On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communis L. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted frommature castor bean cv. Hale seed cotyledons, differ quantitativelyand qualitatively from their counterparts extracted from theendosperm. The soluble fraction contains no glycoproteins, andthe lectins RCA₁ and ricin D are absent. While the insolubleproteins are electrophoretically and immunologically similarto those in the endosperm, they do not form the 100 kD subunitdimers which characterize some of the endosperm insoluble crystalloidproteins. Rapid rates of deposition of all of the soluble andinsoluble proteins present in the mature seed cotyledons commences30–35 d after pollination (DAP) and continues until 45DAP. These proteins are mobilized rapidly beginning 1–2d after seed imbibition and this coincides with an increasein specific activity, in the cotyledons, of two aminopeptidasesand a carboxypeptidase. The soluble and insoluble proteins inthe cotyledons of the mature seed probably function as storageproteins and support the growth of the germinated seed priorto the mobilization of the major protein storage reserves ofthe endosperm. Key words: Ricinus communis, Castor bean, Hale cultivar, Cotyledon, Storage protein, Seed development, Seed germination     

Unsuitable Availability of Nutrients in Germinating Bean Embryos Exposed to Copper Excess

The influence of copper excess on germination rate, growth, minerals, carbohydrates, and amino acids supply in embryonic axis of bean seed was investigated. Compared to the control, Cu treatment caused a reduction in germination percent, embryo length, and accumulation of Ca, Fe, K, Mn, Zn, total soluble sugars, glucose, fructose, sucrose, and amino acids. Moreover, the nutrient concentrations, as well as the electrical conductivity were determined in the germination medium to quantify the extent of solute leakage. Such nutrients were lost in the imbibition medium at the expense of suitable mobilization to the growing embryonic axis. This was associated with an enhancement in accumulation of malondialdehyde, major product of lipoperoxidation process which can be due to the stimulation in lipoxygenase activity in Cu-poisoned tissues.     

Isolation and Mobilization of Storage Proteins from Apple Shoot Bark

Separation of the bark storage protein into three groups was achieved with minimal contamination from interfering cbromophores, using diethylaminoethyl cellulose column chromatography. Two of these groups were shown 10 contain neutral sugars in close association with the proteins. All three groups disappeared rapidly after the onset of growth. Polyacrylamide gel electrophoresis of the storage proteins showed the presence of eight prominent proteins. Ammo acid analysis of hydrolyzates of the proteins indicated that a large percentage of the protein nitrogen was in the form of basic amino acids.     

Lipid Breakdown in Smooth Microsomal Membranes from Bean Cotyledons Alters Membrane Proteins and Induces Proteolysis

The effects of lipid degradation on proteins of smooth microsomalmembranes isolated from young bean cotyledons have been examinedby three techniques, viz. fluorescence energy transfer fromtryptophan to cis-parinaric acid; protein spin-labelling with3-maleimido PROXYL; and SDS-PAGE. Lipid degradation was inducedin isolated membranes by activating phospholipase D and phosphatidicacid phosphatase through the addition of Ca²⁺, by treatmentwith exogenous phospholipase C to simulate the concerted actionsof phospholipase D and phosphatidic acid phosphatase or by treatmentwith exogenous phospholipase A₂ to generate endogenous substratefor lipoxygenase. All of the treatments induced time-dependentchanges in lipid-protein interaction and in protein conformation,and the treatment with phospholipase A₂ also engendered proteolysis.The effects of the Ca²⁺ and phospholipase C treatments on lipid-proteininteraction and protein conformation can presumably be partlyattributed to an accumulation of diacylglycerol in the membrane,whereas the induction of proteolysis by phospholipase A₂ appearsto be due to activated oxygen derived from the lipoxygenasereaction and ensuing lipid peroxidation. Lipid degradation inducedby these treatments simulates that which occurs during naturalsenescence of the cotyledons and hence these observations suggestthat loss of protein function and proteolysis in senescing membranesis facilitated by lipolytic and peroxidative activity withinthe lipid bilayer. Key words: Activated oxygen, lipids, membranes, proteins, senescence     

The Control of Lipid Mobilization in Cucumis Cotyledons

An investigation has been carried out into the role of the axisand testa in the control of lipid mobilization during the germinationof cucumber seedlings. A very close correlation is observedbetween axis growth and lipid depletion. It is suggested thatthis depends upon the rate at which the axis can remove themajor product, sucrose, and not upon a hormonal mode of controlas has been previously suggested. Excised testaleas cotyledons show considerable lipid breakdownand can develop maximum levels of isocitrate lyase and ß-oxidationenzymes even though the axis is not present. The presence ofthe testa inhibits their development however. A novel indirectcontrol mechanism involving the automatic removal of the testa(a phenomenon common amongst most members of the Cucurbitaceae)is suggested.     

Sucrose-Induced Increase of Invertase Synthesis in Castor Bean Cotyledons

Abstract

Aumento della sintesi di invertasi in seguito a trattamento con saccarosio in cotiledoni isolati da semi germinanti di ricino. – L'attività invertasica per cotiledone aumenta durante la germinazione di semi di ricino. In cotiledoni isolati ed incubati in acqua distillata per 15–22 ore, l'aumento di attività invertasica è molto scarso, L'aggiunta di saccarosio 0,1 M al mezzo di incubazione provoca un aumento di circa 40% dell'attività invertasica; aumento che non si riscontra se i cotiledoni vengono incubati in glucosio 0,1 M. La pre-senza di attinomicina D e di puromicina nel mezzo di incubazione previene lo sviluppo dell'attività invertasica. L'apparente specificità del saccarosio nell'indurre l'aumento di sintesi dell'enzima viene brevemente discussa nel quadro piú ampio dei fenomeni di regolazione da substrato delle sintesi di enzimi.     

Water Content and Respiration Rate of Bean Cotyledons

The course of water uptake and respiration rate rise in cotyledonsof Phaseolus vulgaris is divided into three phases. In the first phase lasting 10–16 hrs. respiration rateis controlled by water content; desiccation and reimbibitioninfluence cotyledon water content and respiration rate alikeand the changes are reversible; low temperature prevents watercontent rising above 50 per cent. and also limits respirationrate; both processes have Q₁₀ near unity. The second phase lasting 3–8 hrs. is characterized bya pause in both water uptake and respiration rate rise. In the third phase respiration rate continues to rise untilthe fifth day, after which it falls steadily until eventuallythe cotyledons absciss. The period of rising respiration isone of metabolic activity, the rise having a high Q₁₀ and beingprevented by low temperature. Desiccation at this stage is irreversible.     

Protein Synthesis and Accumulation in Bean Cotyledons during Growth

Analysis of total protein, of specific proteins by gel electrophoresis and immunoelectrophoresis, and of protein synthetic activity in vitro confirmed that intense protein synthesis and accumulation occurred as the French bean (Phaseolus vulgaris L). seed grew from 12 to 20 millimeters. These techniques showed that there was no globulin-1 (G1) fraction (requiring high salt for solubility) present in 6-millimeter seeds, and only very small amounts were synthesized in seeds less than 9 millimeters long. The 7- to 9-millimeter stages represent a 2-day transition period over which genetic information for the G1 protein becomes actively expressed, accounting for at least 50% of all protein synthesized in this tissue during the following 14 days. At maturity, the electrophoretic analysis confirmed that G1 globulin was the major storage protein, representing some 50% of the dry seed protein. Cell-free protein synthesis assays, including immunoprecipitation of the in vitro products, clearly showed G1 polypeptides to be among the polysome-directed products.     

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。     

Respiratory Changes During Seed Germination. Histological Distribution of Respiratory Enzymes and Mobilization of Fat Reserves in Castor Bean Endosperm and Peanut Cotyledons

Germinating peanut cotvledons and germinating castor bean endosperm have been compared with respect to their rates of fat dissimilation and with respect to the anatomical distribution of respiratory activity. The lipid mobilization is much slower in peanut cotyledons than in castor bean endosperm. Light has essentially no effect on either system. As germination progresses, the majority of the succinic dehydrogenase and cytochrome oxidase activities become localized in the vein regions of peanut cotyledons. In the castor bean endosperm these two activities are uniformly distributed throughout the storage parenchyma and increase with germination until the organ becomes soft and visibly senescent.     

Localization of Peroxidized Lipids in Non-Sedimentable Microvesicles of Senescing Bean Cotyledons

Fluorescent peroxidized lipids are present in lipid extractsof microsomal membranes and cytosol from young and senescingbean (Phaseolus vulgaris) cotyledon tissue. In young tissue,the peroxidized membrane lipids are mainly phospholipids, whereasthose in the cytosol are primarily free fatty acids. With advancingsenescence, microsomal peroxidized lipids increase by 200% relativeto membrane protein and by 50% on a per cotyledon basis, andthe increase is mainly attributable to enhanced levels of peroxidizedfree fatty acids. Cytosolic peroxidized lipids expressed ona per cotyledon basis decline by 55% over the same period. Fractionationof the cytosol revealed that, for both young and senescing tissue,about 50% of the cytosolic fluorescent peroxidized lipids areassociated with non-sedimentable microvesicles, which are formedfrom membranes and enriched in phospholipid catabolites. Moreover,the decline in cytosolic peroxidized lipids with advancing senescencecorrelates with progressive impairment of the formation of thesenon-sedimentable microvesicles. Key words: Phaseolus vulgaris, senescence, lipid peroxidation, fluorescence     

Basic Endochitinases Are Major Proteins in Castanea sativa Cotyledons

Basic endochitinases are abundant proteins in Castanea sativa Mill. cotyledons. Three basic chitinases were purified with molecular masses of 25, 26, and 32 kD (Ch1, Ch2, and Ch3) and with isoelectric points between 8 and 9.5. Antibodies raised against Ch1 cross-reacted with Ch2 and Ch3. However, Ch3 showed differences when compared with the other two enzymes, especially in its higher cysteine content. The size, amino acid composition, and N-terminal sequence of Ch1 indicate that it is a class II endochitinase and, therefore, has no cysteine-rich hevein domain. Ch1 inhibits the growth of the fungus Trichoderma viride. The biological role of these endochitinases is discussed.     

Diffusible and Extractable Gibberellins in Bean Cotyledons in Relation to Dwarfisni

Dwarfism in bean plants (Phaseolus vulgaris L.) is investigated in relation to the diffusible and extractable gibberel-lins in cotyledonary tissues. These gibberellins were partitioned into four parts prior to thin layer chromatograplty: non-acidic and acidic ethyl acetate fractions, and non-acidic and acidic butanol fractions. Cotyledonary segments from a tall plant (cv. Kentucky Wonder) seem to diffuse, preferentially acropetally, more gibberellins in each fraction than those from a dwarf plant (cv. Masterpiece). The diffusion increases with the length of the segments and decreases with period after sowing. From experiments on extraetable gibberellins, however, it is concluded that these phenomena actually result from differences in the gibbereliin contents of the tall and the dwarf plants, from differences in the gibberellin contents of the distal and proximal parts of cotyledons, and from the gibberellin contents before and after imbibition. Ten kinds of gibberellin-Iike substances are detectable in the diffusates, exudates and extracts from the cotyledons of both the plants; two in the non-acidic ethyl acetate, three in the acidic ethyl acetate (GA₁, GA₆ and another), two in the non-acidic butanol, and three in the acidic butanol frac—tion. They are almost identical in quality in the dwarf and tall plants, but in the latter they are more abundant in the cotyledons, particularly in their distal part. With respect to the change in content during the period after sowing, the gibberellin-Iike substances are classified in three groups; unchanging, decreasing (GA₁, GA₆, others) and increasing (butanol soluble glucosyl esters and glucosides of gibberellins)-. The increase of glucose-bound gibberellins and the decrease of free gibberellins during the sowing period suggest the occurrence of conversion. This is obvious only in the tall plants. The gibberellin content in cotyledons is higher in the tall plant than in the dwarf plant. Thus, the marked hypocotyl growth in the tall plants may be dependent on the higher content of gibberellins in their cotyledons and on the higner rate of conversion from free to bound forms.     

The Proteins in Mung Bean Mitochondria

The mung bean mitochondria were obtained by sucrose cushion centrifugation in quite pure form. The outer membrane and the mitoplast were separated by digitonin treatment followed by sucrose density gradient centrifugation. SDS gradient polyacrylamide gel electrophoresis showed that the 42–44 KD protein in mung bean mitochondria was located in mitoplast rather than in the outer membrane. Co-electrophoresis with rabbit muscle actin showed that the rabbit muscle actin coincided with the 44 KD band in plant mitochondria. These results suggest that one of the motive force of the swelling and shrinking of the mitochondria may be generated from the mitochondria themselves.     

Structures of Sugar Chains of Water-soluble Glycoproteins in Developing Castor Bean Cotyledons

The structures of sugar chains from the water-soluble glycoproteins in developing castor beans have been identified. The structural analyses were done by a fluorescence method combined with exoglycosidase digestions and ¹H-NMR spectroscopy. The identified structures fell into two categories; one was an oligomannose-type, the other xylomannose-type or xylose-containing type. Among these oligosaccharides, Man₃Fuc₁Xyl₁GlcNAc₂ (M3FX; 38%) and Man₆GlcNAc₂ (M6B; 22%) were the major structures. The higher mannose-content oligosaccharides (Man_{8 ? 7}GlcNAc₂) were only 4.1%, and the further-modified structures (_GNM3FX, M2FX) than M3FX were 22% of the total.     

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。     

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.