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.     

PEARL MILLET (PENNISETUM AMERICANUM (L.) LEEKE) AND GRAIN SORGHUM (SORGHUM BICOLOR (L.) MOENCH) ULTRASTRUCTURE

Ultrastructural features of pearl millet (Pennisetum americanum (L.) Leeke) and grain sorghum (Sorghum bicolor (L.) Moench) caryospses were investigated with thin sections of the dry, mature grain in the transmission electron microscope, and fractured kernels in the scanning electron microscope. The pericarp of those grains is comprised of three distinct layers: epicarp, mesocarp of parenchyma cells, and endocarp of compressed cross and tube cells. Mesocarp cells of grain sorghum contain starch granules embedded in a cytoplasmic matrix. The major constituent of sorghum and millet aleurone cells are aleurone grains (protein bodies) and lipid bodies. Subaleurone cells contain a much higher proportion of protein bodies than starch granules, and the protein bodies are structurally distinct from those in the aleurone. The germ scutellar ultrastructures of the two grains were similar; protein bodies, lipid bodies, epidermal cells and parenchyma cells of the germ are described.     

Fusion of oil bodies in endosperm of oat grains

Few microscopical studies have been made on lipid storage in oat grains, with variable results as to the extent of lipid accumulation in the starchy endosperm. Grains of medium- and high-lipid oat (Avena sativa L.) were studied at two developmental stages and at maturity, by light microscopy using different staining methods, and by scanning and transmission electron microscopy. Discrete oil bodies occurred in the aleurone layer, scutellum and embryo. In contrast, oil bodies in the starchy endosperm often had diffuse boundaries and fused with each other and with protein vacuoles during grain development, forming a continuous oil matrix between the protein and starch components. The different microscopical methods were confirmative to each other regarding the coalescence of oil bodies, a phenomenon probably correlated with the reduced amount of oil-body associated proteins in the endosperm. This was supported experimentally by SDS-PAGE separation of oil-body proteins and immunoblotting and immunolocalization with antibodies against a 16 kD oil-body protein. Much more oil-body proteins per amount of oil occurred in the embryo and scutellum than in the endosperm. Immunolocalization of 14 and 16 kD oil-body associated proteins on sectioned grains resulted in more heavy labeling of the embryo, scutellum and aleurone layer than the rest of the endosperm. Observations on the appearance of oil bodies at an early stage of development pertain to the prevailing hypotheses of oil-body biogenesis.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE CARYOPSIS COAT AND THE ALEURONE CELLS

The results of a light and electron microscopic study of the caryopsis coat and aleurone cells in ungerminated, unimbibed rice (Oryza sativa) caryopses are presented. Surrounding the rice grain is the caryopsis coat composed of the pericarp, seed coat and nucellar layers. The outermost layer, the pericarp, consists of crushed cells and is about 10 μm thick. The seed coat, interior to the pericarp, is one cell thick and has a thick cuticle. Between the seed coat cuticle and endosperm are the remains of the nucellus. The nucellus is about 2.5 μm thick and has a thick cuticle adjacent to the seed coat cuticle. Interior to the caryopsis coat is the aleurone layer of the endosperm. The aleurone completely surrounds the rice grain and is composed of two cell types—aleurone cells that surround the starchy endosperm and modified aleurone cells that surround the germ. The aleurone cells of the starchy endosperm contain many aleurone grains and lipid bodies around a centrally located nucleus. The modified aleurone cells lack aleurone grains, have fewer lipid bodies than the other aleurone cells, and contain filament bundles (fibrils). Plastids of aleurone cells exhibit a unique morphology in which the outer membranes invaginate to form tubules and vesicles within the plastid. Transfer aleurone cells are not observed in the mature rice caryopsis.     

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).     

alpha-Amylase and programmed cell death in aleurone of ripening wheat grains

Late maturity alpha-amylase (LMA) in wheat is a genetic defect that may result in the accumulation of unacceptable levels of high pI alpha-amylase in grain in the absence of germination or weather damage. During germination, gibberellin produced in the embryo triggers expression of alpha-Amy genes, the synthesis of alpha-amylase and, subsequently, cell death in the aleurone. LMA also involves the aleurone and whilst LMA appears to be independent of the embryo there is nevertheless some evidence that gibberellin is involved. The aim of this investigation was to determine whether the increase in alpha-amylase activity in LMA-prone genotypes, like alpha-amylase synthesis by aleurone cells in germinating or GA-challenged grains, is followed by aleurone cell death. Programmed cell death was seen in aleurone layers from developing, ripe and germinated grains using confocal microscopy and fluorescent probes specific for dead or living cells. Small pockets of dying cells were observed distributed at random throughout the aleurone of ripening LMA-affected grains and by harvest-ripeness these cells were clearly dead. The first appearance of dying cells, 35 d post-anthesis, coincided with the later part of the 'window of sensitivity' in grain development in LMA-prone wheat cultivars. No dead or dying cells were present in ripening or fully ripe grains of control cultivars. In germinating grains, dying cells were observed in the aleurone adjacent to the scutellum and, as germination progressed, the number of dead cells increased and the affected area extended further towards the distal end of the grain. Aside from the obvious differences in spatial distribution, dying cells in 20-24 h germinated grains were similar to dying cells in developing LMA-affected grains, consistent with previous measurements of alpha-amylase activity. The increase in high pI alpha-amylase activity in developing grains of LMA-prone cultivars, like alpha-amylase synthesis in germinating grains, is associated with cell death, providing further evidence for the involvement of gibberellin in the LMA response.     

文冠果可孕花与不孕花发育过程的比较研究简

利用半薄切片和透射电镜技术对文冠果可孕花和不孕花的发育过程进行观察和比较。结果显示：(1)小孢子发育初期,两种类型花花药形态无明显差别;小孢子发育双核期,可孕花花药内壁纤维层细胞壁带状加厚,无唇细胞形成。而不孕花花药同侧两个花粉囊之间唇细胞正在分化;小孢子发育成熟期,不孕花花药唇细胞完全形成;散粉期,不孕花花药开裂呈双心形,而可孕花花药则不能开裂散粉。(2)可孕花雌蕊子房内有两室,柱头细胞排列紧密,柱头逐渐发育成圆球形,周围密布乳突细胞,具中空花柱道;不孕花雌蕊柱头停止发育,无中空花柱道,子房室变小,胚囊发育退化。(3)不孕花花药绒毡层中含大量蛋白体,小泡以及乌氏体等细胞器,发育后期绒毡层解体。而可孕花花药绒毡层中细胞器和营养物质积累均较少,发育后期绒毡层解体不完全。(4)可孕花花药内花粉粒细胞壁连续无萌发孔,细胞内含物较少。不孕花花药内花粉出现3个向内凹陷的萌发孔,且花粉内含有大量造粉质体和脂类物质。     

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     

Tissue-specific localization of aspartic proteinase in developing and germinating barley grains

Resting seeds of several plant species, including barley grains, have been reported to contain aspartic proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. aspartic proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.Abbreviations DAA days after anthesis - DTT dithiothreitol - HvAP Hordeum vulgare aspartic proteinase Both authors have contributed equally to this workWe thank Mart Saarma, Pia Runeberg-Roos, Alan Schulman and Yrjö Helariutta for helpful discussions during the study, Tiina Arna and Sari Makkonen for their help in proteinase activity experiments as well as Jaana Korhonen (Department of Pathology, University of Helsinki), Salla Marttila and Ilkka Porali (Department of Biology, University of Jyväskylä, Jyväskylä, Finland) for their advice on microscopical techniques. We also thank Liisa Pyhälä and Leena Liesirova for the production of the antibodies to HvAP at the National Public Health Institute, Helsinki. This study was supported by grants from the Ministry of Agriculture and Forestry and the Academy of Finland.     

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.     

CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as -amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

竹节参雌配子体发育的研究

本文报道了竹节参(Panax japonicus C.A.Mey)雌配子体(胚囊)的发育过程。竹节参大孢子母细胞减数分裂产生线形排列的大孢子四分体。胚囊发育属蓼型,由合点端大孢子发育而成。游离核胚囊时期,胚囊珠孔端的细胞器种类和数量都较胚囊合点端多;胚囊合点端相邻的珠被细胞中有含淀粉粒的小质体,与胚囊珠孔端相邻的退化中的非功能大孢子中则有含淀粉粒的大质体和大类脂体。成熟胚囊中,反足细胞较早退化;极核融合成次生核;卵细胞高度液泡化,细胞器数量较少;助细胞则有丰富的细胞器和发达的丝状器。PAS反应表明,受精前的成熟胚囊中积累淀粉粒。次生核受精后,很快分裂产生胚乳游离核,到几十至数百个核时形成胚乳细胞。卵细胞受精后则要经过较长的休眠期。     

Development of aleurone and sub-aleurone layers in maize

Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10–15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate.     

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.     

Sub-cellular immunolocalization of the glucosinolate sinigrin in seedlings of Brassica juncea

Polyclonal rat antibodies were raised to a bovine serum albumin-sinigrin conjugate and used to immunolocalize sinigrin (2-propenylglucosinolate) in imbibed seeds and developing seedlings of Brassica juncea. (L.) Czern. Sinigrin was localized to protein bodies in aleurone-like cells but shown to be absent from myrosin cells. Double labelling techniques were used to co-localize both myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) and sinigrin. Myrosin grains were labelled only with the anti-myrosinase antibody, but aleurone cells were labelled with both anti-myrosinase and anti-sinigrin antibodies. High-performance liquid chromatographic analysis of conventionally fixed and dehydrated seed tissues (4 h post imbibition in water), indicated a high proportion of sinigrin was retained in fixed tissues. Over a time course of 100 h, protein bodies within aleurone-like cells degraded, fused to form the cell vacuole and lost all myrosinase labelling but retained residual sinigrin labelling. The degradation of protein bodies corresponded to a decrease in retention of sinigrin in the fixed tissues. The results describe for the first time the co-localization of a plant enzyme and its substrate, a secondary metabolite. Received: 8 January 1998 / Accepted: 27 February 1998     

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.     

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.     

外源CO及NO对干旱胁迫下水稻糊粉层细胞死亡的影响

采用荧光显微技术结合药理学方法,以水稻(Oryza sativa L.)种子及其糊粉层为实验材料,研究外源CO、NO对干旱胁迫下水稻种子萌发过程中糊粉层细胞DNA降解及死亡的影响。结果表明:(1)干旱胁迫促进糊粉层细胞的死亡,且近胚端糊粉层细胞的死亡进程早于远胚端的细胞。(2)外源CO及NO供体处理能缓解干旱胁迫下水稻糊粉层细胞DNA的降解,延迟细胞死亡进程;CO专一性抑制剂及NO清除剂能逆转CO及NO的效应,缩短细胞死亡进程。(3)外源CO及NO供体促进干旱胁迫下水稻种子的萌发,CO专一性抑制剂及NO清除剂能抑制干旱胁迫下水稻种子的萌发。(4)CO合成酶抑制剂并不能抑制外源NO对干旱胁迫伤害的缓解效应,即CO能通过NO介导调节干旱胁迫下水稻种子糊粉层细胞的死亡及种子萌发。