Differential leakage of intracellular substances from imbibing soybean seeds

Leakage of electrolytes, substances absorbing UV light, and enzymic activities from imbibing soybean (Glycine max [L.] Merr.) seeds were compared to determine the extent that passive diffusion and cellular rupture contribute to each. Imbibing seeds with testae removed had average Arrhenius energies of activation (5 to 25°C) of 3.0 and 15.8 kilocalories per mole, respectively, for the leakage of electrolytes and embryo malate dehydrogenase activity. Leakage of embryo enzymes from imbibing seeds was dependent on loss of testa integrity and subsequent loss of cellular membrane integrity or inability to seal preexisting membrane discontinuities. These data suggest that electrolyte leakage from imbibing seeds is primarily by passive diffusion, whereas the diffusion of intracellular macromolecules is primarily dependent on physiological phenomena affecting membrane integrity. Kinetic data and examination of the composition of seed leachates indicated that the leakage of substances absorbing UV light during imbibition is due to both passive diffusion of low molecular weight solutes and macromolecules released from ruptured cells.     

Patterns of mitochondrial development in reserve tissues of germinated seeds: A survey

Patterns of mitochondrial development in reserve tissues of several species of seeds were examined during 3 to 4 days after the start of imbibition. In starch-storing seeds (cowpea, kidney bean, Egyptian kidney bean, mung bean, black gram and soybean), mitochondrial activities (state 3 respiration rate, respiratory control ratio) increased during the first 1 to 2 days after imbibition and leveled off thereafter. The initial increase in the activities was little affected by cycloheximide and chloramphenicol. Activities of mitochondrial enzymes (succinate dehydrogenase, EC 1.3.99.1; NAD⁺-malate dehydrogenase, EC 1.1.1.37; cytochrome oxidase, EC 1.9.3.1) did not change much during the experimental period. This suggests that mitochondrial development in starch-storing seeds is primarily due to improvement of pre-existing mitochondria. On the other hand, in the lipid-storing seeds examined (pumpkin, cucumber, okra and castor bean), the rate of mitochondrial respiration and activities of mitochondrial enzymes continued to increase markedly during the experimental period. These increases were strongly inhibited by cycloheximide and chloramphenicol. This indicates that active de novo synthesis of mitochondrial proteins is primarily responsible for the development of organelle activities. The possibility was suggested that the patterns of mitochondrial development in reserve tissues of imbibed seeds might be determined by the kinds of reserve substances.     

Pressure-Driven Extrusion of Intracellular Substances from Bean and Pea Cotyledons during Imbibition

Intracellular substances leak from imbibing cotyledons of grain legumes during imbibition. This work reports the discovery of a biophysical process by which intracellular substances are driven from cotyledons during imbibition. Light and scanning electron microscopy were used to examine bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.) cotyledons and the material released from them into imbibition water. A large fraction of the visible materials released from excised bean and pea cotyledons during the first 30 minutes of imbibition consisted of convoluted or helical streams of material which rapidly emerged from the cotyledons surfaces. Large streams of material from bean cotyledons contained starch grains and protein bodies, and smaller streams from bean and pea cotyledons probably contained protein bodies. The forms of streams were characteristic of a viscous fluid which had been forced by pressure through irregular orifices. The sites of extrusion from bean cotyledons were multicellular blisters which formed on the surfaces of imbibing cotyledons. In 6 hours, pea and bean cotyledons leaked from 1 to 11 micrograms protein per milligram of seed dry weight. The quantities of protein leaked primarily depended on cultivar.     

Leakage during Seed Imbibition

When pea embryos are placed in water solutes leak from themat a rate which declines rapidly at first and then more slowly.Embryos can be dried down over calcium chloride and will thenleak as before when returned to water. Similar results wereobtained with Ricinus seeds after removal of the testa. Pea embryos that have first been allowed to imbibe some waterthrough a small part of their surface (by placing them on dampfilter-paper) leak relatively slowly when subsequently immersedin water; the greater the initial imbibition the slower thesubsequent leakage. Likewise, embryos taken from peas that wereharvested when succulent and tender show only slow leakage It is proposed that as seeds dry out in the course of developmentcell membranes lose their integrity. When such dry seeds areallowed to imbibe water there is a short period, before membraneintegrity is re-established, during which solutes can leak outof the cells.     

ENZYMES OF THE TRICARBOXYLIC ACID CYCLE IN SEA URCHIN EGGS AND EMBRYOS

Changes in the activity of some enzymes of the tricarboxylic acid cycle during development of sea urchins were investigated. Unfertilized eggs showed substantial activity of citrate synthase, aconitase, NAD- and NADP-specific isocitrate dehydrogenases, fumarase and malate dehydrogenase. During development, the activity of citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase and malate dehydrogenase increases gradually, whereas the activity of fumarase remains rather constant. There is no close correlation between changes in the enzyme activity and the increase in oxygen consumption during development. Citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase are mainly localized in the mitochondrial fraction, whereas fumarase and malate dehydrogenase are present in both mitochondrial and cytosol fractions. The intracellular localization of these enzymes does not change during development. A possible mechanism for the regulation of some enzymes of the tricarboxylic acid cycle in sea urchin eggs is discussed.     

Imbibition of white spruce seeds and somatic embryos: A study of morphological changes in an environmental scanning electron microscope and potassium leakage

Summary Potassium leakage and morphological changes during imbibition of white spruce [Picea glauca (Moench) Voss] seeds and somatic embryos were investigated. A single desiccated somatic embryo, a single somatic embryo exposed to a high relative humidity environment for 2 d, and a single dry zygotic embryo leaked similar amounts of potassium over a 120-min period of imbibition in liquid germination medium. A seed without a seed coat leaked two and eight times more potassium than a single whole seed and a single zygotic embryo, respectively. Nearly 50% of the potassium leaked for all tissues was leaked within the first 20 min of imbibition. Exposure of somatic embryos to an environment with high relative humidity resulted in a reduction in the percentage of potassium leaked after 80 and min to levels equivalent to those for zygotic embryos. Using an environmental scanning electron microscope, we found that desiccated somatic embryos and dry zygotic embryos had wrinkled surface cells, with cells in the surface of zygotic embryos being more shrunken in appearance. Imbibition of both types of embryos in water resulted in turgid surface cells after 2 h. Imbibition in liquid germination medium did not cause much hydration of surface cells, which still had wrinkled appearances after 2 h. Finally, imbibition on filter paper on semisolidified germination medium resulted in slower hydration of somatic and zygotic embryos. Cells near the medium appeared hydrated while cotyledon surface cells furthest from the medium resembled cells in desiccated embryos.     

Clinical trial with clotrimazole cream (Bay b 5097) in dermatophytosis and onychomycosis

Growth of Aspergillus flavus and Trichoderma viride on agar media was inhibited around embryos of green peanut seeds but not around embryos of cured seeds, intact peanut seeds, or testae. Both fungi were able to colonize intact seeds and testae. Substances inhibitory to A. flavus and T. viride were extracted with acetone from cotyledons of freshly harvested peanut seeds. Four compounds inhibitory to A. flavus were detected in crude acetone extracts. Three of the compounds demonstrated properties of phenolics. Results of this study suggest that inhibitory compounds in peanut cotyledons may play a role in protecting the peanut embryo from fungal infection.Journal Series No. 486, New Mexico State University, Agriculture Experiment Station, Las Cruces. This research was supported in part by ARS, USDA Grant No. 12-14-100-9899(34) Crop Research Division, Beltsville, Maryland.     

大麦种子前期处理对其外植体性状的调控和对其机制的探讨(Ⅰ)

研究了种壳对大麦种子吸胀生理影响的调节作用 ,同时对其外植体性状的调控作用和其机制也进行了探讨。结果如下 :(1 )种壳显著延缓种子的吸水进程 ;(2 )种壳对分子渗漏影响显著 ;(3 )总 N的渗漏主要来自胚乳 ,Mn+ + 的渗漏在胚中相对较多 ,种壳对 K+ 渗漏影响较大 ;(4 )去壳促进吸胀期间的可溶性蛋白量和过氧化物酶同工酶活性上升 ;(5 )吸胀使 IAA、GA3含量上升 ,使胚乳中的 ABA含量显著减少 ,去壳显著促进吸胀初期胚乳中的 ABA、IAA、GA3含量上升 ,去壳减少吸胀后期胚中的 IAA、ABA、GA3含量 ;(6)去壳减少吸胀 2 4h并且种子进行培养时的培养效果 ,它们具有明显较高的萌发率和愈伤组织诱导率。     

种壳对大麦种子生理和成熟胚培养的影响及机理探讨Ⅱ

用30％PEG6000对大麦种子进行浸种等处理,并对其吸胀生理的多数指标进行了系统分析和检测,研究结果表明：种壳显著延缓种子的吸水进程;种壳对多数分子的渗漏有较大影响。N的渗漏主要来自胚乳,Mn^2 渗漏主要来自胚,种壳显著影响K^ 渗漏。去壳促使吸胀期间可溶性蛋白量和过氧化物酶同工酶的活性显著升高。吸胀使IAA、GA3含量提高,使胚乳中的ABA含量显著减少;去壳促进吸胀初期胚乳中的ABA、IAA、GA3含量,去壳减少吸胀后期胚中的ABA、IAA、GA3含量。种子去壳后吸胀24h有显著较高的萌发率、愈伤组织诱导率。     

Alternative Respiratory Pathway: ITS ROLE IN SEED RESPIRATION AND ITS INHIBITION BY PROPYL GALLATE

Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O₂ uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O₂ uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles.     

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons: Influence of the Axis

Development of mitochondrial activities (state 3 respiration,respiratory control ratio, ADP/O ratio) in peanut cotyledonsoccurs over the first 5 d from the start of imbibition. Mitochondriain cotyledons with the axis attached develop better than inthose from which the axis has been removed. Initially, mitochondriaare deficient in cytochrome c, but after 2 d from the startof imbibition this deficiency is overcome. Mitochondrial developmentin attached cotyledons, as measured by state 3 respiration,respiratory control ratio, ADP/O ratio, and succinate dehydrogenaseand cytochrome oxidase activities, is severely impaired by cycloheximide.This indicates that de novo synthesis of proteins is necessaryfor mitochondria and their enzymes to develop, a situation whichis in sharp contrast to the situation in pea cotyledons. Electronmicroscope studies also show that there is an increase in thenumbers of mitochondria in peanut cotyledons with time afterthe start of imbibition. Two patterns of mitochondrial developmentexist in legumes: in imbibed peanut cotyledons respiratory activitiesincrease due to biogenesis of mitochondria, whereas in pea cotyledonsthe increases are due to improvement of pre-existing organelles     

顽拗性黄皮种子脱水过程中活性氧清除酶活性的变化

以正常性种子花生为对照,研究了顽拗性黄皮种子脱水过程中活性氧清除酶、膜脂过氧化作用以及电解质渗漏率的变化。随着含水量的下降,黄皮胚的电解质渗漏率和膜脂过氧化产物丙二醛(MDA)含量均显著增加;当黄皮胚含水量下降至40％后,SOD活性开始急剧下降,而POD和CAT活性在胚含水量下降过程中呈现出缓慢下降的趋势。花生胚在含水量从45％降至14％的过程中,电解质渗漏率没有明显增加,MDA含量只有少量增加;当含水量降至14％后,电解质渗漏率出现少量增加。花生胚脱水初期,活性氧清除酶活性明显增加,并在整个脱水过程中维持较高的水平。以上结果表明顽拗性种子黄皮的脱水敏感性与活性氧清除酶相对活性变化有关。脱水引起黄皮胚活性氧清除酶活性降低,活性氧清除能力下降,膜脂过氧化作用加强,膜透性增大。黄皮胚的膜系统可能是脱水伤害的靶位之一。     

Culture of pea embryo axes for studies on the reactivation of the cell cycle at germination

Summary A method for culturing excised pea embryo axes, which allows growth of large batches of embryo axes in a reduced volume of medium and is suitable for performing biochemical analyses and early treatment or labeling of embryos with exogenous substances, has been devised. Pea embryo axes were excised from the seeds after 3 h of imbibition and cultured in a mineral medium containing sucrose, vitamins, and casamino acids. Under these conditions, the reactivation of the cell cycle occurs much earlier than in intact seeds: with flow cytometry and bromodeoxyuridine labeling, cells in the S phase were found in the root meristems after only 12 h from the beginning of imbibition. The transition of cells from a quiescent to a proliferative state is accompanied by changes in the electrophoretic pattern of nuclear proteins, particularly with regard to two proteins of apparent molecular weight of 90 and 46 kDa, as shown previously in normally germinating seeds.     

Dual localization of fumarase is dependent on the integrity of the glyoxylate shunt

Fumarase and aconitase in yeast are dual localized to the cytosol and mitochondria by a similar targeting mechanism. These two tricarboxylic acid cycle enzymes are single translation products that are targeted to and processed by mitochondrial processing peptidase in mitochondria prior to distribution. The mechanism includes reverse translocation of a subset of processed molecules back into the cytosol. Here, we show that either depletion or overexpression of Cit2 (cytosolic citrate synthase) causes the vast majority of fumarase to be fully imported into mitochondria with a tiny amount or no fumarase in the cytosol. Normal dual distribution of fumarase (similar amounts in the cytosol and mitochondria) depends on an enzymatically active Cit2. Glyoxylate shunt deletion mutations ( Δmls1 , Δaco1 and Δicl1 ) exhibit an altered fumarase dual distribution (like in Δcit2 ). Finally, when succinic acid, a product of the glyoxylate shunt, is added to the growth medium, fumarase dual distribution is altered such that there are lower levels of fumarase in the cytosol. This study suggests that the cytosolic localization of a distributed mitochondrial protein is governed by intracellular metabolite cues. Specifically, we suggest that metabolites of the glyoxylate shunt act as 'nanosensors' for fumarase subcellular targeting and distribution. The possible mechanisms involved are discussed.     

The Damaging Effect of Water on Dry Pea Embryos During Imbibition

When pea seeds were imbibed in water without their seed coats,vital staining revealed that cells on the abaxial surface ofthe cotyledons were dead. No damage occurred on the surfaceof cotyledons when the seeds were imbibed intact, or beneaththe testa when only half of the testa was removed. Cell deathoccurred as a result of rapid water uptake within the first2 min of imbibition, since reducing the rate of imbibition insolutions of Carbowax 4000 lessened the damage. Cell death wasrestricted to the outer layers of the cotyledons; inner tissuesremained alive. These observations supported the hypothesisthat rapid early leakage during imbibition of dry embryos resultedfrom the death of cells caused by the physical disruption ofmembranes. Imbibition damage resulted in reduced respirationand germination, a decline in the rate of food reserve transferfrom the cotyledons to the growing axis, and a lower growthrate in the seedlings produced. Greater sensitivity of embryosto imbibition damage at low temperature, and similarities betweenfeatures of imbibition damage and chilling injury led to thesuggestion that so-called chilling injury is the result of imbibitiondamage rather than the effects of low temperature.     

The Role of Imbibition Damage in Determining the Vigour of White and Coloured Seed Lots of Dwarf French Beans (Phaseolus vulgaris)

Seeds of dwarf French beans showed evidence of damage alterimbibition in water in the form of dead tissue and high soluteleakage. Damage was more extensive in seeds of a cultivar withwhite testae (Tenderette) both at 20?C and with the initial6 h imbibition at 4?C where damage was more severe, whereasa cultivar with brown testae (Provider) was damaged only alterthe 4?C treatment. Tenderette imbibed more rapidly than Providerin both imbibition treatments. The significant correlationswithin each cultivar of both solute leakage (positive) and theextent of living tissue (negative) with the rate of water uptake,and the reduced damage when seeds imbibed slowly in PEG indicatedthat this was imbibition damage due to rapid water uptake. Differencesin the rates of water uptake were also observed in sand at 15%and 20% mc where rapid imbibition was associated with reducedgermination and the production of more abnormal seedlings. Atemperature of 4?C during the first 6 h germination in sandat 15% mc also reduced germination, particularly in Tcnderette.It is suggested that the more rapid water uptake by white seededcultivars of dwarf French beans and their greater susceptibilityto imbibition damage are responsible for their low vigour. Key words: Imbibition damage, vigour, dwarf French beans     

Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans

Two isoenzymes each of phosphoglucomutase, hexose phosphate isomerase, aldolase, fructose diphosphatase, phosphofructokinase, and 6-phosphogluconate dehydrogenase have been separated by DEAE-cellulose column chromatography of extracts from endosperm of germinating castor beans (Ricinus communis cv. Hale). One of each of the enzymes is localized in the cytosol and the other is confined to plastids. Developmental studies of these isoenzymes were carried out to clarify their roles in the endosperm. In extracts from ungerminated seeds the activities of marker enzymes of mitochondria (fumarase), plastids (ribulose bisphosphate carboxylase), and glyoxysomes (catalase) were low, but phosphoglucomutase, hexose phosphate isomerase, aldolase, and 6-phosphogluconate dehydrogenase were present in relatively high activity. The total amounts of these enzymes increased 3- to 4-fold during the first 5 days of growth. The activities of isoenzymes in the plastids rose in parallel with that of ribulose bisphosphate carboxylase to reach a maximum at day 4, and like the carboxylase they declined sharply thereafter. The activities of the cytosolic isoenzymes peaked at day 5. These changes are consistent with the roles previously proposed for the sequences present in plastid and cytosol.     

Isoenzyme pattern and subcellular localisation of enzymes involved in fumaric acid accumulation by Rhizopus oryzae

Summary Electrophoretic studies of fumarase and nicotine adenine dinucleotide (NAD)-malate dehydrogenase were carried out in the fumaric acid-accumulating fungus Rhizopus oryzae. The analyses revealed two fumarase isoenzymes, one localised solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytosolic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested that de novo protein synthesis is required for increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation. Three distinct isoenzymes of NAD-malate dehydrogenase could be detected in R. oryzae. No changes were observed in the isoenzyme pattern of malate dehydrogenase during fumaric acid production.     

Temperature effects on seed imbibition and leakage mediated by viscosity and membranes

The possible involvement of membranes and water viscosity in the temperature effects on imbibition and solute leakage of radish (Raphanus sativa var. Early Scarlet Globe) seeds and excised sugar pine (Pinus lambertiana Dougl.) embryos was evaluated. In these two seed materials, the temperature effect on initial rates of imbibition and solute leakage could be accounted for primarily by changes in water viscosity, the relationship being approximately linear. It appears that membranes are involved both in water uptake and solute leakage. Heat-killed radish seeds and sugar pine embryos exhibited significantly higher rates of imbibition and solute leakage than did viable ones. In addition, sugar pine embryos exhibited an abrupt change in rates of imbibition and solute leakage between 15 and 20°C, resulting in abnormally high water uptake and solute leakage above this temperature.