Energy-dispersive x-Ray Analysis of Phosphorus, Potassium, Magnesium, and Calcium in Globoid Crystals in Protein Bodies from Different Regions of Cucurbita maxima Embryos

The seeds of Cucurbita maxima contain protein bodies with electrondense globoid crystals. Because of their density globoid crystals are ideal material for energy-dispersive x-ray (EDX) analysis studies of elemental composition. Fixation trials were carried out to test globoid crystal extraction during glutaraldehyde fixation, water washing, and ethanol dehydration. Glutaraldehyde fixation without subsequent washing or dehydration alone produced no significant changes in elemental composition of cotyledon globoid crystals. If glutaraldehyde fixation was followed by water washes or ethanol dehydration there was some loss of the major globoid crystal elements but the relative percentages of the elements P, K, Ca, and Mg remained relatively unchanged. In this paper results of a study of the P, K, Mg, and Ca content of globoid crystals in different tissues of squash embryos are presented. The globoid crystals in the radicle were found to be the least dense in the embryo. Globoid crystals from all embryo regions contained P, K, and Mg. In the various embryo regions P and Mg maintained relatively constant proportions of the globoid crystal composition while K and Ca varied. Of particular significance is the distribution of Ca which is generally an immobile element. Calcium was found in highest amounts in the globoid crystals of the radicle and stem regions while globoid crystals in much of the cotyledon contained little, if any, Ca. The Ca storage thus seems to be spatially arranged in a manner that would aid early growth of the root-shoot axis.     

Mineral reserves in castor beans: the dry seed

Elemental composition and distribution of the mineral reserves in the endosperm and embryo tissues of Ricinus communis cultivars Hale and Zanzibarensis were investigated. Energy dispersive x-ray analysis was used to determine the elemental composition of the globoid crystals, while atomic absorption spectrometry allowed quantification of the elements, particularly Ca, in various seed regions. No major differences were found between the two cultivars with regard to the elemental distribution in globoid crystals. While the majority of globoid crystals contained P, K, and Mg, the occasional one also contained Ca. In extremely rare instances, Fe was detected in globoid crystals. Ca-containing globoid crystals were more common in provascular cell protein bodies in the stem and radicle. Polarized light microscopy, micro-incineration, and acid solubility tests demonstrated the presence of calcium oxalate crystals in the innermost testa which adheres to the endosperm and is often mistakenly identified as endosperm. Atomic absorption spectrometry revealed that most of the calcium present in castor bean seeds is localized in the testa. On a perseed-region basis, the much larger endosperm contains more Ca than does the embryo. However, on a unit-weight basis, the radicle-plus-stem regions contain considerably more Ca than does the cotyledon or endosperm, an observation that is consistent with the observed distribution pattern for Ca-containing globoid crystals.     

Calcium distribution in globoid crystals of cucurbita cotyledon protein bodies

Energy-dispersive x-ray analysis was used to investigate the location of globoid crystals with relatively high Ca levels within cotyledons of Cucurbita maxima, Cucurbita mixta, and Cucurbita andreana. The small globoid crystals in both upper and lower epidermal cells commonly contained Ca. Ca was present in globoid crystals of all provascular regions with the exception of the very small provascular regions of C. maxima. In C. maxima and C. mixta cotyledons, some cases were observed where Ca was found in the globoid crystals of the first layer of mesophyll cells surrounding the provascular region, but in general Ca was absent from globoid crystals of palisade and spongy mesophyll cells. In C. andreana, globoid crystals of palisade and spongy mesophyll cells commonly contained at least some Ca. Cell position and cell type are factors affecting the Ca content of globoid crystals in protein bodies.     

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.     

Protein bodies in seeds

Membrane bound protein bodies (aleurone grains) are thought to be the main subcellular location of protein and mineral storage in seeds. In addition to structurally homogeneous proteinaceous matrix, protein bodies may contain protein crystalloids, electron–dense globoid crystals, electron–transparent soft globoids, and crystals of calcium oxalate. Protein crystalloids vary in shape, size and number. For example, cotyledon mesophyll cell protein bodies in the Cucurbitaceae generally contain protein crystalloids whereas those of Compositae and Cruciferae do not. Globoid crystals, which are rich in phytin, vary greatly in size and number per protein body. Some species have numerous small globoid crystals per protein body whereas others have one or two large globoid crystals per protein body. Phosphorus and various cations (K, Mg, Ca, Fe, Ba, Mn) located in globoid crystals can be studied with an energy dispersive X–ray (EDX) analysis system mounted on a transmission electron microscope. In some cases, cations such as Ca, Mn and Fe are specifically localized in globoid crystals of certain tissues or embryo regions. Further investigations may allow elemental composition of globoid crystals to be used in studies of systematics. Biref–ringent crystals, sometimes in the form of single large crystals but frequently in the form of druses, are present in protein bodies of some species. At least some endosperm protein bodies of all Umbelliferous species examined contain druse crystals. While seed protein bodies of relatively few species have been studied with electron microscopy, there are indications that protein bodies could be a useful character for studies in plant systematics.     

Radicle ofEchinocactus platyacanthus (Cactaceae)

Radicle of matureEchinocactus platyacanthus embryo is approximately 320 m long and represents less then 1/7 of the embryonal axis length. The radicle-hypocotyl boundary can be distinguished according to the striking difference in the size and shape of cells in protoderm and procambium, as well as discontinuity and different number of the cell files in the ground meristem. The root cap is small, consists of 4 layers of cells covering the apex of the radicle. The upper limit of the root cap is approximately 100 m closer towards the radicle tip than the radicle-hypocotyl boundary. Ultrastructure of radicle cells showed numerous lipid bodies as is typical for other oily seeds. Protein bodies of variable structure were also present together with other cell structures. Striking differences in protein body structure were found when protoderm and ground meristem were compared. Several small globoid crystals were present in each protein body of the protoderm, while protein bodies in the radicle ground meristem mostly contained one large globoid crystal. X-ray microanalysis revealed presence of P, K and Mg in all analyzed globoid crystals. Fe, Ca and Zn were detected in some of them.Abbreviations EDX microanalysis energy-dispersive X-ray microanalysis - GC(s) globoid crystals - ICP spectroscopy ion-coupled plasma spectroscopy - LM light microscopy - PB(s) protein bodies - SEM scanning electron microscopy - TEM transmission electron microscopy     

Structure of Protein Bodies and Elemental Composition of Phytin from Dry Germ of Maize (Zea mays L.)

Protein bodies (PBs) from the epidermis and cortex of the radicle, as well as from coleorhizal and scutellar parenchyma of dry maize seeds were studied with the emphasis on presence and elemental composition of globoid crystals. PBs with variable structure were found in investigated tissues. Radicular epidermis was the only investigated tissue containing structurally uniform populations of PBs. They were filled by a homogeneous matrix without globoid crystals or their remnants. PBs structure and amount of proteinaceous matrix varied in the same cell of coleorhiza and scutellum. In the case of radicular cortex, the structure of PBs differed between analyzed individuals. Globoid crystals of various sizes were found in PBs from scutellum, coleorhiza, and from inner layers of radicular cortex. The largest globoid crystals (up to 2.0 μ in diameter) were seen in the scutellum. Energy-dispersive X-ray microanalysis revealed the presence of P,K. Mg, and some traces of Fe and Zn in the majority of globoid crystals from the scutellum. Globoid crystals from coleorhiza and radicular cortex were rich in Zn and Ca. The presence of considerable Zn was usually accompanied by high Ca, at the expense of K and Mg. As EDX analyses revealed significant differences in Zn and Zn/P values between analyzed tissues, it is probable that the majority of Zn is preferably accumulated in globoid crystals of some tissues of maize germ.     

Protein Body Composition in Cuburbita maxima Cotyledons as Determined by Energy Dispersive X-Ray Analysis

Energy dispersive x-ray analysis was used to study the composition of certain protein body components in Cucurbita maxima cotyledons. The globoid crystal was rich in phosphorus, potassium, and magnesium. This elemental composition provides further evidence that the globoid crystal in squash cotyledon protein bodies is composed of phytin, a myoinositol hexaphosphoric acid salt of potassium and magnesium. Calcium, a common component of phytin in many species, was absent or present in only trace amounts in the globoid crystals of squash. Results of analyses of globoid crystals from seeds produced in different parts of North America suggest that there is definite specificity for the cations used in phytin deposition. Variations in soil types and other environmental factors seem not to have influenced the type of cation stored. Energy dispersive x-ray analysis of the proteinaceous regions revealed the presence of phosphorus, sulfur, and a trace of chlorine. Sulfur was expected, due to the presence of some sulfur containing amino acids in the protein.     

Seed Architecture Shapes Embryo Metabolism in Oilseed Rape

Constrained to develop within the seed, the plant embryo must adapt its shape and size to fit the space available. Here, we demonstrate how this adjustment shapes metabolism of photosynthetic embryo. Noninvasive NMR-based imaging of the developing oilseed rape (Brassica napus) seed illustrates that, following embryo bending, gradients in lipid concentration became established. These were correlated with the local photosynthetic electron transport rate and the accumulation of storage products. Experimentally induced changes in embryo morphology and/or light supply altered these gradients and were accompanied by alterations in both proteome and metabolome. Tissue-specific metabolic models predicted that the outer cotyledon and hypocotyl/radicle generate the bulk of plastidic reductant/ATP via photosynthesis, while the inner cotyledon, being enclosed by the outer cotyledon, is forced to grow essentially heterotrophically. Under field-relevant high-light conditions, major contribution of the ribulose-1,5-bisphosphate carboxylase/oxygenase–bypass to seed storage metabolism is predicted for the outer cotyledon and the hypocotyl/radicle only. Differences between in vitro– versus in planta–grown embryos suggest that metabolic heterogeneity of embryo is not observable by in vitro approaches. We conclude that in vivo metabolic fluxes are locally regulated and connected to seed architecture, driving the embryo toward an efficient use of available light and space.     

血皮槭种子休眠机制研究

利用抑制物生物测定法和酸蚀技术研究了血皮槭种子休眠的原因。血皮槭种子吸水是一个非常缓慢的过程,在140 h以后种子含水量才能达到68%左右。酸蚀处理种子3 h,虽然没有加快种子的吸水速率,但能较好得使果皮变薄,也不影响种子的生活力。种子的各部位（果皮、种皮、子叶、胚根）均含有抑制物质,对小白菜种子的发芽率及胚根生长有很强的抑制作用,子叶各种处理水浸提液的抑制作用最强,果皮和种皮次之。血皮槭种子休眠主要由种壳机械障碍和种胚生理休眠两重因素导致,因此如何克服致密果壳而使激素能接触生理休眠的种胚是打破其种子休眠的关键技术。     

Epicotyl morphophysiological dormancy in seeds of Lilium polyphyllum (Liliaceae)

Dormancy-breaking and seed germination studies in genus Lilium reveal that the majority of Lilium spp. studied have an underdeveloped embryo at maturity, which grows inside the seed before the radicle emerges. Additionally, the embryo, radicle or cotyledon has a physiological component of dormancy; thus, Lilium seeds have morphophysiological dormancy (MPD). A previous study suggested that seeds of Lilium polyphyllum have MPD but the study did not investigate the development of the embryo, which is one of the main criteria to determine MPD in seeds. To test this hypothesis, we investigated embryo growth and emergence of radicles and epicotyls in seeds over a range of temperatures. At maturity, seeds had underdeveloped embryos which developed fully at warm temperature within 6 weeks. Immediately after embryo growth, radicles also emerged at warm temperatures. However, epicotyls failed to emerge soon after radicle emergence. Epicotyls emerged from >90% seeds with an emerged radicle only after they were subjected to 2 weeks of cold moist stratification. The overall temperature requirements for dormancy-breaking and seed germination indicate a non-deep simple epicotyl MPD in L. polyphyllum.     

Evolution and ecology of seed internal morphology in relation to germination characteristics in Amaranthaceae

Background and AimsInternal seed morphological traits such as embryo characteristics and nutritive tissue can vary considerably within a plant lineage. These traits play a prominent role in germination processes and the success of seedling establishment, and are therefore under high selective pressure, especially in environments hostile to seedlings, such as arid, saline or highly dynamic habitats. We investigated the relationships of seed internal morphology and germination characteristics of 84 species of Amaranthaceae s.l., a family with numerous lineages that have adapted to stressful growing conditions.MethodsWe used seed cross-sections to assess embryo type and the ratios of embryo to seed surface and radicle to cotyledon length. Furthermore, seed mass, mean time to germination, habitat preferences and further plant traits such as C₃ or C₄ photosynthesis and life form were compiled for each species. Data were analysed using phylogenetic comparative methods.Key resultsWe found embryo type (λ = 1), log seed mass (λ = 0.86) and the ratio of embryo to seed size (λ = 0.78) to be evolutionarily stable, with an annular embryo as ancestral in the family. Linked to shifts to the three derived embryos types (spiral, horseshoe-shaped and curved) is an increase in the ratio of root to cotyledon length and a reduction of nutritive tissue. We observed stabilizing selection towards seeds with relatively large embryos with longer radicles and less nutritive tissue that are able to germinate faster, especially in lineages with C₄ photosynthesis and/or salt tolerance.ConclusionsWe conclude that the evolutionary shift of nutrient storage from perisperm to embryo provides an ecological advantage in extreme environments, because it enables faster germination and seedling establishment. Furthermore, the evolutionary shift towards a higher ratio of root to cotyledon length especially in small-seeded Amaranthaceae growing in saline habitats can provide an ecological advantage for fast seedling establishment.     

黄精种子萌发过程发育解剖学研究

采用石蜡切片技术对成熟黄精种子形态及萌发过程中的形态学变化及解剖结构特征进行了研究,以阐明黄精种子繁殖的生物学机制。结果显示:(1)成熟的黄精种子由外而内依次为种皮、胚乳和胚等3部分组成。其中种皮由一层木质化的细胞组成;胚乳占据种子的大部分结构,胚乳细胞含有大量淀粉,细胞壁增厚;胚处于棒型胚阶段。(2)黄精种子在萌发过程中棒型胚靠近种脐端分化为吸器、子叶联结和子叶鞘,靠近种孔的部位分化出胚根、胚轴和胚芽。(3)黄精种子萌发首先由子叶联结伸长将胚芽和胚根原基推出种孔,紧接着下胚轴膨大形成初生小根茎,吸器留在种子中分解吸收胚乳中的营养物质。(4)通过子叶联结连通吸器和初生小根茎,将胚乳中的营养物质由吸器-子叶联结这个通路转移到初生小根茎中,为初生根茎上胚芽和胚根的进一步分化提供物质保障。(5)黄精种子自然条件下萌发率较低,而且当年不出土。研究表明,黄精种子的繁殖生物学特性是其生态适应的一种重要机制。     

Histochemical and ultrastructural studies on <Emphasis Type="Italic">Salix alba</Emphasis> and <Emphasis Type="Italic">S. matsudana</Emphasis> seeds

Mature seeds of Salix alba L. and Salix matsudana Koidz. are exendospermous and consist of an embryo and a seed coat. Ultrastructural studies show the presence of protein bodies, lipid bodies, chloroplasts, and a nucleus in the cells of most of the embryo tissues. Protein bodies always contain two or more globoid crystals. Energy-dispersive X-ray analysis of globoid crystals revealed the presence of P, K, Mg and Ca as the main constituents in all tissues. The chloroplasts present well-developed grana and, frequently, starch grains in the stroma. In cells of apical meristems, plastid endomembranes are non-organised in grana and deposits of phytoferritin are present in the stroma. Some cells of the subdermal layers of the cotyledons and hypocotyl-radicle axis present a large central vacuole and a narrow peripheral band of cytoplasm within which the protein bodies are scarce. Seeds of the two species studied here have recently been characterised as orthodox with short viability. The present study was carried out in an attempt to advance in the characterisation of these seeds as part of a comprehensive study of Salicaceae seeds.     

Seed Structure and Localization of Reserves inChenopodium quinoa

The three areas of food reserves in quinoa seeds are: a largecentral perisperm, a peripheral embryo and a one to two-celllayered endosperm surrounding the hypocotyl-radicle axis ofthe embryo. Cytochemical and ultrastructural analysis revealedthat starch grains occupy the cells of the perisperm, whilelipid bodies, protein bodies with globoid crystals of phytin,and proplastids with deposits of phytoferritin are the storagecomponents of the cells of the endosperm and embryo tissues.EDX analysis of the endosperm and embryo protein bodies revealedthat globoid crystals contain phosphorus, potassium and magnesium.These results are compared with studies on other perispermousseeds published to date.Copyright 1998 Annals of Botany Company Chenopodium quinoa,EDX analysis, phytoferritin, phytin, protein bodies, quinoa, seed structure, seed reserves, starch grains.     

Ecophysiology of embryo development and seed germination of the European woodland herbaceous perennial Corydalis cava (L.) Schweigg. & Körte subsp. cava (Fumariaceae)

In this study we examined the germination ecology with special reference to the temperature requirements for embryo development and germination of Corydalis cava subsp. cava, under both outdoor and laboratory conditions. Corydalis cava is a spring flowering woodland tuberous geophyte widely distributed across Europe. Germination phenology, including embryo development and radicle and cotyledon emergence, was investigated in a population growing in northern Italy. Immediately after harvest, seeds of C. cava were sown both in the laboratory under simulated seasonal temperatures and naturally. Embryos, undifferentiated at the time of seed dispersal, grew during summer and autumn conditions, culminating in radicle emergence in winter, when temperatures fell to ca 5°C. Cotyledon emergence also occurred at ca 5°C, but first emergence was delayed until late winter and early spring. Laboratory experiments showed that high (summer) followed by medium (autumn) and low temperatures (winter) are needed for physiological dormancy loss, embryo development and germination respectively. Unlike seeds of C. cava that germinated in winter, in other Corydalis species radicle emergence occurred in autumn (C. flavula) or did not depend on a period of high summer temperature to break dormancy (C. solida). Our results suggest that subtle differences in dormancy and germination behavior between Corydalis species could be related to differences in their geographical distribution.     

黄瓜种子萌发过程中Ca~(2 )分布的变化

采用细胞化学方法 ,研究了黄瓜种子中贮藏Ca2 的分布特点及其在萌发过程中的变化动态。干种子的子叶细胞中贮藏有大量的蛋白体、油脂体 ,Ca2 沉淀颗粒大量分布于胞质、胞间隙以及细胞质膜上。大多数蛋白体中有 1至数个圆球形或椭圆体形含Ca2 的球状晶体。相比之下 ,胚芽和胚根细胞中Ca2 较少。种子萌发早期 ,子叶中的贮藏钙及晶体溶解释放出的Ca2 部分转运到生长发育中的胚芽和胚根中。随着萌发的继续 ,胚根和胚芽细胞中的Ca2 不会持续增多 ,反而下降     

Fine structural observations of embryo development inPelargonium XHortorum bailey: with normal and mutant plastids

Summary The ultrastructural changes in the cotyledon, radicle and suspensor haustorium ofPelargonium, containing either normal or mutant plastids, are investigated from the heart stage of embryogenesis to the mature seed. The fine structure of parenchymatous cells from the cotyledon and radicle is essentially similar whereas that of the suspensor haustorium is very different.The cotyledon and radicle develop into one massive storage tissue possessing numerous lipid and several protein bodies per cell, and well developed starch grains. The suspensor haustorium has no storage function, rather it acts as a transitory tissue which dies off as the seed matures. The extensive chloroplast development suggests that, in addition to its traditional role, the suspensor haustorium also acts as a photosynthetic booster for the developing embryo.The development of surviving mutant embryos is similar to normal ones except that in cotyledon and radicle cells plastids develop only to vesicles, which associate into loose prolamellar bodies and sometimes small fenestrated thylakoids, and in the suspensor haustorium cells, only to small compact grana.     

Seed dormancy and germination in three Crocus ser. Verni species (Iridaceae): implications for evolution of dormancy within the genus

The aim of this work was to examine whether seed ecophysiological traits in three closely related Crocus species were associated with ecological niche differentiation and species divergence. Seeds of the temperate tetraploid cytotype of Crocus neapolitanus, the sub‐Mediterranean C. etruscus and the Mediterranean C. ilvensis were placed either on agar in the laboratory under different periods of simulated seasonal conditions or in nylon mesh bags buried outdoors to examine embryo growth, radicle and shoot emergence. In agreement with the phenology observed outdoors, in the laboratory embryos required a cool temperature (ca. 10 °C) to grow to full size (embryo length:seed length, E:S ratio ca. 0.75) but only after seeds received a warm stratification; radicle emergence then followed immediately (November). Shoot emergence is a temporally separated phase (March) that was promoted by cold stratification in C. neapolitanus while in the other two species this time lag was attributed to a slow continuous developmental process. These species have similar embryo growth and radicle phenology but differ in their degree of epicotyl dormancy, which is related to the length of local winter. Conclusions from laboratory experiments that only consider root emergence could be misleading; evaluating the phenology of both root and shoot emergence should be considered in order to demonstrate ecologically meaningful differences in germination behaviour and to develop effective propagation protocols. Although these taxa resulted from recent speciation processes, the outcomes suggest an early onset of adaptation to local ecological factors and that phylogeny may represent a significant constraint in the evolution and expression of seed traits in Crocus.     

The Effect of Stratification on the Endogenous Cytokinin Levels of Seed of Protect compacta and Leucadendron daphnoides

The soybean callus assay was used to study the effect of stratification on the cytokinin levels of the embryo dormant seed of Protea compacta R.Br. and the seed of Leucadendron dapbnoides Meisn., where dormancy is coat imposed. Chilling the seed for 30 days increased germination significantly, and resulted in a simultaneous increase in the butanol soluble cytokinins of both species. It would appear as if these compounds are either synthesized or released from a bound form in embryo dormant seed. In contrast, an interconversion from water soluble to butanol soluble cytokïnins appears to account for the increase where dormancy is coat imposed. The results also indicate that for germination to take place a threshold concentration of cytokinin may be required. It is suggested that the increase in butanol soluble cytokinins may lead to the breaking of dormancy, probably by increasing radicle elongation and/or cotyledon expansion.