Membrane organization in soybean seeds during hydration

The ability of seeds to withstand dehydration indicates that their membranes may maintain structural integrity even when dry. Analysis of polar lipids (the principal lipidic constituents of the membranes) from soybean seeds (Glycine-max (L.) Merr.) by X-ray diffraction indicated that even in the dehydrated state the lipids retained a lamellar (bilayer) configuration. As the degree of hydration was raised, evidence of some structural alteration (apparent as an abrupt increase in bilayer spacing) was obtained from diffraction patterns of both the extracted lipid and particles of seed tissue. In seed tissue this increase in bilayer spacing occurred at a hydration level just above that at which free water could be detected by nuclear-magnetic-resonance analysis. The water content at which the increase in bilayer spacing occurred was higher in the seed tissue than in the extracted polar lipids, probably because other cell components restricted the availability of free water in the seed.Abbreviation NMR nuclear-magnetic resonance     

Role of seed coat in imbibing soybean seeds observed by micro-magnetic resonance imaging

Background and Aims

Imbibition of Japanese soybean (Glycine max) cultivars was studied using micro-magnetic resonance imaging (MRI) in order to elucidate the mechanism of soaking injury and the protective role of the seed coat.

Methods

Time-lapse images during water uptake were acquired by the single-point imaging (SPI) method at 15-min intervals, for 20 h in the dry seed with seed coat, and for 2 h in seeds with the seed coat removed. The technique visualized water migration within the testa and demonstrated the distortion associated with cotyledon swelling during the very early stages of water uptake.

Key Results

Water soon appeared in the testa and went around the dorsal surface of the seed from near the raphe, then migrated to the hilum region. An obvious protrusion was noted when water reached the hypocotyl and the radicle, followed by swelling of the cotyledons. A convex area was observed around the raphe with the enlargement of the seed. Water was always incorporated into the cotyledons from the abaxial surfaces, leading to swelling and generating a large air space between the adaxial surfaces. Water uptake greatly slowed, and the internal structures, veins and oil-accumulating tissues in the cotyledons developed after the seed stopped expanding. When the testa was removed from the dry seeds before imbibition, the cotyledons were severely damaged within 1·5 h of water uptake.

Conclusions

The activation of the water channel seemed unnecessary for water entry into soybean seeds, and the testa rapidly swelled with steeping in water. However, the testa did not regulate the water incorporation in itself, but rather the rate at which water encountered the hypocotyl, the radicle, and the cotyledons through the inner layer of the seed coat, and thus prevented the destruction of the seed tissues at the beginning of imbibition.Key words: Dry seeds, Glycine max, MRI, seed coat, soaking injury, soybean, testa, role of inner layer of seed coat, water uptake     

Imbibition of white spruce seeds and somatic embryos: A study of morphological changes in an environmental scanning electron microscope and potassium leakage

Summary Potassium leakage and morphological changes during imbibition of white spruce [Picea glauca (Moench) Voss] seeds and somatic embryos were investigated. A single desiccated somatic embryo, a single somatic embryo exposed to a high relative humidity environment for 2 d, and a single dry zygotic embryo leaked similar amounts of potassium over a 120-min period of imbibition in liquid germination medium. A seed without a seed coat leaked two and eight times more potassium than a single whole seed and a single zygotic embryo, respectively. Nearly 50% of the potassium leaked for all tissues was leaked within the first 20 min of imbibition. Exposure of somatic embryos to an environment with high relative humidity resulted in a reduction in the percentage of potassium leaked after 80 and min to levels equivalent to those for zygotic embryos. Using an environmental scanning electron microscope, we found that desiccated somatic embryos and dry zygotic embryos had wrinkled surface cells, with cells in the surface of zygotic embryos being more shrunken in appearance. Imbibition of both types of embryos in water resulted in turgid surface cells after 2 h. Imbibition in liquid germination medium did not cause much hydration of surface cells, which still had wrinkled appearances after 2 h. Finally, imbibition on filter paper on semisolidified germination medium resulted in slower hydration of somatic and zygotic embryos. Cells near the medium appeared hydrated while cotyledon surface cells furthest from the medium resembled cells in desiccated embryos.     

Lipid participation in intracellular freezing avoidance mechanisms of lettuce seed

Freeze-fracture electron microscopy was used to study water content related freezing resistance in Grand Rapids lettuce seeds. Consistent and recognizable conformational changes occurred in lipid-water phases of lettuce seeds at different moisture contents. In air-dry lettuce seed cotyledons, the lipids lying in spherical lipid bodies near the cell wall appeared amorphous, while the structure was crystalline above 20% water content. The lipid bodies interassociated into membrane bilayers in seeds containing 20 to 25% water. Such lyotropic phase transitions in membrane lipids during lettuce seed hydration are believed to contribute to the biphasic freezing behavior observed in lettuce seeds at different moisture contents and to provide a natural freezing tolerance mechanism for highly desiccated plant tissues such as seeds.     

聚乙烯醇预处理提高大豆种子活力和抗冷能力的作用

聚乙烯醇预处理大豆种子和聚乙二醇一样,能明显地提高种子的活力、萌发率和抗冷能力。其主要作用可能是:在整个吸胀过程中减少了吸水速度,使膜的修复作用有足够的时间;启动和改善种子内部的生理代谢过程.保证了生长胚根有更多的物质供应。     

Characterization of water distribution and activities of enzymes during germination in magnetically-exposed maize (Zea mays L) seeds

Magnetic seed treatment is one of the physical pre-sowing seed treatments to enhance the performance of crop plants. In our earlier experiment, we found significant increase in germination and vigour characteristics of maize (Zea mays L.) seeds subjected to magnetic fields. Among various combinations of magnetic field (MF) strength and duration, best results were obtained with MF of 100 mT for 2 h and 200 mT for 1 h exposure. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions. Thus, in the present study, changes in water uptake during imbibition and its distribution and activities of germinating enzymes during germination were investigated in maize seeds exposed to static magnetic fields of 100 and 200 mT for 2 and 1 h respectively by nuclear magnetic resonance (NMR) spectroscopy. The magnetically-exposed seed showed higher water uptake in phase II and III than unexposed seed. The longitudinal relaxation time T1 of seed water showed significantly higher values and hence greater molecular mobility of cellular water in magnetically-exposed seeds as compared to unexposed. Component analysis of T2 relaxation times revealed the early appearance of hydration water with least mobility and higher values of relaxation times of cytoplasmic bulk water and hydration water in magnetically-exposed over unexposed seeds. Activities of alpha-amylase, dehydorgenase and protease during germination were higher in magnetically-exposed seeds as compared to unexposed. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions.     

Isoflavonoid biosynthesis and accumulation in developing soybean seeds

Isoflavonoids are biologically active natural products that accumulate in soybean seeds during development. The amount of isoflavonoids present in soybean seed is variable, depending on genetic and environmental factors that are not fully understood. Experiments were conducted to determine whether isoflavonoids are synthesized within seed tissues during development, or made in other plant organs and transported to the seeds where they accumulate. An analysis of isoflavonoids by HPLC detected the compounds in all organs of soybean plant, but the amount of isoflavonoids present varied depending on the tissue and developmental stage. The greatest concentrations were found in mature seeds and leaves. The 2-hydroxyisoflavanone synthase genes IFS1 and IFS2 were studied to determine their pattern of expression in different tissues and developmental stages. The highest level of expression of IFS1 was observed in the root and seed coat, while IFS2 was most highly expressed in embryos and pods, and in elicitor-treated or pathogen-challenged tissues. Incorporation of radiolabel into isoflavonoids was observed when developing embryos and other plant organs were fed with [(14)C]phenylalanine. Embryos excised from developing soybean seeds also accumulated isoflavonoids from a synthetic medium. A maternal effect on seed isoflavonoid content was noted in reciprocal crosses between soybean cultivars that differ in seed isoflavonoids. From these results, we propose that developing soybean embryos have an ability to synthesize isoflavonoids de novo, but that transport from maternal tissues may in part contribute to the accumulation of these natural products in the seed.     

Cracks in the palisade cuticle of soybean seed coats correlate with their permeability to water

BACKGROUND AND AIMS: Soybean (Glycine max) is among the many legumes that are well known for 'hardseededness'. This feature can be beneficial for long-term seed survival, but is undesirable for the food processing industry. There is substantial disagreement concerning the mechanisms and related structures that control the permeability properties of soybean seed coats. In this work, the structural component that controls water entry into the seed is identified. METHODS: Six soybean cultivars were tested for their seed coat permeabilities to water. To identify the structural feature(s) that may contribute to the determination of these permeabilities, fluorescent tracer dyes, and light and electron microscopic techniques were used. KEY RESULTS: The cultivar 'Tachanagaha' has the most permeable seed coat, 'OX 951' the least permeable seed coat, and the permeabilities of the rest ('Harovinton', 'Williams', 'Clark L 67-3469', and 'Harosoy 63') are intermediate. All seeds have surface deposits, depressions, a light line, and a cuticle about 0.2 microm thick overlaying the palisade layer. In permeable cultivars the cuticle tends to break, whereas in impermeable seeds of 'OX 951' it remains intact. In the case of permeable seed coats, the majority of the cracks are from 1 to 5 micro m wide and from 20 to 200 micro m long, and occur more frequently on the dorsal side than in other regions of the seed coat, a position that correlates with the site of initial water uptake. CONCLUSIONS: The cuticle of the palisade layer is the key factor that determines the permeability property of a soybean seed coat. The cuticle of a permeable seed coat is mechanically weak and develops small cracks through which water can pass. The cuticle of an impermeable seed coat is mechanically strong and does not crack under normal circumstances.     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.     

Patterns and kinetics of water uptake by soybean seeds

Soybean [Glycine max (L.) Merr.] plants produce some seeds (called stone or impermeable seeds) that do not take up water for long periods of time. The present investigation confirmed that the stone seed trait is a feature of the seed coat: isolated embryos from both stone and permeable seeds took up water equally quickly. A whole, permeable seed typically imbibed water initially through its dorsal side, forming wrinkles in the seed coat and delivering water to the underlying cotyledons. Later, some lateral movement of water through the coat occurred, presumably through the air spaces of the osteosclereid layer. Imbibition by seeds was a two-phase process, the first dominated by hydration of the seed coat and the second by hydration of the cotyledons, which was rate-limited by the coat. When hydrated, coats of stone seeds were permeable to water but their hydraulic conductivity, as measured with a pressure probe, was smaller than that of coats from permeable seeds by a factor of five. Hydrated coats of both permeable and stone seeds showed weak osmometer properties.     

Organic free radical levels in seeds and pollen: The effects of hydration and aging

In view of their possible role in oxidative deterioration of seeds and pollen, organic free radicals were measured by electron spin resonance in embryonic axes and cotyledons of soybean [ Glycine max (L.) Merr], embryo and endosperm fractions of corn [ Zea mays L.] and pollen of cattail [ Typha latifolia L.]. A pronounced decline in the radical signal ensued when hydration increased above about 7% (wet weight basis) in both the seed materials and in pollen. Moderate hydration of the soybean axis followed by drying led to a small decrease in organic free radicals compared to untreated material, especially if the desiccation step was performed under nitrogen. In a comparison of soybeans of various ages under normal storage, organic free radical levels in the axis showed little or no increase with age. In marked contrast, over 5 days of accelerated aging at 40°C and near-saturating humidity, organic radical levels approximately doubled in the axis. This pronounced increase in free radical content was not associated with a decrease in the proportion of polyunsaturated fatty acids. The data suggest that hydration of seed and pollen causes a release of free radicals from the trapped state.     

Calcium oxalate crystals in the mature seeds of Norway spruce, Picea abies (L.) Karst.

 The morphology and location of crystals encountered in the mature seeds of Norway spruce, Picea abies (L.) Karst., were examined using light and field emission scanning electron microscopy (FESEM). Crystals of various forms and sizes were discovered in different regions and tissues of seeds, particularly in the testa and the nucellus. Both solitary crystals and druses were occasionally enveloped by protrusions of the megaspore membranes or the cuticle of the megagametophyte. Histological studies and acid solubility tests coupled with analysis using energy dispersive X-ray microanalysis and X-ray diffraction evinced the crystals as calcium oxalate, but were unable to identify different hydration forms. Calcium oxalate crystals were most abundant in the damaged and infected tissues, and in the structures that desiccate during the development of the seed. Based on these observations we concluded that the accumulation of calcium oxalate is a regular process belonging to maturation and defense mechanism in spruce seeds. Received: 1 July 1998 / Accepted: 20 September 1998     

The outermost cuticle of soybean seeds: chemical composition and function during imbibition

Seeds of different cultivars of Glycine max (L.) Merr. (soybean) have strikingly different rates of water imbibition. Seeds that readily imbibe water are termed 'soft', while those that remain non-permeable, even after several days in water, are referred to as 'hard', 'stone', or 'impermeable' seeds. What prevents soybean hard seeds from taking up water? Previous work established that the initial imbibition of soft soybean seeds correlates with the presence of small cracks in the outermost cuticle that covers the seed coat, prompting a detailed analysis of soybean seed coat cutin. In this paper, it is shown that the outermost cuticle of the seed coat has an unusual chemical composition, lacking typical mid-chain-hydroxylated fatty acids but being relatively rich in other types of hydroxylated fatty acids. The cuticle of the impermeable cultivar studied contained a disproportionately high amount of hydroxylated fatty acids relative to that of the permeable ones. Moreover, a brief treatment with hot alkali released the omega-hydroxy fatty acid component of the outermost cuticle and created holes in it that caused the seeds to become permeable. This demonstrates that the outermost cuticle of the seed is the critical structure that prevents water uptake by hard seeds.     

不同天然居群小檗种子萌发障碍因子研究

以西天山野果林霍城居群、新源居群和特克斯居群黑果小檗以及特克斯居群红果小檗种子为试验材料,研究了4组野生小檗种子生物学特性、吸水特性以及去种皮、低温层积和不同浓度GA₃处理对小檗种子休眠与萌发特性的影响,结果表明：（1）4组野生小檗种子的吸水率均表现出逐渐增加的趋势,黑果小檗种子与红果小檗种子吸水率差异不明显,其种皮透水透气性相似;（2）霍城居群、新源居群和特克斯居群黑果小檗种子的种皮对萌发存在强烈的抑制作用;而新源居群红果小檗种子的种皮抑制作用不明显;（3）4℃低温层积处理对4组野生小檗种子萌发影响很大,随层积时间的增加小檗种子发芽率均逐渐提高,3组黑果小檗种子层积90 d时,休眠基本被解除;红果小檗种子层积30 d时,休眠基本被解除;（4）浓度为200 mg·L^-1的GA₃溶液处理可显著提高4组小檗种子发芽率,浓度过高或过低均会对小檗种子萌发起到抑制作用。层积60 d时霍城居群、新源居群和特克斯居群黑果小檗去种皮种子用200 mg·L^-1的GA₃溶液处理2 h后,发芽率分别为85.00%、83.33%和86.67%;红果小檗层积15 d时去种皮种子用浓度为200 mg·L^-1的GA₃溶液处理后,发芽率可达86.67%。研究结果将为今后野生小檗的种质资源保护,利用及可持续发展提供技术支持和科学依据。     

ULTRASTRUCTURAL ASPECTS OF CHILLING INJURY IN THE SOYBEAN SEED RADICLE

Imbibitional chilling injury of soybean seeds was studied using light and electron microscopy. Intact seeds were allowed to take up water at 4 C or room temperatures and the ultrastructure of the radicle was examined after hydration. In chilled seeds, mitochondria showed weakly defined envelopes and cristae, in contrast to the well-formed internal structure in the controls. Nuclei of chilled seeds were irregular in shape. Tannic acid was used as a probe of plasmalemma integrity. Most cells in both chilled and control treatments were able to exclude tannin, indicating that chilling injury effects did not damage the plasma membrane enough to allow passage of molecules of that size.     

Quick and accurate detection of Sclerotinia sclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Seed‐borne pathogenic fungi can cause serious damage to soybean crops by reducing the germination, vigour and emergence of the seeds. Special attention should be paid to pathogen detection in seeds to prevent its introduction in disease‐free areas. Considering the importance of rapid and successful diagnosis of seed‐borne pathogenic fungi in soybeans, this study evaluated a method to detect Sclerotinia sclerotiorum and Phomopsis spp. in seeds using quantitative polymerase chain reaction (qPCR). Naturally infested samples were subjected to detection using qPCR and blotter test, and the findings were compared. Using soybean seeds soaked in water, both pathogens were detected at an infestation level up a 0.0625% (one infected seed out of 1,599 healthy seeds) by qPCR. This technique allowed the detection of 300 fg of S. sclerotiorum and 30 fg of Phomopsis spp. DNA in the seed samples. Phomopsis spp. was detected in 40.7% of the evaluated seed batches (81 batches) and S. sclerotiorum was detected in 32.1% of the evaluated batches, although most of the seeds had low infestation levels. It was up to 28.5 times more efficient to use qPCR rather than blotter test to detect pathogens with a low incidence of occurrence in soybean seeds. If routinely used to test healthy seeds, qPCR would contribute to reducing soybean losses due to diseases as well as decreasing the costs required to control those diseases.     

Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms

Internally seedborne microorganisms are those surviving common surface sterilization procedures. Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum. Bacterial isolates from surface-sterilized soybean seed, cv. Williams 82 and cv. Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis. Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G. The effects of this antibiotic on seedling development and on B. japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here. Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development. No differences in nodulation kinetics, nitrogen fixation onset or rates were observed. However, the number of B. japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment. Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B. japonicum to the soybean root, without affecting plant development.     

The unusual tegumental tissues of the Lunaria annua (Brassicaceae) seed: A developmental study using light and electron microscopy

With light, fluorescence, transmission electron, and environmental scanning electron microscopy we studied the development of the Lunaria annua L. (Brassicaceae) seeds in order to reveal basic anatomical information about the unusual tissues of these seeds. In particular the seed tegument tissues possess complex morphological aspects that are relevant to the biology and ecology of this plant. A sclerenchymatic tissue as the innermost layer of the teguments apparently offers robust protection for the embryo, yet is organized to be flexible. This tissue likely controls the passage of water from the tegumental layers towards the embryo. We report here the presence of tannins in the pre-sclerenchymatic layer of the unripe seed. The inner tegument also houses a spongy tissue with wide intercellular spaces. This tissue could impart buoyancy to the seeds, which possibly might be required for water transport. The structural features could indicate that Lunaria may have evolved in a Mediterranean environment, which is characterized by a long dry season, but with a large amount of rainfall concentrated in short periods. Probably, not only the typical enlarged and flattened fruits of Lunaria can easily float and be dispersed away from the mother plant, but also the seeds have this dispersal peculiarity after release from the silicules.