Starch Metabolism in Developing and Germinating Soya Bean Seeds is Independent of {beta}-Amylase Activity

Developing seeds of soya bean cultivars Chestnut and Altonahave only trace amounts of ß-amylase activity. Comparedto a standard variety, Wells, ß-amylase activitieswere 200–300 times lower in Chestnut and Altona. Nevertheless,Chestnut and Altona accumulate starch as a transient reservematerial which is utilized later in development. Seeds of Chestnutand Altona also produce starch early in germination which subsequentlydeclines after the 4th day of germination. Throughout germinationß-amylase levels in these cultivars are about 300-foldlower than that observed in Wells, which has a similar patternof starch metabolism. Widely varying levels of ß-amylasein both developing and germinating seeds appear to be unrelatedto starch metabolism which is very similar in all cultivarsstudied. Consequently, ß-amylase activity seems irrelevantto starch metabolism in the soya bean seed. starch, ß-amylase, Glycine max. (L.), Merr, soya bean     

A Serine Protease in Soybean Seeds That Acts Specifically on the Native {alpha} Subunit of {beta}-Conglycinin

A proteolytic activity directed against the     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

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)     

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     

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.     

Impact of Lead and Cadmium on Enzyme of Citric Acid Cycle in Germinating Pea Seeds

The present investigations were conducted to ascertain the influence of Pb₂₊ and Cd₂₊ both individually and in combination on selected enzymes of tricarboxylic acid (TCA) cycle. All the enzymes of TCA cycle examined (-ketoglutarate dehydrogenase, isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase) were affected deleteriously by Pb₂₊ as well as Cd₂₊ and these metals in combination gave more or less an additive effect.     

Effect of Gibberellic Acid on the Ultrastructure and {alpha}-Amylase Activity of Aleurone Cells of Avena sativa L.

The -amylase activity and ultrastructure of aleurone cells inseeds of Avena sativa L. were studied using seed halves withembryo (embryo seeds) which had imbibed water and seed halveswithout embryo (embryo-less seeds) which had imbibed water withor without GA₃. -Amylase activity was detected in the aleurone layers of embryoseeds that had imbibed water and embryo-less seeds that hadimbibed GA₃-water. The ultrastructure of aleurone cells withdetectable -amylase activity showed marked changes in the roughsurfaced endoplasmic reticulum (rER), in the flattened sacculesforming stacks and in the aleurone grains. The progressive changesin the rER were as follows: first, the number of slender rERincreased; then, the inner space became wider and showed roundor oval profile; and finally, the rER became slender again witha reduced number of adhering ribosomes. The flattened sacculesforming stacks were appressed to the surface of aleurone grains.With time, they decreased in number and finally disappeared.In parallel with the decrease of flattened saccules, digestionof proteinaceous material inside the aleurone grains proceeded. (Received February 24, 1987; Accepted September 3, 1987)     

The Role of Glutamate Decarboxylase and {gamma}-Aminobutyric Acid in Germinating Barley

The variations in glutamate decarboxylase activity and in glutamicacid and -ABA concentration have been measured in barley embryosduring the uptake of water and in the roots and shoots for upto 6 days of growth. Glutamate decarboxylase activity was relativelysteady in the embryos during soaking but rose rapidly once growthbegan. This development paralleled an increase in the concentrationof glutamic acid in both roots and shoots at a time when theconcentration of -ABA was falling. During soaking in aeratedwater, the -ABA content of the embryos rose for 36 h, at whichpoint it accounted for 35 per cent of the soluble amino acids.-ABA was found to be a major free amino acid in roots but notin shoots. Experiments in vivo involving ¹⁴C-labelled glutamicacid and -ABA indicated that carbon from -ABA passed very rapidlyinto the citric-acid cycle intermediates and also that, throughoutthe period studied, -ABA was formed from glutamic acid despitethe alterations in relative concentrations of these amino acidsin the growing tissue.     

Uptake and Effect of Abscisic Acid during Induction and Progress of Radicle Growth in Seeds of Chenopodium album

In the seeds of Chenopodium album L. visible phenomena preceding the final protrusion of the radicle enable a clear distinction between the induction and the progress of growth inside the covering structures. The light-dependent induction of radicle growth is not inhibited by exogenously applied abscisic acid (ABA). Experiments with 1-¹⁴C-ABA ruled out a lack of penetration of the hormone. However, ABA does inhibit the growth of the radicle before final protrusion. This inhibition and the uptake of 1-¹⁴C-ABA are enhanced at lower pH values, indicating absorption of the undissociated molecule. The uptake of labeled hormone strongly increases during the growth of the radicle. This increase is not merely a reflection of extra water uptake. Seeds of different degrees of dormancy contain equallly low levels of endogenous ABA. Much higher levels of ABA in the seeds were obtained by exogenous application of the hormone but these levels stills do not prevent the breaking the dormancy by light. It is concluded that ABA has no function in the regulation of dormancy in C. album seeds.     

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)     

Effect of light treatment and endogenous growth hormones on {alpha}-and {beta}-amylase activities in cotyledons of Phaseolus vulgaris L.

The influence of light and endogenous plant-growth regulatorson the evolution of - and ß-amylases in cotyledonsof Phaseolus vulgaris L. was investigated. Both enzymes, whichare not present in ungerminated seeds, appear during germinationof intact seedlings or incubation of excised cotyledons. -Amylaseactivity decreases upon exposure to light. This inhibition iscorrelated with a drastic increase in chlorophyll content anda change in the endogenous gibberellin-inhibitor balance. ß-Amylaseactivity was not affected by light treatment but was by thepresence of endogenous cytokinins. (Received February 3, 1977; )     

Desiccation and the Control of Expression of {beta}-Phaseolin in Transgenic Tobacco Seeds

Drying of seeds at certain stages prior to maturation, i.e.premature desiccation, will terminate synthetic events uniqueto development, for example, storage protein synthesis, andinitiate processes associated with germination. In this studywe have investigated the role of desiccation in the expressionof a storage protein gene, ß-phaseolin, to determineif such a developmentally-regulated gene remains sensitive todrying when controlled by a promoter that has no known sensitivityto this treatment. We compared, in transgenic tobacco seeds,the effects of maturation and premature drying on the expressionof a full ß-phaseolin gene, and ß-phaseolingenes driven by a cauliflower mosaic virus 35S promoter withor without an alfalfa mosaic virus (AMV) 5' untranslated leadersequence. The results indicate that the ß-phaseolinpromoter is directly down-regulated by desiccation during maturationand, although activated during the drying phase of a prematuredesiccation event, it is not active upon rehydration or imbibition.The 35S promoter is down-regulated also by both maturation dryingand premature desiccation but unlike the ß-phaseolinpromoter it is reactivated upon rehydration or imbibition. Key words: Desiccation, ß-phaseolin, gene regulation, Phoseolus vulgaris, seed development     

Developmental Variation of the Neurotoxin, {beta}-N-Oxalyl-L-{alpha},{beta}-diamino propionic acid (ODAP), in Lathyrus sativus

Several species of the tribe Viceae (Leguminosae) produce non-proteinamino acids that are toxic to man and animals. The neurotoxinß-N-oxalyl-L-,ß-diamino propionic acid (ODAP)in cultivated Lathyrus sativus causes human neurolathyrism,a neurological disease resulting in the paralysis of lower limbs.Surveys have shown that there are large scale variations betweenspecies of Lathyrus and varieties of L. sativus for the amountof cellular ODAP. In the present investigation, thin-layer chromatographyand chemical analysis were used to study developmental variationin the amount of ODAP in tissues and organs of L. sativus. Theresults confirmed that the rate of synthesis and accumulationof ODAP varied during plant development. Increased rates ofsynthesis were confirmed in young seedlings and in the developingfruits of L. sativus.Copyright 1994, 1999 Academic Press Lathyrus sativus, neurotoxin, ODAP, plant development     

The Influence of Boron on the RNA Level, {alpha}-amylase Activity, and Level of Sugars in Germinating Themeda triandra Forsk Seed

The presence of boron in the germination medium markedly increasedthe RNA level and a-amylase activity in the embryo and endospermtissue of dormant 2-month-old seed. In non-dormant 14-month-oldseed the presence of boron slightly increased the level of RNA,but had virtually no effect on the -amylase activity. Gibberellicacid (GA₂) in the germination medium also increased the -amylaseactivity in the 2-month-old seed; the greatest activity wasobtained when both boron and GA₂ were together in the germinationmedium. Changes in starch, sucrose, and reducing sugar levelswere obtained in the seed, in the presence of boron. Indicationsof changes in RNA level, a-amylase activity, and sugar translocationin non-germinating seed occur between the dormant and non-dormantstate. The relationship between boron and gibberellic acid isdiscussed.     

Transcription of the {beta}-galactoside {alpha}2,6-sialyltransferase gene in B lymphocytes is directed by a separate and distinct promoter{dagger}

A single human gene, SIAT1, encodes the ß-galactoside     

The Determination of the Changes in the Free Amino Acid Content of the Eluate from Germinating Seeds of Glycine wightii (L.) and its Effect on the Growth of Lettuce Fruits

A complex pattern of changes in the free amino acid compositionof the eluate from germinating seeds of Glycine wightii wasobserved using a stepped platform apparatus. The growth of lettucefruits was strongly inhibited by the eluate from Glycine wightiiseedlings.     

The primacy of affinity over clustering in regulation of adhesiveness of the integrin {alpha}L{beta}2

Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.     

Three-dimensional EM structure of the ectodomain of integrin {alpha}V{beta}3 in a complex with fibronectin

Integrins are alphabeta heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin alphaVbeta3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn(2+)-bound alphaVbeta3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most alphaVbeta3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound alphaVbeta3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of beta3, with FN9 just adjacent to the synergy site binding region of alphaV. We conclude that the ectodomain of alphaVbeta3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.