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.     

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。     

花生种子贮藏蛋白的氨基酸组成分析及发育过程中17.5 kDa多肽的合成规律

分析了两种不同蛋白质组成类型花生的子叶总蛋白３个主要组分及高甲硫氨酸类型花生的６０．５、４１、３８．５、１８和１７．５ｋＤａ多肽的氨基酸组成,结果表明它们均含有１７种氨基酸,其中天冬氨酸、谷氨酸和精氨酸含量最高,而甲硫氨酸含量和半胱氨酸水平都极低。高甲硫氨酸类型品种的各组分的甲硫氨酸含量均显著高于低甲硫氨酸类型品种的对应组分的甲硫氨酸含量,在这两种类型花生中伴花生球蛋白Ⅱ都是甲硫氨酸含量最高的组分     

One and Two- dimensional Polyacrylamide Gel Electrophoretic Analysis of Seed Polypeptide Composition of Peanut Cultivars

Seed polypeptides from 46 cultivars of peanut (Arachis hypogaea L. ) were compared by SDS-PAGE and two-dimensional pelyacrylamide gel electrophoresis. Arachin, the major seed storage protein of peanut, showed polymorphism. There were four types of arachin pelypeptide pattems. Type Ⅰ consisted of only four major subunits:41 kD,38.5 kD and two of the 18 kD subunits. Type Ⅱ had six major subunits:41 kD,38.5 kD,37.5 kD and three of the 18 kD subunits. Type Ⅲ consisted of 41 kD, 38.5 kD,36.5 kD and three of the 18 kD subunits. And Type Ⅳ consisted of seven major subunits:41 kD,38.5 kD,37.5 kD,36.5 kD and three of the 18 kD subunits. The compositions of conarachin in different cultivars were similar. Amino acid composition analysis of seed protein in 8 peanut cuhivars showed that Type Ⅰ was rich in methionine and cystine.     

Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.)

Vicilin and legumin, the storage globulins of mature dry vetch (Vicia sativa L.) seeds, are found in protein bodies which are present not only in the cotyledons, but also in the radicle, axis and shoot (together, for reasons of simplicity, here called axis). When at 24 h after the start of imbibition (hai) the radicle breaks through the seed coat a major part of the globulins in the axis has already been degraded, whereas in the cotyledons globulin breakdown cannot yet be detected. Globulin mobilization starts with the degradation of vicilin. At 48 hai when globulin mobilization in the cotyledons just begins, the axis is already nearly depleted of globulins. Mobilization of storage globulin is probably brought about by a complex of different cysteine proteinases (CPRs). The papain-like CPR2 and CPR4, and the legumain-like VsPB2, together with their mRNAs, are already present in axes and cotyledons of dry seeds. This means that they must have been formed during seed maturation. Additional papain-like CPRs are formed later during germination and seedling growth. CPR4 and VsPB2 together with their corresponding mRNAs become undetectable as germination and seedling growth proceed. VsPB2 and VsPB2-mRNA are substituted by the homologous legumain-like proteinase B and its mRNA. The composition of stored and newly formed CPRs undergoes developmental changes which differ between axes and cotyledons. It is concluded that storage globulin mobilization in germinating vetch seeds is started by stored CPRs, whereas the mobilization of the bulk of globulin is predominantly mediated by CPRs which are formed de novo.     

Protein composition of cotyledons and aleurone grains inLupinus luteus L. Seeds during Germination

The change in protein composition of whole cotyledons and cotyledon aleurone grains ofLupinus luteus L. during seed germination was studied. SDS-polyacrylamide gel electro-phoresis showed a clear change in composition of cotyledon proteins as well as in composition of the aleurone grains during 5 days of seed germination. At this time, both in whole cotyledons as well as in aleurone grains, two subunits of β-conglutin with mol. m. 53 000 and 39 000 were rapidly hydrolyzed. After 5 days of germination traces of α-conglutin subunits could be detected in the cotyledons, whereas in aleurone grains this globulin fraction disappeared. In whole cotyledons and in cotyledon aleurone grains the γ-conglutin subunits with mol. m. 28 000 and 17 000 were not mobilized during the study period. These results indicate that the protein components with lower mol. m. were degraded later than those withhigher mol. m. during seed germination.     

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.     

Two calcium dependent protein kinases are differently regulated by light and have different activity patterns during seedling growth in <Emphasis Type="Italic">Pharbitis nil</Emphasis>

In this study using biochemical approaches we identified two calcium-dependent protein kinases (CDPKs) named PnCDPK52 and PnCDPK56 in soluble protein extracts from seedlings of Pharbitis nil. Both enzymes phosphorylated the specific substrate histone III-S in the presence of Ca²⁺ and cross-reacted with antibodies against the CDPK. PnCDPKs exhibited quite different activity and protein levels during germination and successive stages of seedling growth. PnCDPK52 protein level was high in seeds and during germination, whereas PnCDPK56 increased in the next stages of seedling growth, being the dominant enzymes in mature seedlings, of the light- and dark-grown plant. In all cases both activity and accumulation of protein PnCDPK56 was higher in dark grown plants whereas exposure to light reduced both factors. When etiolated cotyledons were exposed to light, the activity of PnCDPK56 was reduced to the basal level within 5 h. Conversely, increasing activity of PnCDPK56 in cotyledons of green plants shifted to darkness was extremely rapid, reaching the maximum level after just 1 h of darkness and then gradually decreased. Further lengthening of the darkness to 16 h resulted in a strong increase in activity at 12 h. These data indicate that at least two isoforms of CDPK are involved in germination and seedling growth of P. nil. The differences in PnCDPKs strongly argue for the pleiotropic role of these isoforms. It seems that PnCDPK52 is associated with the germination process and PnCDPK56 with seedling growth. Moreover it suggests that activity of PnCDPK56 is controlled by light via the photoreceptor-dependent pathway.     

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; )     

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; )     

Chlorophyll in desiccated seeds of a euhalophyte, Suaeda physophora, and its significancy in plant adaptation to salinity during germination

The seed cotyledons of a euhalophyte, Suaeda physophora, were found to be dark green. The pigment extracted from the cotyledons was proved to be chlorophyll for the absorption spectra curve of extracts the same as that for leaves. Photosynthetic oxygen-exchanging could be detected after the seeds were moistened for 6 h. Microstructure of organelles in cotyledons of ungerminated seeds was detected by transmission electron microscopy. Histochemical pigmentation was used to investigate the degree of damage on the membrane of radicles. A xerophyte, Haloxylon persicum, whose cotyledons of desiccated seeds also contain chlorophyll was used as a comparative species. The results showed that S. physophora maintained the ultra-structure of chloroplasts, the integrity of plasma membranes of radicles kept much better than that of H. persicum, which showed the great adaptability to salinity of the euhalophyte even at the seed-germination stage. Seeds were incubated in 0 and 700 mmol/L NaCl for 10 days in darkness at 20 ℃ , then ungerminated seeds in NaCl solution were transferred to deionised water and reincubated for another 8 days and was recorded the germination recovery. Dry seeds moistened with deionised water germinated gradually in the dark and germination was maximal after 3 or 4 d. Seeds at 700 mmol/L NaCl for 10 d and transferred back to deionised water germinated abruptly, most seeds germinated in one day and photosynthesis was also detected. It is concluded that recovery germination of S. physophora ensured the seeds could germinate rapidly after salinity declines, for example under the mild but transitory favorable edaphic condition in early spring. Photosynthesis function in seeds might also promoted radicle growth and seedling establishment.     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

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。     

Increased activity of aminopeptidase in the cotyledons of red light-promoted lettuce seeds is controlled by the axis

The first major reserves to be mobilized following germination of light-promoted lettuce seeds ( Lactuca saliva L. cv. Grand Rapids) are the carbohydrates, largely mannans, located within the cell walls of the endosperm. When these have been depleted, the cotyledonary reserves are hydrolysed; the first of these to decline is protein. Water-, salt- and ethanol-soluble proteins are mobilized simultaneously, and coincident with their loss from the cotyledons there is an increase in aminopeptidase activity. The level of enzyme activity increases appreciably in irradiated seeds after about 30 h from the start of imbibition. Essential for this increase, at least initially, is the presence of the axis - first to perceive the light stimulus, and then to produce and/or release a chemical promoter which diffuses into the cotyledons and effects the rise in enzyme activity. Protein synthesis in the cotyledons is a prerequisite for both development and maintenance of the increased aminopeptidase activity.     

Changes in Seed Constituents During Germination and Seedling Growth of Precociously Matured Soybean Seeds(Glycine max)

During 7 d of precocious maturation of soybean seed (Glycinemax), the starch content declined and soluble sugar levels increasedin patterns similar to natural seed dehydration and maturation.Total seed protein content and total seed dry weight increasedwhereas oil content remained relatively unchanged. Overall,the proportions of the constituents in precociously maturedseeds were comparable to naturally mature seeds. Precociouslymatured soybean seeds showed much the same germination and seedlinggrowth frequency patterns as naturally matured seeds. Duringgermination and seedling growth of precociously matured seeds,starch, soluble sugar, protein and oil levels followed patternssimilar to naturally mature, germinating seeds and seedlings.Therefore, precocious maturation may be used as a model systemto investigate the control of the physiological and biochemicalevents occurring during seed maturation which lead to germinationand subsequently, seedling growth. Glycine max (L.) Merr., soybean, cotyledons, maturation, germination/seedling growth     

Allocation of proteolytic activity in the seedling of Cynara cardunculus L. in the initial growth stages

Cynara cardunculus L. seeds were germinated in vitro under environmentally controlled conditions. Seeds showed a 60% germination rate, and three growth stages were established based on the seedling mean relative growth rate (RGR). Root, stem and cotyledons were compared in these stages with respect to the emergence of total proteases and cardosin activity and its allocation in the seedling. In growth stage I (1st-5th post-germinative days), seedlings grew very slowly. Total proteases and cardosins were already active at the onset of seedlings in the stem. Total soluble protein remained constant in cardoon seedlings during stage I, and the content of all free amino acids (aa) but proline (Pro) was equally allocated on the 1st post-germinative day. In growth stage II (5th-10th post-germinative days), seedlings grew intensively and exhibited fully developed cotyledons. A pronounced increase in the content of all free aa up to the middle of growth stage II in both stems and roots was observed. In addition, the allocation of the total proteolytic activity and cardosins followed a gradient from the root to the seedling shoot. However, the whole seedling soluble protein remained constant up to the 7th day in and tended to peak on the 10th post-germinative day, being allocated mainly to the seedling stem. In growth stage III (10th-15th post-germinative days), cardoon seedlings exhibited the lowest mean RGR and the highest R/S growth ratio. An intensive degradation of total soluble protein present in the whole seedling except for cotyledons (ca. 5-fold) was observed. Nevertheless, in growth stage III, both the gradients exhibited by total proteases and cardosins activities between the root and the seedling shoot were enhanced, as were contents of all aa except Pro, exhibiting the highest levels in cotyledons on the 15th post-germinative day.     

蚕豆蛋白质亚基分析与特异种质鉴定

采用SDS-PAGE方法,对112份不同基因型蚕豆的清蛋白和球蛋白亚基的差异性进行分析。结果显示:(1)蚕豆清蛋白和球蛋白亚基的有效等位变异分别为1.750 0±0.452 3、1.545 5±0.522 2,多态性比率分别为75.00%、54.55%,清蛋白的亚基遗传多样性指数较球蛋白亚基高。(2)蚕豆清蛋白和球蛋白分别含有在不同基因型蚕豆中构成不同的12和10个亚基,其中清蛋白含有9个基本亚基,116kD、96kD、45kD为清蛋白的3个特异亚基;球蛋白含有8个基本亚基,58kD、35kD为球蛋白的2个特异亚基;研究共鉴定筛选出42个含有清蛋白特异亚基和21个含有球蛋白亚基的种质资源。(3)清蛋白的97kD、63kD基本亚基和球蛋白的97kD、56kD、47kD基本亚基存在一定的缺失现象,共鉴定出19个清蛋白亚基和21个球蛋白亚基缺失的优异种质。研究表明,蚕豆清蛋白和球蛋白亚基构成在不同种质之间具有差异性,除基本亚基外,部分种质还含有特异亚基或缺失亚基。     

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.     

Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism.     

Proteolysis of the main storage protein of buckwheat seeds at the early stage of germination

The changes in the main storage protein of seeds of buckwheat ( Fagopyrum esculentum Moench cv. Shatilovskaya 5), 13S globulin, were studied during seed germination. During the first three days of germination the 13S globulin is subjected to a limited proteolysis, which consists in the splitting of some of its subunits into large polypeptide fragments. Insignificant changes in the sedimentation coefficient of the 13S globulin during the first days of germination as well as immunochemical data, indicate that the limited proteolysis of the 13S globulin does not cause any major changes in its structure.
Dormant buckwheat seeds contain a proteolytic enzyme (a metalloproteinase), which can cause limited proteolysis of 13S globulin. The proteinase hydrolyzed some subunits of the 13S globulin to high molecular weight fragments. In the presence of sodium dodecylsulphate the electrophoretic pattern of 13S globulin, isolated from 3-day-old buckwheat seedlings, was almost identical to that of 13S globulin from dormant seeds hydrolyzed with metalloproteinase. It is suggested that the proteolysis of 13S globulin observed in vitro may also take place in vivo in the course of seed germination.