Occurrence of Multiple Forms of {alpha}-Amylase and Absence of Starch Phosphorylase in Soya Bean Seeds

Soya bean cultivars ‘Altona’ and ‘Chestnut’have active but quite low levels of -amylase. Activity was assayedwith specific substrates, oxidized amylose and ß-limitdextrin, which were resistant to attack by ß-amylase.During seed development -amylase activity increased to a maximumin both cultivars and then declined towards maturity. Matureand germinating seeds retain low activities of -amylase. Gelelectrophoresis separated the -amylase activity into six majorbands which occurred in both cultivars. The isozyme patternwas quite similar for developing, mature and germinating seed.although the relative proportion of activity in the variousbands changed somewhat. Starch phosphorylase was not detectedin any soya bean seed samples tested, but good activity wasfound in potato tuber extracts used as a control. Mixing experimentsusing soya bean and potato extracts indicated there were noinhibiting factors in soya bean seed extracts. Soya bean seedextracts probably do not contain starch phosphorylase. Glycine max (L.), Merr, soya bean, -amylase, isozymes, phosphorylase     

Starch Deposition and Carbohydrase Activities in Developing and Germinating Soya Bean Seeds

Immature soya bean seeds accumulate starch as a transient reservematerial which is utilized later in development. Germinatingseeds also accumulate starch reserves, probably as a resultof gluconeogenesis from storage lipid. Developing beans showa rapid increase in ß-amylase activity which continuesinto early germination before declining. Distribution of ß-amylaseactivity is not consistent with its supposed role in starchdegradation. Soya bean seeds also contain -amylase and -glucosidaseactivities which could be responsible for starch mobilization. Glycine max (L.) Merr., soya bean, starch, carbohydrase, amylase, -glucosidase     

Metabolism of Abscisic Acid in Developing Seeds of Phaseolus vulgaris L. and its Correlation to Germination and {alpha}-Amylase Activity

Bean seeds were unable to germinate during their developmentwhich was characterized either by an increasing ABA content(expressed as ng ABA/seed) or by a fairly high and constantABA concentration (expressed as ng ABA g^–1 fr. wt). During seed maturation the mother plant induces a dormant stateby depressing the ABA catabolism and keeping the endogenousABA at a high level to prevent both the premature hydrolysisof starch (-amylase activity) and the germination of the morphogeneticallyadult yet immature seeds. The depth of this induced dormancyis positively correlated with endogenous ABA concentration. Application of exogenous ABA to fully mature seeds, which containno endogenous ABA and show a very active ABA catabolism, onlyprolonged the lag phase but had no influence on the furtherdevelopment of the -amylase activity.     

{beta}-Amylase Activity as an Index for Germination Potential in Rice

Seeds of different vigour also differ in their germination ability.In rice (Oryza sativa), this difference was correlated withthe level of incorporation of ³⁵S-methionine into 25-60% ammoniumsulphate precipitable material that was rich in amylase proteins.This protein fraction, from dry seeds, contained no -amylaseactivity. In contrast, ß-amylase activity was presentin all seed stocks capable of 99% germination, although thelevel was lower in seeds that grew slowly when germinated. Inlow viability low vigour stock (i.e. extensively deterioratedseeds) ß-amylase activity was absent. Alpha-amylaseactivity in all stocks was detected only after 24 h from thestart of imbibition. These results indicate that ß-amylaseactivity is reliable indicator of the germination ability ofrice seed stocks and of their vigour during germination.Copyright1995, 1999 Academic Press Rice (Oryza sativa L.,), germination, ß-amylase, -amylase, seed vigour     

Changes in {alpha} and {beta}-Amylase Activities during Germination of Seeds of Alfalfa (Medicago sativa L.)

We examined the methods available for the assay of -amylasein alfalfa (Medicago sativa) and found the Phadebas test mostsuitable. The Phadebas assay and activity staining on ampholinegels after isoelectrofocusing revealed that an amylase is presentin the dry seeds of alfalfa and that its activity decreasesrapidly after the second day of seed germination. An amylasewas purified by affinity chromatography and gel filtration.The kinds of sugar generated from soluble starch by the purifiedamylase resembled those generated by other -amylases from plants,in particular those from mung bean (Vigna radiata). These resultsindicate that the amylase in alfalfa seeds belongs to the familyof -amylases. The molecular weight and isoelectric point ofthe -amylase were determined to be 43 kDa and 4.92, respectively. The Pantrac assay and activity staining on immobiline gels afterisoelectrofocusing revealed that the activities of ß-amylasesincreased during the initial 4 to 5 days of germination. Furthermore,treatment of whole seedlings with cycloheximide or actinomycinD inhibited the increase in activity of ß-amylasesbut did not affect the reduction in activity of -amylase. During germination of alfalfa seeds, -amylase activity decreaseswhile, in contrast, ß-amylase activity increases (inthe cotyledons of germinating seeds), changes that are specificto the germinating seeds of alfalfa. (Received September 8, 1990; Accepted February 20, 1991)     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)     

Loss of Starch and Increase of {alpha}-Amylase Activity in Leaves of Flooded Tobacco Plants

The effect of flooding on the contents of chlorophyll, proteinand starch and the activity of     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

A Note on the Presence of {beta}-Amylase in Moulds

Evidence is given for the presence, in the mould Mucor Rouxianusand in several Rhisopus species, of an amylolytic enzyme producingglucose directly from starch. This glucosidase probably possessesa wide specificity range and attacks maltose as well. It doesnot destroy the iodine reaction of starch, but as a rule itis accompanied by another amylase which does destroy thb reaction.Of the mould amylases investigated, Mucor-amylase containedthe highest proportion of the glucosidase.     

Starch Degradation Metabolism towards Sucrose Synthesis in Germinating Araucaria araucana Seeds

As starch is the main seed reserve material in both species of Araucaria of South America, A. araucana and A. angustifolia, it is important to understand starch breakdown in both embryo and megagametophyte tissues of Araucaria seeds. Sugar analysis by thin layer chromatography indicates that sucrose is the main sugar produced in both tissues. Enzyme reactions coupled to benzidine oxidation indicate that sucrose is the main sugar moved from the megagametophyte to the growing regions of the embryo via the cotyledons.

Phosphorylase was detected in both embryo and megagametophyte tissues by the formation of [³²P]glucose-1-P and by formation of [¹⁴C] amylopectin from [¹⁴C]glucose-1-P. The enzyme activity increases 5-fold in both embryo and gametophyte to a peak 18 hours after the start of imbibition. Debranching enzyme, α-glucosidase, and hexokinase are also present in both embryonic and megagametophytic tissues.

Branched glucan oligosaccharides accumulate during this time, reaching a maximum 40 hours after imbibition starts, and decline after germination occurs.

The pattern of activity of the enzymes studied in this work suggests that starch degradation is initiated by α-amylase and phosphorylase in the embryo and by phosphorylase mainly in the megagametophyte. Sucrose-P synthase seems to be the enzyme responsible for sucrose synthesis in both tissues.

     

Synthesis and Turnover of {alpha}-Amylase in Cotyledons of Germinating Vigna mungo Seeds: Effects of Exogenously Applied End-Products and Plant Hormones

Pulse-chase experiments indicated that the higher levels ofa-amylase in detached and incubated cotyledons of Vigna mungothan those in cotyledons attached to the embryonic axis weredue to both faster synthesis and slower degradation of the enzymein the detached cotyledons than in the attached cotyledons.Levels of a-amylase in the cotyledons were examined in termsof possible effects of end-products and the effects of exogenouslyapplied plant hormones and growth regulators. Levels of a-amylaseactivity and content were reduced by high concentrations ofglucose and sucrose, and it is suggested that this effect wascaused mostly by osmotic stress and partly by end-product repression.The level of a-amylase was nearly twice that in controls after1 to 10µM GA₃ had been applied to the cotyledons. In addition,0.1 mM kinetin, 0.1 mM 2,4-D and 0.1 to 0.S mM naphthaleneaceticacid also increased the level by 34% to 66% as compared to thecontrol. ABA and uniconazole both prevented the synthesis ofa-amylase. (Received July 4, 1994; Accepted November 14, 1994)     

RNA Polymerase II Activity in Developing and Germinating Cowpea Seeds

RNA polymerase II was partially purified from germinating cowpeaseeds and characterized. RNA polymerase II activity per seedwas low soon after flowering but increased thereafter, reachinga maximum after about two weeks. Upon desiccation, the activitydecreased and was very low in quiescent seeds. After germination,the activity increased rapidly. (Received July 23, 1990; Accepted November 30, 1990)     

Blue Light Mediated Regulation of {beta}-Amylase Activity in Mustard (Sinapis alba L.) Cotyledons

In the cotyledons of mustard (Sinapis alba L.) seedlings grownunder continuous blue light, ß-amylase activity increasedbetween 42–96 h from sowing and thereafter the ß-amylaseactivity abruptly declined. Preirradiation with blue light didnot increase the responsivity of the subsequent phytochrome-mediatedß-amylase increase in the cotyledons. The run-offkinetics of ß-amylase increase in seedlings transferredfrom blue light to darkness indicated that the components ofthe blue light-triggered signal chain are kinetically identicalto those of the phytochrome-mediated signal chain. Far-red reversibilityexperiments showed that the above blue light response is eithermediated by phytochrome directly or the blue light photoreceptorrequires the coaction of phytochrome. (Received November 11, 1987; Accepted March 23, 1988)     

Apoplastic {alpha}-Amylase in Pea is Enhanced by Heat Stress

The apoplastic -amylase (EC 3.2.1.1 [EC] ) in pea (Pisum sativum L.cv. Laxton's Progress No. 9) seedlings was enhanced by a 4-to 6-fold with heat stress. Increased levels of the enzyme persistedfor 48 h at least after the stress and subsequent heat treatmentsenhanced the activity further. These results indicate that thefunction of apoplastic -amylase in vegetative tissues may berelated to plant stress. (Received October 14, 1996; Accepted February 20, 1997)     

Part of the Lysyl Residues in Wheat {alpha}-Amylase is Methylated as N-{varepsilon}-Trimethyl Lysine

Amino acid analyses of wheat -amylase purified from germinatingseeds by affinity chromatography showed a high content of amodified lysyl residue. The modified residue was identifiedas N--trimethyl lysine. The presence of trimethyl lysine in-amylase is discussed in terms of isozymes. ¹ Present address: National Institutes of Health, Bldg. 10,Rm. 9B-15, Bethesda, MD 20205, U.S.A. (Received August 20, 1981; Accepted March 19, 1982)     

Cyclic AMP Promotion and Abscisic Acid Inhibition of {alpha}-Amylase Activity in the Seeds of Rice (Oryza sativa L.)

Cyclic AMP and GA₃ stimulated both -amylase activity in riceendosperm and the germination of the seed. In combination theyalso induced germination of ABA-treated seeds but cyclic AMPalone failed to neutralize the inhibitory effect of ABA; withadded kinetin, however, it promoted the -amylase activity ofthe dormant seeds. The enzyme activity decreased as the storageperiod of seeds increased. Cyclic AMP and GA     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea     

Effects of Nitrate Level on Nitrogen Metabolism in Winged Bean and Soya Bean

The effects of high (15 mM) and low (0.75 mM) solution nitratelevels on nitrogen metabolism in three genotypes (IL 7A, IL13 and IL 21) of winged beans [Psophocarpus tetragonolobus (L.)DC.] and one genotype (Williams) of soya bean [Glycine max (L.)Merrill] were investigated. Plants were grown for 42 days ina greenhouse in solution culture prior to sampling. The 15 mM nitrate treatment resulted in greater growth of allplant parts except roots. Growth of soya beans was more responsiveto nitrate level than was growth of winged beans. The high nitratelevel inhibited nodulation in all plants. The IL 13 and IL 21winged bean genotypes had similar nitrogenase activity (acetylenereduction per plant) as the soya bean and IL 7A winged beangenotype had lower activity. However, the IL 13 winged beangenotype had higher nitrogenase activity (acetylene reductionper unit nodule mass) than the other three genotypes which allhad similar activity. The 15 mM solution nitrate level stimulatedleaf and root nitrate reductase (NR) activity for all plants.All winged bean genotypes had higher leaf NR activity and higherpercentage reduced- and nitrate-nitrogen contents of leavesand stems compared with soya beans. However, total protein (reducednitrogen) was greater in soya beans when sampled indicatingthat more nitrate had been metabolized by soya beans than bywinged beans during the 42-day growth period. Psophocarpus tetragonolobus (L.) DC., winged bean, Glycine max (L.) Merrill, Soya bean, nitrate reductase, nitrogen fixation, nitrogenase activity, nodulation     

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein contentof pea seeds (Pisum sativum L.) as compared to soya bean seeds(Glycine max L. Merrill) might be due to faster degradationof the pea storage proteins during development of the seed.Pea and soya bean cotyledons were subjected to a ‘pulse-chase’experiment using [³H]glycine in in-vitro cultures. In peas,legumin had a half-life of 146 days, while vicilin had a half-lifeof 39 days. There was no measureable degradation of soya beanstorage proteins. Even with the pea storage proteins, the half-liveswere so much longer than the maturation time of seeds that degradationof storage proteins could not account for the lower proteincontent of peas as compared to soya beans. The validity of theseresults was indicated by the finding that non-storage proteinshad much shorter half-lives and that omission of a carbon ora nitrogen source greatly accelerated degradation. Labelledglycine was found to be a good probe for protein turnover studiesbecause it was very rapidly metabolized. Glycine max L. Merrill, soya bean, Pisum sativum, L. pea, protein turnover, storage proteins, legumin, vicilin