Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

Hydrolysis of Crystalloid Storage Protein in Castor Bean Seed Endosperm During and Following Germination

Hydrolysis of the insoluble crystalloid storage proteins ofcastor bean endosperm during germination released buffer-solublepolypeptides with molecular weights in the presence of sodiumdodecyl sulphate of 30000–40000. These polypeptides appearto be dimers since the addition of 2-mercaptoethanol decreasestheir molecular weights to 15000–22000. Hydrolysis ofthe crystalloid proteins was detected 12–18 h after seedimbibition (HAI), which is before the completion of germination;maximum rates were attained at 30 HAI. During this period, parallelincreases in free amino acids were observed. Hydrolysis of thecrystalloid proteins during early germination was insensitiveto cycloheximide treatment and therefore did not require newlysynthesized proteases. Hydrolysis was effected by proteaseswhich were made in an inactive form during seed developmentand activated upon seed imbibition. Key words: Castor bean, crystalloid storage protein hydrolysis, seed germination, endosperm     

Proteases and Peptidases of Castor Bean Endosperm: Enzyme Characterization and Changes during Germination

The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.     

Mobilization of Endosperm Reserves and Uptake of Sucrose and Valine by the Cotyledons of Euphorbia lathyris L.

Upon germination, the endosperm triacylglycerols and proteinswere converted to sucrose and amino acids. During early postgerminativegrowth, the rate of sucrose and amino acid production exceededthe rate of uptake by the cotyledons. As a result, the levelsof total amino acid and sucrose in the endosperm increased;maximum levels were reached at 7 d and 10 d after imbibition(DAI), respectively. Intact seedlings were used to measure thedevelopment of valine, arginine, glutamic acid, and sucroseuptake rate throughout the course of endosperm depletion. Maximumamino acid uptake rates were measured at around 9 DAI, the highestuptake rate for sucrose was obtained at 12 DAI (just beforedepletion of the endosperm). The daily increase of sucrose andamino acid uptake could be manipulated, by replacing the endospermwith a pre-incubation solution during 1 d. The increase in sucroseuptake in vitro was equal to that measured with intact seedlingswhen the cotyledons were pre-incubated in 10 mol m^–3 sucrose.Higher sucrose concentrations reduced the increase of sucroseuptake; at 300 mol m^–3 sucrose (corresponding to the meanendosperm sucrose concentration) sucrose uptake after pre-incubationwas even lower than before. This reduction was largely counteractedwhen the pre-incubation solution was supplemented with minerals.The development of the valine uptake was hardly affected bysucrose, but was inhibited by several amino acids. Key words: Euphorbia lathyris seedling, sucrose uptake, amino acid uptake, reserve mobilization     

花生种子萌发过程中胚轴多胺氧化酶的活性变化

研究花生(Arachis hypogaea)种子萌发过程中胚轴多胺氧化酶(PAO)的活性变化及其与种子萌发的关系表明：胚轴中的PAO活性是在种子萌发过程中逐渐形成的,而黑暗条件更有利于该酶的活性形成;放线菌素D(10mg／L)、环己酰亚胺(10mg／L)处理对种子萌发的抑制率分别为26.3％和87.3％,对胚轴PAO活性的抑制率分别为41.1％和94.0％,显示胚轴中的PAO很可能参与花生种子的萌发过程,且其mRNA在种子发育过程中已合成并贮存于种子中,萌发时PAO活性的出现主要是由于这些mRNA转译合成了PAO。     

萌发绿豆子叶衰老期间蛋白质代谢的变化

萌发绿豆子叶自然衰老过程中可溶性蛋白质含量一直下降;从衰老开始到衰老前期,总游离氨基酸含量明显上升;但游离氨基酸各组分在子叶衰老期间的变化趋势并不相同。~3H-亮氨酸掺入蛋白质试验和多聚核糖体的相对量及其与总核糖体的比值(P/T)测定都证明在子叶衰老前期有蛋白质的新合成。子叶衰老期间。氨肽酶活性明显降低;而以酪蛋白为底物的蛋白水解酶活性却急剧上升,承担着催化蛋白质降解的主要功能。     

Protein Bodies from the Endosperm of Castor Bean: Subfractionation, Protein Components, Lectins, and Changes during Germination

Protein bodies from the storage endosperm of dry castor bean (Ricinus communis L.) were isolated by successive nonaqueous linear density gradient centrifugation. The isolated protein bodies were lysed by the addition of water, and the various structural components of the organelles were separated by sucrose gradient centrifugation. The matrix protein remained at the top of the gradient while the membrane, the crystalloids, and the globoids migrated to densities 1.15 g/cm³, 1.30 g/cm³, and > 1.46 g/cm³, respectively. The protein of the protein bodies was distributed evenly between the crystalloids and the matrix, and little protein was present in the globoids or the membrane.     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Phosphatidylcholine Synthesis in Castor Bean Endosperm : I. Metabolism of l-Serine

Three levels of free amines and the activities of their biosynthetic enzymes were measured in subcellular fractions of two cell lines of Nicotiana tabacum L. cv Xanthi. The TX4 cell line, a p-fluorophenylalanine resistant culture which accumulates high levels of cinnamoylamides, was compared to the wild-type culture TX1. In cells harvested on day 6 of the growth cycle, nearly all free putrescine, spermidine, and tyramine was found in the supernatant fraction of both cell lines. Although a consistent portion of ornithine decarboxylase activity was detected in the nuclear-enriched fractions of TX1 and TX4, the largest levels of activity were in the supernatants of both lines. In TX1, arginine decarboxylase activity was low relative to that of ornithine decarboxylase, but, in the TX4 line arginine decarboxylase levels in the cytosol were substantially elevated. Tyrosine decarboxylase was not detected in 6-day-old TX1 cells, but significant amounts of activity were measured in the 1000g and supernatant fractions of TX4. S-Adenosylmethionine decarboxylase activity was low in both cell lines and was located predominantly in the supernatant.     

不同成熟度花生胚萌发时子叶中贮藏蛋白质的降解

花生(Arachis hypogaea L.)汕油71果针入土20d(20 DAP)的种子剥去种皮后,10％的胚可以萌发,至40 DAP发芽率达98％。不同发育时期的花生胚萌发 10d后子叶盐溶蛋白质和花生球蛋白降解表明,20和32 DAP胚萌发后,子叶中这些蛋白质只有部分降解。随着胚成熟度增加,子叶中降解这些蛋白质的能力不断提高。20～40 DAP胚萌发4d时,子叶的BAPAase和GHE活性较低。50～80DAP胚萌发 4d,子叶中上述两种酶均显示较高的活性。     

绿豆子叶衰老期间核酸代谢的变化

萌发绿豆的子叶自然衰老期间,核酸含量降低,RNA降低的幅度比DNA大。电泳分析结果表明,子叶衰老期间细胞核主带DNA明显降低;而迁移慢的卫星带DNA变化不大。在RNA各组分中,18S rRNA从衰老前期就开始降低;25S rRNA和4～5S小分子RNA到衰老后期才缓慢下降。DNase和RNase活性在子叶整个衰老期间都明显升高,是导致核酸含量下降的主要原因。~3H-核苷掺入试验表明,核酸的合成速率在子叶衰老前期有所上升,到衰老后期又降低。poly(A)~ -mRNA含量在子叶开始衰老时明显上升。     

Changes in Antioxidative Protection in Bean Cotyledons During Natural and Continuous Irradiation-Accelerated Senescence

We employed continuous irradiation (CL) for induction of premature senescence caused by enhanced production of reactive oxygen species. As a model plant we used bean (Phaseolus vulgaris L. cv. Jantar) cotyledons because they have well defined and a quite short life span. Senescence of bean cotyledons induced by CL progressed more rapidly than natural senescence: the life span of CL cotyledons was 13 d compared to 16 d in controls (C). Chl content was significantly lower in 10- and 13-d-old CL plants than in C plants and the change with age was not statistically significant. Activities of all antioxidative enzymes declined either with senescence onset or during whole life span. Activity of antioxidative enzymes, except ascorbate peroxidase, was lower in CL plants compared to C plants. On the contrary, contents of non-enzymatic antioxidants -carotene and ascorbate were higher in CL plants than in C plants. No significant difference, except in the youngest cotyledons, was observed in glutathione content.     

Temperature-induced Phase Changes in the Membranes of Glyoxysomes, Mitochondria, and Proplastids from Germinating Castor Bean Endosperm

Sucrose-gradient purified mitochondria, glyoxysomes, and proplastids from germinating castor bean (Ricinus communis L.) endosperm were examined by electron-spin resonance spectroscopy. A temperature-induced phase change was demonstrated in all of these organelles, their derivative membranes, and in micelles formed from the membrane phospholipids. The apparent transition temperature of the membrane lipids varied slightly between the samples, but in all cases, fell within the temperature range around 10 C where physiological and biochemical changes in the response to temperature for most chilling-sensitive plants occur.     

Mobilization of Reserve Proteins During Early Stages of Seed Germination in Fagopyrum Esculentum

Hydrolysis of 13S globulin, the main storage protein in grains of common buckwheat (Fagopyrum esculentum Moench), proceeds in at least two phases during germination. The first stage, involving a limited proteolytic cleavage of the protein, is associated with increased activity of proteases having maximum activity at pH 7.6. The second stage, involving further hydrolysis of the partially cleaved protein, starts after 12 h of imbibition. During this phase, activity of proteases increased and activity maximum shifted to pH 5.6. Nevertheless, 13S globulin retains its antigenic identity till the emergence of radicle and plumule. Thus, it may not be the major source of amino acids utilized by the germinating seed during the initial stages of imbibition. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

萌发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L—苹果酸、a—酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP／O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

Biosynthesis of Cytidine 5'-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm

Cytidine 5′-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn²⁺ being preferred and giving maximum activity at 2.5 millimolar. The estimated K_m for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).

The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 millimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K_m for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K_m estimated for CTP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold.

     

Starch Mobilization in Ultradried Seed of Maize （Zea mays L.） During Germination

The effects of ultradry storage on the starch mobilization in maize (Zea mays L.) seed after aging were investigated. The results indicated that there were no significant differences in the content of ATP, starch, and soluble sugar, as well as the activity of amylase, between ultradried seeds and seeds stored at -20℃ during germination. These results were consistent with the higher level of vigor of the ultradried seed. Sieve tube introduction of a fluorescence dye (carboxyl fluoresceindiacetate) and laser confocal microscopy were used to study the development of plasmodesmata in the ultradried seeds. The results indicated that plasmodesmata developed well in ultradried seeds. Fluorescence analysis also showed that the fluorescence intensity in the radicle of ultradried seeds was stronger than that in seeds with a higher moisture content. This suggests that ultradry treatment has no adverse effects on the seeds. After seed imbibition, cell orgaelles could be resumed. It is concluded that ultradry seed storage is beneficial for maintaining seed vigor and that starchy mobilization proceeds regularly during germination.