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.     

Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.     

Localization of major endopeptidase activities in maize endosperms

Using a tissue print method, major endopeptidase activitieswere observed in the aleurone layer and along parts of scutellumsurface 1 d after imbibition. By day 2 the zone of activityhad spread into the subaleurone and starchy parenchyma cellsof the endosperm. Three days later, activity was detected throughoutthe endosperm tissue, but not in the embryo. Endosperm tissues,aleurone layers and scu-tella were dissected from the seedlingsat different stages after imbibition and endopeptidase activitywas analysed by an activity stain after native PAGE. At leastten different endopeptidase activities were detected in theendosperm tissues during the initial 5 d. Activities similarto these ten enzymes were also detected in aleurone layers.These results suggest that the main source of these endopeptidasesin the endosperm is the aleurone layer. The scutellum had adifferent spectrum of endopeptidases. One of these alternativeendopeptidases, which was detected on the first day after theaddition of water, was a metallo-enzyme with electrophoreticproperties similar to an activity found in endosperm tissueshortly after imbibition. Key words: Zea mays, endopeptidase localization, seed germination     

Expression of the 30 kD cysteine endoprotease B in germinating barley seeds

The expression of a 30 kD cysteine endoprotease (EP-B) was studied by in situ hybridization and immunomicroscopy to clarify its role in germinating barley grains. At the beginning of germination, EP-B mRNA was expressed in the scutellar epithelium and aleurone cells next to the embryo. Later, mRNA levels were highest in the aleurone layer proceeding to the distal end of the grain. During the first day of germination, EP-B protein was strongly localized to the germ aleurone and scutellar epithelium from where the secretion into the starchy endosperm began. Secretion was also observed to proceed along the aleurone layer to the distal end. These results show that EP-B is differentially localized during germination, and both scutellum and aleurone layer are able to synthesize and secrete EP-B protein.     

Changes in cell wall polysaccharides of germinating barley grains

Decorticated barley grains were germinated at 25° for 6 days, until the endosperm reserves were nearly exhausted. The neutral monosaccharide components of the hydrolysates of the cell walls and gums from the embryo, aleurone layer and starchy endosperm and the endospermic starch were determined at daily intervals. The amount of embryo cell wall polysaccharide increased 40 times and glucose became the major component, followed in abundance by xylose and arabinose. The cell wall and gum polysaccharides of the aleurone layer (plus testa) and the starchy endosperm declined during germination and their compositions altered. The endospermic starch also decreased. In the early stages of germination the apparent composition of the cell walls of the aleurone layer and starchy endosperm depended upon how they had been prepared. After 6 days the cell walls and gums had provided a significant carbohydrate supply to the living tissues, equivalent to 18.5% of the endospermic polysaccharide degraded during growth, starch having provided the remaining 81.5%.     

Asparaginyl endopeptidase during maturation and germination of durum wheat

Asparaginyl-endopeptidase activity was detected in endosperms of maturing and germinating wheat seeds. The highest activity was found during maturation before the maximal accumulation of storage proteins. The enzyme activity then decreased in the dry seeds and increased again during germination. The increase of activity during germination required the presence of the embryo. In fact, the activity found in detached endosperms was lower than that found in attached ones. The localization at tissue level of the enzyme reveals differences between maturation and germination: the enzyme was about equally located in the aleurone layer and starchy endosperm during maturation, but solely in the aleurone layer during germination. The asparaginyl enzymes from maturing and germinating seeds had many similar properties, such as pH optimum, pH stability, thermal stability and sensitivity to thiol reagents and to thiol compounds. The results suggest that asparaginyl endopeptidases may be involved in the modification of proproteins of storage proteins during seed maturation and in the degradation of storage proteins deposited in the aleurone layer during germination.     

The function of the aleurone layer during galactomannan mobilisation in germinating seeds of fenugreek (Trigonella foenum-graecum L.), crimson clover (Trifolium incarnatum L.) and lucerne (Medicago sativa L.): A correlative biochemical and ultrastructural study

Summary The reserve endosperm galactomannans of fenugreek (Trigonella foenum-graecum L.), crimson clover (Trifolium incarnatum L.) and lucerne (Medicago sativa L.) are broken down to free galactose and mannose in dry-isolated endosperms (devoid of embryo) incubated under germination conditions. Breakdown is prevented by inhibition of protein synthesis or of oxidative phosphorylation in the aleurone layer. Resting aleurone cells contain inter alia a large number of ribosomes more or less regularly distributed in the ground plasma. At the onset of germination, before galactomannan breakdown begins, polysomes are formed and seem, at least partly, to become associated with vesicles and flat cisternae both probably newly formed and derived from ER. Concurrently with galactomannan breakdown in the reserve cells, wall corrosion occurs in the aleurone layer, the contents of the aleurone grains disappear and the rough vesicles and cisternae proliferate. Later a large central vacuole is formed which incorporates smaller vacuoles emerging from the cytoplasm, and at the same time the rough ER vesicles and cisternae become highly distended.It is concluded that the cells of the aleurone layer are responsible for the synthesis and secretion into the storage cells of the enzymes necessary for galactomannan degradation. The physiology of galactomannan breakdown is compared and contrasted with that of starch mobilisation in the endosperm of germinating cereal grains.This is part three in a series of papers dealing with galactomannan metabolism. Part two: Planta (Berl.) 100, 131–142 (1971).     

Secretion of basic and acidic chitinases from salt-adapted and -unadapted winged bean cells

Northern hybridizations were used to study the site of synthesis of three carboxypeptidases (Cpases I-III) which occur in the starchy endosperm of germinating barley grain ( Hordeum vulgare L.). Further evidence was obtained by studying secretion of these enzymes from scutella or aleurone layers separated from germinating grains. Messenger RNA for Cpase II was detected only in developing grain, and the bulk of the mRNA was localized in the starchy endosperm. This suggests that Cpase II is synthesized at the site of its accumulation, the starchy endosperm. In contrast, Cpase I is expressed during germination and the predominant site of synthesis is the scutellum, from which it is secreted into the starchy endosperm. Cpase III is also synthesized during germination, but the bulk of it is synthesized in and secreted from the aleurone layer. Thus, the three carboxypeptidases, all of which seem to play a role in hydrolysis of the reserve proteins in the starchy endosperm during germination, have different sites of synthesis.     

Amino acids and quaternary nitrogen compounds in the germinating wheat grain

The tissues of the quiescent wheat grain contained free amino acids and quaternary nitrogen compounds. During germination the amino acid levels increased several fold. In the aleurone tissue and starchy endosperm glutamine was the predominant amino acid. Asparagine was predominant in the seedling tissues. Choline and glycine betaine were the principal quaternary nitrogen compounds present. The aleurone tissue and the embryo/seedling contained large quantities of glycine betaine. The increase in free amino acid levels in the aleurone tissue during the first 2 days of germination occurred independently of the embryo. After the second day, the further increase in levels was dependent upon the presence of the embryo and of gibberellic acid (GA). Estimation of the individual amino acids and quaternary nitrogen compounds released from incubating aleurone layers into aqueous media revealed a selective release of some compounds and retention of others. The process was regulated by GA. Possible mechanisms for the release of amino acid and its control by GA are discussed.     

Embryology in Trithuria submersa (Hydatellaceae) and relationships between embryo, endosperm, and perisperm in early-diverging flowering plants

? Premise of the study: Despite their highly reduced morphology, Hydatellaceae bear the unmistakable embryological signature of Nymphaeales, including a starch-rich maternal perisperm and a minute biparental endosperm and embryo. The co-occurrence of perisperm and endosperm in Nymphaeales and other lineages of flowering plants, and their respective functions during the course of seed development and embryo germination, remain enigmatic. ? Methods: Development of the embryo, endosperm, and perisperm was examined histologically from fertilization through germination in flowers and fruits of Trithuria submersa. ? Key results: The embryo of T. submersa initiates two cotyledons prior to seed maturity/dormancy, and their tips remain in contact with the endosperm throughout germination. The endosperm persists as a single layer of cells and serves as the interface between the embryo and the perisperm. The perisperm contains carbohydrates and proteins, and functions as the main storage tissue. The endosperm accumulates proteins and aleurone grains and functions as a transfer cell layer. ? Conclusions: In Nymphaeales, the multiple roles of a more typical endosperm have been separated into two different tissues and genetic entities: a maternal perisperm (nutrient acquisition, storage, mobilization) and a minute biparental endosperm (nutrient transfer to the embryo). The presence of perisperms among several other ancient lineages of angiosperms suggests a modest degree of developmental and functional lability for the nutrient storage tissue (perisperm or endosperm) within seeds during the early evolution of flowering plants. Finally, we examine the evolutionary developmental hypothesis that, contrary to longstanding assumptions, an embryo-nourishing perisperm along with a minute endosperm may represent the plesiomorphic condition for flowering plants.     

Modification patterns in germinating barley--malting II

Modification refers to breaking down of cell walls and the conversion of starch-to-sugars in the endosperm of grains by the action of enzymes released from the aleurone layer and possibly the scutellum during germination. Experimentalists have observed two completely different modification patterns in germinating barley. Based on an enzyme reaction, strongly nonlinear diffusion model developed in Part I of this series of papers on malting we examine simple models which predict modifications patterns similar to both observed patterns. We show that one of the observed patterns represents a natural propagation mode that acts as an attractor for the system. The speed of approach to this mode is strongly effected by initial conditions, a consequence of the enzymic nature of the reaction and the dramatic change of diffusivity brought about by the reaction.     

Gibberellin-induced separation of cells in isolated endosperm of celery seed

Summary The mature seed of celery (Apium graveolens, L.) contains a small axile linear embryo surrounded by endosperm which occpies the bulk of the seed. The endosperm is living and consists of mostly large angular thick-walled cells containing aleurone grains (often with globoids) and lipid droplets. — Using de-embryonated seeds, it has been shown that the endosperm was induced to break down by gibberellin. The aleurone grains became swollen and lost their contents and the bulk of each cell wall was hydrolyzed. However, a thin resistant layer of wall remained around each protoplast. The wall hydrolysis caused the endosperm to break down into individual cells which could be plasmolyzed and therefore appeared to be still living. All cells of the endosperm responded to gibberellin in a similar way although the cells near the radicle appeared to degrade more rapidly than those elsewhere. There was no change in the absence of the hormone. The response was apparently specific to gibberellin and did not occur in the presence of ethylene, kinetin, abscisic acid and indole acetic acid. The results were the same in light and in darkness. — It has been thought that endosperm breakdown during germination of seed such as celery involved release of hydrolases from the expanding embryo. The results of this study indicate that endosperm breakdown might be caused by hydrolases arising in the endosperm itself in response to gibberellin released from the embryo.Abbreviations GA gibberellic acid - PAS periodic acid-Schiff reagent Most of this work was performed in the Department of Vegetable Crops at the Volcani Center, Bet Dagan, while J.V. Jacobsen was a Research Fellow there.     

The role of the sucrose transporter, OsSUT1, in germination and early seedling growth and development of rice plants

Using expression analysis, the role of the sucrose transporter OsSUT1 during germination and early growth of rice seedlings has been examined in detail, over a time-course ranging from 1 d to 7 d post-imbibition. Unlike the wheat orthologue, TaSUT1, which is thought to be directly involved in sugar transfer across the scutellar epithelium, OsSUT1 is not expressed in the scutellar epithelial cell layer of germinating rice and is, therefore, not involved in transport of sugars across the symplastic discontinuity between the endosperm and the embryo. OsSUT1 expression was also absent from the aleurone cells, indicating it is not involved in the transport of sucrose in this cell layer during germination. However, by 3 d post-imbibition, OsSUT1 was present in the companion cells and sieve elements of the scutellar vascular bundle, where it may play a role in phloem loading of sucrose for transport to the developing shoot and roots. This sucrose is most likely sourced from hexoses imported from the endosperm. In addition, sucrose may be remobilized from starch granules which are present at a high density in the scutellar ground tissues surrounding the vasculature and at the base of the shoot. OsSUT1 was also present in the coleoptile and the first and second leaf blades, where it was localized to the phloem along the entire length of these tissues, and was also present within the phloem of the primary roots. OsSUT1 may be involved in retrieval of sugars from the apoplasm in these tissues.     

Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley

During germination of barley grains, the appearance of DNA fragmentation started in aleurone cells near the embryo and extended to the distal end in a time-dependent manner. DNA fragmentation was demonstrated to occur only after the expression of -amylase mRNA in the aleurone layer. In addition, cell wall degradation started in cells near the embryo on the sides facing the endosperm. Subsequently cell wall degradation extended to the lateral cell walls and to cells more to the distal end of the grain. A typical alteration of the nucleus was observed by electron microscopy and an almost complete degradation of DNA was found in the nucleus while the nuclear envelope remained intact. The results indicate that programmed cell death occurred in aleurone cells during germination. A model is proposed for the regulation of programmed cell death in aleurone cells during germination involving ABA levels and cell wall degradation.     

Differentiation of a Functional Aleurone Layer Within the Seed Coat of Sinapis alba L.

The hitherto unresolved ontogenetic origin of the aleurone layerin mustard (Sinapis alba L.) seeds was investigated with lightand electron microscopy. Contrary to previous views, this layerof storage cells is neither derived from the endosperm nor fromthe nucellus, but from a particular cell layer within the innerintegument of the seed coat. These cells differentiate and becomefilled with storage protein and fat concurrently with the maturationof the embryo. They survive seed desiccation and become depletedof storage materials during seed germination. Temporally correlatedwith the germinating embryo, the aleurone cells produce microbodyenzymes, which are controlled by light in a similar fashionin both tissues. Sinapis alba L., mustard, aleurone layer, seed coat, seed formation, germination     

Differentiation mechanism and function of the cereal aleurone cells and hormone effects on them

The cereal aleurone cells differentiate from the endosperm epidermis with the exception of endosperm transfer cells. Aleurone cells contain proteins, lipids, and minerals, and are important for digesting the endosperm storage products to nurse the embryo under effects of several hormones during the seed germination. The differentiation of aleurone cells is related to location effect and special gene expression. Moreover, the differentiation of aleurone cells is probably affected by the cues from maternal tissues. In the paper, differentiation mechanism and function of aleurone cells and hormone effects on them are reviewed. Some speculations about the differentiation mechanism of aleurone cells are given here.     

The diffusive transport of gibberellins and abscisic acid through the aleurone layer of germinating barley grain: a mathematical model

A mathematical model of the diffusive transport of abscisic acid (ABA) and gibberellins (GAs) through the aleurone layer of barley (Hordeum vulgare L.) grain is presented. The model consists of two partial differential equations describing the accumulation of phytohormone in the apoplastic and symplasmic compartments of the aleurone layer, both spatially and temporally. The mathematical model contains the morphology of the barley grain and the physicochemical properties of the two phytohormones. A mathematical derivation of the accumulation ratios for the two phytohormones between the symplast and apoplast under equilibrium conditions resulted in different distribution mechanisms for GAs and ABA. A sensitivity analysis of the accumulation ratio for GAs indicated high sensitivity to the apoplastic pH and the membrane potential, whereas the accumulation ratio for ABA proved to be most sensitive to the pH difference between the apoplast and symplast. The diffusive transport time for GAs to the basal site of the aleurone layer as calculated with the mathematical model is within a physiologically plausible timescale according to experimental data from the literature. Abscisic acid cannot be transported by diffusion to the end of the aleurone layer as quickly as GAs, according to model simulations. Therefore, the functional role of ABA in germination is likely to be in the vicinity of the embryo.     

Soluble and particulate lysophospholipase in the aleurone and endosperm of germinating barley

Lysophospholipase was measured in extracts of germinating barley by determining the amount of free [¹⁴C]palmitate released from [1-¹⁴C] 1-palmitoyl-lysophosphatidylcholine (LPC). Soluble and particulate lysophospholipase activity was measured at 1-day intervals in extracts from the aleurone and endosperm of barley seeds germinated for 8 days. The soluble and particulate activities of the aleurone increase approximately in parallel with one another and after 8 days of germination have 20–30 times more activity than at day 1. The activity profiles and the distribution of the activity between the soluble and particulate forms of lysophospholipase in the endosperm are markedly different. With the exception of the first 2 days when the aleurone activity is low, the endosperm activity is less than that associated with the aleurone. The soluble activity increases during the first 3 days and is more active than that of the aleurone. Thereafter it diminishes and remains low. The particulate enzyme, however, increases dramatically between days 4 and 5 and remains moderately high. The fourth and fifth day represent that stage of germination when starch-bound LPC is released in concert with the increase in amylase activity. It is proposed that it is this particulate form of the endosperm activity which may be responsible for maintaining the level of free LPC low in the endosperm of the germinating seed.     

X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains

Energy-dispersive x-ray analysis was used to investigate the elemental storage within protein bodies, specifically the globoid crystals, in grains of wheat. Areas of the grain investigated included various parts of the embryo, the aleurone layer plus starchy endosperm near the embryo and the aleurone layer plus starchy endosperm farthest from the embryo. Variations did occur grain-to-grain, cell-to-cell and, in certain regions, intracellularly. No protein bodies with electron-dense globoid crystals were found in the starchy endosperm. Generally globoid crystals contained P, K, and Mg in all areas investigated. Globoid crystals from the aleurone layer farthest from the embryo on occasion contained Ca, whereas aleurone globoid crystals near the embryo sometimes contained Fe. In most of the embryo regions examined, a few globoid crystals contained Ca along with P, K, and Mg. No specific pattern to the Ca distribution could be found. Welldefined elemental distribution occurred with Mn. Manganese was found only in globoid crystals located in the base and midregions of the stele in the radicle. Thus, in wheat there is some specific distribution of minerals dependent upon cell type and/or position in the grain.