Globoid Crystals Research Articles

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60–80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

Raman and Fourier transform infrared spectroscopy application to the Puno and Titicaca cvs. of quinoa seed microstructure and perisperm characterization

The aim of this study was to investigate the quinoa fruit and seed microstructure, as well as to determine the qualitative composition of quinoa whole seed spatial localisation of food reserves in cultivars Puno and Titicaca using two complementary spectroscopic techniques (Fourier Transform infrared and Raman). The analyses of the seeds also included measurements of the crude proteins and starch contents. The experiment was carried out during the 2016 growing season in rain-fed conditions in the north of Serbia. The analysis of the scores of the principal components based on the Raman spectra revealed two groups in both seed parts (cotyledons and perisperm). The analysis of the loadings highlighted the spectrum region that contributed to the differentiation, e.g. the band at 472 cm−1 was related to the amylopectin content in the perisperm region. As for the cotyledons, the spectral range from 1100 to 1650 cm−1 was responsible for genotype differences and it included both the most important bands derived from Amide I, II and quinoa protein with globoid crystals composed of phytin. IR analysis, similar to the analyses of the crude proteins and starch contents in the seeds, failed to reveal any differences in biochemical composition between two analyzed genotypes.

Preparative Procedures Markedly Influence the Appearance and Structural Integrity of Protein Storage Vacuoles in Soybean Seeds

In legumes, vacuoles serve as the final depository for storage proteins. The protein storage vacuoles (PSVs) of soybean contain electron-transparent globoid regions in which phytic acid ( myo-inositol-1,2,3,4,5,6-hexakisphosphate) is sequestered. This paper reports the effect of preparative procedures on the appearance and ultrastructural integrity of PSVs in soybeans. Electron microscopy examination of both developing and mature soybean seeds that were postfixed with osmium tetroxide revealed PSVs that had a homogeneous appearance with very few globoid crystals dispersed in them. Numerous electron-dense lipid bodies were readily seen in these cells. Omission of osmium tetroxide strikingly altered the appearance of PSVs and aided the visualization of the location of the globoids in the PSVs. In contrast to the osmicated tissue, lipid bodies appeared as electron-transparent spheres. The choice of dehydration reagent or staining procedure had little influence on the appearance of the PSVs. The results of this study demonstrate the profound effect of osmium tetroxide on the appearance and structural integrity of PSVs in soybean.

Ultrastructure and development of seedlings of the parasitic weedCuscuta japonica

Shoot subapical cells in the parasitic angiospermCuscuta japonica seedlings were ultrastructurally studied. Seedlings were grown for 3 d in the dark and then for an additional 3 d in sunlight. Under either type of illumination, most cells in the primary meristem contained several vacuoles with or without electron-dense particles. These vacuoles were believed to be derived from degraded protein bodies with globoid crystals that were stored in the embryos. As growth progressed, the reserves were gradually depleted, while various cell organdies increased. This indicated that those storage reserves were utilized for seedling development and that, concurrently, cellular metabolism in the seedling cells converted from a quiescent to an active state. When seedlings were exposed to sunlight, etioplasts with prolamellar bodies developed into chloroplasts possessing thylakoids that were well-organized into grana. These observations suggest that C.japonica seedlings might exist autotrophically and photosynthesize during a free-living stage prior to parasitizing their hosts.

Ultrastructure of Mature Embryos in the Parasitic Flowering PlantCuscuta japonica

The ultrastructural features of embryos were studied from mature dry and soaked seeds of the parasitic angiospermCuscuta japonica. Outer tangential walls in the protoderm cells were thickened and covered by a thin cuticle layer. These walls could play important roles in preventing water loss from theCuscuta seedling surfaces after germination and in strengthening the surfaces against various environmental stresses. In the protoderm cells of soaked embryos, lipid materials were released into the thick outer walls through the fusion of lipid bodies with the plasma membrane. In the dry embryos were stored a large number of protein bodies with globoid crystals and lipid bodies. Numerous lipid bodies also were aligned under the plasma membrane. In both dry and soaked embryos, protein bodies were digested and transformed into small vacuoles. The degraded reserves of the lipid and protein bodies could then be mobilized to nourish subsequent germination and seedling growth. Proplastids in the soaked embryo cells contained a few thylakoids and electron-dense plastoglobuli, and crystallized phytoferritin. The phytoferritin, an iron-protein complex, would also be utilized in chloroplast development for autotrophic seedling growth.

Histochemical and ultrastructural studies on Salix alba and S. matsudana 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 large central perisperm, a peripheral embryo and a one to two-cell layered endosperm surrounding the hypocotyl-radicle axis of the embryo. Cytochemical and ultrastructural analysis revealed that starch grains occupy the cells of the perisperm, while lipid bodies, protein bodies with globoid crystals of phytin, and proplastids with deposits of phytoferritin are the storage components of the cells of the endosperm and embryo tissues. EDX analysis of the endosperm and embryo protein bodies revealed that globoid crystals contain phosphorus, potassium and magnesium. These results are compared with studies on other perispermous seeds published to date.

The mineral nutrient composition of wheat grains from ears grown to maturity in the field or cultured in media containing different concentrations of P, Mg, K and Ca

AbstractA study was conducted to assess the effect of the supply of phosphorus and the major cations on the mineral nutrient composition of whole mature wheat grains and the electron-dense globoid crystals found in the aleurone cells. Ears of wheat plants, growing in the field, were detached after anthesis. The ears were cultured to maturity in a sterile control solution which contained all essential elements. Other ears were cultured in solutions which contained no phoshorus, no calcium, no magnesium, combinations of these (−P, −Ca; −P, −Mg) or a solution which contained additional potassium but no phosphorus, calcium or magnesium. Ears cultured in the control solution produced grains with higher concentrations of P, Mg, K and Ca than grains from ears which matured on plants in the field. All the −P treatments produced grains with lower concentrations of P and higher concentrations of Mg, K and Ca than the plants grown in the field. The appearance and elemental composition of the globoid crystals in the aleurone cells support the hypotheses that the size of globoid crystals is regulated, in part, by the amount of phosphorus deposited in the grain and also by the relative proportions of K, Mg and Ca in the tissue. The culture of individual ears of cereal plants is a useful technique by which to study the transfer to and deposition of mineral nutrients in cereal grains.

The influence of experimentally induced changes in the (Mg + Ca):K balance on protein bodies formed in developing Cucurbita seeds

Phytate, the main mineral nutrient storage compound in seeds, is stored inside protein bodies. Phytate is usually concentrated in dense bodies called globoid crystals. In 1985, Lott and co-workers proposed that the balance of (Mg + Ca):K may be important in controlling globoid crystal formation and provided some experimental evidence to support this proposal. To test this hypothesis further, developing Cucurbita fruits were injected with sterile K salt solutions. Squash cotyledons generally have large globoid crystals and a relatively high (Mg + Ca):K ratio. We hypothesized that experimental reduction of the ratio by the addition of K would result in the alteration of the size and number of globoid crystals. Developing seeds were remarkably resistant to attempts to alter the elements taken up for storage. The elemental content of embryo tissues was measured quantitatively with neutron activation analysis. Controls plus those few samples showing a distinct shift in the ratio owing to elevated K content were analyzed further. Energy dispersive X-ray analysis of cryogenically prepared samples was used to study the elemental content of globoid crystals and transmission electron microscopy was used to study the ultrastructure of the protein bodies. The results provide additional support for the hypothesis being tested. Key words: phytate, globoid crystals, Cucurbita, seeds, mineral nutrients, protein bodies.

Studies of mature seeds of eleven Pinus species differing in seed weight. II. Subcellular structure and localization of elements

Seed tissues of 11 species of Pinus, varying greatly in seed weight, all contained protein bodies with one or more protein crystalloids and (or) one or more globoid crystals. Protein body structure and the types of elements stored in globoid crystals of protein bodies were similar between female gametophytes and their embryos. Energy dispersive X-ray analysis of globoid crystals revealed the presence of P, K, and Mg, a finding consistent with their being phytate rich. Traces of Ca and Fe were also detected in globoid crystals of some tissues. The P and Mg levels in globoid crystals of embryo and female gametophyte tissues decreased as interspecific seed tissue weight increased. High levels of Fe and significant levels of P, K, and Mg were detected in small, often ≤ 0.33 μm, naturally electron-dense particles distributed in all tissues of the embryo and female gametophyte. Unlike conventional phytate-rich globoid crystals, these Fe-rich particles were not contained in typical protein bodies but were contained within membrane-bound structures that may be plastids. Iron levels of the Fe-rich particles of Pinus seed tissues decreased as seed tissue weight increased. Key words: Pinus, seed weight, energy dispersive X-ray analysis, globoid crystals, Fe-rich particles.

Studies of storage reserves in seeds of the marine angiosperm Zostera capricorni

The structure of Zostera capricorni Aschers, seeds was determined to be similar to that of Zostera marina L. Staining with Lugol's iodine revealed large quantities of starch in all tissues of the embryo. Other stains revealed smaller amounts of lipids and proteins. Electron-dense globoid crystals were present in all tissues of the embryo. Energy dispersive X-ray (EDX) analysis revealed the presence of varying amounts of P, Mg, K and Ca in the globoid crystals. Small amounts of Ca were detected in globoid crystals of all tissues of the embryo. Neutron activation analysis (NAA) results showed elevated contents of Na and Cl in whole seed samples. Varying amounts of these two elements were found in globoid crystals of the embryo.

Studies of Capsicum annuum seeds: structure, storage reserves, and mineral nutrients

Storage reserves of Capsicum annuum (sweet pepper) seeds were studied using light microscopy, histochemical techniques, and electron microscopy. The seed had mainly protein and lipid as storage reserves. Starch was not detected. Thickened endosperm cell walls likely are a store of nonstarch polysaccharide. Protein bodies had protein crystalloids and globoid crystals as inclusions in the proteinaceous matrix. Protein bodies in embryo tissues varied greatly in size both inter- and intra-cellularly, while endosperm protein bodies were similar in size. Energy dispersive X-ray analysis and neutron activation analysis revealed the element content of C. annuum seeds. The presence of P, K, and Mg in all globoid crystals is consistent with their being phytin-rich. The average ratios of Mg:K, based on peak-to-background values for globoid crystals, were lowest in provascular tissues, where most globoid crystals were very small, and were highest in ground meristem tissues, where globoid crystals were large. Fe, Zn, Mn, Ca, S, and Cl were detected in some globoid crystals. In globoid crystals of both the cotyledon and hypocotyl – radicle axis, Fe was always detected in protoderm cells, was occasionally detected in ground meristem cells, and was never detected in provascular cells. Neutron activation analysis results showed that Mg, K, S, and Cu were higher in the embryo than in the endosperm, while Cl, Na, and Ca were higher in the endosperm. Mn had similar concentrations in both tissues. Key words: Capsicum annuum, storage reserves, protein body, globoid crystal, phosphorus, seed.

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

AbstractProtein 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.

Seed reserves and early symbiotic protocorm development of Platanthera hyperborea (Orchidaceae)

Seeds of the terrestrial orchid Platanthera hyperborea consist of a thin testa and a simple embryo without a cotyledon. Epidermal and parenchyma cells of the embryo contained lipid and protein as storage reserves. Many of the protein bodies had globoid crystals identified by their P, Ca2+, Mg2+, and K+ content. Germination occurred with either Rhizoctonia cerealis or Ceratorhiza goodyerae-repentis as the fungal symbiont on Warcup's medium. The fungus entered through dead suspensor cells and triggered protocorm development and concomitant utilization of lipid and protein reserves. Fungal hyphae formed pelotons with protocorm cells initially, and these went through stages of vacuolation and collapse. Some hyphae stored small numbers of polyphosphate bodies. Clumps of degenerated hyphae were usually encased within material that stained positive with aniline blue, presumably callose. Key words: Platanthera, orchid, seed, protocorm, endophytic fungus.

Protein bodies in Datura stramonium seeds: structure and mineral nutrient composition

The structure of protein bodies in the endosperm and embryo of Datura stramonium was studied with a variety of light-and electron-microscopic techniques. Protein bodies had one to several globoid crystals and one or two protein crystalloids in the proteinaceous matrix. Although the embryo protein bodies rarely had more than two globoid crystals, the endosperm protein bodies had varying sizes and numbers of globoid crystals, even within the same cell. Energy-dispersive X-ray analysis of globoid crystals revealed the presence of P, K, and Mg in all cases. Traces of Fe, Mn, and Zn were also found in globoid crystals of protein bodies from certain cell types. The distribution patterns of these three elements were quite specific; for example, Mn traces were found only in the globoid crystals of the protoderm. Neutron-activation analysis of endosperm and embryo tissues was used to quantitatively measure the concentration of Ca, Cl, Cu, I, K, Mg, Mn, Na, and S. The results from structural studies and the element analysis studies are discussed in the context of solanaceous seeds in particular but also with relation to seeds in general. Key words: protein bodies, Datura stramonium, seed, globoid crystals, energy-dispersive X-ray analysis, Solanaceae.

A histological and elemental analysis study of the mature seed of Begonia semperflorens

The total element content and the subcellular distribution of mineral nutrients were investigated in a species that has very minute seeds. The major storage substances in the seed of Begonia semperflorens were lipids sequestered in lipid vesicles and proteins concentrated in protein bodies. Transmission electron microscopy revealed the presence of protein crystalloids and electron-dense globoid crystals in the protein bodies. These inclusions varied both in size and number in protein bodies from different tissues. Energy dispersive X-ray analysis of the globoid crystals showed the high Mg, K, and P levels characteristically found in phytin from large-sized seeds. Small traces of Ca were found in globoid crystals within protein bodies found in the provascular tissue of the embryo. Neutron activation analysis was used to measure the elemental composition of the whole seed and these results were correlated with the findings of energy dispersive X-ray analysis studies of globoid crystals. Key words: Begonia seed, energy dispersive X-ray analysis, protein body, globoid crystal, protein crystalloid, neutron activation analysis.

Sensitivity of various phytates to beam damage during x-ray analysis

Phytic acid (myoinositol hexaphosphoric acid) has 12 hydrolyzable hydrogens and forms salts with a variety of cations. Phytic acid salts are stored in seeds and act as nutrient reserves of elements such as Mg, K, Ca and P. In diets heavily dependent on grains, phytates have been implicated in reducing the bioavailability of elements such as zinc and iron. The complexing properties of phytates have made these compounds useful in a number of medical and industrial procedures.Phytin reserves in seeds often are localized in electron dense spherical particles called globoid crystals located within protein bodies. The characteristics of globoid crystals remain poorly defined. Energy-dispersive x-ray analysis of individual globoid crystals from various species has shown that there is considerable variation in the relative proportions of Mg, K, Ca and P species-to-species.

Elemental storage in Cucurbita embryos: X-ray microanalysis of magnesium, potassium, calcium, and phosphorus within globoid crystals

Embryos of Cucurbita maxima, Cucurbita andreana, and their reciprocal hybrids store phosphorus, magnesium, potassium, and calcium in distinctive globoid crystals within protein bodies found in cotyledon mesophyll cells. Samples of cotyledon tissue from each of the four kinds of embryos were cryogenically prepared and analyzed by X-ray microanalysis in the frozen state in a scanning electron microscope. The relative proportions of phosphorus, magnesium, potassium, and calcium were expressed as peak to background ratios. Total concentrations of the four elements in whole cotyledons, as measured by neutron activation analysis, were similar to the trends shown by the peak to background ratios for the same elements. This supports the concept that the bulk of the elements P, K, Mg, and Ca in seed tissues is stored in globoid crystals.

Identification of zinc-containing globules in roots of a zinc-tolerant ecotype of Deschampsia caespitosa

Abstract In previous work on Zn‐tolerant and non‐tolerant ecotypes of Deschampsia caespitosa root elongation was shown to be a sensitive parameter of Zn toxicity. Laser microprobe mass analysis of small volumes in thin sections from freeze substituted root tips of seedlings grown on nutrient solutions containing high levels of Zn, showed the sporadic presence of Zn. This Zn seemed to be associated with particles, but the optical resolution of the method was insufficient to establish the nature of the intracellular Zn distribution. Subsequent electron probe X‐ray microanalysis showed the presence of Zn in globular bodies of about 1 μm diameter, which were frequent in the inner cortical cells of the elongation zone of root tips of the Zn‐tolerant ecotype. The globules were located within the cytoplasm in small vacuoles, and, within each treatment, contained stable proportions of Zn, Mg, and K with relatively large amounts of P. It is suggested that their properties are similar to the globoid crystals of phy...

Distribution de quelques oligo-éléments dans des cristaux de graines de Raphanus

Globoid crystals were studied in the cotyledonary parenchyma of anhydrous seeds of radish (Raphanus sativus L., Brassicaceae) in ultrathin sections after aldehydic fixation. Crystals derived from the same seeds were also isolated, by print techniques without fixation or aqueous phase. Eleven elements (Mg, Al, Si, P, S, K, Ca, Mn, Fe, Cu, Zn) were looked for using transmission electron probe microanalysis and a wave length dispersive X-ray spectrometry (Camebax microprobe). Crystals of various sizes, rich in P, Mg, and Ca, could contain some micro-elements (Mn, Fe, Zn … ) which could be included in the phytine. Other small crystals, stable under radiation and rich in Fe, were low in P. These two sorts were characterized in globoids, in sections and in prints. Other crystals, isolated from dry seeds, rich in Ca, K, and Mg, were poor in P. Finally, long or angular very stable crystals, rich in Al, lacking P, and hence without phytine, could contain other metals (Fe, Cu, Zn … ) which could be included in nonphytinic molecules. A given metal (Fe or Mn … ) can be found in several types of crystals. Several groups of crystals with similar characters (morphology, composition) were defined, either in protein bodies, in ultrathin sections, or in prints.