene production in plant tissue

(+)-5-Hydroxy-dioxindole-3-acetic acid ( 1) was isolated from rice bran as a substance synergistic with auxin in the auxin induced ethylene production by etiolated mungbean hypocotyl segments. 5-Hydroxy-oxindole-3-acetic acid ( 4) and IAA were also obtained. The importance of a hydroxyl group in the 5-position in the two compounds was suggested since synthesized (±)-dioxindole-3-acetic acid ( 6) was inactive.

Relationships in actions of indoleacetic acid, benzyladenine and abscisic acid in ethylene production1

The relationships between IAA and ABA, and between BA and ABA in their effects on ethylene production were examined with etiolated mungbean hypocotyl segments. When ABA and IAA were simultaneously applied to the tissues, ABA inhibited IAA-induced ethylene production and the degree of inhibition was solely determined by the ABA concentrations. Increasing concentrations of BA did not affect ABA inhibition. Low concentrations of ABA slightly increased endogenous ethylene production. When ABA and BA were applied together in the presence of IAA, the degree of ABA inhibition was again determined by the ABA concentrations regardless of the BA concentrations. BA did not recover ABA inhibition and ABA did not inhibit the stimulative effect of BA on both endogenous and IAA-induced ethylene production. Almost the same results were obtained with ABA and BA pretreatment of the tissues. This indicates that in the processes of IAA-induced ethylene production, IAA and ABA act in series, but that the actions at their respective sites are independent.

Physical Nature of Irreversible Deformation of Plant Cells

Etiolated mung bean hypocotyl segments were incubated in 0.25 m mannitol solutions with indoleacetic acid. They were then deformed mechanically with a longitudinal tensile force at a constant strain rate. The magnitudes of the mechanical forces were comparable to those of the hydrostatic forces existing in normally growing tissues. Each segment was repeatedly deformed and returned to zero force. The total deformation was increased at each cycle.The irreversible and elastic changes in length and diameter were measured for each deformation and the changes in surface area and volume calculated. In addition the applied stress and the work of irreversible and of elastic deformation were determined as functions of deformation.It was found that irreversible elongation, irreversible change in surface area and total change in surface area all were linear functions of total imposed elongation. However, very little change in volume occurred during the deformations.The work of irreversible deformation was found to be independent of temperature between 8 degrees and 25 degrees . It was also virtually independent of rate of deformation measured over a 5-fold range of deformation rates.From these results it is concluded that the irreversible deformation of mung bean hypocotyl tissue occurs by plastic deformation rather than by viscous flow. Thus, the irreversible deformation occurred as a result of breaking cross-links of a cross-linked polymer system.