Accumulation pattern of arachin and its subunits in maturation of groundnut seeds

The accumulation pattern of arachin and its subunits in growinggroundnuts was investigated. Soluble proteins were extractedfrom the kernels at twelve different stages of maturation (4–16weeks after pegging). Fractionation showed arachin, conarachinII, 5S and 2S protein components with sucrose gradient centrifugation.Ten weeks after pegging, only 35% of the maximum amount of arachinhad accumulated, whereas conarachin II was 85%, the 5S component89%, and the 2S component 76%. Arachin, however, increased rapidlyin the later stage of maturation. No change in the subunit ratioin arachin during seed growth was observed on the patterns ofsodium dodecyl sulfate-gel electrophoresis and gel isoelectricfocusing in the presence of urea. The ratio of the arachin subunitscontained in urea-extractable fraction of the kernels was constantthroughout seed development and was consistent with the subunitratio in arachin. On the other hand, the arachin subunits inthe free forms, if any, accounted for less than 1% of the associatedarachin subunits. Probably, the arachin subunits synthesizedin equimoles are associated into arachin without individualdeposition and are accumulated as arachin associates in growingseeds. (Received July 17, 1980; )     

花生种子贮藏蛋白质合成和累积与活力的关系

花生种子发育过程中活力的形成在时间上是不均衡的,果针入土后４０ｄ内活力水平很低,４０ｄ之后活力水平才急剧上升,这和贮藏蛋白迅速合成的时期吻合．随着贮藏蛋白质的合成和累积,由发育转向萌发时其被降解的速度加快,花生球蛋白被优先降解．和贮藏蛋白质其它组分相比较,花生球蛋白和种子活力有更密切的关系．     

The Relation of Storage Protein to Vigor and Its Mobilization Pattern in Germinating Peanut Seeds

The peanut (Arachis hypogaea L.) seeds harvested at the last stage of maturation were divided into five grades by size. The content of total protein, salt-soluble protein, arachin, conarachin I and 2s globulin in these seeds were measured. No obvious differences in germination percentage and the length of radicle and hypocotyl within 3d germination in dark were observed among the five grades of seeds. But there were significant differences in the seedling growth after two weeks of germination in light. There was a very close correlation between the storage protein in cotyledons and the seedling growth. When seeds germinated in light, the efficiency of mobilization of the salt-soluble protein in the cotyledons was higher than that in the cotyledons of the seeds germinating in dark. All of the salt-soluble protein in cotyledons was used up after 14d seedling growth in light. SDS-PAGE of salt-soluble protein showed that 23.5, 38.5 and 41 kD subunits of arachin were first mobilized during germination. The 18 kD subunits of arachin were not mobilized until the above-mentioned subunits were used up. The 60.5 kD subunit of conarachin I and 2s globulin were degradated within 2 to 3 days during germination.     

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。     

萌发中花生胚轴的耐干性与热稳定蛋白

成熟花生种子吸胀１８ ｈ 发芽率达１００ ％ 。在这１８ ｈ 的范围内,胚轴即使经干燥处理,萌发生长率仍保持１００ ％ ,而热稳定蛋白含量变化很小。吸胀２４ ｈ 后,经干燥的花生胚完全丧失萌发生长能力。ＳＤＳＰＡＧＥ和双向电泳表明,花生胚轴的热稳定蛋白主要是贮藏蛋白,该蛋白中的花生球蛋白大亚基,伴花生球蛋白Ｉ和２Ｓ 蛋白的降解与胚轴的耐干性丧失有关。     

Variation and inheritance of the arachin polypeptides of groundnut (Arachis hypogaea L.)

Summary Variation in the arachin polypeptides of groundnut genotypes was observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Three regions could be observed on the electropherogram. Region 1, corresponding to conarachin, did not show any variation; region 2, consisting of arachin acidic subunits, showed variation; region 3, containing the arachin basic subunits, did not show any variation. There are four varietal classes of arachin polypeptide patterns: class A comprised three acidic subunits of arachin of molecular weights 47.5, 45.1 and 42.6 kd and a basic subunit of 21.4 kd; class B, with three acidic subunits of molecular weights 47.5, 45.1 and 41.2 kd and a basic subunit of 21.4 kd; class C of an additive pattern of class A and class B; class D, of two acidic polypeptides of 47.5, 45.1 kd and the basic 21.4 kd subunit. Of the 90 genotypes studied, 73% belong to class A, 15% to class B and 6% each to class C and D. Analysis of F₂ seeds from a cross between class A and class B genotypes showed that the two polypeptides (42.6 kd and 41.2 kd) are coded by nonallelic genes and also revealed that class C and class D patterns arose as a result of hybridisation between class A and class B. A. monticola, the progenitor of A. hypogaea, showed a pattern similar to the additive pattern of class A and class B while some diploid Arachis species had the 41.2 kd polypeptide. Based on arachin polypeptide patterns the probable origin of A. hypogaea has been suggested.     

Influence of Different Storage Conditions on the Mycotoxin Production and Quality of Fusarium-Infected Wheat Grain

Wheat seed samples with different initial infection levels of Fusarium culmorum were kept under different storage conditions for 36 weeks. Samples for analysis were drawn before storage and at intervals of 6‐8 weeks to determine the mycotoxin contents, seed health and seed quality. Zearalcnone (ZEA) accumulated to higher kernel contents towards the end of storage, when the seed was stored under warm and humid conditions [25°C/90% relative humidity (RH)], whereas the deoxynivalenol (DON) content of severely infected kernel samples (> 50%) remained unchanged under any of the conditions. On the other hand, DON contents increased in samples with a slight (4%) or moderate (15%) Fusarium infection level. when the seed was stored under Warm and humid conditions. Nivalenol (NIV) was not found in any samples immediately after harvest but later on in storage, and only under cool or warm but very humid conditions (15°C/84% RH and 25°C/90% RH). During storage, the mycotoxin contents of the samples did not reflect the percentage of Fusarium infected kernels. Under warm but dry conditions (25°C/62% RH) the seed germination rate showed a slight increase or remained nearly constant; at the same time the Fusarium infection level of the kernels decreased fairly fast. Cool and dry conditions (15°C/56% RH) maintained good seed quality but the Fusarium infection level of the kernels remained largely the same. Warm and humid conditions are not appropriate to maintaining quality of both seed and grain product.     

Reconstitution of Arachin from Isolated Subunits

Six subunits of arachin were isolated in urea solution. They were then reassociated by removing urea by co-dialysis against 20 mM sodium phosphate buffer (pH 7.9), containing 30% sucrose, 0.1 M> sodium chloride and 7 mM β-mercaptoethanol, without agitation at 25°C. The reconstitution yield was greater than 90%. The reconstituted molecule was indistinguishable from intact arachin in disc electrophoretic mobility, subunit composition, sedimentation behavior depending upon ionic strength, circular dichroism, ultraviolet absorption and fluorescence emission spectra, and stabilities against heating, proteases and guanidine hydrochloride. The reconstituted arachin was, therefore, suggested to be in native state.

On the other hand, we found that co-dialysis of four or five subunits of arachin formed hexamer which contained the corresponding four or five subunits. These hexamers were more labile than intact arachin against heating. These facts suggest that the assembly of all six subunits to a hexamer will most advantage the quaternary structure of arachin.     

Cytokinin Activity in Lupinus albus L: IV. Distribution in Seeds

Endogenous levels of cytokinin activity were examined in Lupinus albus L. seed at intervals of 2 weeks after anthesis using the soybean callus bioassay. High levels of cytokinin activity per gram seed material were present in the seeds at 2, 4, and 6 weeks after anthesis. The cytokinin activity per gram seed material was low at 8 and 10 weeks after anthesis. Cytokinin activity associated with each seed was greatest at 6 weeks after anthesis. The majority of the activity in the seeds at 4, 6, and 8 weeks after anthesis was in the endosperm. Cytokinin activity was also detected in the testas and embryos at 4, 6, 8, and 10 weeks, and the suspensors at 4 weeks. Column chromatography of extracts of the different seed fractions on Sephadex LH-20 indicated that the cytokinins present coeluted with zeatin, zeatin riboside, and the glucoside cytokinins. It is suggested that cytokinins are accumulated in the seeds and are stored in the endosperm mainly in the form of ribosides and glucosides of zeatin. The reduction in cytokinin activity in the seed coincides with the reduction in endosperm volume and embryo growth and suggests that these compounds are utilized during the course of seed maturation.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels.

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.     

Changes of Endogenous ABA Content in Relevance to the Vigor of Peanut Seeds

During the development of peanut (Arachis hypogaea L. ) seed, the endogenous ABA content increased steadily'in hypocotyl, increased to a peak at 40 d after pegging with a drastic decline afterwards in testa; and in cotyledon, increased to a peak at 60 d after pegging but with a slight fall afterwards. There seemed to be a close relationship between the increose of vigor index and net loss of endogenous ABA content in the peanut seed germinating in vitro. Osmoticum (mannitol) promoted the endogenous ABA in the cotyledon and hypocotyl. and Fluridone inhibited that in the cotyledon. There were two different paths of the endogenous ABA synthesis in peanut seed, C40 in the cotyledon and C15 in the hypocotyl. When peanut seeds were put in the conditions of precocious maturation or germination tine endogenous ABA content fell down. Result from this experiment concluded that the hypocotyls played an important role in the transition from development to germination of. peanut seed.     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

花生种子内源ABA含量变化及其与活力的关系

花生（Ａｒａｃｈｉｓ ｈｙｐｏｇａｅａ Ｌ．）种子发育过程中,胚轴内源ＡＢＡ 含量一直是增加的;种皮内源ＡＢＡ含量在果针入土后４０ ｄ 最大,然后急剧下降;子叶内源ＡＢＡ 含量在果针入土后６０ ｄ 出现高峰,然后有轻微下降。种子活力指数和萌发时内源ＡＢＡ 的净下降量有密切关系。甘露醇可促进离体胚内源ＡＢＡ 合成,１－甲基－３－苯基－５（３－［三氟甲基］－苯基－４－（１氢）－吡啶）抑制子叶内源ＡＢＡ 的合成,子叶和胚轴存在不同的ＡＢＡ 合成途径。种子早熟和早萌处理时,内源ＡＢＡ 含量都下降,胚轴在种子由发育向萌发转换中起着十分重要的作用     

Seeds of the annual grasses Schizachyrium spp. as a food resource for tropical granivorous birds

Seed dynamics of the annual tropical grasses Schizachyrium fragile (R. Br.) A. Camus, S. pachyarthron C. Gardner and S. pseudeulalia (Hosok.) S.T. Blake were studied with the aim of documenting fluxes in granivore food resources. In S. fragile, seed production began in the early dry season, and seed output was primarily influenced by seedling survival. Following seed-fall, there were 651 S. fragile seeds/m² (393 kernels/m²⁾ and 1014 S. pachyarthron seeds/m² (593 kernels/m²) across the study area, with a combined kernel biomass of 14.1 × 10³ g/ha. Seed densities remained stable through the dry season, then declined rapidly after wet season rain. Initial wet season rains of up to 25 mm, 40 mm and 50 mm resulted in seed-bank germination of < 5%, 57%, and 93%, respectively. Some seeds were buried by rain and resurfaced or germinated later, but about 30% was lost. Widespread and abrupt depletion of the seed-bank is likely to occur at the start of about 8% of wet seasons on central Cape York Peninsula, leaving little seed, either for subsequent germination or as food for granivores. Burning early in the dry season, when most seeds were still on the plants, reduced seed densities by 85%. The proportion of seeds with sound kernels was reduced in areas burnt by dry season fires, both directly after the fires and, subsequently, as a result of preferential granivore activity. Areas burnt in the dry season were thus depleted of seed earlier than were unburnt areas. Food availability for granivores should therefore be optimized by fire regimes that include a range of burning histories, including fires in both early dry and early wet seasons as well as keeping other areas unburnt.     

Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (a_w) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an a_w of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.     

Accumulation pattern and identification of seed storage proteins in zygotic embryos of Pinus strobus and in somatic embryos from different maturation treatments

Somatic embryogenesis (SE) of Pinus strobus L. has been greatly improved over the last few years with respect to both the initiation frequencies from a number of seed families and production of mature somatic embryos that readily convert to plants. However, there are no data on biochemical characterization of somatic embryos in relation to zygotic embryos of eastern white pine and on the optimal duration of the maturation stage. It is believed that somatic embryos closely resembling zygotic embryos not only morphologically but biochemically would display more vigorous growth. Hence, in this study the accumulation pattern of the most abundant seed storage proteins in zygotic and somatic embryos were characterized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and identified by amino acid sequencing and tandem mass spectrometry (MS/MS). This showed that somatic embryos accumulated storage proteins in a similar manner to zygotic embryos and that the most abundant were the buffer‐insoluble 11S‐ globulins MW 59.6 kDa, which dissociated under reduced conditions to 38.2–40.0 and 22.5–23.5 kDa range polypeptides, and buffer‐soluble 7S vicilin‐like proteins MW 46.0–49.0 kDa, which did not separate under reduced conditions. Other relatively abundant soluble proteins were in the ranges of 25–27 and 27–29 kDa. The only group of proteins that showed different migration profiles in the presence of β‐mercaptoethanol (ME) were the low molecular mass proteins of 14.6–16.5 kDa. Somatic embryos that matured for 9 weeks on medium with 6% sucrose accumulated more storage proteins than those matured on medium with 3% sucrose and the extension of the maturation period to 12 weeks resulted in significant reduction of the storage proteins on both media. As expected, somatic embryos matured on medium with 6% sucrose had lower water potential (Ψ) than those from medium with 3% sucrose. Nonetheless, the somatic embryos matured under the best of tested conditions (6% sucrose for 9 weeks) had slightly higher water content; 1.35 ± 0.28 g H₂O g^?1 DM (mean ± sd ) than the mature non‐dried zygotic embryos; (1.16 ± 0.09 g H₂O g^?1 DM), and accumulated less storage proteins, whose amounts were either similar to (7S‐vicilins) or below (11S‐globulins) those found in the immature zygotic embryos collected 2 weeks prior to the usual cone collection. The implications of these results for further research and development of viable artificial seed is discussed.     

花生种子发育和萌发过程中贮藏蛋白的合成和降解

以花生品种汕油5 2 3种子为材料,分离纯化花生球蛋白的41 kD和38.5 kD两种主要亚基及伴花生球蛋白的6 0.5 KD亚基并制备抗体.We stern blot分析表明,3种亚基在花生胚组织分化期的胚轴和子叶中就开始合成,其中60.5 kD亚基是最先在胚轴和子叶中大量合成和积累的贮藏蛋白,41 kD和38.5 kD亚基在随后的发育中积累量不断增加;种子萌发时这3种亚基的降解进程不一样,胚轴和子叶中41 kD和38.5kD亚基的降解均先于60.5 kD亚基.     

Structure of Malonogalactan: Carbon-13 Nuclear Magnetic Resonance Spectra of Malonogalactan

Six kinds of arachin subunits were isolated by isoelectric focusing in sucrose density gradient in the presence of 6 m urea and 0.2 m β-mercaptoethanol. These subunits (S₁ to S₆) had different isoelectric points (pI 5.8, 6.0, 6.3, 7.1, 7.4 and 8.3) and they were found in parent arachin in the weight ratio of 2.5 : 2.2 : 2.6 : 1.6 : 1.1 : 1.0. The molecular weights and N-terminal amino acids of the isolated subunits were 35,500 and valine (S₁), 37,500 and isoleucine (S₂), 40,500 and isoleucine (S₃) and 19,500 and glycine (S₄, S₅ and S₆). While, the parent arachin contained approximately one mole of valine, two moles of isoleucine and three moles of glycine as the N-terminal amino acids per 180,000 g. It is, therefore, concluded that arachin consists of six different subunits to form the molecular weight 180,000. No disulfide bond takes part in the subunit association of arachin, because the dissociation in a urea solution occurred in the absence of β-mercaptoethanol.     

Precursor Ribosomal Ribonucleic Acid and Ribosome Accumulation In Vivo During the Recovery of Salmonella typhimurium from Thermal Injury

When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA.