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)     

In Vivo Synthesis and Turnover of alpha-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds

α-Amylase activity increased in attached cotyledons of germinated Vigna mungo seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double immunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. α-Amylase contents, determined by single radial immunodiffusion method, changed in parallel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of α-amylase in V. mungo cotyledons.

The rate of incorporation of the label from [³H]leucine into α-amylase and the ratios of dpm in α-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of α-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons α-amylase was synthesized and accumulated, because of low degrading activity during incubation.

     

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)     

The Yellowing of Attached and Detached Cucumber Cotyledons

Measurements of various constituents and the activities of variousenzymes have been made in attached and detached cotyledons ofCucumis sativus. The complex changes observed suggest that thereis a reproducible pattern. Comparison of results for attachedand detached material shows that, although both become yellow,the other biochemical changes associated with yellowing arenot the same.     

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)     

Imunohistochemical Localization of alpha-Amylase in Cotyledons of Vigna mungo Seedlings

We studied the localization of α-amylase with indirect fluorescence microscopy in transversely sectioned cotyledons of Vigna mungo seedlings. Tissue sections were fixed in periodate-lysine-paraformaldehyde and treated with anti-α-amylase immunoglobulin G followed by fluorescein isothiocyanate labeled goat anti-rabbit immunoglobulin G. α-Amylase appeared in the cells farthest from vascular bundles on the second day of growth and appeared gradually closer to the vascular bundles as growth progressed. The pattern of α-amylase appearance was similar in detached cotyledons, indicating that attachment of the embryonic axis has no effect on this pattern. However, in attached cotyledons, α-amylase disappeared from the regions where starch grains had been digested, but in detached cotyledons there was no disappearance of α-amylase, and digestion was slower than in intact cotyledons.     

The Respiration of Mature Field Bean (Vicia faba L.) Leaves During Prolonged Darkness

Dark respiration in attached and detached mature leaves of thefield bean (Vicia faba L.) was studied whilst leaves experiencedup to 60 h of darkness. The results showed: (1) the initialrespiration rate to vary according to the irradiance duringthe previous photoperiod; (2) the dark respiration rate (perunit area) of attached leaves to be essentially constant duringa normal 12 h night although there was a rapid loss in leafd. wt during this time; (3) after 12 h, the respiration rateof attached leaves decayed to an asymptotic value at about 36h; (4) the respiration rate of leaves detached at the end ofthe photoperiod and maintained in the dark on deionised water,decayed only after 36 h of darkness; (5) there was no differencebetween the respiration rate of attached and detached leavesduring the normal 12 h night. It is concluded that the dark respiration of attached fieldbean leaves is intially related to the synthesis and translocationof sucrose in addition to maintenance. After about 36 h, whenthe rate of CO₂ efflux is more or less steady, the CO₂ effluxreflects the intensity of maintenance processes only. The maintenancerespiration rate (determined after 60 h in the dark) rangedfrom 062 to 151 mg CO₂ (g d. wt)^–1 h^–1 but wasrelatively unaffected by several applied treatments. Vicia faba L., field bean, respiration, maintenance, nitrate, non-structural carbohydrate, export     

Synthesis of DNA and the Development of Amylase and Phosphatase Activities in Cotyledons of Germinating Seeds of Vaccaria pyramidata

As in cotyledons of Agrostemma githago, synthesis of DNA takesplace after germination in cotyledons of Vaccaria pyramidataand is followed by the formation of hydrolases, in particular,-amylase and acid phosphatase. If DNA synthesis is inhibitedby hydroxyurea, no, or only slight, enzyme activity develops.The possible role of this DNA synthesis is discussed. Key words: DNA synthesis, amylase activity, phosphatase activity, seed germination, cotyledons, Vaccaria pyramidata     

Evidence that Ethylene is not Involved in Red-light-induced Stimulation of Chlorophyll Formation in Etiolated Cucumber Cotyledons

The involvement of ethylene in red-light-induced stimulationof chlorophyll (Chl) formation was studied because one of thered-light effects on Chl formation (the lateappearing effect)interacts with the ethylene effect in 3-day-old excised etiolatedcotyledons of cucumber (Cucumis sativus L. cv. Aonagajibai).Ethylene production by etiolated cotyledons of intact seedlingsin the dark is enhanced by a red-light pulse, but the effectdoes not occur in excised cotyledons. Application of ethylenein the dark to 3-day-old intact seedlings has little effecton Chl formation in the cotyledon during subsequent continuousillumination, although ethylene pretreatment of 5-day-old seedlingssignificantly stimulates Chl formation. Removal of endogenousethylene by mercuric perchlorate [Hg(ClO₄)₂] does not specificallysuppress the red-light action on Chl formation in both attachedand excised cotyledons. Inhibition of ethylene synthesis byaminoethoxyvinylglycine does not affect the red-light effecton Chl formation in excised cotyledons. These facts indicatethat ethylene does not operate as a mediator of red light instimulating Chi formation in either attached or excised cotyledons. (Received December 13, 1981; Accepted March 30, 1981)     

cytokinin-induced amylolytic activity in bean cotyledons: Identification of the regulated enzyme

Separation of an extract of cotyledons of Phaseolus vulgarison a column of Sephadex G-200 revealed three amylolytic fractions.The slower migrating fraction hydrolyzed/ß-Limit dextrinazure and was inhibited by EDTA. The activity of this fractionwas enhanced by the embryo axis and this effect could be fullyreplaced by kinetin or benzyladenine. These results suggestthat the bean embryo axis exerts a promotive influence on theactivity of a-amylase in the cotyledons and that this effectis mediated by cytokinins. The other two amylolytic fractions did not show a-amylase activity.No effect of the embryo axis or of cytokinins on their activitycould be noted. ¹Present address: The Thimann Laboratories, University of California,Santa Cruz, California, U.S.A. (Received June 27, 1979; )     

Stimulation of dark CO2 fixation by ammonia in isolated mesophyll cells of Papaver somniferum L.

Addition of 2 mM ammonium ion to isolated mesophyll cells ofPapaver somniferum resulted in a 3-fold or greater increasein their rate of dark ¹⁴C fixation, while even 0.1 mM NH₄⁺ nearlydoubled that rate. The most rapid increase in the labeling ofa metabolite occurred in aspartate, and was accompanied by adecrease in the steady-state level of labeled phosphoenolpyruvate.No change in labeled pyruvate level occurred, and alanine labelingdeclined. Ammonium ion addition had no effect on the respiratoryrate of these cells in the dark. We conclude that NH₄⁺ stimulatesphosphoenolpyruvate carboxylation in this system, but has nodetectable effect on the pyruvate kinase reaction in the dark.Our results are compared with earlier findings and the possibleregulatory function of ammonia is discussed. (Received July 23, 1979; )     

The Inhibition of Growth by Gravitropic Stimulation in Darkness in Agravitropic Primary Roots of Zea and the Role of Calcium

The primary roots of the "Golden Cross Bantam 70" cultivar ofZea mays are agravitropic in darkness and their orthogravitropismis light-dependent. Analysis of the agravitropic roots providesimportant information about the mechanism of orthogravitropism.However, the underlying mechanism of the agravitropic responsein darkness is unknown. We found that the growth of intact primaryroots was inhibited by gravitropic stimulation (i.e., changingthe orientation of the roots from vertical to horizontal) indarkness, but that of detipped roots was not. The role of calciumin this gravistimulation-dependent inhibition of growth wasinvestigated using apical 5-mm segments of the primary roots.The gravistimulation-dependent inhibition of growth was preventedby applying 10 mM MES-KOH buffer at pH 6.0 to the root cap.By contrast, the application of 0.1–1 mM buffer at pH6.0 and 10 mM buffer at pH 4.5–5.0 allowed the gravistimulation-dependentinhibition of growth. Furthermore, when the buffer of 10 mM(pH 6.0) contained 1–5 mM CaCl₂, the gravistimulation-dependentinhibition of growth was apparent. By contrast, when weak (1mM) buffer at pH 6.0 or 10 mM buffer at pH 4.5 contained 5 mMEGTA, no gravistimulation-dependent inhibition of growth wasobserved. Thus, the gravistimulation-dependent inhibition ofgrowth in darkness seemed to be mediated by an increase in thelevel of free Ca²⁺ in the root tip. These results suggest thatfree Ca²⁺ in the apoplast of the root tip plays an importantrole in the agravitropic response in darkness as well as inorthogravitropism under light of the roots of this cultivarof Zea mays. (Received March 21, 1994; Accepted July 25, 1994)     

The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L

The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6-day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination.     

A Novel Isourazole Herbicide, Fluthiacet-Methyl, is a Potent Inhibitor of Protoporphyrinogen Oxidase after Isomerization by Glutathione S-Transferase

A novel isourazole herbicide, fluthiacet-methyl (methyl [[2-chloro-4-fluoro-5-[(5,6,7,8-tetrahydro-3-oxo-lH,3H-[l,3,4]thiadiazolo[3,4-a]pyridazin-l-ylidene)amino]phenyrjthio]acetate;experimental code name, KIH-9201) promoted the leakage of electrolytesfrom cotyledons of velvetleaf (Abtilon theophtasti Medic) andcotton (Gossypium hirsutum L.) plants that are sensitive tothis compound. It induced the accumulation of protoporphyrinIX in cotyledons of cotton and inhibited Chl biosynthesis incotyledons of velvetleaf and cotton at low concentrations (I₅₀values, 10–12 nM). Fluthiacet-methyl was converted toits urazole by glutathione S-transferase that had been partiallypurified from velvetleaf. The urazole inhibited protoporphyrinogenoxidase (Protox, EC 1.3.3.4 [EC] ) from some plants, including velvetleaf,at low concentrations (I₅₀ values, 5.1–11 nM), whereasfluthiacet-methyl was not as potent. The effects in vivo (electrolyteleakage and inhibition of Chi biosynthesis) of fluthiacet-methylwere correlated with the inhibition of Protox activity by theurazole and not with the action of fluthiacet-methyl itself.From these results, it is concluded that fluthiacet-methyl inhibitsProtox activity after conversion to the corresponding urazoleby glutathione S-transferase. It is in this way that fluthiacet-methylexerts its effect as a light-dependent peroxidizing herbicide. (Received November 1, 1994; Accepted March 6, 1995)     

The Utilization of Cotyledonary Reserves in Phaseolus vulgaris L. cv. Carioca: I. CHANGES IN TOTAL AMYLOLYTIC AND PROTEOLYTIC ACTIVITY AND THE EFFECTS OF 6-BENZYLADENINE AND GIBBERELLIC ACID UPON WHOLE SEEDLINGS

Axis growth commenced only 20 h from imbibition in Phaseolusvulgaris when both uptake of water and oxygen were levellingoff. Cotyledonary dry material was exhausted by day 9, the greatestrates of transfer occurring from day 2. Utilization of reserveprotein proceeded relatively faster than dry matter and proportionatelymore was translocated to the shoot, particularly the leaf blades.Lamina protein and chlorophyll content showed a biphasic increasewith rapid synthesis prior to day 7 being followed by slowerrates up to maximum leaf area on day 17. The level of free aminoacids in the cotyledons fell continuously from day 3 and roseto peak levels in the axis between days 5 and 7. Soluble sugarsincreased throughout the period examined in the axis and accumulatedin the cotyledons prior to day 7. Exogenous application of GA₃ had marked morphological effectsupon the seedling though did not significantly alter the distributionof dry matter or protein reserves: 6-BA, in addition to distinctmorphological effects, delayed the mobilization of reservesto the axis. Total amylolytic activity in the cotyledon, optimal at pH 5.5,increased continually from day 3 to 9 whereupon activity abruptlydeclined. This increase was due to the appearance of -amylase;ß-amylase, while present, remained at a constant andcomparatively low level. Peak proteolytic activity occurredprior to that for amylases between days 5 and 7. 6-BA significantlyincreased both amylolytic and proteolytic activities in vitrothough did not alter the changes in levels of free amino acidsor sugars in the reserve tissue and axis. The discrepancy betweenin vivo and in vitro effects of 6-BA may be attributed to concomitanteffects upon seedling development whereby sink capacity is reduced.     

Peak power output is maintained in rabbit psoas and rat soleus single muscle fibers when CTP replaces ATP

The chemomechanicalcoupling mechanism in striated muscle contraction was examined bychanging the nucleotide substrate from ATP to CTP. Maximum shorteningvelocity [extrapolation to zero force from force-velocity relation(V_max) andslope of slack test plots (V₀)], maximumisometric force (P_o), power, andthe curvature of the force-velocity curve[a/P_o(dimensionless parameter inversely related to the curvature)] weredetermined during maximumCa²⁺-activated isotoniccontractions of fibers from fast rabbit psoas and slow rat soleusmuscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTPas the nucleotide substrate. In addition to a decrease in the maximumCa²⁺-activated force in both fibertypes, a change from 4 mM ATP to 10 mM CTP resulted in a decrease inV_max in psoasfibers from 3.26 to 1.87 muscle length/s. In soleus fibers,V_max was reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by thechange in nucleotide as the result of a three- to fourfold decrease inthe curvature of the force-velocity relationship. The results areinterpreted in terms of the Huxley model of muscle contraction as anincrease in f₁and g₁ coupled toa decrease in g₂(where f₁ is therate of cross-bridge attachment and g₁ andg₂ are rates ofdetachment) when CTP replaces ATP. This adequately accounts for theobserved changes in P_o,a/P_o,and V_max.However, the two-state Huxley model does not explicitly reveal thecross-bridge transitions that determine curvature of the force-velocityrelationship. We hypothesize that a nucleotide-sensitive transitionamong strong-binding cross-bridge states followingP_i release, but before the release of the nucleotide diphosphate, underlies the alterations ina/P_o reported here.

     

Inhibition of Gibberellic Acid-induced Starch Mobilization by Salicylhydroxamic acid in Avena fatua L. Seed

Inhibition of GA₃-induced endosperm mobilization in Avena fatuaL. by salicylhydroxamic acid (SHAM), a widely used alternativerespiration inhibitor, was studied. SHAM strongly inhibitedthe GA₃-induced release of reducing sugars in the incubationmedium by 3 mm de-embryonated endosperm segments; at 4 mM SHAM,GA₃-induced sugar release was inhibited by 66–79 per cent.Extracts prepared from segments incubated in 0.05 mM GA₃ with2, 5 and 10 mM SHAM showed 30, 53 and 71 per cent lower -amylaseactivity, respectively, compared to the GA₃-alone treatment.Addition of SHAM (0.5–5 mM) during the enzyme assay hadno effect on the activity of -amylase. Thus, the inhibitionof starch mobilization in endosperm by SHAM is due to inhibitionof the production and not the activity of -amylase. The inhibitionof Avena fatua seedling growth by SHAM reported earlier may,in part, be due to its effect on endosperm mobilization. Since (1) Avena fatua seeds have been shown to have little orno SHAM-sensitive respiration, and (2) concentrations of SHAMnecessary for inhibiting endosperm mobilization were significantlyhigher than those generally necessary for inhibiting alternativerespiration, the inhibition of endosperm mobilization by thiscompound does not appear to involve its effect on alternativerespiration. Avena fatua L., wild oat, -amylase, endosperm, gibberellic acid, salicylhydroxamic acid, seed     

Changes in Levels of Cytokinins in Etiolated Squash Seedlings after Illumination

When etiolated seedlings of squash (Cucurbita maxima Duch. cv.Houkou-aokawaamaguri) were exposed to light, their cotyledonsbegan to accumulate chlorophyll at a low rate for the firsthour but at a high rate therafter. After 0, 1 and 2 h of illumination,cytokinin levels in cotyledons, hypocotyls and roots were analyzedby ELISA (enzyme-linked immunosorbent assay) and GC-SIM (gaschromatography-selected ion monitoring). Cytokinin levels measuredby ELISA were about 10 times higher than those measured by GC-SIM,suggesting that ELISA was not suitable for the measurementsof cytokinin levels in the extracts from these tissues. Cytokininsfound by GC-SIM were t-RZ (t-ribosyl zeatin), c-RZ, t-Z (t-zeatin),c-Z, RiP (ribosyl isopentenyladenine) and iP (isopoentenyladenine).Levels of biologically active cytokinins (t-RZ and t-Z) didnot show marked changes after illumination. Levels of c-RZ andc-Z did, however, decrease in cotyledons and increase in hypocotylsafter illumination. Promotion of the accumulation of the chlorophyllin detached squash cotyledons by exogenously applied t-Z waspartially inhibited by the addition of c-Z, suggesting thatthe decrease in the endogenous level of c-Z in cotyledons ofintact seedlings after illumination permits the light-inducedformation of chlorophyll. (Received August 8, 1990; Accepted April 4, 1991)     

The influence of the embryonic axis and cytokinins on reserve mobilization in germinating lupin seeds

The influence of the embryonic axis and cytokinins (CKs) onreserve mobilization has been examined in yellow lupin (Lupinusluteus L. cv. JSG 6167) seed during germination and during earlygrowth for up to 9 d in the dark. The study included determinationof starch, soluble sugars, proteins, and amino acid content.Amylolytic and proteolytic enzyme activity was also measuredin untreated cotyledons with intact embryo (attached) or detachedcotyledons (embryo removed), and in detached cotyledons followingtreatment with CKs namely, dihydrozeatin, (diH)Z, and 6-benzylaminopurine,BAP. Generally, the detached cotyledons showed reduced mobilizationand decreased enzymatic activity in comparison to attached cotyledons,indicating the importance of the embryonic axis in this process.However, a rise in protease activity and free amino acid contentwas detected in 9-d-old detached cotyledons suggesting thatthe end products do not necessarily inhibit enzyme activity.While (diH)Z was partially effective in inducing reserve mobilizationand enzymatic activity in detached cotyledons, the effect ofBAP was more pronounced and appeared to replace the embryonicaxis. The embryonic axis of this species has recently been shownto synthesize CKs which are transported to the cotyledons, arehighly stabe and induce cotyledon expansion and chlorophyllsynthesis. The results of the present investigation and previousstudies from this laboratory collectively indicate that theregulation of reserve mobilization in yellow lupin seeds appearsto be mediated, at least in part, by a stimulus, probably aCK, emanating from the embryonic axis. Key words: Lupinus luteus, cytokinins, benzylaminopurine, dihydrozeatin, embryonic axis, lupin seeds, reserve mobilization