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     

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     

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     

Genotypic Differences in Leaf Water Relations between Brassica juncea and B. napus

Various kinds of measurement of tissue water status were madeseveral times during water stress and recovery in Brassica juncea(cv Canadian Black) and B napus (cv Drakkar) Unstressed plantsof the two species had similar leaf water potentials (_w), solute(_s) and turgor potentials (_p) Values of relative water content(RWC) and the slope of the linear relationship between _p andRWC (_p/RWC) were greater in B napus than in B juncea Statistical correlations of pooled data for the watered andstressed treatments differentiated the relationships among RWC,_w and its components in the two species The major statisticaldifference was that _p/RWC was related to RWC in B napus andto _w and _s in B juncea A decline in _p/RWC with decreasing _sin B juncea may be a mechanism for maintaining _p at low soilwater potentials through maintenance of more elastic cell walls. Brassica juncea, Brassica napus, osmotic adjustment, tissue elasticity, water relations     

Osmotic Properties of Drought Stressed Periwinkle (Catharanthus roseus) Genotypes

The pressure-volume technique was employed to compare waterrelations and moisture stress-induced osmotic adjustment ofPeriwinkle (Catharanthus roseus) cv. Pink (PC), Oscillatus (REC)and White (WC). Leaf water potential (_w), osmotic potential(_s), turgor potential (_p), bulk modulus of elasticity (), boundwater (RWC_w) and leaf hydration (H), were estimated by exposingthe plants to a drying cycle during which well watered plantswere dehydrated to zero turgor, and then irrigated. Osmoticadjustment (_w ¹⁰⁰) was calculated by comparing _a at full hydration(_a ¹⁰⁰) in stressed plants after recovery, with _a ¹⁰⁰ in controlplants. Values of ₂¹⁰⁰ were 0.76, 0.33 and 0.11 MPa in cv. PC,REC and WC, respectively. Maintenance of _p at lower ₃ and relativeleaf water content (RWC) in prestressed PC was attributableto a higher alkaloid content and greater leaf cell wall elasticity.RWCW was plotted against _p to determine its contribution tohydration maintenance at lower _p. Genotype PC showed greaterRWC_w at lower _p compared with REC and WC. The present studyhas demonstrated that there are cultivar differences in alkaloidaccumulation and water relations in acclimated plants and thatthe relative ranking for drought resistance within periwinkleappeared to correspond with the changes in osmotic properties. Medicinal plant, drought resistance, alkaloids, periwinkle [Catharanthus roseus (L.) G. Don]     

Wilting of Leaves in Vicia faba L.

Wilting of leaves of Vicia faba L., which occurs when the pressurepotential (_p) is zero, and the leaf-water potential () at wiltingboth depend entirely upon the solute potential at incipientplasmolysis (_so) and not on soil-water status. Wilting in V.faba is acropetal; this is consistent with the hypothesis thatthere is a gradient of decreasing _so up the plant and that wateris transferred from the lower to the upper leaves, hasteningthe overall water loss from the lower leaves to the point when_p is zero. The gradient in _so up the plant is of the order of3–8 bar. It is proposed that wilting when _p>0 (i.e. > _so) shouldbe ‘apparent wilting’ and that when _p0 (i.e. _so),‘true wilting’.     

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; )     

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     

Water-Relations Parameters of the Phloem. Determinations of Volumetric Elastic Modulus and Membrane Conductivity using an Applied Force Method and Shrinkage and Swelling of Tissues in Solutions at Differing Osmotic Pressure

Water-relations parameters were measured on sections of secondaryphloem from red oak (Quercus borealis michx. f.) and white ash(Fraxinus americana var. biltmoreana [Beadle] J. Wright) usinga linear displacement transducer. Changes in tissue thicknessin response to changes in the osmotic pressure of the bathingsolution were used to calculate the volumetric elastic modulusplus osmotic pressure (_v + ) of the tissue, and an applied forcemethod was used to estimate the time constant for water equilibration(T). The hydraulic conductivity of the cell membranes (L_p) wascalculated utilizing _v + and r values. The time-dependent behaviour of the tissue was much more complexthan originally expected. A correction for a time-dependentprocess that we call ‘drift’ was required to obtainnumbers for _v + . Furthermore, _v + was calculated on two assumptionsin order to relate changes in tissue dimensions to sieve elementparameters. In the first case, a lower limit for _v + of thesieve elements was determined by attributing all changes intissue dimensions to these cells. For red oak the average _v+ on this assumption is 72 bars. Assuming that all cell typeswere equally responsible for the changes in tissue dimensionsresulted in an _v + value of 192 bars for oak. If _v + and rare the same for all cells in the tissue, L_p for the sieve elementsof oak is 9.6 x 10^–8 cm s^–1 bar^–1. Exudationfrom the sieve elements of white ash during excision of thephloem led to artificially high values of _v + for that species. Quercus borealis michx. f., Fraxinus americana var, biltmoreana (Beadle) J. Wright, red oak, white ash, water relations, phloem, volumetric elastic modulus, membrane hydraulic conductivity     

{gamma}-Amiobutyric Acid Metabolism in Plants: Part 1. Metabolism in Yeasts

The metabolism of -aminobutyric acid (AB) by two yeasts, Saccharomycescerevisiae and Torulopsis utilis, was investigated. Both yeastsgrew well upon AB as a sole source of nitrogen (N), and thelag phase for Torulopsis was shorter than when provided the N-source. The metabolism of AB by Torulopsis, whichwas associated with an increased O₂ uptake, was adaptive incharacter. The enzyme whose formation was induced by the supplyof AB was a transaminase, which was apparently specific forAB as the amino donor. Small amounts of transaminase were presentin unadapted, -grown cells. The optimum pH, equilibrium constant, Michaelis' constant, and coenzyme requirementwere investigated for the transamination reaction involving-ketoglutaric acid (KG) as amino group acceptor. Succinic semi-aldehyde(SSA) was a product of this transamination reaction.The possibility;that some AB was converted into SSA by a direct oxidative deaminationremained unconfirmed. The further conversion of SSA into succinic acid was establishedusing intact. cells for both yeasts. This oxidation processwas shown to be linked to the reduction of pyridine nucleotidesvising extracts of Saccharomyces as a source of SSA dehydrogenase.Dehydrogenase activity could be ascribed to two separate enzymes,one linked to DPN, and the other utilizing TPN and requiringMg⁺⁺ as an activator. The properties of the former enzyme, whichwas more important quantitatively, were investigated and comparedwith those described in the literature for an aldehyde dehydrogenaseof baker's yeast and for SSA dehydro-genases of Pseudomonas.Torulopsis extracts could catalyse the reduction of SSA to -hydroxybutyricacid (OHB); the OHB dehydrogenase involved required TPNH asa coenzyme. Certain other properties of this enzyme are recorded. The possibility is discussed that AB and SSA act as intermediatesin a metabolic pathway that may form a by-pass of the KG-succinatestage of the tricarboxylic acid cycle.     

Abscisic Acid and Water Relations of Rice (Oryza sativa L.): Sequential Responses to Water Stress in the Leaf

Water stress was imposed by withholding water at an early vegetativestage from plants of two rice cultivars (IR20 and 63–83)grown in pots. As stress intensified the following sequenceof responses of the leaves was observed: (i) rise in abscisicacid (ABA) content, (ii) closure of stomata, (iii) initiationof leaf rolling. In both cultivars, turgor (_p) declined linearly with total waterpotential () of the leaf. Bulk leaf ABA content increased linearlyas _p declined, and attained twice the control (unstressed) levelfollowing a reduction in _p of about 0.12 MPa. Stomatal conductance exhibited a sigmoidal relationship to _p,declining abruptly when a particular ‘critical’_p was reached (threshold response). The critical potentialsvaried considerably between experiments, but were closely correlatedwith control potentials and with the potentials at which ABAconcentration doubled relative to controls. Leaf rolling was initiated at s near to zero _p. Increases inthe ratio of adaxial to abaxial conductance were associatedwith rolling. Variations in the above responses could be accounted for byvariations in the rate of stress development, which in termsof reduction ranged from 0.38 to 0.86 MPa day^–1. Fastdrying rates resulted in: (a) reduced osmotic adjustment, (b)increased amounts of ABA in the leaf at a given level of or_p, (c) an increase in the ABA concentration present at 50 percent stomatal closure, and (d) initiation of leaf rolling ata higher . Oryza sativa L., rice, water stress, stomata, leaf rolling, abscisic acid     

{gamma}-Aminobutyric Acid Metabolism in Plants: Part 2. Metabolism in Higher Plants

The metabolism of -aminobutyric acid (AB) has been studied inhigher plants, particularly in peas and peanuts. Transaminationappeared to form the first step in AB degradation although transaminaseactivities were very low. The relatively active AB transaminaseassociated with whole pea plants possessing nodulated rootsappears to reside almost entirely within the nodules. AB transaminationwas demonstrated conclusively in extracts of mitochondria fromcotyledons of peanut seedlings; pyruvic acid acted as a betteramino-group acceptor than -ketoglutaric acid (KG). AB transaminaseactivity present in the microsomal and soluble cytoplasmic fractionsof the cells was very low AB was not metabolized perceptibly by intact mitochondria frompeanut, but when various organic acids were supplied simultaneously,an extra uptake of oxygen occurred and was associated with ABdisappearance. Aspartate, alanine, and ammonia were formed usingthe nitrogen atom of AB. The metabolic pathway followed by the carbon skeleton of ABwas traced by supplying C¹⁴-labelled material to leaf discsof peas and to mitochondria from peanut cotyledons. Radioactivitywas incorporated into organic acids, amino-acids, and respiratorycarbon dioxide in a manner suggesting that AB was convertedinto succinate which was then metabolized by the enzymes ofthe Krebs cycle present in the plant mitochondria. Glutamic decarboxylase was shown to be present largely in thenon-particulate (soluble) cytoplasm of cells. The enzymes responsiblefor AB synthesis and degradation, glutamic decarboxylase, andAB transaminase, respectively, therefore largely reside in differentsub-cellular fractions.     

An Analysis of Seed Development in Pisum sativum III. The Relationship Between the r Locus, the Water Content and the Osmotic Potential of Seed Tissues in vivo and in vitro

The water content and osmotic potential (₂) of the differentparts of the pea fruit have been measured during developmentof the seed in two lines near-isogenic except for the r locus.During the early development of both genotypes, the testa possesseda more negative ₂ than either embryo, endosperm or pod while,at stages when liquid endosperm was present, the embryo alwaysmaintained ₂, more negative than the endosperm. A clear effectof the r locus on water content and ₂ was only observed in embryotissue — wrinkled (rr) embryos possessing more water andmaintaining a more negative ₂ than round (RR) for most of thedevelopmental period studied. The more negative ₂ of wrinkledembryos correlated with their greater uptake of water in vivo. When cultured in vitro, the embryos of both genotypes showedmaximum growth (fresh or dry weight) if 10 per cent sucrosewas added to the medium (equivalent to about — 1.2 MPa).Round embryos, however, increased in weight more than wrinkledat all sucrose concentrations examined. Cultured embryos maintaineda similar or more negative ₂ than their surrounding medium;wrinkled more negative than round. Embryo culture, osmotic potential, Pisum sativum, pea, r locus, seed development, tissue culture, water content     

Activity of Wall-bound Enzymes in Callus Cultures of Gossypium hirsutum L. During Growth

Activities of - and ß-glucosidase, - and ß-galactosidase,-mannosidase, ß-1,3-glucanase, acid and neutral invertaseswere detected in the cytoplasmic fraction as well as in cellwalls isolated from callus cultures of cotton. Activity of ß-mannosidase,however, could not be detected in the cell walls. Transfer ofcallus to a fresh medium did not immediately influence the activitiesof -glucosidase and ß-galactosidase but increasedsignificantly ß-glucosidase, -mannosidase, acid andneutral invertases. Addition of cycloheximide (1 and 100 mgl^–1) further stimulated acid and neutral invertases butnot other enzymes tested. Sodium chloride (NaCl) was effectivein extracting a-glucosidase, ß-glucosidase, ß-galactosidase,acid and neutral invertases. EDTA extracted most of the -galactosidase,-mannosidase, ß-1,3-glucanase and some -glucosidase.But, NaCl and EDTA could not extract some of the - and ß-glucosidasesand also acid and neutral invertases as evidenced from the residualand extra cellular activity. Studies with whole cells as a sourceof enzyme revealed that some of these enzymes were associatedwith the cell surface. Callus, glycosidases, glucanase, growth, Gossypium hirsutum     

Photosynthetic Oxygen Evolution at Low Water Potential in Leaf Discs Lacking an Epidermis

Land plants encountering low water potentials (low _w) closetheir stomata, restricting CO₂ entry and potentially photosynthesis.To determine the impact of stomatal closure, photosyntheticO₂ evolution was investigated in leaf discs from sunflower (Helianthusannuus L.) plants after removing the lower epidermis at low_w. Wounding was minimal as evidenced by O₂ evolution nearlyas rapid as that in intact discs. O₂ evolution was maximal in1 % CO₂ in the peeled discs and was markedly inhibited when_w was below –1·1 MPa. CO₂ entered readily at all_w, as demonstrated by varying the CO₂ concentration. Resultswere the same whether the epidermis was removed before or afterlow _w was imposed. Due to the lack of an epidermis and readymovement of CO₂ through the mesophyll, the loss in O₂ evolvingactivity was attributed entirely to photosynthetic metabolism.Intact leaf discs showed a similar loss in activity when measuredat a CO₂ concentration of 5 %, which supported maximum O₂ evolutionat low _w. In 1 % CO₂, however, O₂ evolution at low _w was belowthe maximum, presumably because stomatal closure restrictedCO₂ uptake. The inhibition was larger than in peeled discs at_w between –1 and –1·5 MPa but became thesame as in peeled discs at lower _w. Therefore, as photosynthesisbegan to be inhibited by metabolism at low _w, stomatal closureadded to the inhibition. As _w became more negative, the inhibitionbecame entirely metabolic.     

Shrinkage of Attached Roots of Opuntia ficus-indica in Response to Lowered Water Potentials--Predicted Consequences for Water Uptake or Loss to Soil

Attached 2-month-old roots of the succulent plant, Opuntia ficus-indica,shrank 0.4% radially during periods of maximal transpirationunder wet conditions. In contrast, reversible decreases in diameterof nearly 20% occurred for these roots as their ambient waterpotential () in the vapour phase decreased from –0.01to –10 MPa over 8 d, the changes being slightly more rapidat 40 °C than at 10 °C. Such substantial diameter changesbecame progressively less with root age, from a 43% decreasein diameter at 3 weeks to a 6% decrease at 12 months Root shrinkagewas slight when was decreased from –0.01 to –0.3MPa, the latter being similar to the root water potential.As was further decreased from –0.3 to –10 MPa,water movement out of cortical cells caused considerable rootshrinkage. The root hydraulic conductivity (L_p) decreased only30 to 60% for a change in from –0.01 to –10 MPacompared with a decrease of over 10⁶-fold for the soil hydraulicconductivity over this range. The overall conductivity of thesoil, the root-soil air gap, and the root was predicted to bedominated by L_p for _soil above –0.3 MPa. As simulated_soil decreased below –0.3 MPa, the root-soil air gap initiallybecame the primary limiter of water loss from the roots. Below–5 MPa for 1-month-old roots and below –2 MPa for12-month-old roots, the soil became the main limiter of waterloss. Thus, water uptake from wet soils apparently was mainlycontrolled by root properties Water loss to drying soils wascontrolled by the development of a root-soil air gap aroundshrinking roots during the initial phase of soil drying andby the reduction of the soil hydraulic conductivity at evenlower _soil. Root diameter, root hydraulic conductivity, root-soil air gap, soil hydraulic conductivity     

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.     

Water Relations of Potato Leaves. II. Pressure--Volume Analysis and Inferences About the Constancy of the Apoplastic Fraction

Water relations characteristics of potato (Solanum tuberosumL. cv. Bintje) leaves were determined from pressure—volumeanalysis using a pressure chamber. Turgor was 077 MPa and thebulk volumetric modulus of elasticity 81 MPa at full turgidity;turgor loss occurred when water potential () had declined to–087 MPa at a relative water content (RWC) of 0912;the apoplastic water fraction (A) was 0235. As is usually found,there was a linear relation between 1/ and RWC beyond turgorloss. This finding supports the assumptions of the constancyof A during leaf dehydration. Beyond turgor loss the difference between and [measured afterfreezing and thawing (_d)] was about 01 MPa. This differencedid not increase as the leaf water content decreased. This resultcontradicts the constancy of A. It was concluded from calculations with a simple model of leafdehydration that analysis of the relation between and _d providesmore insight in the changes in the apoplastic fraction thanthe relation between 1/ and RWC. Research on the size of theapoplastic fraction and its changes with water potential wouldcomplement current understanding of leaf water relations. Solanum tuberosum, L., water potential, pressure chamber, osmotic potential, pressure potential, relative water content, apoplast, symplast     

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