Expression of a-Amylase in Phaseolus vulgaris and Vigna mungo Plants

Levels of starch and soluble sugar in pods of Phaseolus vulgarisand Vigna mungo plants were analyzed during the course of maturationof fruits. The results suggest that the immature pods of P.vulgaris function to some extent as temporary reservoirs ofcarbohydrates for growth of seeds. A less clear pattern of accumulationof starch was observed in pods of maturing fruits of Vigna mungo.Measurements of a-amylase activites in pods of maturing fruitsand immunoblotting with an antiserum against     

Alicyclic acid metabolism in plants 7. Metabolism of 14G-labelled alicyclic acids in germinating Phaseolus mungo seeds

When Phaseolus mungo seeds were allowed to germinate in 5–20mM quinate solution in the dark, a marked increase in the endogenousshikimic acid level occurred in their tissues. The acid levelrose distincdy on the 2nd day of germination and reached a maximumon the 4th day. The quinate-¹⁴C fed to germinating seeds waspredominantly converted to shikimic acid, and little radioactivitywas found in 3-dehydroshikimic acid. When quinate-¹⁴C was suppliedsimultaneously with relatively high concentrations of 3-dehydroquinicacid or 3-dehydroshikimic acid, its conversionto shikimic acidwas restrained, but hardly any radioactivity was trapped ineither of the dehydro compounds. 3-Dehydroquinic acid-¹⁴C or3-dehydroshikimic acid-¹⁴C fed to the seeds was metabolizedpreferentially to shikimic acid. The experimental results arediscussed with respect to the metabolic relationship betweenquinic acid and odier alicyclic acids in the aromatic biosynthesisof P. mungo seedlings. (Received October 16, 1975; )     

Further Studies on Glucose Catabolism in the Early Phases of Germination of Phaseolus mungo Seeds

In the early stage of germination of Phaseolus mungo seeds,oxygen uptake was little inhibited by iodoacetate and sodiumfluoride, yet carbon dioxide output was strongly inhibited.The inhibitors also decreased alcohol formation severely inthis stage. It was shown that NaF attacked its target (enolase)and curtailed the glycolytic activity. On the basis of theseresults, a mechanism for the insensitivity of oxygen uptakeis proposed.     

Cytokinins in Root Nodules of Phaseolus mungo

Four cytokinins have been separated from extracts of root nodulesof Phaseolus mungo by thin-layer chromatography. Their activitywas determined on the basis of their ability to induce betacyaninsynthesis in cotyledons of Amaranthus caudatus. Zeatin and itsriboside showed greater activity than N⁶ (²-(isopentenyl)) aminopurine and its riboside in the bioassay. Phaseolus mungo, mung bean, cytokinins, isopentenyl, amino-purine, zeatin, betacyanin synthesis, Amaranthus caudatus     

Physiological Studies on Germination of Phaseolus mungo Seeds: II. GLUCOSE AND ORGANIC-ACID METABOLISMS IN THE EARLY PHASES OF GERMINATION

The metabolism of glucose and organic acids was studied in theearly phases of germination of Phaseolus mungo seeds. Duringthe first 5–6 h of imbibition, ethanol fermentation activelyoccurred and in the following stage the TCA cycle seemed tobe markedly activated. CO₂ fixation seemed to be low, at leastin the early stage of germination. Aspartic acid was convertedactively into malic acid. This seemed to be a cause of the activesynthesis of malic acid observed.     

Physiological Studies on Germination of Phaseolus mungo Seeds: I. DEVELOPMENT OF RESPIRATION AND CHANGES IN THE CONTENTS OF CONSTITUENTS IN THE EARLY STAGES OF GERMINATION

Changes in respiration rate and in the contents of various constituentsduring the early period of germination of Phaseolus mungo seedswere studied. The course of the respiration developed in threephases. A sharp rise was observed in the first phase (Phasea), followed by the second phase (Phase b) of fairly constantrespiration rate. The respiration rate increases again in thethird phase (Phase c). The O² uptake in Phases a and b was notinhibited by iodoacetate and fluoroacetate, while that in Phasec was inhibited. The contents of aspartic and glutamic acidsand amides were very high. A decrease of aspartic acid contentwas notable during the first few hours of germination. Citricand malic acids were the major organic acid constituents. Citricacid content in the seeds decreased, while that of malic acidremained unchanged. The leaching of malic acid into the soakingmedium was remarkable during the first 6 h of imbibition     

Development of Glycolytic and Mitochondrial Activities in the Early Phase of Germination of Phaseolus mungo Seeds

The course of development of mitochondrial activities was studiedin the early stage of germination of Phaseolus mungo seeds.Mitochondrial activities (state 3 oxygen uptake rate, respiratorycontrol, and ADP/O values) increased, while the activities ofglycolytic enzymes (aldolase, glyceraldehyde-3-phosphate dehydrogenase,and pyruvate kinase) hardly changed, during the early periodof imbibition. The activities of mitochondrial enzymes (malateand succinate dehydrogenases, and cytochrome oxidase) increasedduring the period, but the rates were still low compared withthose of glycolytic enzymes. On the basis of these results,a significant difference in activation patterns between glycolyticand mitochondrial activities is discussed.     

Synergistic effect of ribose and 2-deoxy-ribose with nutrition and auxin in rooting hypocotyl cuttings of Phaseolus mungo

Exogenously supplied ribose and deoxyribose in a medium containingsucrose+ IAA considerably enhanced the formation of roots onhypocotyl cuttings of Phaseolus mungo with intact apex andleaves.The effect increased with increasing concentration of pentosesugars and was more pronounced with deoxyribose than with ribosesugar. (Received October 25, 1975; )     

Structure and Expression of a-Amylase Gene from Vigna mungo

A single copy of the a-amylase gene, composed of three intronsand four exons, was found in Vigna mungo. Examination of levelsof a-amylase and its mRNA in detached cotyledons indicated thatattachment of the embryonic axis is not required for expressionof the gene in cotyledons of germinating seeds. (Received December 21, 1993; Accepted March 14, 1994)     

1,3,7-Trimethylxanthine,an allelochemical from seeds ofCoffea arabica: Some aspects of its mode of action as a natural herbicide

Summary Certain aspects of selective toxicity of 1,3,7-trimethylxanthine (1,3,7-T), a natural herbicide, were studied inPhaseolus mungo andAmaranthus spinosus. Treatment of seeds ofA. spinosus with 1,3,7-T (sub-lethal concentration) caused a marked decrease (29.5%) in amylase activity. Kinetic studies with respect to substrate saturation and Km values as well asin vitro treatments of the cell-free enzyme indicated no effect on its catalytic property. The inhibitory effect of 1,3,7-T on amylase activity could not be counteracted with treatments of GA₃. A general biochemical analysis revealed that the starch/sugar ratio increased in the seedlings grown from treated seeds. Analysis also showed a higher ratio for insoluble/soluble nitrogen. It is suggested that inhibition of germination is caused by reduced synthesis of amylase which is not mediated through gibberelic acid. Treatment of seeds ofPhaseolus mungo with, 1,3,7-T. caused no change in amylase activity,in vivo nitrate reductase activity, ethanol soluble and insoluble nitrogen and protein content.     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

p. 86, line 6, should read: These patterns of soluble protein synthese are similar to thoseobserved after precocious maturation and subsequent rehydrationof castor bean (Kermode and Bewley, 1985a, b, 1986), and withinaxes of Phaseolus vulgaris L. seeds (Dasgupta and Bewley, 1982). instead of: These pattern of protein synthesis. Unlike castor bean thoseobserved after precocious maturation and subsequent rehydrationof castor bean (Kermode and Bewley, 1985a, b, 1986), and withinaxes of Phaseolus vulgaris L. seeds (Dasgupta and Bewley, 1982).     

Alicyclic acid metabolism in plants 8. Association, of two enzymes of the shikimate pathway in shoots of Phaseolus mungo seedlings

The association of two enzymes involved in the shikimate pathway,3-dehydroquinate hydro-lyase (EC 4.2.1.10 [EC] ) and shikimate: NADPoxidoreductase (EC 1.1.1.25 [EC] ), was studied with shoots of etiolated4-day-old Phaseolus mungo seedlings. The enzymes were not separableby ammonium sulfate fractionation, sucrose density gradientcentrifugation, polyacrylamide gel electrophoresis and chromatographyon Sephadex G-100 and DEAE-Sephadex A-50. The results are discussedin relation to the channelling function of metabolites in thealicyclic acid metabolism in higher plants. (Received October 28, 1975; )     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

Acid Phosphatase in Cotyledons of Germinating Vigna mungo Seeds

A marked increase in acid phosphatase activity took place incotyledons of germinating Vigna mungo seeds. The attachmentof axis organs was not required for this development of enzymeactivity in cotyledons. DEAE-cellulose column chromatographyrevealed that the phosphatase is composed of at least threeforms. (Received August 19, 1981; Accepted October 30, 1981)     

Inhibition of Synthesis of Peroxidases and Some Proteins by Heliangine in Phaseolus mungo Hypocotyl Sections

The peroxidase activity in Phaseolus mungo hypocotyl sectionsfloated on water decreased during the first 3 h and then increasedagain. The activity of this enzyme was reduced by heliangineand cyeloheximide in vivo, but not in vitro. Peroxidase activityis inhibited by heliangine and cycloheximide since the enzymeis not synthesized. Disk gel electrophoresis studies revealed that at least sixspecies of proteins, soluble in Tris-HCl, pH 7.6, were synthesizedin Phaseolus hypocotyl sections during 24 h incubation on water.Heliangine inhibited the synthesis of two of them, a speciesof peroxidase and another protein. Heliangine inhibited the incorporation of radioactive leucineinto the cell wall fraction, suggesting that it inhibits synthesisof cell wall proteins. It did not, however, inhibit the incorporationof labelled proline into the cell wall fraction. The resultssuggest that heliangine inhibits the synthesis of only someproteins.     

Regeneration of Fertile Plants from Callus in Phaseolus polyanthus Greenman (Year Bean)

For the first time, plant regeneration of several domesticatedgenotypes of Phaseolus polyanthus Greenman (year bean) has beenachieved. Thidiazuron in combination with indole-3-acetic acidwas used to induce morphogenic, green nodular callus from explantsthat had been obtained either from greenhouse-grown plants orfrom in vitro -germinated seeds. Of the six genotypes of P.polyanthus tested, five produced shoots in vitro. Regeneratedshoots that formed roots in vitro were established in the greenhouse,whereas non-rooted shoots could be established in vitro by grafting.Morphologically normal progeny plants were obtained from thegreenhouse-established regenerants. However, by using the sameprocedure, no regeneration response was observed in two domesticatedand two wild genotypes of P. coccineus L. (runner bean). Thisprotocol should help achieve Agrobacterium - or particle bombardment-mediatedgenetic transformation to improve this important food legume.Copyright 2001 Annals of Botany Company Callus, genetic transformation, grafting, legumes, organogenesis, Phaseolus coccineus L., Phaseolus polyanthus Greenman, Phaseolus vulgaris L     

Control of the Hypocotyl Hook Angle in Phaseolus mungo L.: the Role of Parts of the Seedling

Phaseolus mungo L. seedlings were grown in the dark for 5 d.They were either kept in the dark or exposed to 5 min red light.The location of the light-sensitive region (the hook) and theanatomy of light-induced opening are described. The responsewas phytochrome-mediated. The time course of light-induced openingand its far-red reversal are described and the implicationsfor the mechanism of light-induced opening discussed. The lagphase between irradiation and the start of opening was >8h, for 3 h of which the response remained completely photoreversible.This implies that some critical level of a factor is requiredbefore hook opening can be initiated. The terminal parts (leavesand cotyledons) kept the hooks shut in the dark, but did notdirectly affect the light response. The necessity for distinguishingbetween treatment effects on hook angle and on light-inducedopening is noted. We hypothesize that the terminal parts produceda hook-maintaining factor whose level declined after their removal.This factor was dispersed or inactivated following irradiation,or the hook cells were rendered less sensitive to it. Differencesbetween the responses of P. mungo and those reported for otherspecies are discussed.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.