The origin of protein bodies in developing soybean cotyledons: a proposal

Summary The biogenesis of protein bodies is examined in cotyledons of soybean (Glycine max, Merr) at the time when reserve protein is beginning to accumulate in the cotyledons. Reserve protein is deposited in the central vacuoles of parenchyma cells and new protein bodies arise from the central vacuole by pinching-off small masses of reserve protein surrounded by a portion of the tonoplast.Supported by a grant to MJC from the National Science Foundation (Metabolic Biology) and a grant (to BYY) from the National Research Council of Canada.On leave from the Department of Biology, University of New Brunswick, N.B., Canada.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.     

Sucrose uptake by cotyledons of Ricinus communis L.: Characteristics,mechanism, and regulation

Cotyledons of Ricinus communis take up externally supplied sucrose at a rate of up to 150 mol/h/g fresh weight, which is very high when compared with other sugar transport systems of higher plants. The uptake of sucrose is catalysed with a K _m of 25 mmol l^–1; at high sucrose concentrations a linear (diffusion) component becomes obvious. Other mono-, di-, or trisaccharides do not compete for sucrose uptake. Sucrose is accumulated by the cotyledons up to 100-fold, whereby most of the transported, externally supplied sucrose mixes with sucrose present in the tissue. At low sucrose concentrations, however; a small unexchangeable internal pool of sucrose becomes evident. Poisons of energy metabolism such as FCCP inhibit uptake and accumulation of sucrose. The transport of sucrose induces an increase of respiration, from which an energy requirement of 1.4 ATP/sucrose taken up can be calculated. Sucrose is taken up together with protons at an apparent stoichiometry of 0.3 protons/sucrose. Other sugars do not cause proton uptake. The K _m for sucrose induced proton uptake is 5 mmol l^–1; the discrepancy to the K _m for sucrose uptake as well as the low proton: sucrose stoichiometry might possibly be caused by a large contribution of diffusion barriers. The estimated proton-motive potential difference would by sufficient to explain an electrogenic sucrose accumulation. The rate of uptake of sucrose is subject to feedback inhibition by internal sucrose. It is also regulated during growth of the seedlings since it develops rapidly during the first days of germination and declines again after the 4th day of germination, though no substantial increase of passive permeability resistance was observed.Abbreviations DMO dimethyloxazolidinedione - FCCP trifluoromethoxy (carbonyl-cyanide) phenylhydrazon - fr. wt. fresh weight     

Ultrastructure of Protein Bodies and Plastids in Cotyledon of Nelumbo nucifera Gaertn.at Seed Germination

The present article deals mainly with the formation and dissolution of protein bodies and development of plastids in cotyledon cells of Nelumbo nucifera during seed germination. Electron microscopic studies reveal that protein bodies are formed after imbibition of the cotyledons before germination. They are produced through accumulation of protein material in small vacuoles delivered from the exudates of endoplasmic reticulum or by fragmentation of endoplasmic reticulum itself. In the period of germination, most of the material in the protein bodies dissolute and they coalesce with each other forming large vacuoles. The protein residue of the vacuoles condenses into small blocks with high electron density adhering to the tonoplast or freely floating in the vacuole. Thus, it suggests that the protein bodies of the germinating N. nucifera cotyledons are originated from vacuoles formed by endoplasmic reticulum. Part of the plastids found in cotyledonous cells of mature N. nucifera seeds exists as proplastids. They develop continuously after imbibition of the cotyledons. During the period of seed germination, many concentric lamellae are developed along the plastid membrane on which they later coalesce with the neighboring concentric lameUae forming loosely organized prolamellar bodies which condense into paracrystalline lattices. No ribosomes are present in the inter spaces of paracrystatline lattice. One to several prolamellar bodies can be developed in one plastid.     

Advantage of early seedling emergence in <Emphasis Type="Italic">Fagus crenata</Emphasis>: importance of cotyledon stage for predator escape and pathogen avoidance

To examine how early-emerging seedlings take advantage of establishment in a deciduous forest, we explored the relationships among the emergence date, growth stage and major causes of mortality (damping-off by fungi and predation by rodents) in seedlings of Fagus crenata. The emergence of current-year seedlings and their survival and damage were followed at short (3–7 days in early spring) intervals for a growing season. The growth stage was divided into two stages, with only cotyledons (CT stage) and with true leaves (TL stage). The survival rate was negatively correlated with the emergence date, indicating the advantage of early seedling emergence. This advantage was largely explained by the lower occurrence of damping-off. In contrast, seedling predation occurred independently of the emergence date, but depended strongly on the developmental stage. Rodents consumed a considerable number of seeds during the early period after emergence, and strongly preferred CT-stage over TL-stage seedlings throughout the growing season. Therefore, seedling predation was inferred to be concentrated in a relatively short period while the remaining seeds were depleted and CT-stage seedlings were abundant. The seedling stage synchronously shifted from the CT to TL stage. This growth-stage transition was independent of the emergence date and appeared to correspond with the timing of seed depletion. Delayed stage transition resulted in a disproportionately high risk of damping-off later in the season. Our results indicate that early seedling emergence is advantageous for F. crenata for resistance to pathogens and that the timing of growth-stage progression of seedlings appears important to escape predation because of the distinct food preference of predators.     

Light and Electron Microscopic Observation on the Developing Pattern of Cotyledons of Nelumbo nucifera

The whole growing period (from the formation of cotyledon primordia till ripe) of cotyledons of Nelumbo nucifera takes about 30 days, but can be varied in 3–5 days according to the varieties, temperature, light duration and its intensity during the blooming season. The observations by light and electron microscopy show that the feature and structure of mesophyllous cell are changed greatly during its development. The developing pattern of the cotyledons is similar to that of dicotyledonous plants. For comparitive analysis the authors divide the whole developing process into four developing phases: Ⅰ, the phase of cell division and organ formation; Ⅱ, the phase of cell vacuolation, elongation and swelling; Ⅲ, the phase of main physiological function in which the materials are largely synthesized and accumulated and Ⅳ, the phase of dehydration, contraction, maturation and dormancy. The development of mesophyllous cells in different part of cotyledons is not simultaneous and the duration of each phase is also different. In general, the developing order is from the base to the top and first the outlayer then the centre. On the 25–26th days after fertilization almost all mesophyllous cells are developing into maturation and dormancy by order. This is the first report about the developing pattern of cotyledons and the ultrastructural changes in mesophyllous cells of Netumbo nucifera.     

Seed reserve-protein glycosylation in an in vitro preparation from developing cotyledons of Phaseolus vulgaris

A particulate preparation from developing cotyledons of Phaseolus vulgaris L. was incubated with uridine-5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc; [6-³H]glucosamine), and by polyacrylamide gel electrophoretic analysis it was shown that the labeled (N-acetyl)glucosamine (GlcNAc) was incorporated into the principal reserve protein of the cotyledons, vicilin, and also into phytohemagglutinin. Some of the labeled product also reacted with antiserum to vicilin from mature seeds. In contrast it was not possible to detect the incorporation of labeled mannose from guanosine-5-diphospho-D-mannose (GDP-mannose; [U-¹⁴C]mannose) into either of these proteins by gel-electrophoretic analysis of the mannose-labeled products, but we did observe a low incorporation of mannose into material which reacted with antiserum to vicillin. The predominant glycosylation reaction in vitro was therefore probably a transfer of GlcNAc alone, rather than in combination with mannose as preformed oligosaccharide.Abbreviations GlcNAc N-acetyl-D-glucosamine - GDP guanosine 5-diphospho - IEF isoelectric focusing - PHA phytohemagglutinin - SDS sodium dodecylsulfate - UDP uridine-5-diphospho     

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)     

The endoplasmic reticulum of mung-bean cotyledons

Germination and seedling growth of mungbean (Vigna radiata (L.) Wilczek) are accompanied by the incorporation of radioactive amino acids, glycerol, galactose, and glucosamine in an organelle fraction of the cotyledons which co-equilibrates with NADH-cytochrome-c-reductase activity at 1.13 g·cm^–3 on isopycnic gradients containing 1 mM EDTA. Up to 20% of the newly synthesized proteins accumulate in this organelle fraction. The organelle fraction has been identified as rough endoplasmic reticulum (ER) on the basis of its increased density (1.16 g·cm^–3) when 3 mM MgCl₂ is included in all media. Seedling growth is also accompanied by a marked rise (more than 5-fold) in ER-associated NADH- and NADPH-cytochrome-c-reductase activity, and by the incorporation of⁵⁹Fe into ER-associated heme. Other manifestations of the reorganization of the ER in the cotyledons include a relative increase in membrane-associated RNA (from 12% of total RNA after 12 h of imbibition to 23% after 6 d of growth), and a change in the pattern of polypeptides associated with the ER. These results provide further evidence for the extensive reorganization of the ER of the cotyledons which accompanies seedling growth. The reorganization includes the simultaneous breakdown of the pre-existing tubular ER and the biosynthesis of new ER components.This is the fourth paper in a series on the endoplasmic reticulum of mung-bean cotyledons. The first three papers are referenced as Gilkes and Chrispeels (in press); Harris and Chrispeels 1980; Van der Wilden et al. (in press)