花生种子的发育与贮藏蛋白质的合成和积累

花生果针入土后16天(16 DAP),种子干重和鲜重开始迅速增加。整个发育阶段可分为5个时期:组织分化期(0～20 DAP)、成熟前期(21～28 DAP)、成熟中期(29～40DAP)、成熟中后期(41～62 DAP)和成熟后期(63～88DAP)。种子发芽率在成熟前期和中期迅速提高并到达最大值,而苗成活率在成熟中后期达到最大值,苗鲜重则以88 DAP种子的为最大。种子发育过程中,贮藏蛋白质的合成与积累模式与种子干重变化相似。SDS-PAGE分析表明,种子发育初期(16 DAP)子叶中已积累花生球蛋白和伴花生球蛋白I。双向凝胶电泳显示花生球蛋白各个亚基在20DAP时均已存在,伴花生球蛋白I的主要亚基在整个发育过程中其等电点有所变化,含量也逐渐增加。其他蛋白质在种子发芽力形成阶段(20～40 DAP)的变化较为显著。     

ATP Synthesis in Cell-Free Extracts of Peanut (Arachis hypogaea L.) Seeds

Cell-free extracts of peanut (Arachis hypogaea L., cv. Shulamit)seeds, incubated with various substrates, synthesized ATP. Significantsynthesis occurred in the presence of AMP + PEP, NADH₂ + PEPand NAD + PEP. When the activities were examined in extractsprepared with 0.3 M mannitol, the rates were 0.6, 0.1 and 0.04nmol min^–1 mg^–1 protein, respectively. The activitiesunder such conditions were linear with time up to 90 min incubationat 30 °C. In the presence of PEP + NADH₂ there was a higherspecific activity in extracts from non-dormant seeds than fromdormant seeds. No such difference was found when PEP + AMP orNAD + PEP was used as the substrate. The temperature dependenceof the activity showed a relatively high energy of activation(Ea) for AMP + PEP and a low one if NADH₂ + PEP or NAD + PEPwas used as substrate. In buffer extracts of seeds ATP was synthesizedin the presence of the above-mentioned substrate combinationsbut the rate of activity exhibited a lag phase at the earlytime of incubation, after which higher rates of activities (ascompared with mannitol extracts) were obtained. The activitieswere Co⁺-dependent, with a K_m of about 0.7 mM. In the bufferextracts relatively high activities of adenylate kinase (EC2.7.4.3 [EC] (AK) and pyruvate kinase (EC 2.7.1.50 [EC] ) (PK) were found.AK was stimulated by ethephon (ethylene). This effect is temperature-dependentand occurs in both directions: in the presence of ADP (ATP +AMP) as well as if ATP + AMP is used as substrate to synthesizeADP. PK is Co⁺-dependent, and unaffected by ethephon. Both activitieswere stimulated by malonate. Key words: Adenylate Kinase, Arachis hypogaea, ATP synthesis, Peanut, Pyruvate kinase, Seed     

花生种子萌发早期胚轴细胞DNA合成的特点

花生种子吸胀6h后胚轴DNA中有~3H-胸苷掺入。咖啡因和羟基脲均对6～12h的~3H—胸苷掺入具强烈的抑制作用;当12～24h时,咖啡因的抑制作用较大;但30h以后,羟基脲的抑制作用超过咖啡因。双链DNA放射性从种子吸胀9h后迅速上升,单链DNA放射性在吸胀12h后出现一个明显的峰。但在吸胀12h后,单链DNA形成和存在的时间是短暂的。     

The Lack of Correlation between ATP Accumulation in Seeds at the Early Stage of Germination and Seed Quality

Seeds of various species were treated with polyethyleneglycolin the absence or presence of AMP and phosphoenolpyruvate, redriedand examined for ATP accumulation at the early stage of germinationand for the rate of germination under suboptimal temperatures.The pretreatments resulted in seeds with various levels of accumulatedATP but in most comparative experiments no correlation was foundbetween the ATP accumulation and the rate of germination. Similarpretreatments with naturally aged seeds led to the same conclusion.The fact that ATP accumulation is a result of ATP synthesisas well as ATP utilization was demonstrated by a protein inhibitionexperiment which showed a possibility to produce seeds withrelatively high ATP accumulation levels at the early stage ofgermination, but with very low germination ability. Key words: AMP, PEP, Polyethyleneglycol, Pretreatment     

Ditylenchus destructor in Hulls and Seeds of Peanut

The potato rot nematode, Ditylenchus destructor Thorne, is reported for the first time in hulls and seeds of peanut. The populations found differed from D. dipsaei and D. myceliophagus in habitat, number of lateral incisures, shape of tail tip, and length of postvulval sac. Infected hulls had brown necrotic tissue at the point of connection with the peg, and a black discoloration appeared first along the longitudinal veins. Infected seeds were usually shrunken, and testae and embryos had a yellow to brown or black discoloration. Of 877 seed samples graded "damaged" from all major peanut producing areas of South Africa, 73% were infected.     

Changes in Endogenous Growth Kegulators in Loblolly Pine Seeds During the Process of Stratification and Germination

The seeds of loblolly pine (Pirns taeda L.) were cold-stratified for 0, 14, 28, 42, and 56 days. Endogenous growth regulators were extracted from these seeds, and also from the germinating and the 28-day warm stratified seeds. Partially purified extracts were separated on chromatographic paper. The chromatograms were cut into 10 equal segments, and these were tested for biological activity using three different btoassays. The results indicated that the unstratified seeds and those stratified up to a period of 28 days contained very little or no growth promoter (GA-like substances), and a relatively high concentration of an inhibitor (presumably abscisic acid), Following 42-day stratification, the promoter concentration gradually increased while the inhibitor level fell almost to zero. A high level of promoter but no inhibitor was detected in germinating seeds. No auxin-like activity was noted in the unstratified seeds. This activity slowly increased up to a period of 28 days and remained at this level for the subsequent stratification periods. However, the activity greatly increased in the germinating seeds. Very little changes in the levels of growth regulators were noted in warm-stratified seeds as compared to the unstrati-fied controls.     

Changes in Some ATP-Dependent Activities in Seeds during Treatment with Polyethyleneglycol and during the Redrying Process

During the imbibition of seeds in polyethyleneglycol (PEG),increasing amounts of ATP accumulated up to 24 h. Similar amountsaccumulated in the seeds during 4–5 h of imbibition inwater. Radioactive amino acids were increasingly incorporatedin the acid-insoluble fraction up to 24 h imbibition in PEG,as well as in water, after which a sharp decrease occurred upto 5 d of imbibition. If seeds were imbibed in PEG or waterin the presence of radioactive acetate, water-insoluble radioactivityincreased linearly in seeds during 5 d of imbibition. The amountsof incorporated amino acids or acetate were about double inPEG-imbibed as compared with in water-imbibed seed. The incorporationof AMP into the acid-insoluble fraction in seeds imbibed inPEG in the presence of radioactive AMP levelled off after 24h followed by a sharp decrease of up to 10% of the peak 5 dafter the start of imbibition. In water-imbibed seeds the incorporationof AMP continued to increase during at least 5 d of imbibition.During redrying of PEG-treated seeds (24 h), at least 80% ofthe accumulated ATP decreased during 18 d. The total radioactiveamino acids and nucleotide decreased during 3 d of redryingby 20% and 60%, respectively. At that time, the acid-insolubleincorporates increased by 20% and 50%, respectively. Some ofthe AMP was released as CO₂. Key words: AMP, Germination, Nucleic acid synthesis, Osmoconditioning, PEG, Protein synthesis     

ATP Synthesis in Cotyledons of Cucumber and Mung Bean Seeds during the First Hours of Imbibition

ATP concentration increased rapidly during the first 6h of imbibitionin cotyledons of cucumber and mung bean seeds. The increasewas strongly inhibited by 1-h treatment of tissues with cyanidein both species. Carbonylcyanide m-chlorophenylhydrazone, anuncoupler of oxidative phosphorylation, showed little effectduring the first hour of the treatment, but its inhibitory effecton ATP synthesis became significant after 3 h. Mitochondrialfractions prepared from 6-h-old cucumber cotyledons were capableof phosphorylating ADP to ATP. These results suggest that mitochondrialoxidative phosphorylation may be involved in ATP synthesis duringthe early hours of imbibition in both cucumber and mung beanseeds. (Received December 7, 1987; Accepted April 9, 1988)     

Factors Influencing Aflatoxin Accumulation in Peanut Kernels and the Associated Mycoflora

Accumulation of aflatoxin in Spanish peanut kernel samples from different geographical areas in Texas during 1966, as detected by the thin-layer chromatographic method, was relatively low. Analysis of samples obtained from growers using artificial drying equipment (forced air and supplemental heat), when windrow conditions were unfavorable for rapid drying, suggests that this practice reduces the possibility of aflatoxin accumulation. In general, peanuts harvested from land planted to peanuts the previous year were more highly infested with fungi and contained more aflatoxin than peanuts grown on land planted with rye, oats, melons, or potatoes the previous year. Aflatoxin incidence tended to decrease from south to north Texas. These findings verify previous research observations that moist tropical climates are conducive to fungal infestation and aflatoxin accumulation. Detection of aflatoxin in sound mature kernels (kernels screened for minimal size) indicates that the practice of screening for removal of small immature kernels and removal of obviously damaged kernels does not completely eliminate aflatoxin contamination.     

Physiology of Oil Seeds: IV. Role of Endogenous Ethylene and Inhibitory Regulators during Natural and Induced Afterripening of Dormant Virginia-type Peanut Seeds

To further elucidate the regulation of dormancy release, we followed the natural afterripening of Virginia-type peanut (Arachis hypogaea L.) seeds from about the 5th to 40th week after harvest. Seeds were kept at low temperature (3 ± 2 C) until just prior to testing for germination, ethylene production, and internal ethylene concentration. Germination tended to fluctuate but did not increase significantly during the first 30 weeks; internal ethylene concentrations and ethylene production remained comparatively low during this time. When the seeds were placed at room temperature during the 30th to 40th weeks after harvest, there was a large increase in germination, 49% and 47% for apical and basal seeds, respectively. The data confirm our previous suggestion that production rates of 2.0 to 3.0 nanoliters per gram fresh weight per hour are necessary to provide internal ethylene concentrations at activation levels which cause a substantial increase of germination. Activation levels internally must be more than 0.4 microliter per liter and 0.9 microliter per liter for some apical and basal seeds, respectively, since dormant-imbibed seeds containing these concentrations did not germinate. Abscisic acid inhibited germination and ethylene production of afterripened seeds. Kinetin reversed the effects of ABA and this was correlated with its ability to stimulate ethylene production by the seeds. Ethylene also reversed the effects of abscisic acid. Carbon dioxide did not compete with ethylene action in this system. The data indicate that ethylene and an inhibitor, possibly abscisic acid, interact to control dormant peanut seed germination. The inability of CO₂ to inhibit competitively the action of ethylene on dormancy release, as it does other ethylene effects, suggests that the primary site of action of ethylene in peanut seeds is different from the site for other plant responses to ethylene.     

Ethylene as a Component of the Emanations From Germinating Peanut Seeds and Its Effect on Dormant Virginia-type Seeds

The embryonic axes of Spanish-type peanut seeds that do not exhibit dormancy to any extent were found to produce ethylene during germination. Virginia-type peanut seeds of the extremely dormant variety NC-13 produced low levels of ethylene when imbibed but not germinating. Treatments that released dormancy of NC-13 peanut seeds resulted in increased ethylene production by the embryonic axis. The estimated internal concentration of ethylene in Virginia-type peanut seeds was 0.4 ppm at 24 hr of germination. Fumigation with an external concentration of 3.0 to 3.5 ppm for 6 hr was sufficient to break dormancy of Virginia-type peanut seeds. These results suggest that ethylene is associated with the germination processes of non-dormant seeds and participates in the breaking of seed dormancy of dormant peanut varieties.     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

ATP Production from Adenine by a Self-coupling Enzymatic Process: High-level Accumulation under Ammonium-limited Conditions

To improve ATP production from adenine, we optimized cultivation and reaction conditions for the ATP producing strain, Corynebacterium ammoniagenes KY13510. In the conventional method, 28% NH₄OH has been used both to adjust pH during cultivation and reaction, and to provide nitrogen for cell growth. In the ATP-producing reaction, high concentrations of inorganic phosphate and magnesium ion are needed, which form magnesium ammonium phosphate (MgNH₄PO₄) precipitate. To keep inorganic phosphate and magnesium ions soluble in the reaction mixture, it was indispensable to add phytic acid as a chelating agent of divalent metal ions. Under such conditions, 37 mg/ml (61.2 mM) ATP was accumulated in 13 h (Appl. Microbiol. Biotechnol. 21, 143 1985). If ammonium ion was depleted from the reaction mixture to avoid MgNH₄PO₄ formation, we expected that there was no need to add phytic acid and ATP accumulation might be improved. Therefore, we obtained the cultured broth of C. ammoniagenes KY13510 strain with low ammonium ion content (less than 1 mg/ml as NH₃) by the method that a part of alkali solution (28% NH₄OH) for pH control was replaced with 10 N KOH. Using this culture broth, ATP producing reaction was done in 2-liter jar fermentor, controlling the pH of the reaction mixture with 10 N KOH. Under these conditions, the rate of ATP accumulation improved greatly, and 70.6 mg/ml (117 mM) ATP was accumulated in 28 h. The molar conversion ratio from adenine to ATP was about 82%. Phytic acid was slightly inhibitory to ATP formation under these ammonium-limited conditions.     

Proteins of Soybean Seeds: II. Accumulation of the Major Protein Components during Seed Development and Maturation

Fresh weight and dry weight as well as quantitative and qualitative protein changes in the developing soybean (Glycine max) seed were described from 12 days after flowering until maturity. The seed proteins were separated on sucrose density gradients into three major fractions, having average sedimentation coefficients of 2.2S, 7.5S, and 11.8S. The 2.2S sedimenting proteins predominated at very early stages of development (12 days after flowering) and decreased proportionately throughout maturation. The 7.5S and 11.8S components appeared to be synthesized later in maturity and in larger amounts than the 2.2S proteins. Electrophoretic studies on extracts from whole seeds and on isolated protein fractions confirmed the early abundance of proteins in the 2.2S fraction and revealed temporal differences in the accumulation of three components of the 7.5S fraction. The 11.8S sedimenting fraction appeared throughout seed development as a homogeneous protein which accumulated in the seed with a time course similar to that of the total 7.5S protein fraction.     

Purification, Characterization and Cloning of Phospholipase D from Peanut Seeds

We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K _m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl₂ and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.     

Seed Dormancy in Red Rice : VIII. Embryo Acidification during Dormancy-Breaking and Subsequent Germination

Exposure of dehulled, dormant red rice (Oryza sativa) seeds to dormancy-breaking treatments (10 mm sodium nitrite, 20 mm propionic acid, 30 mm methyl propionate, 40 mm propionaldehyde, or 70 mmn-propanol) induced tissue pH acidification during chemical contact at least 12 h before visible germination. During chemical contact, the onset of embryo acidification occurred before or coincident with the chemical contact interval necessary for subsequent germination. Upon seed transfer to H₂O following chemical contact, embryo pH also decreased coincident with visible germination. During this period, the percentage of germination and embryo pH were closely linked irrespective of the dormancy-breaking compound used. Therefore, tissue acidification during the breaking of seed dormancy and the germination process may be analogous to similar tissue pH changes associated with the termination of developmental arrest in other multicellular systems, such as brine shrimp cysts and nematode larvae.     

Accumulation of Vacuolar H+-Pyrophosphatase and H+-ATPase during Reformation of the Central Vacuole in Germinating Pumpkin Seeds

Protein storage vacuoles were examined for the induction of H+-pyrophosphatase (H+-PPase), H+-ATPase, and a membrane integral protein of 23 kD after seed germination. Membranes of protein storage vacuoles were prepared from dry seeds and etiolated cotyledons of pumpkin (Cucurbita sp.). Membrane vesicles from etiolated cotyledons had ATP- and pyrophosphate-dependent H+-transport activities. H+-ATPase activity was sensitive to nitrate and bafilomycin, and H+-PPase activity was stimulated by potassium ion and inhibited by dicyclohexylcarbodiimide. The activities of both enzymes increased after seed germination. On immunoblot analysis, the 73-kD polypeptide of H+-PPase and the two major subunits, 68 and 57 kD, of vacuolar H+-ATPase were detected in the vacuolar membranes of cotyledons, and the levels of the subunits of enzymes increased parallel to those of enzyme activities. Small amounts of the subunits of the enzymes were detected in dry cotyledons. Immunocytochemical analysis of the cotyledonous cells with anti-H+-PPase showed the close association of H+-PPase to the membranes of protein storage vacuoles. In endosperms of castor bean (Ricinus communis), both enzymes and their subunits increased after germination. Furthermore, the vacuolar membranes from etiolated cotyledons of pumpkin had a polypeptide that cross-reacted with antibody against a 23-kD membrane protein of radish vacuole, VM23, but the membranes of dry cotyledons did not. The results from this study suggest that H+-ATPase, H+-PPase, and VM23 are expressed and accumulated in the membranes of protein storage vacuoles after seed germination. Overall, the findings indicate that the membranes of protein storage vacuoles are transformed into those of central vacuoles during the growth of seedlings.