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)     

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; )     

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     

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)     

Localizing a-Amylase Gene Expression in Germinated Rice Grains

In situ hybridization was used to localize the sites of expressionfor two a-amylase genes (RAmy1A and RAmy3D) in rice (Oryza sativaL.) over five days of germination. Messenger RNAs from bothgenes were initially detected in the scutellar epithelium andappeared at later stages of germination in the aleurone layer.RAmy3D mRNA reached its peak of accumulation 2 days(d) earlierin the scutellar epithelium than RAmy1A mRNA. Both mRNAs continuedto accumulate in the aleurone layer up to 5 d of germination,although RAmy1A mRNA reached significantly higher levels thanRAmy3D mRNA. Overall, the aleurone layer was responsible forproducing the majority of the total grain a-amylase mRNA. (Received July 27, 1991; Accepted November 6, 1991)     

Effects of 5-Aminolevulinic Acid on the Accumulation of Chlorophyll b and Apoproteins of the Light-Harvesting Chlorophyll a/b-Protein Complex of Photosystem II

The effects were examined of 5-aminolevulinic acid (ALA) onthe accumulation of Chl and apoproteins of light-harvestingChl a/b-protein complex of photosystem II (LHCII) in cucumbercotyledons under intermittent light. A supply of ALA preferentiallyincreased the accumulation of Chl a during intermittent illumination.However, when cotyledons were pretreated with a brief exposureto light or benzyladenine (BA), the stimulatory effect of ALAon the increase in the level of Chl b was greater than thatin the level of Chl a, resulting in decreased ratios of Chla/b. Time-course experiments with preilluminated cotyledonsrevealed that LHCII apoproteins accumulated rapidly within thefirst 30 min of intermittent illumination with a decline duringsubsequent incubation in darkness. A supply of ALA did not affectthe accumulation of LHCII apoproteins during the intermittentlight period, but it efficiently inhibited the decline in theirlevels during the subsequent darkness. After exposure to a singlepulse of light of BA-treated cotyledons, the prompt increasein levels of LHCII apoproteins was not accompanied by the formationof Ch b, which began to accumulate later. The pattern of changesin levels of LHCII apoproteins was quite similar to that inlevels of Chl a. These results suggest that LHCII apoproteinsare first stabilized by binding with Chl a and that an increasedsupply of Chl a and the accumulation of LHCII apoproteins areprerequisites for the formation of Chl b. ¹Present address: Department of Chemistry, Faculty of Scienceand Technology, Meijo University, Aichi, 468 Japan.     

Formation of Chlorophyll-Protein Complexes during Greening 1. Distribution of Newly Synthesized Chlorophyll among Apoproteins

The relationship between the accumulation of Chl and the apoproteinsof the light-harvesting Chl a/b-protein complex of PS II (LHCII)during the greening of cucumber cotyledons was studied. LHCIIapoproteins were not detected in etiolated cotyledons. Uponillumination, Chl a was formed as a result of photoconversionof protochlorophyllide (Pchlide) which had accumulated in thedark. During the lag period that preceded the accumulation ofChl, a small amount of LHCII apoproteins appeared. The amountof LHCII apoproteins increased with increases in levels of Chlb, though somewhat more rapidly during the first 10 h of greening.Treatment with benzyladenine (BA) or levulinic acid (LA) wasused to vary the supply of Chl a for apoproteins by promotingor inhibiting the synthesis of Chl a, respectively. LA decreasedbut BA increased the rate of accumulation of Chl b and LHCIIapoproteins. Only small amounts of Chl b and LHCII apoproteinswere formed under intermittent illumination. However, in thepresence of chloramphenicol (CAP), which inhibits the synthesisof plastome-coded proteins including apoproteins of the P700-Chla-protein complex (CP1) and a Chl a-protein complex of PS II(CPa), we observed the accumulation of Chl b and LHCII apoproteins,both of which are of nuclear origin. During incubation in thedark after intermittent exposure to light, CAP alone allowedneither destruction nor accumulation of Chl b and LHCII apoproteins,but it did enhance the effect of CaCl₂ in inducing both Chlb and these apoproteins. These results can be explained by assumingthat apoproteins of CP1 and CPa have a higher affinity for Chla than do LHCII apoproteins. When the availability of Chl ais limited, these apoproteins compete with one another for Chla, with the resultant preferential formation of CP1 and CPa.However, when the supply of Chl a becomes large enough for saturationof apoproteins of CP1 and CPa, some of the Chl a is incorporatedinto LHCII apoproteins either directly or after conversion toChl b. Thus, the formation of different Chl-protein complexes(CPs) is regulated by the relative rates of synthesis of Chla and apoproteins and by differential affinities of the apoproteinsfor Chl a. ⁴Present address: Kyowa Hakko Co., Ltd., 4041, Ami-machi, Inashiki,Ibaraki, 300-03 Japan (Received September 14, 1989; Accepted April 26, 1990)     

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)     

CHLOROPHYLL FORMATION IN ISOLATED PUMPKIN COTYLEDONS IN THE PRESENCE OF KINETIN AND CHLORAMPHENICOL

Investigations have been made on the changes in the levels ofprotochlorophyll, chlorophyll a and chlorophyll b in relationto the kinetin induced expansion of isolated pumpkin cotyledonsin the presence and absence of chloramphenicol. It has been shown that rise in pigment level keeps pace withexpansion growth of the cotyledons. Kinetin markedly promotes the synthesis of protochlorphyll withoutmuch affecting the rate of its photoreduction to chlorophyll. Chloramphenicol strongly inhibits the development of both chlorophylla and b. The inhibition seems to be due to its interferenceboth with the synthesis of protochlorophyll and its subsequentconversion to chlorophyll. The inhibitory effect of chloramphenicol on the formation ofchlorophyll a is greater than on that of chlorophyll b, suggestingthereby the probability of divergent pathways for the formationof the two chlorophylls. (Received December 21, 1966; )     

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.

     

SPECIFIC RNA FROM PHOTOPERIODICALLY INDUCED COTYLEDONS OF PHARBITIS NIL

The nucleotide ratio of several RNA species from cotyledonsof Pharbitis nil subjected to a single 16 hr night with or withouta 15 min light-break, or to continuous light was investigated.RNA species examined were RNAs from nuclear, mitochondrial,microsomal, and supernatant fractions separated by differentialcentrifugation, and s-, r-, and m-RNAs fractionated by methylatedalbumin column chromatography. Of the RNAs examined, m-RNA alone was found to change its nucleotideratio with photoperiod applied. Thus as compared with m-RNAfrom non-induced cotyledons (exposed to continuous light oran interrupted night), m-RNA from cotyledons induced by an uninterruptednight contained significantly reduced guanylic and cytidylicacids on molar ratio basis. A working hypothesis was proposed that floral stimulus productionin cotyledons may be directed by gene DNA derepressed photoperiodically. (Received October 18, 1966; )     

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.     

粘虫中肠α-淀粉酶活性测定方法的参数优化

针对粘虫Mythimna separata中肠α-淀粉酶筛选了11种不同参数组合的3，5－二硝基水杨酸活性测定方法，并对其中最适组合的各个参数进行了优化。结果表明：在离体测定条件下，粘虫中肠α-淀粉酶活性的最优化测定参数为0.03 mol/L 磷酸盐缓冲液（pH 8.0，含有55 mmol/L NaCl）、温度45℃、吸收波长480 nm。Ca²⁺对α-淀粉酶活性具有抑制作用。该优化法能够显著降低粘虫、德国小蠊Blattella germanica、黄粉虫Tenebrio molitor、淡色库蚊Culex pipiens pallens和家蝇Musca domestica等昆虫α-淀粉酶的米氏常数K_m值，且粘虫和德国小蠊α-淀粉酶的V_max值增大，但黄粉虫、淡色库蚊和家蝇α-淀粉酶的V_max值均明显减小。结果说明，在该优化体系下，粘虫α-淀粉酶与底物的亲和力增强，最大反应速度增大，测定酶活性的准确性和灵敏度显著提高；同时该优化体系也可作为测定德国小蠊α-淀粉酶活性的优化方法，但不适合作为黄粉虫、淡色库蚊和家蝇α-淀粉酶的最优化测定方法。     

Control of alpha-Amylase Development in Cotyledons during and following Germination of Mung Bean Seeds

Developmental patterns of α-amylase in Vigna radiata cotyledons during and following germination were quite different depending on the differences in the treatments of cotyledons during the imbibitional stage. When axis-detached cotyledons were imbibed in water with seed-coats attached, α-amylase activity did not increase and remained low. On the other hand, when the cotyledons were imbibed in water after seed-coat removal, the enzyme activity increased markedly. If the axis was attached to the cotyledons, α-amylase showed a marked development even under the former imbibition conditions. These changes in the enzyme activity were in parallel with those in the enzyme content, and the content, in turn, was dependent upon the availability of mRNA for α-amylase. We propose that the regulation of the development of α-amylase in cotyledons may involve some factor(s) inhibitory to accumulation of α-amylase mRNA, which is present in dry cotyledons and can be removed from cotyledons by leakage or by the presence of the axis.     

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.     

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)