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.     

Amaranthus hypochondriacus: Seed Structure and Localization of Seed Reserves

The mature seed of Amaranthus hypochondriacus L. consists ofa peripheric embryo surrounding the nutritive tissue which isthe perisperm. Endosperm remnants are close to the root tip.Cytochemical analysis revealed that the embryo and endospermcells had a quite homogenous internal organization, with proteinbodies embedded in a lipid matrix. The embryo, however, appearsvariable in tissue organisation, due to the differentiationof the three primary meristematic tissues: the procambium appearsas a single bundle in the embryonic axis or as small bundlesthroughout the cotyledons length, these provascular cells aresmall and elongated and with fewer reserves and more cellularorganelles than the large protoderm and ground meristem cells.These latter cells have more protein bodies, and they show ahigher number of larger globoid crystal inclusions than theothers. The perisperm is a starchy tissue, and its cells havethin walls and are full of angular starch grains.Copyright 1994,1999 Academic Press Amaranth, Amaranthus hypochondriacus, seed structure, seed reserves     

Protein Bodies of Developing Seeds of Vicia faba

Changes in fine structure and starch, nitrogen, and solublesugar content were followed through to maturation in developingcotyledons of Vicia faba. Various ultrastructural changes wereobserved in the developing cotyledons, notably an increase inthe number of membrane-bound ribosomes which corresponded withthe onset of storage protein deposition. The build-up of storageprotein was shown to occur in the cytoplasm within membrane-boundvacuoles which subsequently became the protein bodies of themature seed, retaining the original tonoplast as the boundingmembrane of the protein body. Nuclei became lobed during thelater phases of maturation; phytoferritin was observed in plastidsof mature seeds. The deposition of reserves in the cotyledonswas complete by 85–90 days after flowering, followingwhich water was lost until the seed became hard and ‘ripe’by no days after flowering.     

Respiration Rate, Mitochondrial Activity and Mitochondrial Structure in the Cotyledons of Phaseolus vulgaris L. during Germination

Parallel observations have been made on changes in respirationrate, mitochondrial activity, and the fine structure of mitochondria,in the cotyledons of Phaseolus vulgaris germinating at 25^? Cin the dark. While oxygen uptake of the intact cotyledons risesto a peak between the third and fifth days of germination, mitochondrialactivity falls from 36 hours onwards. In most of the storagecells, mitochondrial cristae have become swollen and the matrixhas darkened by the third day. The continulng rise in respirationrate beyond 36 hours could be accounted for by the increasein soluble oxidative activity, which occurs while mitochondrialactivity is falling. Alternatively, it could be mediated bymitochondria from the vascular bundle cells, where swellingand darkening are delayed till after the fifth day of germination,and where there is proliferation of cristae till the fourthday.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. II. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the radicle tips of germinatingPhaseolus vulgaris seeds revealed that cytokinins are transportedrapidly from the embryonic axis to the cotyledons, and are utilizedand metabolized extensively in these organs. The informationobtained on the transportation between the different parts ofthe embryo is consistent with the view that the mobilizationof food reserves from the cotyledons is controlled by cytokininswhich originate in the embryonic axis. Tentative identificationof the radioactive metabolites formed indicate that the appliedzeatin was altered by side-chain cleavage and by substitutionto the adenine ring. Phaseolus vulgaris, bean, germination, cytokinins, transport, radicle     

Ultrastructural Observations on Proliferating Storage Cells of Mature Cotyledons of Phaseolus vulgaris L. Cultured in vitro

Explants of mature cotyledons of Phaseolus vulgaris form callusrapidly when cultured in vitro with their adaxial surfaces embeddedin a solidified nutrient medium containing coconut milk, kinetinand 2,4-D. Proliferation is confined to the highly polyploidstorage cells and commences near the adaxial epidermis, whichis soon ruptured by the callus developing internally. Callusformation progresses to the abaxial tissue and within 3–4weeks sub-culturing is possible. The in vitro grown storage cells undergo thinning of their walls,loss of food reserves, hypertrophy, development of various new-wallsand nuclear activation leading to division. The induction ofnuclear and cell divisions within this mature storage tissuecontrasts with normal germination in which these cells undergorapid senescence after depletion of their food reserves. Nuclear division in early callus growth is apparently mainlyamitotic. It is preceded by the development of multiple nucleoli.The nuclear envelope also becomes more complex and deeply lobed;leading to formation of a nuclear isthmus and final separationinto two nuclei. No chromosomes are visible during nuclear fragmentation.Amitosis is accompanied by freely-forming walls, which may developadjacent to a nuclear isthmus and perhaps participate in nuclearfragmentation. Large labyrinthine wall bodies frequently occuron these walls. Mitoses are only observed in already dividedstorage cells. A cell plate forms between the two daughter nuclei,and microtubules are present at its margins in contrast to freely-formingwalls where none are evident. Phaseolus vulgaris L., bean, in-vitro culture, cotyledon, ultrastructure     

Some Effects of Potassium Deficiency on Seedling Development

Seedlings with leaf-like photosynthetic cotyledons such as Cucumissativus, Sinapis alba and Trifolium pratense have higher growthrates and a greater requirement for an external supply of potassiumthan hypogeal species (Pisum sativum, Vicia faba) or epigealspecies with very fleshy cotyledons (Phaseolus vulgaris, Lupinusalbus, Lupinus angustifolius). The initial stages of seedlingdevelopment in the latter two groups involve mainly transferof reserves from cotyledons to axis and a relatively small increasein total seedling dry weight takes place during the first 2to 3 weeks. Potassium may be drawn either from the supply already presentin the cotyledons or from an external source. Species with expandingphotosynthetic cotyledons utilize the reserve potassium duringcotyledon development and it is not transported to the epicotyl.Thus, in these species an external supply of potassium is essentialfor full development of the photosynthetic system and the roots.In species whose cotyledons are not major photosynthetic organsthe reserve potassium is transported from the cotyledons tothe developing axis. In these species culture in a nutrientsolution deficient in potassium or in distilled water increasesroot dry weight but reduces plumule weight (although to a lesserextent than in species with leaf-like cotyledons). In general,the response of seedlings to culture in a nutrient solutiondeficient in potassium was very similar to culture in distilledwater only. Thus, although all the species studied had similar levels ofpotassium in the seed (µg potassium/mg dry weight) itssite of utilization differs. It appears that seedlings withleaf-like cotyledons are more susceptible during early seedlinggrowth to deficiencies in external factors, such as potassium,than are species with fleshy cotyledons.     

Growth Analysis of Soybean Seedlings During the Lifespan of the Cotyledons

Patterns of seedling growth of Glycine max in light and darknesswere compared during the period from germination to cotyledonabscission. Fitted growth curves and the derived functions,relative growth rate, unit leaf rate, leaf area ratio and specificleaf area, were used to assess the relative importance in seedlinggrowth of cotyledon storage reserves, cotyledon photosynthesisand leaf photosynthesis. The cotyledons are of an intermediatetype with a predominant storage and a minimal photosyntheticfunction. Cotyledon reserves support seedling growth until theprimary leaves expand, after which growth depends on leaf photosynthesis. Glycine max (L.) Merr., soybean, cotyledons, growth analysis, seedling development     

Cytochemical Localization of Reserves during Seed Development in Arabidopsis thaliana under Spaceflight Conditions

Successful development of seeds under spaceflight conditionshas been an elusive goal of numerous long-duration experimentswith plants on orbital spacecraft. Because carbohydrate metabolismundergoes changes when plants are grown in microgravity, developingseed storage reserves might be detrimentally affected duringspaceflight. Seed development in Arabidopsis thaliana plantsthat flowered during 11 d in space on shuttle mission STS-68has been investigated in this study. Plants were grown to therosette stage (13 d) on a nutrient agar medium on the groundand loaded into the Plant Growth Unit flight hardware 18 h priorto lift-off. Plants were retrieved 3 h after landing and siliqueswere immediately removed from plants. Young seeds were fixedand processed for microscopic observation. Seeds in both theground control and flight plants are similar in their morphologyand size. The oldest seeds from these plants contain completelydeveloped embryos and seed coats. These embryos developed radicle,hypocotyl, meristematic apical tissue, and differentiated cotyledons.Protoderm, procambium, and primary ground tissue had differentiated.Reserves such as starch and protein were deposited in the embryosduring tissue differentiation. The aleurone layer contains alarge quantity of storage protein and starch grains. A seedcoat developed from integuments of the ovule with gradual changein cell composition and cell material deposition. Carbohydrateswere deposited in outer integument cells especially in the outsidecell walls. Starch grains decreased in number per cell in theintegument during seed coat development. All these characteristicsduring seed development represent normal features in the groundcontrol plants and show that the spaceflight environment doesnot prevent normal development of seeds in Arabidopsis. Arabidopsis ; spaceflight; embryo; endosperm; seed coat; storage reserves     

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     

Importance of Endosperm for Nutrition of Fraxinus pennsylvanica Seedlings

Contact between endosperm and cotyledons of germinating Fraxinuspennsylvanica Marsh. seedlings was not essential for continuationof seedling growth or adaptation of cotyledons for photosynthesis.However, when cotyledons were in contact with endosperm, thecotyledons had faster rates of elongation and dry weight increase,slower depletion of reserves, and higher chlorophyll contents.Photosynthetic contributions by cotyledons may be enhanced ifenviron mental conditions support rapid emergence of cotyledonsfrom surrounding seed structures.     

An Ultrastructural Investigation of Protein and Lipid Reserves in the Endosperm of Pinus pinea L.

Proteins and lipids are the two main storage substances in theendosperm of Pinus pinea L. In imbibed seeds, lipid and proteinbodies almost completely fill the cell volume and only a fewpoorly structured organelles can be detected. During germinationthe storage substances are reabsorbed whilst plastids, roughendoplasmic reticulum, ribosomes and polysomes, microbodies(glyoxysomes, most likely) become evident. Starch is also formed.When the cotyledons emerge completely from the endosperm envelopethe latter shows highly vacuolate cells but still rich in cytoplasmicorganelles, lipids, protein bodies and starch grains. Different patterns of protein body degradation are discussed. Pinus pinea, endosperm, ultrastructure, protein, lipid, stored reserves     

Utilization of Seed Reserves and Currently Produced Photosynthates by Embryonic Tissues of Pine Seedlings

The importance of seed reserves for growth of Pinus resinosaAit. during and shortly after seed germination was studied undercontrolled conditions. Tissues in the resting embryo were notcompletely differentiated. Many small, presumably reserve particleswere present in the embryo in addition to reserves in the megagametophyte.During seed germination, procambia in the embryo first differentiatedprotophloem 2 days after seeds were sown. The radicle beganto emerge from the seed coat at 5 days, at which time initialxylem formation was observed. Also, at approximately the sametime, primordia of primary needles were forming in the peripheralzone of the apex. Elements of the photosynthetic apparatus,including stomata and mesophyll with chloroplasts, were differentiatedfirst in the hypocotyl and then in cotyledons between 5 and8 days after seeds were sown. Photosynthetic rates of youngseedlings were correlated with rates of cotyledon expansion.During early developmental stages, reserve particles in megagametophytecells and embryo cells gradually disappeared. Surgical removalof megagametophytes at various stages of seed germination resultedin subsequent growth inhibition of the hypocotyl-radicle axis,with early removal of cotyledons suppressing most growth. Growthof primary needles appeared to be influenced indirectly by megagametophytereserves, probably by changes in amount of photosynthetic tissue.The embryo alone possessed capacity to differentiate such tissuesas primary needle primordia, stomata, and primary and secondaryvascular systems. Megagametophyte reserves appeared to contributeto growth of embryonic tissues only after the embryo itselfinitiated growth. Both current photosynthesis of seedlings andseed reserves contributed importantly to seedling development.     

Histochemical studies on mobilization of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark at 27 C, and time-sequence changes of mobilization of protein and starch reserves in cotyledons were observed by histochemical techniques. The distributions of amylase and protease activities in cotyledon sections were also examined during germination by use of the starch-polyacrylamide gel film and India ink-gelatin film methods, respectively. Amylolytic and proteolytic processes occurred more or less simultaneously during the germination. At the day 2 stage, low levels of hydrolytic enzyme activities were observed throughout cotyledon sections. At day 4, both amylase and protease activities appeared to increase in tissue areas farthest from vascular bundles, and the mobilization of starch and protein reserves also proceeded in these areas. At day 6, the reserves were found to remain only in the cells around vascular bundles. When cotyledons were detached from axis organs, allowed to imbibe water and incubated for 4 days at 27 C, the breakdown of reserves was markedly retarded and the patterns of enzyme localization in cotyledon sections appeared not as conspicuous as those in the sections from intact cotyledons. These histochemical results are discussed with reference to the previous results ofin vitro experiments.     

Changes in Nuclear DNA Content, Cell and Nuclear Size, and Frequency of Cell Division in the Cotyledons of Brassica napus L. during Embryogenesis

Cellular behaviour was examined during embryogenesis in Brassicanapus to test whether or not polyploidy occurs in the cotyledonsduring the phase of oil deposition. Nuclear DNA content, nuclearand cell size, and the mitotic index were measured in the cotyledonson various days post anthesis (dpa). In squashed monolayersfrom 15 dpa cotyledons, a polyploid (>5C) population wasdetected together with a substantial number of cells in G2 (4C).Nuclear volume was measured on sectioned tissues and, at 15dpa, the range of values from the cotyledons (40–500 *m³)contrasted with that in the vestigial suspensor and endosperm(50–> 600 µm³). At 15 dpa the nuclear volumedata suggest that whilst cells in the cotyledons were in Gland G2 many endosperm and suspensor cells were polyploid. Thus,polyploidy observed in the squashed monolayers was probablydue to contaminating endosperm/suspensor cells. At 25 and 35dpa, polyploidy was not detected; all cells were in Gl (2C)and cell area increased. The mitotic index peaked at 20 dpabefore declining and given the narrower distribution of nuclearvolumes at 25 and 35 dpa (50–300 µm³), these dataare consistent with cell arrest in Gl. Thus, polyploidy wasnot detected in the cotyledons of B. napus which differs fromwhat is known about cellular development in legume cotyledons. Key words: Brassica napus L., DNA, nuclear volume     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, germination     

Analysis of Lipids of Citrullus lanatus (cv. Sugar baby) During Seed Germination and Seedling Growth

Changes in the dry weight (dry wt.), total and neutral lipids,and fatty acid composition were determined in the cotyledonsand axis of Citrullus lanatus cv. Sugar baby seedlings duringtheir first 12 d growth in the dark, at 25 ? 1 ?C. The major stored reserves were mobilized between days 3 and8. The lipid concentration after day 4 decreased rapidly inthe cotyledons. On the first day the lipid concentration inthe axis was higher than in the cotyledons but decreased rapidlyand from day 5 up to the end of the experiments it remainedat very low values. In the cotyledons, the change in the fattyacid composition was relatively small compared to the significantchange observed in the axis. The neutral lipids of the cotyledonsdecreased sharply between days 2 and 6 while their fatty acidcomposition suffered no significant change. The axis, comparedto the cotyledons, contained a small but concentrated amountof neutral lipids, which decreased up to day 6. Thereafter,an increase was observed. The fatty acid composition of theaxis neutral lipids changed significantly, linoleic and oleicacid decreased while palmitic and linolenic acid increased. Key words: Citrullus lanatus, seed germination, seedling growth, lipids, fatty acids     

Gibberellin-induced Changes in Ultrastructure of Lettuce Cotyledons

Seeds of lettuce (Lactuca saliva L. cv. Grand Rapids) were incubatedin water or in gibberellin A₃ (GA). Some ultrastructural differencesbetween the cotyledons of 145 hour-old seedlings resulting fromthese two treatments are reported. The cotyledons of GA-treatedseedlings contained many plastids with poorly developed grana,while the control cotyledons contained only well-developed plastids.The frequency of grana in developed chloroplasts was also reducedby the hormone treatment. Effects of GA on the nucleus includeda decrease in heterochromatin and an increase in nucleolar volume.After GA treatment, the nucleolar matrix had a largely fibrillarappearance in contrast to the predominantly granular structureof the control nucleoli. Ultrastructural changes are discussedin relation to the GA-induced variations in the biochemicalcomponents previously reported.     

Inhibition of Zeatin-Induced Growth of Cucumber Cotyledons by Sulfite Ions

The inhibitory effects of sulfite ions on zeatin-induced cellexpansion in cotyledons excised from dark-grown seedlings ofcucumber (Cucumis sativus L.) were examined. With 50 µMzeatin the growth rate in white light was about twice that ofthe control. Addition of Na₂SO₃ in the growth medium inhibitedthe zeatin-induced growth of cotyledons. Zeatin-treatment increasedthe osmotic potential in cell sap of cotyledons, while sulfitedecreased it. These treatments had no significant effect onpotassium concentration. Sulfite inhibited the zeatin-inducedincrease in contents of fructose and glucose, but did not affectsucrose content. The relative contents of non-cellulosic constituentsof cell walls fell with the advance of culture. This decreasewas repressed by sulfite, indicating that inhibition of expansiongrowth in cucumber cotyledons by sulfite ions was the resultof alterations in the cell wall structure due to changes inthe cell wall metabolism. (Received June 12, 1984; Accepted October 24, 1984)     

Changes in Levels of Cytokinins in Etiolated Squash Seedlings after Illumination

When etiolated seedlings of squash (Cucurbita maxima Duch. cv.Houkou-aokawaamaguri) were exposed to light, their cotyledonsbegan to accumulate chlorophyll at a low rate for the firsthour but at a high rate therafter. After 0, 1 and 2 h of illumination,cytokinin levels in cotyledons, hypocotyls and roots were analyzedby ELISA (enzyme-linked immunosorbent assay) and GC-SIM (gaschromatography-selected ion monitoring). Cytokinin levels measuredby ELISA were about 10 times higher than those measured by GC-SIM,suggesting that ELISA was not suitable for the measurementsof cytokinin levels in the extracts from these tissues. Cytokininsfound by GC-SIM were t-RZ (t-ribosyl zeatin), c-RZ, t-Z (t-zeatin),c-Z, RiP (ribosyl isopentenyladenine) and iP (isopoentenyladenine).Levels of biologically active cytokinins (t-RZ and t-Z) didnot show marked changes after illumination. Levels of c-RZ andc-Z did, however, decrease in cotyledons and increase in hypocotylsafter illumination. Promotion of the accumulation of the chlorophyllin detached squash cotyledons by exogenously applied t-Z waspartially inhibited by the addition of c-Z, suggesting thatthe decrease in the endogenous level of c-Z in cotyledons ofintact seedlings after illumination permits the light-inducedformation of chlorophyll. (Received August 8, 1990; Accepted April 4, 1991)