Endoplasmic Reticulum of Mung Bean Cotyledons: ACCUMULATION DURING SEED MATURATION AND CATABOLISM DURING SEEDLING GROWTH

Homogenates of mung bean cotyledons were subjected to equilibrium density centrifugation on linear sucrose gradients and the positions of the various organelles determined by assay of marker enzymes. Measurement of phospholipid distribution on such gradients showed that the major peak of phospholipid at a density of 1.11 to 1.13 grams per cubic centimeter coincided with the position of the endoplasmic reticulum (ER), confirming ultrastructural evidence that storage parenchyma cells are rich in ER. Germination and seedling growth were accompanied by a rapid decline in ER-associated phospholipid but a marked increase in the ER marker enzyme NADH cytochrome c reductase. Similar experiments with developing seeds indicated that the amount of ER-associated phospholipid increases during cotyledon expansion reaching a maximum during seed maturation. There was no subsequent decline during seed desiccation, instead ER-associated phospholipid levels were maintained in the dry seed until germination when catabolism was initiated 12 to 24 hours after the start of imbibition. This timing indicates that the observed ER breakdown is not an expression of the overall senescence of the cotyledons, but may represent the dismantling of the extensive rough ER used for reserve protein synthesis during cotyledon development.     

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.     

The endoplasmic reticulum of mung-bean cotyledons: Quantitative morphology of cisternal and tubular ER during seedling growth

The ultrastructure of the endoplasmic reticulum (ER) in storage parenchyma cells in the cotyledons of mung beans (Vigna radiata L.) was examined during germination and seedling growth. Two different methods were used to visualize the ER: thin (0.08 m) sections of tissue fixed in formaldehyde and glutaraldehyde and post-fixed with osmium tetroxide, and thick (1 m) sections of tissue fixed in buffered aldehyde and post-fixed with zinc iodide-osmium tetroxide (ZIO). Changes in relative amounts of ER were quantified by morphometry (stereology).The ER occurs in two forms: a cisternal form with associated ribosomes which can be seen at all stages from imbibition to cotyledon senescence, and a tubular form which initially has associated ribosomes. Stereoscopic images of thick sections of cotyledons of 2-day-old seedlings show that the tubular ER consists of a three-dimensional array of interconnecting tubules which have numerous connections with the cisternal ER. The network of tubules and cisternae extends throughout the cytoplasm enveloping the protein bodies. Germination and seedling growth are accompanied by a reduction in the total volume occupied by the ER. This reduction is the result of a preferential loss of tubular ER and occurs largely before protein mobilization. Cisternal ER decreases during the first 48 h of imbibition and seedling growth, but storage cells subsequently show an increase in cisternal ER just prior to and during the period of protein mobilization. Cisternal ER remains conspicuous during the last phase of reserve mobilization when starch is broken down and the cells are starting autophagy.Abbreviations ER endoplasmic reticulum - ZIO zinc iodide-osmium tetroxide This is the second in a series of papers on the endoplasmic reticulum of mung bean cotyledons. The first paper is referenced herein as Gilkes and Chrispeels (1980)     

Seed reserve translocation and early seedling growth of eight tree species in a tropical deciduous forest in Mexico

Seed reserves play an essential role during germination and seedling establishment and are particularly important for species that grow in seasonal ecosystems with a short growing season. In this study, we examined (a) how and when the seedlings change their dependence from seed resources to external resources, (b) the lipid, nitrogen, and non-structural carbohydrate reserve translocation from seeds to seedlings over time, and (c) whether reserve translocation may be correlated to cotyledon and leaf lifespan of seedlings for eight tree species in a tropical deciduous forest in north-western Mexico. Our results showed that the cotyledon lifespan was not related to the cotyledon type (photosynthetic or reserve) and that the cotyledon biomass did not decrease significantly until germination. In six of the eight studied species, biomass allocation to the leaves was favored; lipids were the first reserve exhausted before the first leaves were totally expanded in seven of the eight study species. Species with the highest N concentration had expanded leaves and lost their cotyledons faster than species with a low N concentration. Our results suggest that tropical deciduous forest species employ different strategies to survive the dry season and re-sprout in the next growing season mediated by seed reserve concentrations, translocation patterns and subsequent biomass allocation.     

Changes in Cell Fine Structure in the Cotyledons of Phaseolus vulgaris L. during Germination

When germination begins, the storage cells of Phaseolus vulgariscotyledons are packed with starch grains and protein bodies.Digestion of these reserves starts in cells furthest away fromthe vascular bundles and is practically completed in eight daysat 25° C. After the reserves are hydrolysed, the storagecells die. The changes in fine structure during the processof digestion and protoplasmic breakdown are described. Vascularbundle and epidermal cells survive till the cotyledons absciss,but in these tissues also profound changes occur in cellularorganization. The observations on fine structure are discussedwith reference to the metabolic activities of the cotyledons.     

Metabolism of carbohydrate and lipid reserves in germinated cotton seeds

Utilization of reserve lipid and carbohydrates during germination (0–12 h) and postgerminative growth (12–48 h) was studied in cotton (Gossypium hirsutum L.) seedlings. Raffinose and stachyose were utilized during the germination period and early growth; mobilization was associated with -galactosidase (EC 3.2.1.22) activity. Results from pulse-chase experiments with [³H]raffinose supplied exogenously to 4-h soaked seeds indicated that raffinose-derived catabolites contributed to the coincident increase in cotyledon sucrose and starch, and to the small increase in axis dry weight. Starch appears to be an alternative sink for end products of hydrolysis of reserve carbohydrates prior to the onset of rapid axis growth and cotyledon expansion. Mobilization of neutral lipid commenced at about 16 h after soaking, concomitant with development of key glyoxylate-cycle and other gluconeogenesis-related enzyme activities. Axis dry weight increased three-fold between 24 and 48 h. Results from pulse-chase (3 h, 16 h) experiments in which [2-¹⁴C]acetate was supplied to cotyledons of intact 22-h-old seedlings showed that acetate-derived metabolites were not transported exclusively to the axes, but were partitioned between axes and cotyledons. Only 27% of total incorporated radioactivity was recovered in axes following the chase, 18% was evolved as CO₂, and the rest was recovered in water-soluble substances (20%) and polymers (31%) within the cotyledons. Of the polymers, 55% of the activity was in polysaccharides (Starch, pectic substances, hemicellulose, cellulose), 25% in protein, and 20% in unidentified neutral and acidic compounds. Considering these data, the amount of lipid mobilized, and various routes by which supplied [2-¹⁴C]acetate could be metabolized, it appears that lipidderived compounds contribute only 25–40% of axis dry-weight gain. Lipid-derived substances retained in the cotyledons likely are utilized for expansion and differentiation of the cotyledons into photosynthetic organs.     

The Formation of Polyploid Cells in Ripening Cotyledons of Pisum sativum L. in Relation to Ribosome and Protein Synthesis

The present paper deals with the formation of polyploid nucleiand the synthesis of RNA and protein in the parenchyma cellsof developing cotyledons of Pisum sativum L. During seed formationthese cells synthesize large amounts of reserve proteins andstarch, which later on are used up by the embryo during seedgermination. The changes of the amount of DNA per cell in the ripening cotyledontissue have been estimated by Feulgen histophotometry. The amountsof DNA, RNA, and protein in the whole cotyledons have been estimatedby chemical methods. In this way it was possible to correlatethe fluctuations of the amount of DNA, RNA, and protein withchanges at the cellular level. During a preparatory phase, preceding the phase of real cellexpansion and intensive accumulation of seed globulins and starch,the storage cells attain a high level of polyploidy: nucleiwith up to a 64 C DNA content are formed. The results indicate a correlation between the high degree ofpolyploidy in the parenchyma cells of the cotyledon and thehigh rate of RNA and protein synthesis (seed globulins) in thisstorage organ (gene dosage effect).     

Distribution and Isoenzymes of Aspartate Aminotrans-f erase in Cotyledons of Germinating Pumpkins

During germination a steady decline in the reserve protein occurred in dark grown pumpkin cotyledons. By 9 days, 80% of this nitrogen reserve was hydrolyzed but only 50 % was removed from the cotyledons. The remaining nitrogen (30 %) was incorporated into water soluble protein which reached a maximum 9 days after germination. The increase in water soluble protein in pumpkin cotyledons parallel the increase in soluble and particulate aspartate aminotransferase (E.C.2.6.1.1.), suggesting that this enzyme is involved in nitrogen metabolism during germination. Little enzyme activity was found in pumpkin tissues other than the cotyledons. Four anodally moving isoenzymes were found in the soluble aspartate aminotrans-ferase fraction and 3 anodally moving isoenzymes were found in the particulate fraction. The slowest moving isoenzymes disappeared first during germination.     

Protein Bodies of Germinating Seeds of Vicia faba: CHANGES IN FINE STRUCTURE AND BIOCHEMISTRY

Changes in fine structure and starch, total nitrogen and solublesugar contents were followed during the first 3 weeks of germinationin Vicia faba cotyledons. Following hydration the reserves were mobilized and loss ofprotein from the cotyledons began after 4 days; concomitantfine structural changes included the swelling and coalescenceof protein bodies, the disappearance of their contents, andthe differentiation of the provascular tissue in the cotyledonwithin which ‘transfer cells’ developed. There wassome development of rough endoplasmic reticulum during the earlypart of germination. A pattern of protein-body degradation appeared in the cotyledonsduring germination, those cells nearest to the vascular bundlesand to the epidermis being the first to lose their reserves.After 3 weeks' growth the parenchyma cells of the cotyledonwere very vacuolate and typical of senescent tissue, while thevascular bundle cells still retained their contents and remainedactive-looking.     

黄瓜种子萌发过程中Ca~(2 )分布的变化

采用细胞化学方法 ,研究了黄瓜种子中贮藏Ca2 的分布特点及其在萌发过程中的变化动态。干种子的子叶细胞中贮藏有大量的蛋白体、油脂体 ,Ca2 沉淀颗粒大量分布于胞质、胞间隙以及细胞质膜上。大多数蛋白体中有 1至数个圆球形或椭圆体形含Ca2 的球状晶体。相比之下 ,胚芽和胚根细胞中Ca2 较少。种子萌发早期 ,子叶中的贮藏钙及晶体溶解释放出的Ca2 部分转运到生长发育中的胚芽和胚根中。随着萌发的继续 ,胚根和胚芽细胞中的Ca2 不会持续增多 ,反而下降     

大豆子叶衰老的细胞学观察

以萌发后不同时期的大豆子叶为材料,通过普通光镜和荧光显微镜观察,分析了不同时期子叶细胞的结构变化及子叶细胞内蛋白质和淀粉含量的组织化学变化.结果表明,随着种子萌发时期的延长,子叶细胞内的蛋白质和淀粉含量逐渐减少,在子叶衰老过程中,细胞内蛋白质首先消耗殆尽,淀粉的消耗速度较蛋白质慢;大豆子叶细胞在萌发后第18天时出现典型的植物编程性死亡的形态学特征,子叶细胞内营养物质的消耗诱发子叶细胞发生细胞凋亡.     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质, 主要为蛋白质、淀粉和淀粉质体DNA。这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料。通过组织化学和显微镜观察, 研究莲从种子萌发到植株生长至具有4个节时, 子叶中贮藏物质消耗的全过程。在此过程中, 子叶中的贮藏物质不断降解,营养物质发生转运。蛋白体首先发生降解, 其大量降解主要发生在幼苗三叶期。淀粉质体降解时会聚 集成团, 之后体积逐渐减小, 最后完全降解。种子萌发后65天是子叶贮藏物质消耗末期, 淀粉质体DNA的含量比萌发后20天的三叶期明显减少。细胞壁的形态结构发生多种形式的变化, 细胞壁发生的这些变化与子叶细胞间物质的运输有关。含多糖的球形颗粒通过维管束在子叶中运输。     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质,主要为蛋白质、淀粉和淀粉质体DNA.这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料.通过组织化学和显微镜观察,研究莲从种子萌发到植株生长至具有4个节时,子叶中贮藏物质消耗的全过程.在此过程中,子叶中的贮藏物质不断降解,营养物质发生转运.蛋白体首先发生降解,其大量降解主要发生在幼苗三叶期.淀粉质体降解时会聚集成团,之后体积逐渐减小,最后完全降解.种子萌发后65天是子叶贮藏物质消耗末期,淀粉质体DNA的含量比萌发后20天的三叶期明显减少.细胞壁的形态结构发生多种形式的变化,细胞壁发生的这些变化与子叶细胞间物质的运输有关.含多糖的球形颗粒通过维管束在子叶中运输.     

In Vitro Stability and Inactivation of Peptide Hydrolases Extracted from Phaseolus vulgaris L.

Endopeptidase activity against azocasein had a higher temperatureoptimum (50°C) in leaf extracts than in cotyledon extracts(37°C). The temperature optima for aminopeptidase (46°C)and for carboxypeptidase (53°C) were similar in leaf andcotyledon extracts. The endopeptidase activity showed an excellentstability in crude extracts from leaves even at 37°C, whilethe endopeptidase in cotyledon extracts was less stable. Carboxypeptidasewas very stable in both leaf and cotyledon extracts. Aminopeptidasewas the least stable of the enzymes investigated and its inactivationrate depended on the source of the extract. A moderate stabilitywas observed in extracts of leaves or of ungerminated seeds,but this enzyme was rapidly inactivated in cotyledon extractsat pH 5.4. At pH 7.5 aminopeptidase remained active longer thanat pH 5.4. From experiments with mixed extracts it could beconcluded that in cotyledons an aminopeptidase inactivatingfactor was formed during germination. This factor was heat sensitive,excluded by Sephadex G-25, precipitated by 75% ammonium sulfateand inhibited by tosyl-L-lysine chloromethyl ketone. These datasuggest that the factor is a protein and considering the similarproperties it appears possible that it is the endopeptidaseformed during germination. (Received May 15, 1981; Accepted July 18, 1981)     

Changes in nitrogen fractions and proteolytic activities in the cotyledons of germinating lentils

Changes in ninhydrin positive material, free amino acids and protein content during germination of seeds of Lens culinaris Med. have been studied. Ninhydrin positive material and free amino acids reached their highest concentration at the fifth day of germination. Total protein which represents 21% of the total dry weight of the lentil cotyledons, suffers a degradation of only 24% in seven days of germination; in the same period of time, reserve proteins underwent a degradation of 69%, legumin being the more abundant at the start, and the more rapidly depleted. Five different classes of proteolytic activities have been reported in lentil cotyledons: caseinolytic, active against the reserve proteins of the lentil cotyledons themselves, aminopeptidase, peptidehydrolase, carboxypeptidase and dipeptidase. The removal of the axis did not seem to exert any significant influence on the enzymatic activity.     

DNA and protein content relationship in the cells of mature cotyledons ofPisum sativum

Summary The relationship between the DNA and reserve protein contents have been investigated in the cells of mature cotyledons ofPisum sativum. The study was made on two seeds chosen, on preliminary analysis, as having a markedly different protein content per cotyledon. The seed with the higher protein content was of theCameor variety, the other was of theAmino variety. The DNA content per cell was measured with a scanning microspectrophotometer and the protein content per cell using morphometric methods. In both seeds, nuclei of the storage parenchyma were polyploid in a range from 8 to 64 C levels, but the average DNA content per cell was higher in the Cameor specimen. The protein content also, whether expressed per unit of protoplasm or per cell, was always higher in the Cameor seed. The greater protein content of this seed was due to a higher content of protein per cell. In both seeds the level of ploidy and the reserve protein content per cell increased in the same way from the peripheral zone to the centre of the cotyledon. A high positive correlation was found for the two seeds between the DNA content and the protein content per cell.     

Subcellular Localization Studies Indicate That Lipoxygenases 1 to 6 Are Not Involved in Lipid Mobilization during Soybean Germination

Soybean (Glycine max) lipoxygenase (LOX) has been proposed to be involved in reserve lipid mobilization during germination. Here, subcellular fractionation studies show that LOX1, -2, -3, -4, -5, and -6 isozymes were associated with the soluble fraction but not with purified oil bodies. The purified oil bodies contained small amounts of LOX1 (<0.01% total activity), which apparently is an artifact of the purification process. Immunogold labeling indicated that, in cotyledon parenchyma cells of LOX wild-type seeds that had soaked and germinated for 4 d, the majority of LOX protein was present in the cytoplasm. In 4-d-germinated cotyledons of a LOX1/2/3 triple null mutant (L0), a small amount of label was found in the cytoplasm. In epidermal cells, LOX appeared in vacuoles of both wild-type and L0 germinated seeds. No LOXs cross-reacting with seed LOX antibodies were found to be associated with the cell wall, plasma membrane, oil bodies, or mitochondria. Lipid analysis showed that degradation rates of total lipids and triacylglycerols between the wild type and L0 were not significantly different. These results suggest that LOX1, -2, -3, -4, -5, and -6 are not directly involved in reserve lipid mobilization during soybean germination.     

Ultrastructural and Biochemical Changes in Cotyledon Reserve Tissues3

Changes in major protein, lipid and carbohydrate reserves duringthe germination of Citrus limon (L.) Burm. f. seeds have beenstudied. The rate of release of amino acids and soluble sugarshas been evaluated. Mobilization of protein reserves began 4 d after the onset ofimbibition. The main period of hydrolysis occurred between 8d and 24 d after the start of germination. Ultrastructural studies showed the presence of protein bodiesin quiescent cotyledon cells. These bodies virtually disappeared14 d after the start of germination. The nitrogenous compoundsthat were liberated and subsequently translocated were predominantlyin the form of asparagine, arginine, and proline. The cotyledonshad a lipid content representing 51.7% of their dry weight.Lipid reserves in quiescent cotyledons were laid down in theform of oil-bodies. These organelles rapidly disappeared asgermination progressed, and were replaced by vacuoles. The starch content of quiescent cotyledons is very low, butit increased considerably up to 20 d after germination started. Key words: Proteins, lipids, carbohydrates