IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.     

Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca²⁺ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl₂ significantly promoted the formation and growth of adventitious roots. EGTA (a Ca²⁺ chelator) or ruthenium red (a Ca²⁺-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl₂. Furthermore, inclusion of 5 mM CaCl₂ significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl₂ decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl₂-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl₂. In addition, CaCl₂ increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl₂. These results imply that the stimulative effect of Ca²⁺ on adventitious rooting is partially related to Ca²⁺-induced changes in the activities of antioxidative enzymes and IAA oxidase.     

Diheteroarylurea derivatives as adventitious rooting adjuvants in mung bean shoots and M26 apple rootstock

The present research investigates the biological profile of eight symmetrical diheteroarylureas and phenylheteroarylureas, testing their hypothetical cytokinin-like activity and rooting activity. Cytokinin-like activity was assayed by the betacyanin (so-called amaranthin) accumulation test and by the tomato regeneration test. The rooting activity was assessed using the mung bean rooting test, the apple stem slice test and the rooting of apple microcuttings. Three compounds, 1,3-di(pyrazin-2-yl)urea (3a), 1,3-di(benzo[d]oxazol-5-yl)urea (3b) and 1,3-di(benzo[d]oxazol-6-yl)urea (3c), enhanced adventitious root formation in apple stem slice test, but only 3b and 3c were active in the mung bean rooting test. Compound 3b, that showed the best rooting activity, was also able to enhance the adventitious root formation in apple microcuttings. None of the compounds showed cytokinin-like activity.     

Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study we demonstrated that the exogenous H₂O₂ was able to promote the formation and development of adventitious roots in mung bean seedlings. Treatments with 1–100 mM H₂O₂ for 8–18 h significantly induced the formation and development of adventitious roots. Catalase (CAT) and ascorbic acid, which are H₂O₂ scavengers or inhibitors, eliminated the adventitious root-promoting effects of exogenous H₂O₂. H₂O₂ may have a downstream signaling function in the auxin signaling pathway and be involved in auxin-induced adventitious root formation. 2,3,5-Triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport, strongly inhibited adventitious rooting of mung bean seedlings; however, the inhibiting effects of TIBA on adventitious rooting can be partially reversed by the exogenous IBA or H₂O₂. Diphenylene iodonium (DPI) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H₂O₂ formation in plant cells. DPI treatment strongly inhibited the formation of adventitious roots in mung bean, but the inhibitory effects of DPI on rooting can be partially reversed by the exogenous H₂O₂ or IBA. This indicates that the formation of adventitious roots was blocked once the generation of H₂O₂ through NADPH oxidase was inhibited, and H₂O₂ mediated the IBA-induced adventitious root formation. Furthermore, a rapid increase in the endogenous level of H₂O₂ was detected during incubation with water 12–36 h after the primary root removal in mung bean seedlings. Three hours after the primary root removal, the generation of endogenous H₂O₂ was markedly induced in IBA-treated seedlings in comparison with water-treated seedlings. This implies that IBA induced overproduction of H₂O₂ in mung bean seedlings, and that IBA promoted adventitious root formation via a pathway involving H₂O₂. Results obtained suggest that H₂O₂ may function as a signaling molecule involved in the formation and development of adventitious roots in mung bean seedlings.     

Arginine and ornithine decarboxylases, the polyamine biosynthetic enzymes of mung bean seedlings

General properties and relative activities of l-arginine decarboxylase (ADC) (EC 4.1.1.19) and l-ornithine decarboxylase (ODC) (EC 4.1.1.17), two important enzymes in putrescine and polyamine biosynthesis, were investigated in mung bean (Vigna radiata L.) tissues. Both activities increase linearly with increasing concentrations of crude enzyme, but the increase in ADC activity is considerably greater. The decarboxylation reaction is linear for up to 30 to 60 minutes, and both enzymes have a pH optimum of 7.2. alpha-Difluoromethyl-ornithine inhibits ODC activity of excised roots, while increasing ADC activity.High specific activity of both enzymes is detected in terminal buds and leaves, while root and hypocotyl activity is low. Different ADC-to-ODC activity ratios are found in various tissues of mung bean plants. Substantial increase in the activity of both enzymes is detected in incubated sections as compared with intact plants. A comparison of several plant species indicates a wide range of ADC-to-ODC activity ratio.It is suggested that both ADC and ODC are active in plant tissues and that their relative contribution to putrescine biosynthesis is dependent upon the type of tissue and growth process.     

Distribution of hydrogen peroxide during adventitious roots initiation and development in mung bean hypocotyls cuttings

Previous studies have shown that hydrogen peroxide (H₂O₂) may mediate the auxin response during the formation of adventitious roots (AR). However, the mechanism and distribution of H₂O₂ during AR formation remains unclear. In this study, we investigate the spatiotemporal changes and role of H₂O₂ in AR initiation and development. Application of 5?C100 mM H₂O₂ to Mung bean (Phaseolus radiatus L.) hypocotyl cuttings induced AR formation in a dose-dependent manner. The effect was blocked by ascorbic acid (AA), an important reducing substrate for H₂O₂ reduction. Depletion of endogenous H₂O₂ by AA resulted in the significant reduction of AR emergence, suggesting a physiological role for H₂O₂ in the regulation of AR formation. Determination of H₂O₂ content showed that the level of H₂O₂ increased gradually and reached the highest value 60 h after induction of AR. Further detection of endogenous H₂O₂ by the specific fluorescent probe dichlorofluorescein diacetate (H₂DCF-DA) and 3,3??-diaminobenzidine (DAB) staining in transverse sections of the basal region of cuttings revealed that obvious H₂O₂ signals were observed in the pericycle cells between the vascular bundles 24 h after the primary roots were removed. With the development of root primordia, H₂O₂ signals increased gradually and were mainly distributed in the root meristem. AA significant inhibited the H₂O₂-dependent fluorescence and the formation of AR, suggesting an essential role of H₂O₂ generation during AR initiation and development. Furthermore, the involvement of Ca²⁺ during H₂O₂-mediated AR formation was evaluated. Ca²⁺ channel inhibitors LaCl₃ and ruthenium red (RR) and Ca²⁺ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N??,N??-tetraacetic acid (EGTA) prevent H₂O₂-induced AR formation, which indicate that the hypocotyl cuttings response to H₂O₂ depends on the availability of both intracellular and extracellular Ca²⁺ pools, and Ca²⁺ is a downstream messenger in the signaling pathway triggered by H₂O₂ to promote adventitious root formation.     

Ethylene and rooting of mung bean cuttings. The role of auxin induced ethylene synthesis and phase-dependent effects

We have re-examined the role of ethylene during rooting of mung bean cuttings. Cuttings were treated for 5 days with a low or a high concentration of NAA (naphthaleneacetic acid). During this 5 days period, we also applied STS (silverthiosulfate, an inhibitor of ethylene action) or ACC (1-aminocyclo-propane-l-carboxylic acid, a direct precursor of ethylene). At high NAA concentration, STS promoted and ACC inhibited rooting, respectively. At low NAA concentration, the effects were opposite, STS being inhibitory and ACC promotive. AVG (aminoethoxyvinylglycine, an inhibitor of ethylene synthesis) gave similar results as STS. Together, these data suggest supraoptimal and suboptimal ethylene levels in the tissue at high and low NAA concentration, respectively. We also examined whether the effect of ethylene varied during the successive phases of the rooting process. Thus, we gave 24 h pulses with either STS or ACC during the rooting treatment. During the first two days (0–48 h), ACC-pulses were promotive and STS-pulses inhibitory. Later on (48–168 h), the ACC-pulses were inhibitory and the STS-pulses promotive. Whether this effect was observed or not was dependent on the NAA concentration. These data indicate that ethylene promotes or inhibits rooting depending on the stage in the rooting process. When ACC was added only during the initial period, rooting was increased at all NAA concentrations in a NAA dose-response curve and the optimal NAA concentration remained the same. This suggests that ethylene renders more cells responsive to NAA.     

铅对绿豆幼苗生长的影响

Effects of Pb^2 on the growth of mung bean(Phaseolus radiatus L.)seedlings were conducted with an experiment.The results showed that the dry weight of roots increased by 13.01%,the dry weight of shoots decreased by 12.80%,the free proline and MDA content in leaves increased specially by 2.07% and 5.82%,the activity of POD decreased by 37.32% compared to control under 0.05mmol/L Pb^2 treatment.Under 0.5mmol/L Pb^2 treatment,the dry weights of roots and shoots decreased specially be 14.45% and 29.36%,the free proline and MDA content in leaves increased specially by 43.55% and 28.50,the activity of POD decreased by 59.37% compared to control.The results indicated that Pb^2 interfered the growth of mung bean seedlings by destroying cell membrane intergrity and decreasing the activity of POD in leaves.     

Influence of ethylene on adventitious root formation in mung bean hypocotyl cuttings

The relationship between ethylene and adventitious root formation in mung bean hypocotyl cuttings was studied.Ethephon, an ethylene-releasing compound, at 5 x 10 -5 M increased root number and root dry weight on hypo-cotyl cuttings. When ethephon was applied to hypocotyl at different times after excision, there were two effectivetimes for root production i.e. between 06 h and 18-24 h. These two time periods correspond to the induction phase and the late initiation phase of root development, respectively. After excision, three peaks of ethylene productionwere observed. The first peak commencing at 6 h started the sequence of reactions leading root formation, the second peak appearing at 12 h coincided with the beginning of the increase of the IAA level during primordia initiation, and the third peak showing at 48 h played a role in root differentiation and growth. Ethylene stimulated rooting by enhancing the increase in auxins. Thus it appears that the IAA-induced ethylene production may be a factor involved in the stimulation of adventitious root formation.     

钙在IAA诱导绿豆下胚轴原生质体膨大过程中的作用

This paper studied on the role of calcium in IAA-induced swelling of protoplasts isolated from hypocotyl in etiolated mung bean (Phaseolus radiatus L.) seedlings. Protoplasts incubated in CaCl2-bearing medium without hormone maintained a constant volume and a consistent intensity of 45Ca2+ radioactivity. To treat with IAA, they began to swell and continually swelled to the maximum volume 30 minutes later (Fig. 2). However, the protoplasts could not swell when IAA was added into the medium without CaCl2 (Fig. 1). It was suggested that Ca2+ may be necessary for IAA to induce protoplast swelling. And also, IAA enabled the protoplasts to swell in less extent with K+, Zn2+, Ba2+ or Mg2+ instead of Ca2+ (Fig. 3). Radioisotope experiments showed that K+ influx increased when K+ replaced Ca2+ (Fig. 4), and water absorption plays a role in the swelling (Fig. 5). 45Ca2+ accumulation in protoplasts treated by IAA was much higher than that of control, and the time course of 45Ca2+ accumulation was similar to that of protoplasts swelling (Fig. 6). 45Ca2+ level and the swelling of protoplasts sharply declined when EGTA, verapamil or LaCl3 was added into the medium (Table 1, 2 and 3). These results indicated that Ca2+ may play an important role in IAA-induced swelling.     

The activity of antioxidant enzymes in Arabidopsis thaliana exposed to colchicine and H2O2

Studies on the possible interference of colchicine and H2O2 with the activity of some antioxidant enzymes were carried out on Arabidopsis thaliana v. Columbia grown in Murashige and Skooge nutrient medium. Measurements of superoxide dismutase (SOD), guaiacol peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) activities were conducted spectrophotometrically. In the presence of colchicine, SOD activity increased, while CAT, APX and POX activities decreased. Inhibitory H2O2 effects on the activity of the enzymes were found. Colchicine pre-treatment resulted in an increase in CAT activity and a further increase in SOD activity in plants treated with H2O2.     

The dynamic distribution of NO and NADPH–diaphorase activity during IBA-induced adventitious root formation

Nitric oxide (NO) is a multifunctional molecule involved in numerous physiological processes in plants. In this study, we investigate the spatiotemporal changes in NO levels and endogenous NO‐generating system in auxin‐induced adventitious root formation. We demonstrate that NO mediates the auxin response, leading to adventitious root formation. Treatment of explants with the auxin indole‐3‐butyric acid (IBA) plus the NO donor sodium nitroprusside (SNP) together resulted in an increased number of adventitious roots compared with explants treated with SNP or IBA alone. The action of IBA was significantly reduced by the specific NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO), and the nitric oxide synthase (NOS, enzyme commission 1.14.13.39) inhibitor, N^G‐nitro‐l ‐arg‐methyl ester (l ‐NAME). Detection of endogenous NO by the specific probe 4,5‐diaminofluorescein diacetate and survey of NADPH–diaphorase activity (commonly employed as a marker for NOS activity) by histochemical staining revealed that during adventitious root formation, NO and NADPH–diaphorase signals were specifically located in the adventitious root primordia in the basal 2‐mm region (as zone I) of both control and IBA‐treated explants. With the development of root primordia, NO and NADPH–diaphorase signals increased gradually and were mainly distributed in the root meristem. Endogenous NO and NADPH–diaphorase activity showed overall similarities in their tissue localization. Distribution of NO and NADPH–diaphorase activity similar to that in zone I were also observed in the basal 2–4‐mm region (zone II) of IBA‐treated explants, but neither NO nor NADPH–diaphorase signals were detected in this region of the control explants. l ‐NAME and c‐PTIO inhibited the formation of adventitious roots induced by IBA and reduced both NADPH–diaphorase staining and NO fluorescence. These results show the dynamic distribution of endogenous NO in the developing root primordia and demonstrate that NO plays a vital role in IBA‐induced adventitious rooting. Also, the production of NO in this process may be catalyzed by a NOS‐like enzyme.     

Radiation inactivation analysis of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings

Radiation inactivation analysis was employed to determine the functional masses of enzymatic activity and proton translocation of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings. The activities of H(+)-pyrophosphatase decayed as a simple exponential function with respect to radiation dosage. D(37) values of 6.9+/-0.3 and 7.5+/-0.5 Mrad were obtained for pyrophosphate hydrolysis and its associated proton translocation, yielding molecular masses of 170+/-7 and 156+/-11 kDa, respectively. In the presence of valinomycin and 50 mM KCl, the functional size of H(+)-pyrophosphatase of tonoplast was decreased, while that of submitochondrial particles remained the same, indicating that they are two distinct types of proton pump using PP(i) as their energy source.     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.     

Anatomical and biochemical changes during adventitious rooting of apple rootstocks MM 106 cultured in vitro

Adventitious rooting in microcuttings of Malus rootstocks MM106 was studied as regards their histological and biochemical aspects. Microcuttings from shoots raised in Murashige and Skoog's (1962) medium were transferred into a rooting medium containing IBA in the dark, then fixed 0, 3, 5, 7 and 10 days after. Some cambial zone and adjacent phloem cells became dense cytoplasm, nuclei with prominent nucleoli and the first cell divisions were observed at day 3. Meristemoids became individualized, consisting of densely staining cells (with enlarged nucleoli) formed outside the xylem by day 5. Identifiable root primordia with a conical shape and several cell layers were present at day 7. Roots with organized tissue system emerged from the stem 10 days after the root induction treatment. From these histological observations, it can be established that the rooting induction stage ended before day 3. The initiation stage, with the first histological modifications to the formation of meristemoids, would correspond to the transient increase of our biochemical marker (peroxidase activity) until day 5. The best rooting percentage obtained with cultures in the presence of auxin during 5 days confirms this hypothesis. The expression of rooting can then take place.     

The interaction between H<Subscript>2</Subscript>O<Subscript>2</Subscript> and NO,Ca<Superscript>2+</Superscript>, cGMP,and MAPKs during adventitious rooting in mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In the present study, we present a signaling network involving H₂O₂, nitric oxide (NO), calcium (Ca²⁺), cyclic guanosine monophosphate (cGMP), and the mitogen-activated protein kinase (MAPK) cascade during adventitious rooting in mung bean seedlings. Both exogenous H₂O₂ and the NO donor sodium nitroprussiate were capable of promoting the formation and development of adventitious roots. H₂O₂ and NO signaling pathways were elicited in parallel in auxin-induced adventitious rooting. Cytosolic Ca²⁺ was required for adventitious rooting, and Ca²⁺ served as a downstream component of H₂O₂, as well as cGMP or MAPK, signaling cascades. cGMP and MAPK cascades function downstream of H₂O₂ signaling and depend on auxin responses in adventitious root signaling processes.     

The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting

The recently discovered group of plant hormones, the strigolactones, have been implicated in regulating photomorphogenesis. We examined this extensively in our strigolactone synthesis and response mutants and could find no evidence to support a major role for strigolactone signaling in classic seedling photomorphogenesis (e.g. elongation and leaf expansion) in pea (Pisum sativum), consistent with two recent independent reports in Arabidopsis. However, we did find a novel effect of strigolactones on adventitious rooting in darkness. Strigolactone‐deficient mutants, Psccd8 and Psccd7, produced significantly fewer adventitious roots than comparable wild‐type seedlings when grown in the dark, but not when grown in the light. This observation in dark‐grown plants did not appear to be due to indirect effects of other factors (e.g. humidity) as the constitutively de‐etiolated mutant, lip1, also displayed reduced rooting in the dark. This role for strigolactones did not involve the MAX2 F‐Box strigolactone response pathway as Psmax2 f‐box mutants did not show a reduction in adventitious rooting in the dark compared with wild‐type plants. The auxin‐deficient mutant bushy also reduced adventitious rooting in the dark, as did decapitation of wild‐type plants. Rooting was restored by the application of indole‐3‐acetic acid (IAA) to decapitated plants, suggesting a role for auxin in the rooting response. However, auxin measurements showed no accumulation of IAA in the epicotyls of wild‐type plants compared with the strigolactone synthesis mutant Psccd8, suggesting that changes in the gross auxin level in the epicotyl are not mediating this response to strigolactone deficiency.     

The effect of ethylene on adventitious root formation in mung bean (Vigna radiata) cuttings

A promotive effect of ethylene on the formation of adventitious roots by mung bean cuttings was demonstrated using a recirculating solution culture system to apply dissolved ethylene. The number of roots increased in proportion to the length of exposure to the gas. Mean root numbers per cutting for a 4-day exposure to ethylene and an air control were 45 and 19, respectively. The tissue was most sensitive to a 24-h ethylene “pulse” 2–3 days after taking cuttings. Rooting was maximal at a concentration of 13 μl 1^?1 ethylene. The ethylene treatment inhibited the growth of roots and terminal buds. Application of Ag⁺, as silver thiosulfate, reversed the effect of ethylene on the two growth responses but had no effect on root numbers. Norbornadiene, another inhibitor of ethylene action, reversed all three ethylene responses.     

Purification and characterization of isoforms of beta-galactosidases in mung bean seedlings

Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.