Involvement of photobleaching and inhibition of protochlorophyll(ide) accumulation in tentoxin effects on greening of mung bean seedlings

Several types of evidence indicate that tentoxin-caused reduction of chlorophyll accumulation in greening primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] is due to both photobleaching and decreased protochlorophyll(ide) synthesis. Greening was greater under dim (2.5 μmol m_-2 s_-1) far-red or white light than under bright (180 to 200 μmol m_-2 s_-1) white light in tentoxin-treated tissues, whereas there was a positive correlation between fluence rate and greening in control tissues. Under continuous white light (100 μmol m_-2 s_-1) chorophyll(ide) accumulation was slower in tentoxin-treated than in control tissues. This was caused by greater photobleaching of newly formed chlorophyll(ide), as well as by decreased protochlorophyll(ide) synthesis. Photobleaching did not affect protochlorophyll(ide) synthesis in control or tentoxin-treated tissues. Chlorophyll(ide) was less stable in tentoxin-treated than in control tissues during a 24 h period of darkness. Plastids of tentoxin-treated tissues had all of the chlorophyll-proteins of control plants. Etioplasts of tentoxin-treated plants contained normal galactolipid contents, but galactolipids in these plants were greatly reduced in white light. Reduced chlorophyll accumulation caused by tentoxin is apparently the result of both photodestruction and of reduced synthesis of chlorophyll.     

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.     

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.     

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.     

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.     

Chill-induced wilting and hydraulic recovery in mung bean plants

• 1 The hydraulic conductance of roots of chilling-sensitive mung bean plants is reduced markedly at low temperatures. When roots are chilled suddenly under high irradiance, or when plants with roots chilled in the dark are exposed to a natural dawn, the stomata remain open for several hours. During this period the plants may wilt severely if the evaporative demand is sufficiently large. Under lower evaporative demand and less severe wilting, the plants may subsequently rehydrate.
• 2 Following root chilling there is a rapid (> 30 min) initial change in root conductivity (3. 2- fold).
• 3 Within hours the hydraulic conductivity of the pathway from stem xylem to leaf tissue decreases dramatically.
• 4 Within 1 d, the hydraulic conductivity of the roots decreases further (4. 5-fold).
• 5 Over 5 d these large resistances disappear and conductivity recovers to a value greater than at the start of chilling. This response and stomatal closure allow the plant to rehydrate to a condition similar to that of controls.
• 6 There is no simple relation between this hydraulic recovery and the accumulation of abscisic acid in the roots.

     

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.     

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.     

Water and solute flow through mung bean roots under applied pressure

The technique of applying hydrostatic pressure on the root medium to study water and solute flows through excised plant roots and to study various characteristics of roots in relation to flow has been used by many workers but flows in excised roots have not been compared with those in intact transpiring plants. In the present study this comparison has been made using mung bean roots. Results show that excised roots under pressure lack the ion selectivity which is observed in intact plant roots and conduct salt many times higher than salt flows through intact plant roots. The role of stem resistance in the rates of water and salt flow through roots has been discussed. The suitability of this technique for solute flow studies through mung bean roots is questioned.     

Effect of water and salt stresses on growth, chlorophyll, mineral ions and organic solutes contents, and enzymes activity in mung bean seedlings

The experiment was made by using different concentrations of polyethylene glycol (PEG) or salt solutions to decrease the osmotic potential of the growth medium to reveal the response of mung bean (Vigna radiata) to water and salt stresses. No germination (emergence of the seedling) occurred at medium osmotic potential lower than -1.0 MPa in all treatments. It was found that the activity of α-amylase and protease, and contents of proline, saccharides and the soluble proteins decreased in the germinating seeds during 3-d stress. However, after 10-d stress, the contents of organic solutes and the activity of the hydrolytic enzymes increased. Growth, chlorophyll content and mineral uptake were also significantly reduced under stress. The seedlings under water stress induced by PEG were affected much more than under salinity. This may be due to the maintenance of a higher succulence under salt stress than under PEG-induced water stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Pullulanase in mung bean cotyledons. Purification, some properties and developmental pattern during and following germination

Starch debranching enzyme was purified from mung bean ( Vigna radiata ) cotyledons to investigate its properties and developmental pattern during and following germination. A debranching enzyme was purified up to the step where only a doublet of polypeptides with molecular masses of 99 and 101 kDa, respectively, was detected by SDS-PAGE. The enzyme is thought to be a single chain monomer, as the molecular mass of the enzyme determined by gel filtration was 72 kDa. Monoclonal antibodies raised against the purified preparation recognized the doublet, indicating that the two polypeptides have immunological homology to each other. The enzyme preparation showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with glycogen. These substrate specificities indicate that the debranching enzyme from mung bean cotyledons is of the pullulanase type. Immunoblotting profiles revealed that the enzyme is present in dry seeds and decreases gradually after imbibition, suggesting the possibility that the pullulanase plays a role in developing mung bean cotyledons.     

Tentoxin does not cause chlorosis in greening mung bean leaves by inhibiting photophosphorylation

Effects of the fungal toxin, tentotoxin, on development and chlorophyll accumulation of plastids of primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] were studied using spectrophotometric, electrophoretic, and microscopic procedures. In etioplasts of control tissues both prolamellar bodies and prothylakoids occurred, whereas small vesicles were associated with structurally distinct prolamellar bodies in tentoxin-affected etioplasts. As determined by in vivo spectrophotometry, tentoxin-affected etioplasts had 25% less phototransformable protochlorophyll(ide) and 35% less non-phototransformable protochlorophyll(ide) than had control etioplasts after 5 days of dark seedling growth. Tentoxin had no effect on the rate of the Shibita shift. Protochlorophyll(ide) resynthesis in the dark immediately after protochlorophyll(ide) phototransformation was five to six times slower in tentoxintreated than in control tissues. Effects on chlorophyll(ide) content were observed within 30 min of the beginning of continuous white light exposure. In vivo measurement of cytochrome f redox activity revealed that this cytochrome was linked to light-driven electron flow in control tissues within 20 min of the beginning of continuous white light, whereas in the tentoxin-treated tissues there was no linkage (despite the presence of cytochrome f ) at any time. Coupling factor 1 was present and had potential ATPase activity in both control and tentoxin-affected plastids. There was about sixteen times more chlorophyll in control than in tentoxin-treated tissues in continuous as well as in intermittent (2 min light/118 min dark) light. These data are consistent with the view that tentoxin disrupts normal etioplast and chloroplast development through a mechanism unrelated to photophosphorylation.     

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.     

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     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

Comparison of the developmental patterns of mitochondrial malate dehydrogenase between mung bean and cucumber cotyledons during and following germination

The development of mitochondrial NAD⁺-malate dehydrogenase (EC 1.1.1.37) in mung bean and cucumber cotyledons was followed. using the antibody raised against it, during and following germination. The developmental patterns were quite different between the two. In cucumber, the content of mitochondrial malate dehydrogenase continued to increase through 3–4 days after the beginning of imbibition. This was, at least in part, due to active synthesis of the enzyme protein, and the synthesis seemed to be regulated by the availability of the translatable mRNA for the enzyme. In mung bean, on the other hand, the enzyme was present in dry cotyledons at a rather high concentration, and remained at a constant level between day 1 and day 3 after the reduction of the content to one-half its initial level during the first day. De novo synthesis of the enzyme could not be detected in mung bean cotyledons by pulse-labeling experiment.