Premature Drying Increases the GA-Responsiveness of Developing Aleurone Layers of Barley (Hordeum vulgare L.) Grain

The effect of premature drying on the sensitivity of aleuronelayer cells of developing barley (Hordeum vulgare L.) grainto gibberellic acid (GA₃) was investigated. The capacity ofbarley aleurone layer cells to respond to GA₃, as indicatedby -amylase synthesis and secretion by de-embryonated grain,increased during the later stages of development. Aleurone layersof immature grain (younger than 30 d after anthesis; DAA) werenot capable of producing amylase in response to GA₃; however,premature drying at this stage promoted GA-responsiveness resultingin the induction of mRNA for -amylase and increased -amylasesynthesis and secretion. Preincubation of the immature grainor its maintenance at 100% relative humidity prior to exposureof the de-embryonated grain to GA₃ also led to an enhanced capacityof the aleurone layer to produce amylase and its mRNA as comparedto the fresh, untreated grain. However, the amount of mRNA andenzyme produced was much lower than that induced by prematuredrying. Moreover, following these nondrying treatments, thealeurone layer cells remained unresponsive to exogenous GA₃;the same amount of enzyme was produced in the absence of appliedGA₃. Transient expression of chimeric gene constructs in aleuronelayer cells of de-embryonated grain suggest that drying up-regulatesthe -amylase gene promoter in response to GA₃. We conclude thatdesiccation is required for barley aleurone layer cells to becomeresponsive to GA₃ and hence express their full potential foramylase synthesis and secretion. ³Present address: Department of Biochemistry, University ofMissouri, 117 Schweitzer Hall, Columbia MO 65211, U.S.A.     

Promotion and inhibition of {alpha}-amylase production in barley endosperm by cyclic 3',5'-adenosine monophosphate and adenosine diphosphate

The possibility that gibberellin-induced -amylase synthesisin barley endosperm might be mediated by cyclic-3',5'-adenosinemonophosphate (3',5'-AMP) was examined. Promotion of -amylasesynthesis by 3',5'-AMP (5 mM) was observed in the absence ofgibberellic acid (GA₃) and in combination with GA₃ at concentrationsbelow 2 mµM. When combined with gibberellin at concentrationsabove 2 mµM, however, 3',5'-AMP reduced the amount of-amylase obtained. The cyclic nucleotide showed slight activityat concentrations as low as 0.05 mM. These promotions were shownto be due to increased synthesis of -amylase rather than toan increased secretion of the enzyme. Of a variety of adeninecompounds and nucleoside diphosphates tested only 3',5'-AMPand adenosine diphosphate (ADP) induced -amylase synthesis.Longer incubation times were necessary to obtain maximal -amylaseinduction with the nucleotides than with GA₃. ADP and 3',5'-AMPwere about one third and one fifth as active, respectively,as GA₃ in promoting -amylase synthesis, although GA₃ was morethan 10⁷ times more effective. AMO-1618 did not inhibit theaction of the nucleotides and methanolic extracts of the nucleotidesshowed no gibberellin-like activity. Both nucleotides were synergisticwith GA₃ in overcoming the inhibitory effects of acetate andcitrate buffers on -amylase synthesis. (Received February 24, 1969; )     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

Pumpkin (Cucurbita sp.) seed globulin III. Comparison of subunit structures among seed globulins of various Cucurbita species and characterization of peptide components

Various Cucurbita seed globulins showed patterns similar toone another on SDS-gel electrophoresis, and ß bandsfor unreduced globulins and , ', and ' bands for reduced ones.On gel electrophoresis in 6 M urea, reduced globulin gave twoacidic and two basic bands. These corresponded to and ' chainsand ₁ and ₂ chains, respectively, identified by two-dimensionalurea-SDS gel electrophoresis. The compositions of the and ßsubunits were proposed. (Received September 8, 1977; )     

ON THE EFFECTS OF PLANT EXTRACTS ON THE {alpha}-AMYLASE ACTIVITY IN RICE ENDOSPERM

Effects of ethanol extracts obtained from several plant materialson -amylase activity in rice endosperm were examined. Promotingeffect on the -amylase activity was estimated in terms of gibberellinA₃ equivalence using the embryoless endosperm. Inhibiting effectwas examined using intact seed. On chromatography of the extractfrom Prunus, the zones showing promotion of the -amylase activityalso exhibited the activity for the elongation of the leaf sheathof rice seedling. But this was not the case with the extractsfrom Pharbitis, Allium and Lupinus. (Received April 13, 1966; )     

The Effect of Calmodulin Antagonists on Gibberellic Acid-induced Enzyme Secretion in Barley Aleurone Layers

Calmodulin activity was detected and assayed in barley aleuronecells. The effect of calmodulin antagonists on GA₃-induced enzymesynthesis and secretion in barley aleurone layers was also investigated.These calmodulin antagonists (chlorpromazine, haloperidol) inhibitedonly GA₂-induced -amylase secretion. This inhibitory effectwas intensified after 6 h of GA₃-incubation. This leads us tosuggest that some calmodulin-controlled mechanism is involvedin GA₂-induced -amylase secretion. Hordeum vulgare L., barley aleurone cells, gibberellic acid, -amylase secretion, calmodulin, calmodulin antagonist     

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)     

Enzymatic Degradation of Chlorophyll in Chenopodium album

The breakdown of chlorophyll (Chi) in crude extracts of Chenopodiumalbum (white goose foot) in the dark was examined. Derivativesof pheophorbide were formed when Chi or chlorophyllide wasincubated with depigmented crude extracts. The formation ofpheophorbide was completely prevented by heat treatment of extracts,indicating that the reaction was enzymatic, and the presenceof a Mg-releasing enzyme, the so called Mg-dechelatase, waspostulated. This hypothesis was strongly supported by the observationthat the formation of pheophorbide was inhibited by 51% by 10mM MgCl₂. Analysis by high-performance thin-layer chromatography(HPTLC) and liquid chromatography (HPLC) showed that the appearanceof chlorophyllide , pheophorbide 13²-hydroxychlorophyllide and pyropheophorbide was accompanied by a concomitant decreasein levels of Chi The formation of 13²-hydroxychloro-phyllide was not clearly an enzymatic reaction and requires furtherexamination. It appears that Chl is degraded in a crude extractof C. album via the following enzymatically catalyzed reactions (Received September 10, 1990; Accepted November 15, 1990)     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

Brassinosteroid-Induced Bending of the Leaf Lamina of Dwarf Rice Seedlings: An Auxin-Mediated Phenomenon

A synthetic brassinosteroid (BR), 2,3,22ß,23ß-tetrahydroxy-24ß-methyl-B-homo7-oxa-5-cholestan-6-one, an isomer of the growth promoter brassinolide,when applied to seedlings of dwarf rice Oryza sativa var. Tan-ginbozuand Waito-C, induced a significant bending of the second leaflamina at 100 ng/plant and higher dosages. Promotion of thesecond leaf sheath elongation, the characteristic response ofdwarf rice varieties to gibberellins, was significantly butmodestly enhanced by BR at a dosage of 10,000 ng/plant, fiveorders of magnitude higher than the minimal dosage responseto GA₃. Gibberellin A₃ had no significant effect on the bendingof the second leaf lamina, nor did any synergism exist betweenBR and GA₃ in leaf lamina bending or leaf sheath elongation.Neither ethylene nor (2-chloroethyl)phosphonic acid (ethephon)caused the bending of the second leaf lamina, and neither synergizedthe BR effect. However, IAA and -naphthaleneacetic acid causedsignificant bending at 5,000 ng/plant, and both auxins significantlysynergized the effect of BR on the bending, IAA being effectiveat 500 ng/ plant in this regard. The antiauxins, 2,3,5-triiodobenzoicacid (TIBA) and -(p-chlorophenoxy)isobutyric acid (PCIB) completelynullified both the BR-induced bending and the BR$IAA-synergizedbending. The BR-induced bending response may thus be mediatedthrough endogenous auxin. (Received May 11, 1982; Accepted August 25, 1982)     

An {alpha}-Glucan from Suspension-Cultured Rice Cells

An -glucan was isolated from 11-day-old suspension-culturedrice cells by extraction with hot Na-phosphate buffer (pH 6.8).The -glucan had []_D=+234? (C = 0.14, in water) and its averagemolecular weight was estimated to be about 1.4 ? 10⁴, basedon elution characteristics on acalibrated Sepharose CL-6B column.Upon partial acid hydrolysis, the -glucan gave mainly malto-oligosaccharides.The maximum absorption of the iodine complex of the -glucanin the presence of Na₂SO₄ was at 470 nm. The results of hydrolysisby , ß- and iso-amylases and methylation analysisindicated that the isolated -glucan is a highly branched polysaccharidewith an average chain length of 9. The exterior and interiorchain lengths of the -glucan were calculated to be 5 and 3,respectively. (Received July 23, 1986; Accepted February 7, 1987)     

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)     

{alpha}-Amylase synthesis by a cell free system and plant growth substances

A microsomal preparation from the aleurone layer pre-treatedwith GA or H-ol had the ability to synthesize -amylase whenit was incubated with an appropriate medium. -Amylase synthesiswas inhibited by the addition of p-fluorophenylalanine or bypre-treatment with RNase. The synthesized -amylase was separatedinto three isozymes by disc-electrophoresis (Received July 25, 1970; )     

Induction of sexual agglutinability of {alpha} mating-type cells as the primary action of the peptidyl sex factor from {alpha} mating-type cells in Saccharomyces cerevisiae

The effect of a pure preparation of substance-I_A (S-I_A) whoseamino acid sequence is identical to that of one of the factorpeptides (2), on sexual agglutinability and DNA synthesis wascomparatively studied. The optimum concentration of S-I_A forthe induction of sexual agglutinability of cells of an inducible strain was 1 ng/ml. The inducing action of S-I_A was detectedin 20 min and reached a maximum in 60 min. Only 8.7% inhibitionof DNA synthesis by S-I_A in the same strain was detected in1 hr and 40.4% inhibition in 2 hr at a concentration of 1 µg/ml.These results suggest that the primary action of the peptidyl sex fractor on a mating-type cells is the induction of sexualagglutinabiity. (Received October 25, 1977; )     

Effect of preincubation UV irradiation of abscisic acid and gibberellin A3 on {alpha}-amylase induction in barley half-seeds

A 24 hr preincubation UV (254 nm) irradiation of RS-ABA andGA₃ solutions completely negated the inhibitory effect of ABAon GA₃ induced -amylase production with barley half-seeds. Thisirradiation, however, caused no significant inhibition of GA₃-induced-amylase production. UV treatment of an extract from Tulipaindicated that this technique can be used to photooxidize ABA-likesubstances present in plant extracts. ¹ Michigan Agricultural Experiment Station Journal Article No.5774. This research was supported in part by a grant from theNetherlands Flower-Bulb Institute, New York and the OrnamentalMarketing Board, The Hague. ² Present address: Istituto di Orticoltura e Floricoltura, Universitàdegli Studi di Pisa, 56100, Italy. (Received February 26, 1972; )     

Pumpkin (Cucurbita sp.) seed globulin IV. Terminal sequences of the acidic and basic peptide chains and identification of a pyroglutamyl peptide chain

Pumpkin seed globulin is composed of heterogeneous polypeptidechains, acidic and chains and basic ₁ and ₂ chains (12). This study showed that the basicchains had similar N-terminal sequences, Gly-Leu-Asp-Glu-Thr-Ile-for the ₁ chain and Gly-Leu-Glu-Glu-Thr-Ile- for ₂. On the contrary,the N-terminal sequences of the acidic and chains were dissimilar, Ile-Gln-Gly-Tyr- for the chain and no N-terminal residue for the chain, according to routine terminal analysis. Pyrrolidonylpeptidase digestion of the chain and its thermolysin digestion followed by Edman degradationsrevealed that the N-terminal sequence of the chain was < Glu-Ile-Glu-Gln-Gln-Glu-Pro(Trp,Ser)-. The N-terminal sequences and the C-terminal residuesindicated that the acidic and chains were more heterogeneous than the basic ₁ and ₂ chains.A preliminary study on the degradation of storage globulin isalso presented. (Received November 9, 1979; )     

Penicillin Induction of Gibberellin and {alpha}-Amylase Biosynthesis in Rice Endosperm

Penicillin induces the synthesis of -amylase in embryoless riceendosperm and enhances the gibberellin-induced response. Penicillininduction of -amylase can be prevented by inhibitors of nucleicacid and protein synthesis, CCC and 2,4-DNP. A characteristic gibberellin-like activity in the extracts frompenicillin-treated endosperms becomes detectable after 12 hfrom the addition of penicillin. This gibberellin-like activityis located on paper chromatograms at the R_F typical for GA₃and its formation is blocked by CCC, an inhibitor of GA biogenesis.Glucose has no effect on the biosynthesis of either gibberellinor -amylase induced by penicillin. The time-course study of the levels of different constituentsshows that penicillin probably induces RNA and DNA synthesisin the first place, which results in gibberellin biosynthesis,which in turn stimulates the synthesis of -amylase. The possiblemode of action of penicillin in higher plants is discussed.     

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)     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

An Assessment of Gibberellin Structure-activity Relationships

Information on the biological activities of gibberellins (GAs)in the barley aleurone, Tangin-bozu dwarf rice, dwarf pea, lettucehypocotyl and cucumber hypocotyl bioassays is reviewed and discussedin the context of GA structure-activity relationships. The barley aleurone bioassay exhibits a limited response toGAs and it is suggested that this may be because the aleuronecells are able to carry out few GA interconversions. Consequentlyactivity is determined by the degree of compatibility betweenthe GAs and a receptor site. In this assay high biological activityis associated with GAs having a 3ß-hydroxy--lactonestructure. This activity is substantially enhanced by the additionalpresence of a 13-hydroxyl group. The substitution of a -lactoneor a -lactol for a -lactone results in reduced activity while3ß,13-dihydroxy GAs with either 20-carboxyl or 20-methylfunctions are completely inactive. The Tanginbozu dwarf ricebioassay responds to many more GAs than the barley aleuronesystem possibly because the rice seedlings can carry out extensiveGA interconversions. Under these circumstances GAs that areinactive per se can be metabolically converted to active forms.There is no interaction between the 3ß- and 13-hydroxyfunctions of GA molecules in the rice assay. Activity appearsto be determined by the degree oxidation of the C-20 group.The order of activity is usually -lactone > -lactol >-lactone > methyl > carboxyl. It is suggested this mayindicate that in rice seedlings C₂₀-GAs are converted to C₁₉-GAsvia a Baeyer-Villiger type oxidation. Activity in the dwarfpea bioassay is dependent upon GAs possessing both 3ß-and 13-hydroxyl groups and is again related to the state ofoxidation at the C-20 locus. In the lettuce bioassay activityis restricted to GAs with a -lactone function. In some instancesa -lactone, but not a -lactol, can substitute effectively. Thismay imply that the applied C₂₀-GAs are not converted to C₁₉-GAsand that the response to the -lactone results from the six-memberedring mimicking the -lactone at the receptor site. Only GAs havinga 19,10 or a 19,20 lactonic bridge show substantial activityin the cucumber bioassay. The additional presence of eithera 12- or a 13-hydroxyl group severely reduces activity.