Inhibition of auxin-induced ethylene production by salicylic acid in mung bean hypocotyls

Salicylic acid (SA), a common plant phenolic compound, influences diverse physiological and biochemical processes in plants. To gain insight into the mode of interaction between auxin, ethylene, and SA, the effect of SA on auxininduced ethylene production in mung bean hypocotyls was investigated. Auxin markedly induced ethylene production, while SA inhibited the auxin-induced ethylene synthesis in a dose-dependent manner. At 1 mM of SA, auxininduced ethylene production decreased more than 60% in hypocotyls. Results showed that the accumulation of ACC was not affected by SA during the entire period of auxin treatment, indicating that the inhibition of auxin-induced ethylene production by SA was not due to the decrease in ACC synthase activity, the rate-limiting step for ethylene biosynthesis. By contrast, SA effectively reduced not only the basal level of ACC oxidase activity but also the wound-and ethylene-induced ACC oxidase activity, the last step of ethylene production, in a dose-dependent manner. Northern and immuno blot analyses indicate that SA does not exert any inhibitory effect on the ACC oxidase gene expression, whereas it effectively inhibits both the in vivo and in vitro ACC oxidase enzyme activity, thereby abolishing auxin-induced ethylene production in mung bean hypocotyl tissue. It appears that SA inhibits ACC oxidase enzyme activity through the reversible interaction with Fe²⁺, an essential cofactor of this enzyme. These results are consistent with the notion that ethylene production is controlled by an intimate regulatory interaction between auxin and SA in mung bean hypocotyl tissue.     

Purification and partial characterization of an aminopeptidase from mung bean cotyledons

An aminopeptidase (EC 3.4.11.-) was purified to homogeneity, as judged by SDS-PAGE. from mung bean ( Vigna radiata ) cotyledons. The molecular mass of this peptidase was estimated as 75 kDa by gel filtration. When an oligopeptide consisting of 5 amino acid residues was used as substrate, amino acids were released in the order of the N-terminal sequence of the oligopeptide chain. This enzyme apparently requires free sulfhydryl for its activity, as judged by the effects of various proteinase inhibitors. Among aminoacyl- p -nitroanilides examined for the availability as substrates of the enzyme, p -nitroanilides with hydrophobic amino acids were preferred substrates. According to western immunoblot profiles, the enzyme level in cotyledons was high at the early stage of imbibition and declined rapidly after germination.     

Partial purification and properties of a 36-kDa 1-aminocyclopropane-1-carboxylate N-malonyltransferase from mung bean

A 36-kDa 1-aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase, which converts the ethylene precursor ACC into the conjugated derivative malonyl-ACC (MACC), has been isolated from etiolated mung bean ( Vigna radiata ) hypocotyls, and partially purified in a four-step procedure. The enzyme is stimulated about 7-fold by 100 m M K⁺ salts or 0.5 m M Co²⁺ salts, and is inhibited competitively by D-phenylalanine (K_i= 1.3 m M ) and non competitively by CoASH (0.3 m M ). Beside malonyl-CoA, it is capable to use succinyl-CoA as an acyl donor. The 36-kDa enzyme described here exhibits a lower optimum temperature (40°C) and a 7- or 3-fold lower apparent K_m for ACC (68 μ M ) and malonyl-CoA (74 μ M ), respectively, when compared with its 55 kDa isoform already isolated from the same plant material. This data support the idea that several isoforms of ACC N-malonyltransferase exist in plants. These isoforms may play a differential role in regulating the availability of ACC, and consequently the rate of ethylene production, as well as detoxifying cells from D-amino acids.     

Aminoethoxyvinylglycine, cobalt and ascorbic acid all reduce ozone toxicity in mung beans by inhibition of ethylene biosynthesis

Evidence is presented in support of the hypothesis that stress ethylene formation determines ozone toxicity in plants. In studies with mung beans ( Vigna radiata ) ozone toxicity was reduced not only when plants had been pretreated with aminoethoxyvinylglycine (AVG) but also after pretreatment of plants with CoCl₂ and ascorbic acid. While AVG prevents the enzymatic conversion of S-adenosylmethionine (SAM) to I-aminocyclopropane-I-carboxylic acid (ACC), cobalt and free radical scavengers such as ascorbic acid inhibit the subsequent conversion of ACC to ethylene. Stomatal opening was not affected by pretreatment of plants with inhibitors of ethylene biosynthesis.     

Relationship of steroidal structure to ethylene production by etiolated mung bean segments

Several brassinosteroid (BR) analogues, cholesterol and aldosterone were evaluated for their effectiveness alone and in combination with indole-3-acetic acid (IAA) in stimulating ethylene production by etiolated mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Changing the conformation of the two hydroxyl groups on C-22 and C-23 positions from α to β did not greatly reduce the efficiency of these compounds to stimulate ethylene production alone or in combination with IAA. There was little difference in activity observed when the conformation of the methyl group in the C-24 position was changed from α to β. However, when hydroxyls were deleted from the side chain in the C-22 and C-23 positions, the compound was rendered inactive alone or in combination with IAA. The compound was also inactivated by removing the 7-oxa function on the B-ring and by substituting an ethyl group for the methyl group in the C-24 position. Both aldosterone and cholesterol were ineffective in promoting ethylene production. This study shows that very stringent structural features are required for a steroid to have BR-like activity and to act synergistically with auxin in the promotion of ethylene synthesis.     

Characterization of new microsatellite markers in mung bean,Vigna radiata (L.)

The present work reports the isolation and characterization of new polymorphic microsatellites in mung bean (Vigna radiata L.). Of 93 designed primer pairs, seven were found to amplify polymorphic microsatellite loci, which were then characterized using 34 mung bean accessions. The number of alleles ranged from two to five alleles per locus with an average of three alleles. Observed and expected heterozygosity values ranged from 0 to 0.088 and from 0.275 to 0.683, respectively. All seven loci showed significant deviations from Hardy–Weinberg equilibrium, whereas only one pairwise combination (GBssr‐MB77 and GBssr‐MB91) exhibited significant departure from linkage disequilibrium. These newly developed markers are currently being utilized for diversity assessment within the mung bean germplasm collection of the Korean Gene Bank.     

芦丁对绿豆幼苗营养生长的影响及其与IAA的相互作用

观察了植物体内的天然黄酮芦丁和吲哚乙酸(IAA)对绿豆幼苗营养生长的影响并测定胚轴中的芦丁和IAA含量.光照条件下芦丁(60μg/mL以下)处理对绿豆幼苗生长有一定促进作用,表现为胚轴和主根伸长加快、侧根数目增多、鲜重或干重增加;而光照条件下更高浓度芦丁(80μg/mL以上)处理及黑暗条件下芦丁(20～100μg/mL)处理对绿豆幼苗生长有抑制作用.当培养基中的芦丁浓度为60～80 μg/mL时,光照下的幼苗比暗处理的幼苗在胚轴中积累更多的芦丁;而芦丁浓度为40μg/mL以下和接近100μg/mL时幼苗在光照下累积的芦丁较暗处理的幼苗更少.0.1μg/mL以上的IAA促进芦丁的累积而进一步抑制幼苗胚轴和主根的伸长.当培养基中含有40 μg/mL的芦丁和0.5μg/mL的IAA时,胚轴中累积的芦丁达到高峰.芦丁降低黄化幼苗内源性IAA在胚轴中的累积,并抑制幼苗对IAA的吸收.     

The effect of brassinosteroid and 2,4-D-L-amino acid conjugates on ethylene production by etiolated mung bean segments

Brassinosteroid (BR) an analogue of brassinolide was tested in combination with thirteen 2,4-dichlorophenoxyacetic acid-L-amino acid conjugates for its possible synergistic effects on ethylene production by etiolated mung bean (Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. When BR was used in combination with 2,4-D-L-amino acid conjugates the degree of enhanced ethylene production varied with the conjugate tested. In fact, the activity of the conjugate alone was directly related to its activity with BR.     

Inhibitory effect of polyamines on the activity of endopeptidase in mung bean cotyledons

The activity of cysteine endopeptidase (EP) in the cotyledons of mung bean seeds increased with time after germination. When cotyledons were excised from the embryonic axis in the course of seedling growth, the activity of EP in the excised cotyledon markedly dropped during the following incubation of 1 d. However, the level of EP protein in excised cotyledons, as examined by immunoblotting, was similar to that in axis-attached cotyledons at the corresponding stage. Thus, it seems that the low activity of EP in excised cotyledons is not due to a decrease in the content of EP protein, but due to a loss of the activity of existing EP. Treatment of attached cotyledons with polyamines (PAs; putrescine and spermidine [Spd]) resulted in a decrease in EP activity, while the same PA-treatment brought about little alteration in the level of EP protein. This indicates that PAs somehow produce an inhibitory effect on the activity of EP. Axis-removal resulted in an accumulation of Spd in the cotyledon. The possibility is suggested that PA, especially Spd, is involved in the inhibition of EP activity in excised mung bean cotyledons.     

Tentoxin-induced loss of plastidic polyphenol oxidase

Tentoxin-treated mung bean plants are shown to lack chloroplast polyphenol oxidase (PPO) by enzymatic, electrophoretic and cytochemical analysis. Incorporation of PPO (a protein coded by nuclear DNA) into the plastid may occur via concentration of the protein into inner envelope-derived vesicles. PPO integration into the plastid is apparently blocked by a tentoxin treatment although fraction I protein (and hence the proteins for chloroplast ribosome production) is not affected by this fungal toxin. Both apical and etiolated plastids from teotoxin-treated plants lack PPO. Thus, it is unlikely that the primary effect of tentoxin is due to the binding of the chloroplast coupling factor, as previously supposed.     

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 m M to 0.1 m M and 0.01 m M to 1.0 m M , respectively. The optimum concentration for inhibition of ethylene production by TIBA was 0.05 m M and CMPA was 0.5 m M . 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 m M TIBA for 6 h or 0.5 m M 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 (0.05m M ) or CMPA (0.5 m M ) 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.     

Individual and interactive effects of temperature,carbon dioxide and abscisic acid on mung bean (Vigna radiata) plants

We studied the effects of temperature, carbon dioxide and abscisic acid on mung bean (Vigna radiata). Plants were grown under 26/22°C or 32/28°C (16?h?light/8?h?dark) at 400 or 700?μmol?mol^?1 CO₂ and received ABA application of 0 or 100?μl (10?μg) every other day for three weeks, after eight days of initial growth, in growth chambers. We measured 24 parameters. As individual factors, in 16 cases temperature; in 8 cases CO₂; in 9 cases ABA; and as interactive factors, in 4 cases, each of temperature?×?CO₂, and CO₂?×?ABA; and in 2 cases, temperature?×?ABA were significant. Higher temperatures increased growth, aboveground biomass, growth indices, photochemical quenching (qP) and nitrogen balance index (NBI). Elevated CO₂ increased growth and aboveground biomass. ABA decreased growth, belowground biomass, qP and flavonoids; increased shoot/root mass ratio, chlorophyll and NBI; and had little role in regulating temperature–CO₂ effects.

Abbreviations: A_N: net CO₂ assimilation; E: transpiration; F_v/F_m: maximum quantum yield of PSII; g_s: stomatal conductance; LAR: leaf area ratio; LMA: leaf mass per area; LMR: leaf mass ratio;φPSII: effective quantum yield of PSII; qNP: non-photochemical quenching; qP: photochemical quenching; SRMR: shoot to root mass ratio; WUE: water use efficiency     

Effect of water stress on the enzymes of nitrogen metabolism in mung bean (Vigna radiata Wilczeck) nodules

Abstract. Water stress created by withholding irrigation in mung bean resulted in decreased leaf water potential and nodule moisture content. Decreased leaf water potential was associated with decreased activity of nitrogenase, glutamine synthetase (GS), asparagine synthetase (AS), aspartate amino transferase (AAT), xanthine dehydrogenase (XDH) and uricase. However, the activity of glutamate dehydrogenase increased three-fold under severe stress. The activity of allantoinase and allantoicase was not affected by moderate stress but decreased under severe stress. The in vitro production of allantoic acid from allantoin and uric acid in the cytosol fraction decreased more than its production from xanthine and hypoxanthine. The production of NADH also decreased under stress.
During recovery from severe stress, the activity of XDH and uricase further decreased, whilst that of allantoinase and allantoicase increased compared to the control. This corresponded with the higher content of ureides during recovery. The recovery in other enzymes was not complete although leaf water potential and nodule moisture content recovered fully within 24 h.     

Bacterial phytotoxins, syringomycin, syringostatin and syringotoxin, exert their effect on the plasma membrane H+-ATPase partly by a detergent-like action and partly by inhibition of the enzyme

Syringostatin is a newly discovered phytotoxin produced by a phytopathogenic bacterium Pseudomonas syringae pv. syringae lilac isolate. The effects of syringostatin and the similar phytotoxins, syringomycin and syringotoxin, on H⁻-ATPase activity were investigated using cultured mung bean ( Vigna radiata L. cv. Ryokuto) cells or plasma membrane vesicles isolated from mung bean hypocotyls. ³¹P-NMR analysis of cultured cells treated with syringostatin revealed that the cytoplasmic pH was decreased. When plasma membrane was prepared by a two-step method (Dextran gradient followed by a sucrose gradient). syringostatin, syringomycin and syringotoxin inhibited the H⁺-ATPase activity in a dose-dependent manner. In contrast, these toxins stimulated H⁺-ATPase activity when plasma membrane was prepared by a one-step method (sucrose gradient). While these toxins inhibited the H⁺-ATPase activity of inside-out plasma membrane vesicles, the H⁺-ATPase activity of right-side-out vesicles was stimulated. The detergent. Triton X-100, abolished this stimulatory effect of the toxins on the H⁺-ATPase of right-side-out vesicles and of one-step purified plasma membrane. The toxins also inhibited the activity of the plasma membrane H⁺-ATPase solubilized with deoxycholate and Zwittergent 3–14. Taken together, these results indicate that these toxins exert their effects partly by a detergent-like action on the plasma membrane and partly by inhibition of the enzyme.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Heat shock increases chilling tolerance of mung bean hypocotyl tissue

The effects of heat shock on the chilling tolerance of mung bean [Vigna radiata (L.) Wilczek] seedling tissue were studied by using two measurements of chilling injury: increased 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and solute leakage. ACC oxidase activity (measured as ACC-induced ethylene production) of freshly excised mung bean hypocotyl segments was highly dependent on the temperature at which the seedlings were grown. However, this highly temperature-dependent level of ACC oxidase activity was probably a wound response since it was almost entirely eliminated by incubating the excised segments at 20°C for 3 h. In contrast, heating of excised segments to 40°C for up to 4 h resulted in a time-dependent increase in ACC oxidase activity which was sensitive to cycloheximide, indicating rapid protein synthesis during the heat treatment. ACC oxidase activity fell sharply during subsequent chilling at 2. 5°C. After 3 days of chilling, all treated segments, regardless of their initial ACC oxidase activity, showed a decline to the same low activity level and ACC oxidase activity continued to fall slowly for up to 9 days at 2. 5°C. Hypocotyl segments excised from seedlings held at 15°C showed no change in solute leakage, but leakage increased rapidly when seedlings were either chilled at 2. 5°C or heated to 32°C (just below the heat shock temperature). Chill-induced leakage from non-heat-shocked segments increased steadily with chilling duration and was unaffected by cycloheximide concentration up to day 6. Within the elevated rate of leakage on day 9, however, leakage was lower from segments exposed to 10 and 50 μM cycloheximide. Solute leakage was markedly reduced for up to 9 days when segments were heat shocked at 40°C for 3 or 4 h with or without 10 M cycloheximide, but the presence of 50 μM cycloheximide caused an initial doubling of solute leakage and a 3-fold increase after 3 days of chilling. Cycloheximide prevented formation of heat shock protection against chilling from the start at 50 μM and after 9 days at 10 μM. These results indicate that the protection afforded by heat shock against chilling damage is quantitative and probably involves protein synthesis.     

Effects of spermidine on the synthesis of α-amylase in cotyledons of mung bean seedlings

When cotyledons of mung bean [ Vigna radiata (L.) Wilczek] were treated with spermidine (3 m M ) during the first 6 h of imbibition, the development of α-amylase activity in cotyledons during the following 3 days was severely inhibited (75%) This inhibition was due to a slower accumulation of α-amylase protein, which in turn resulted from an inhibition of α-amylase synthesis. The rise in the level of α-amylase mRNA in cotyledons was also inhibited by spermidine treatment. However, the degree of inhibition of mRNA accumulation (40%) was not so marked as that of the activity of α-amylase synthesis (80%). These results are discussed in relation to the mode of action of spermidine on α-amylase expression.     

Isolation of plasma membranes from mung bean hypocotyl sections by two-phase partitioning: suitability of the technique for ageing tissues

This paper discusses the application of a particular two-phase partitioning system to the isolation of plasma membranes from heterogeneous starting material, differing in physiological age. Plasma membranes were isolated from hypocotyl segments of mung beans ( Vigna radiata L. Wilczek) on four successive days in order to examine the variation caused by ageing of the seedling. Additionally, the segments were cut at different positions of the hypocotyl to measure variation caused by position-related ageing. To assess purity and degree of contamination of the plasma membrane-enriched preparations, a series of membrane enzyme markers were screened for all isolated fractions. Glucan synthetase II activities were enriched in the plasma membrane fractions, but enrichment and recovery became less pronounced with increasing age. Plasma membrane ATPase activity affected by VO₄^3-, Ca²⁺ and K⁺ was similar in all segments throughout the time-course of the experiment (4 days). However, control ATPase activity varied with segment origin: the physiologically oldest segments showed only 75% activity compared to the youngest ones. K_m and V_max values indicated a smaller proportion of active enzyme but higher substrate affinity as the age of the segments increased. Contamination by intracellular membranes was minimal and unrelated to tissue age.     

Competition of unsaturated compounds with ethylene for binding and action in plants

The compounds 2,5-norbornadiene, cyclopentadiene, furan, pyrrole, thiophene, 1-methylpyrrole, dicyclopentadiene, methylcyclopentadiene (dimer), and cycloheptatriene have been tested for competition with ethylene for binding and for biological activity using banana fruit. In addition, acetylene, allene, and 1,3-butadiene were tested. All of these compounds competed with ethylene for binding in vitro in a Triton X-100 extract of mung bean sprouts and in vivo in tobacco leaves. Only acetylene, allene, and furan are active in giving an ethylene response in banana. the others all suppress the ethylene response. Only acetylene and allene stimulate ethylene synthesis. Most of the compounds diffuse away from the binding site upon exposure to air.R.J. Reynolds Research Apprentice