Proteomic Profiling of the Aleurone Layer of Mature Arabidopsis thaliana Seed

The aleurone layer of mature Arabidopsis thaliana seed plays important roles in seed germination and dormancy. However, the proteomic profile of this cell layer is unknown partly because it is difficult to separate this thin cell layer from the mature seeds. In this study, we have used a simple technique to separate the aleurone layer along with the seed coat following germination of seeds and determined for the first time the putative protein composition of this cell layer. By subjecting the total proteins extracted from the seed coat to 2D gel electrophoresis followed by liquid chromatography/tandem mass spectrometry, we identified four AGI loci, AT4G28520, AT5G44120, AT1G03880, and AT1G03890; all of which belong to the seed storage family of proteins. Because in Arabidopsis the diploid aleurone cells of the seed coat perform protein storage functions similar to that of triploid endosperm of other plant species, it is assumed that the above AGI loci are associated with the aleurone layer of the seed coat.     

Anatomical and ultrastructural changes in aleurone and myrosin cells of Sinapis alba during germination

Summary The cells of the embryo of Sinapis alba L. include either aleurone or myrosin grains and all cells contain oil bodies. Aleurone grains and oil bodies are degraded during germination. The myrosin grains of each myrosin cell, on the other hand, gradually turn into one big vacuole containing the myrosin. Probably very little, if any, new myrosin is formed in the cotyledons and hypocotyl of the seedling after germination. No difference was found between aleurone and myrosin cells in the development of organelles. The cells of provascular bundles of the mature embryo contain different amounts of aleurone grains in different stages of development, and their organelles are more developed than those of all other cells.     

Studies on Mature Seeds of Cuscuta pedicellata and C. campestris by Electron Microscopy

The seeds of Cuscuta pedicellata have been investigated by transmissionand scanning electron microscopy. Additional observations havebeen made on seeds of C. campestris by SEM only. The seed coatconsists of an outer single epidermis, two different palisadelayers, and an inner multiparenchyma layer. The outer epidermalwall in C. pedicellata has a thick cuticle and zones rich inpectic substances. The thicker ‘U-shaped’ cell wallsin the outer palisade layer are strengthened by a wall layerof hemicellulose. The inner palisade layer has thick walledcells with a ‘light line’. The inner cell wall ofthe compressed multiparenchyma layer has a thin cuticle. A fairlythick cuticle is positioned directly on the endosperm surface.The aleurone cell walls are different from the remaining endospermwalls. The latter are thick and believed to be of galactomannans.There is a ‘clear’ zone between the plasmalemmaand the cell wall in the aleurone cells. The embryo cells arepacked with lipids and proteins. In Cuscuta campestris mostendosperm has been absorbed during the seed development. Theembryo apex has two minute leaf primordia. The features of theCuscuta seeds are discussed in relation to functional and environmentalconditions. Cuscuta pedicellata, Cuscuta campestris, seed, seed coat, cuticle, cell walls, endosperm, aleurone cells, galactomannan, embryo, TEM, SEM     

Control of Seed Germination by Abscisic Acid: I. Time Course of Action in Sinapis alba L

The germination process of mustard seeds (Sinapis alba L.) has been characterized by the time courses of water uptake, rupturing of the seed coat (12 hours after sowing), onset of axis growth (18 hours after sowing), and the point of no return, where the seeds lose the ability to survive redesiccation (12 to 24 hours after sowing, depending on embryo part). Abscisic acid (ABA) reversibly arrests embryo development at the brink of radicle growth initiation, inhibiting the water uptake which accompanies embryo growth. Seeds which have been kept dormant by ABA for several days will, after removal of the hormone, rapidly take up water and continue the germination process. Seeds which have been preincubated in water lose the sensitivity to be arrested by ABA after about 12 hours after sowing. This escape from ABA-mediated dormancy is not due to an inactivation of the hormone but to a loss of competence to respond to ABA during the course of germination. The sensitivity to ABA can be restored in these seeds by redrying. It is concluded that a primary action of ABA in inhibiting seed germination is the control of water uptake of the embryo tissues rather than the control of DNA, RNA, or protein syntheses.     

The development of protein and oil bodies in the seed of Sinapis alba L.

Summary The cotyledons of Sinapis alba L. seed are the storage organs and first photosynthetic organs. The development of the cotyledon cell contents was studied using electron and light microscopy. From the heart shaped embryo (11 days from petal fall) to the mature seed, nine stages were examined.Both types of protein grains (designated aleurone grains and myrosin grains) were found to form within vacuoles, but the mode of protein accumulation differed with each type of grain.Oil bodies were apparent with the EM from 18 days onwards, but could not be seen to arise from the ER. They were granular in appearance at early stages, but later became electron transparent.     

Allelopathic effects of sunflower extracts on mustard seed germination and seedling growth

The allelopathic potential of two cultivars of sunflower (Helianthus annuus L.) Lech and Ogrodowy was studied. Leaf aqueous extracts of sunflower at 2.5, 5, and 10 % (m/v) concentrations were applied to determine their effect on mustard (Sinapis alba) seed germination and seedlings growth under laboratory conditions. Increasing concentration of aqueous extracts of sunflower inhibited seed germination, up to almost complete failure of germination, observed in the presence of 10 % extract from sunflower cv. Ogrodowy. The decrease in germinability was well correlated with increased membrane deterioration, assayed as electrical conductivity and enhanced lipid peroxidation, detected as increased malondialdehyde content.     

Comparative Requirement for Endogenous Ethylene during Seed Germination

Requirement for endogenous ethylene during seed germinationof the following ten species was determined: Lycopersicon esculentumMill, (tomato), Allium cepa L. (onion), Avena fatua L., dormantpure line AN-51 (wild oats), Cucumis sativus L. (cucumber),Sinapis arvensis L. (wild mustard), Tagetes erecta L. (marigold),Raphanus sativus L. (radish), Triticum aeslivum L. (wheat),Catharanthus roseus L. (periwinkle), and Phaseolus aureus L(mung bean). Experiments were done under controlled conditionssuited for the germination of each species. Criteria used todetermine the need for endogenous ethylene were: (i) temporalrelationship between ethylene production and seed germination;(ii) parallel inhibition of ethylene synthesis and seed germinationby L-     

Barbiturate-induced Disorientation of Root and Shoot Geotropism in Germinating Cruciferous Seedlings

Cruciferous seedlings germinated on glass-distilled water andon barbital manifested normal root and shoot geotropisms whereastwisted roots and shoots were evident in seedlings germinatedon amobarbital and secobarbital, the roots often being completelysuspended in the air. The marked difference in germination anddevelopment behaviour of cress and white mustard seedlings germinatedon barbital compared with that on amobarbital and secobarbitalindicate that the Meyer-Overton principle of drug potency andlipid solubility was operative in both species of germinatingseedlings. Lepidium sativum L., Sinapsis alba L., seed germination, geotropism, barbiturates     

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     

The fine structure of aleurone cells in the soybean seed coat

Summary The morphology and fine structure of aleurone cells of soybean [Glycine max (L.) Merr.] seed coats were analyzed with transmission electron microscopy for the period of rapid seed fill up to physiological maturity. Thin sections and freeze-fracture replicas were prepared for each stage. The aleurone is a tissue lining the embryo sac and consists of a single layer of cells attached to the aerenchyma of the seed coat proper. During seed fill, aleurone cells contained numerous Golgi-derived vesicles in the basal region of the cytoplasm that were either free or attached to the plasma membrane along the lateral and basal regions of the cell wall. Correspondingly, the Golgi apparatus were well developed with individual dictyosomes having 5 to 8, highly fenestrated stacked cisternae. The degree of fenestration along the periphery of each cisterna increased from the cis to trans region. Rough endoplasmic reticulum (RER) was also abundant, often consisting of up to 30, stacked swollen cisternae which occupied large regions of cytoplasm. Plasmodesmata which connected adjacent aleurone cells was not observed along the dorsal walls of aleurone cells that faced aerenchyma. At physiological maturity, dictyosome cisternae were less fenestrated and had fewer associated secretory vesicles. Stacked lamellae of RER were absent, being replaced by short tubular cisternae and small vesicles. At physiological maturity, the aleurone cells had thick walls, and contained numerous lipid bodies in apposition to the plasma membrane. The cytoplasm appeared densely stained in thin-sections and contained protein bodies and amyloplasts with large starch grains. We conclude that during the period of rapid seed fill aleurone cells produce, package, transport and secrete vesicular contents toward the embryo, that is followed at physiological maturity by the storage of lipid, protein and starch in the same cells. The embryo is the most likely destination for secretory products during the period of rapid seed fill. The fate of the stored food reserves in aleurone cells at physiological maturity may be analogous to that of aleurone tissue of grasses, being utilized during imbibition for processes important to germination.     

Localization of the beet mild yellowing virus inSinapis alba L.

Virus particles of isometric shape with a diameter of 26 nm were found in the sieve tubes and accompanying phloem cells in ultrathin sections prepared from the nerves of white mustard (Sinapis alba L.) leaves and roots infected with the beet mild yellowing virus (BMYV). BMYV particles were much more frequent in the roots ofSinapis alba plants. Isometric particles were not found in the leaves and roots of healthy mustard plants.     

Early Development of the Seed Coat of Soybean (Glycine max)

Although the development of the soybean ovule has been fairlywell studied, knowledge of the sequence of events in the seedcoat during the first 3 weeks after flowering is incomplete.The goal of the present study was to document, using light microscopy,the early development of the soybean seed coat with respectto changes in structure and histochemistry. At anthesis, theseed coat consists of an outer layer of cuboidal epidermal cellssurrounding several layers of undifferentiated parenchyma (whichtogether constitute the outer integument), and an inner layerof cuboidal endothelial cells (the inner integument). At 3 dpost anthesis (dpa), the inner integument has expanded to includethree to five layers of relatively large cells with thick, heavily-stainingcell walls immediately adjacent to the endothelium. By 18 dpa,the outer integument has developed into a complex of tissuescomprised of an inner layer of thick-walled parenchyma, an outerlayer of thin-walled parenchyma containing vascular tissue whichhas grown down from the lateral vascular bundles in the hilumregion, a hypodermis of hourglass cells, and palisade layer(epidermis). The thick-walled parenchyma of the inner integumenthas become completely stretched and compressed, leaving a single,deeply staining wall layer directly above the endothelium. At21 dpa, the outermost cells of the endosperm have begun to compressthe endothelium. At 45 dpa (physiological maturity) the seedcoat retains only the palisade layer, hourglass cells, and afew layers of thin-walled parenchyma. The innermost layer ofthe endosperm, the aleurone layer, adheres to the inside ofthe seed coat. This knowledge will be invaluable in future studiesof manipulation of gene expression in the seed coat to modifyseed or seed coat characteristics. Copyright 1999 Annals ofBotany Company Soybean, Glycine max, seed coat, development, aleurone.     

Effects of Ageing on Amylase Activity and Scutellar Cell Structure during Imbibition in Wheat Seed

In wheat seed the scutellum plays an important role in the hydrolysisof stored substrate during germination. This layer is activatedfirst, whilst the aleurone becomes activated later. A good correlationexists between the initiation of visible germination and theappearance of enzyme activity in the scutellum. Enzyme activityin the aleurone becomes apparent only when the germinating seedlingreaches the rapid growth phase. Electron microscopic observationsshow that during the later stages of germination the scutellarcells develop finger like projections. These may serve to absorbendospermic reserves hydrolysed by aleurone amylase. The scutellumof aged non-germinating seeds showed no amylase activity andno finger like projections were produced even after prolongedimbibition.Copyright 1993, 1999 Academic Press Wheat (Triticum aestivum L.), deteriorated, germination, scutellum, scanning electron microscopy, aleurone     

Structure and Histochemistry of Embryo Envelope Tissues in the Mature Dry Seed and Early Germination of Phacelia tanacetifolia

The embryo envelope tissues in both mature dry seed and duringearly germination of Phacelia tanacetifolia were investigatedby bright-field and fluorescence light microscopy and scanningelectron microscopy. The ruminate seed had an irregularly reticulatesurface owing to the presence of polygonal areas, correspondingto the cells of the seed coat. The raised margins of these cellsjoined at the lobe tips, where radially arranged thickeningsoccurred. The unitegmic seed coat was made up of three distinctlayers: the frayed outer layer, the middle layer with portionsrising outwards to form the radial thickenings, and the innerlayer, the thickness of which was greatest in the micropylarzone. The endosperm tissue had two regions, the micropylar andthe lateral endosperm, which differed in polysaccharide composition,thickness and metachromasy intensity, and presence (in the lateralendosperm) or absence (in the micropylar endosperm) of birefringenceof the cell walls. Moreover, in the micropylar region, wherethe embryo suspensor remnant was found, Ca-oxalate crystalswere scarce or absent. The presence of a partially permeablecuticle covering the seed endosperm was observed. Incubationof seeds in Lucifer Yellow CH indicated that water was ableto penetrate quickly into the seed coat along the pathway formedby the radial thickenings, the raised margins of the polygonalcells and the middle layer. Afterwards, LY-CH readily infiltratedthe apical portions of the seed lobes and then the whole endosperm.Following imbibition, morphological changes were found in themicropylar endosperm, such as the initial digestion of proteinbodies. In addition, both in the seed coat and in the endosperm,a weaker fluorescence, probably due to leaching of polyphenolicsubstances, was observed. Once the seed coat was broken at themicropylar end of the seed, the endosperm cap surrounding theradicle tip had to be punctured by it so that complete germinationcould occur. Weakening and rupture of the micropylar endospermare briefly discussed. Copyright 2000 Annals of Botany Company Phacelia tanacetifolia, seed coat, micropylar endosperm, endosperm cap, early germination, structure, histochemistry     

Observations on the Germ Aleurone of Barley. Morphology and Histochemistry

The germ aleurone over the embryonic axis of barley was examinedin strips of tissue peeled off harvest-ripe grains. The germaleurone is only one cell thick but resembles 'normal' aleuronein being composed of living cells with dense, lipid-rich cytoplasmand thick walls containing phenolic material. In contrast tothe cells of the 'normal' aleurone, germ aleurone cells containvery few phytin or protein deposits. When the germ aleuroneis ruptured during germination, the walls at the torn edge becomethickened with shiny golden-brown material, and 'sealed' tothe testa. Two days after germination, lignin can be detectedin the walls of a single row of germ aleurone cells adjoiningthe scutellum. The role of the germ aleurone in defence againstmicroorganisms is discussed. It is suggested that the metabolicactivities in the germ aleurone in imbibed grains compete withthe embryo for oxygen, and thus may be one of the factors whichdetermine whether a grain germinates or remains dormant.Copyright1994, 1999 Academic Press Barley, Hordeum vulgare L., germ aleurone, histochemistry, defence mechanism, lignin, dormancy, microorganisms, pre-mature germination     

Water uptake and splitting of wild oat caryopsis

Imbibition and germination experiments were conducted on the caryopses of wild oats (Avena fatua L.). The embryo envelopes, pericarp and aleurone layer, which completely cover the embryo-endosperm, do not form barriers against water uptake. The initial uptake of water is passive and the water moves across the pericarp with ease as it contains cracks; it is, however, transported across the aleurone layer through its cell walls into the endosperm and embryo of the caryopsis. The starchy endosperm enlarges due to water uptake causing the pericarp to rupture, thus exposing the aleuronelayer-covered seed. The aleurone layer is structurally heterogenous consistings of radially compressed irregular cells and cuboidal or radiallys tretched cells; the latter contains thicker walls. The former type is present along the abaxial side of the embryo and in the crease on the adaxial side of the caryopsis; the latter type covers the endosperm. The physical distention of the endosperm due to water uptake causes the rupture of the pericarp and the aleurone layer, and facilitates the emergence of the radicle and coleorhiza of the embryo during caryopsis germination.     

The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.     

Embryo Dormancy and Light Requirements in the Germination of Helipterum craspedioides (Compositae), an Arid-zone Annual

Seeds of Helipterum craspedioides require light for germination,and this may be given while the seed is not fully imbibed. Removalof the seed coat, or leaching, does not bring about germinationin the dark. Gibberellin enhances dark germination to some extent,whether or not the seed coat is removed. The light requirementtherefore resides in the embryo. Fresh seed is dormant in lightor dark. Excised embryos of these dormant seeds grow slowly,rarely giving mature plants, but a larger number of normal plantsis produced after gibberellin treatment. Dormancy is lost afterhigh temperature storage, but not stratification.     

Seed Coat Dormancy in Two Species of Grevillea(Proteaceae)

The role played by the seed coat in seed dormancy of Grevillealinearifolia(Cav.) Druce and G. wilsonii(A. Cunn.) was testedby a series of manipulations in which the seed coat was dissectedand removed, dissected and returned to the decoated seed, ordissected, removed and given a heat shock, and returned to thedecoated seed. Germination of intact seeds of both species wasalso examined after exposure to heat shock, smoke, or heat shockand smoke combined. Water permeability of the seed coat wasinvestigated by examining imbibition. For intact seeds, virtuallyno germination occurred under any treatment (G. wilsonii), orgermination was increased by exposure to either heat or smoke(G. linearifolia). Removal of the seed coat led to germinationof all decoated seeds for G. linearifolia, or a proportion ofdecoated seeds for G. wilsonii. Inclusion of smoked water inthe incubation medium led to a higher proportion of decoatedseeds germinating for G. wilsonii. Returning the seed coat,either with or without heat shock to the seed coat, did notsignificantly affect germination in either species. Seed coatswere permeable to water in both species. For the two Grevilleaspecies, there were different dormancy mechanisms that werecontrolled by the seed coat (G. linearifolia) or by both theseed coat and embryo (G. wilsonii). Copyright 2000 Annals ofBotany Company Grevillea linearifolia, Grevillea wilsonii, dormancy, seed coat dormancy, seed coat permeability, smoke, heat shock, germination     

Callose Deposits make Clover Seeds Impermeable to Water

The permeability status of the seed from ten genotypes of subterraneanclover, determined by soaking, is shown to be related to callosedeposition in the seed coat. Large amounts of callose were foundin the nutrient (parenchyma) layer of impermeable but not permeableseeds. It is suggested that the nutrient layer controls permeabilityand thus the ease of germination of seeds. Trifolium subterraneum L., clover, seed coat impermeability, callose