The glassy state and accelerated aging of soybeans

Deteriorative changes in seeds may be expected to reflect changes in physical state as well as in chemical composition. In the present study, measurements of changes in glass transition, lipid phase transition and sugar content were made during accelerated aging of two cultivars of soybeans ( Glycine max Merrill cv. Chippewa 64 and cv. Hodgson 78). The glass transition in axes, as measured by the thermally stimulated depolarization current method, showed gradual decreases in both magnitude and transition temperature during accelerated aging, and eventually, axes of seeds lost their ability to enter the glassy state. Sucrose, raffinose and stachyose contents in seed axes showed little or no change during the aging treatment. Membrane lipids in aged axes retained the liquid crystalline phase during aging. These data suggest that the changes of glass transition during accelerated aging occurred without associated changes in soluble sugar contents or changes in the liquid crystalline state of membrane lipids. The loss of the glass transition during accelerated aging could be a consequence of the annealing effect due to elevated temperature and moisture content. We propose that a loss of the glassy state during accelerated aging leads to an enhanced rate of subsequent deteriorative reactions in seeds and accelerates the loss of viability.     

杂交水稻和不育系种子的劣变与生理生化变化

杂交水稻和不育系种子在人工加速老化处理后,其发芽率、发芽指数和活力指数均比常规水稻种子下降迅速。在人工老化过程中,前者种子浸泡液的外渗氨基酸和钾离子含量均比后者高,游离的有机酸、氨基酸和脂肪酸含量增加的速度相似,均大于常规水稻种子。蛋白酶活性和可溶性蛋白质含量的增减,在易发生劣变种子与常规水稻种子之间均有显著差异。     

Lipid peroxidation and peroxide-scavenging in soybean seeds during aging

The possible role of lipid peroxidation in seed deterioration was investigated during natural aging and accelerated aging of seeds of edible soybean ( Glycine max [L], Merr. cv. Kaohsiung Selection No. 1). Natural aging was achieved by sealing the seeds in aluminum foil bags coated with polyethylene and storing the seeds at room temperature for 3 to 12 months. Accelerated aging was obtained by incubating the seeds at 45°C and close to 100% relative humidity for 3 to 12 days, after which the seeds were air dried to their original moisture level (8%). The results indicate that both natural and accelerated aging enhanced lipid peroxidation, as germination was depressed. Aging also inhibited the activity of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase. The changes in germination and physiological activities, expressed as a function of aging duration, were somewhat similar in the two aging treatments.     

Patterns of protein synthesis during the germination of pea axes,and the effects of an interrupting desiccation period

As germination of axes of Pisum sativum L. seeds progressed, profound quantitative and qualitative changes occurred in the patterns of protein synthesis. This was shown by fluorography of gels following two-dimensional polyacrylamide gel electrophoresis separation of [³⁵S]methioninelabelled proteins. The effects of desiccation during germination on these in-vivo protein-synthesis patterns were followed. Desiccation differentially affected the synthesis of proteins. Usually, however, upon rehydration following desiccation the types of proteins being synthesized were recognizable as those synthesized earlier during imbibition of control, once-imbibed axes: seeds imbibed for 8 h, and then dried, did not recommence synthesis of proteins typical of 8-h-imbibed control seeds, but rather of 4-h-imbibed control seeds. Seeds imbibed for 12 h, and then dried and rehydrated, synthesized proteins typical of 4-h-and 8-h-control seeds. Thus drying of germinating pea axes caused the proteinsynthesizing mechanism to revert to producing proteins typical of earlier stages of imbibition. Drying during germination never caused the seed to revert to the metabolic status of the initial mature dry state, however.Abbreviation DR dried and rehydrated     

Protein repair L-isoaspartyl methyltransferase in plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley seeds.

Protein L-isoaspartate (D-aspartate) O-methyltransferases (MTs; EC 2.1.1.77) can initiate the conversion of detrimental L-isoaspartyl residues in spontaneously damaged proteins to normal L-aspartyl residues. We detected this enzyme in 45 species from 23 families representing most of the divisions of the plant kingdom. MT activity is often localized in seeds, suggesting that it has a role in their maturation, quiescence, and germination. The relationship among MT activity, the accumulation of abnormal protein L-isoaspartyl residues, and seed viability was explored in barley (Hordeum vulgare cultivar Himalaya) seeds, which contain high levels of MT. Natural aging of barley seeds for 17 years resulted in a significant reduction in MT activity and in seed viability, coupled with increased levels of "unrepaired" L-isoaspartyl residues. In seeds heated to accelerate aging, we found no reduction of MT activity, but we did observe decreased seed viability and the accumulation of isoaspartyl residues. Among populations of accelerated aged seed, those possessing the highest levels of L-isoaspartyl-containing proteins had the lowest germination percentages. These results suggest that the MT present in seeds cannot efficiently repair all spontaneously damaged proteins containing altered aspartyl residues, and their accumulation during aging may contribute to the loss of seed viability.     

Delayed luminescence of air-dry soybean seeds as a measure of their viability

Delayed luminescence (DL) excited by visible light has been observed in air-dry seeds of various plants. Changes in seeds of soybeans ( Glycine max L. W.) which occur during natural and accelerated aging were reflected in subsequent loss of vigor and DL increase. The increase of the luminescence is caused by the changes in seed biopolymers, leading to a decrease in their ability to take up water. The method makes it possible to measure DL from a single seed. With aging, the DL was shifted toward higher intensities and there was greater variability. By monitoring the DL of seeds it may be possible to evaluate their quality and storage capability.     

Biochemical Changes in Sorghum Seeds Affected by Accelerated Aging

Examination of the initial rates of enzyme activities in variousseed lots demonstrated that amylase, glutamic-pyruvic-transaminase,RNase, and glutamate decarboxylase follow the vigour profile,namely, an increase after 6 d of aging treatment followed bya decrease up to 48 d of aging time. Acid phosphatase, tetrazoliumreduction, and [¹⁴C]leucine incorporation decline directly withthe time of aging and no increase in those activities at 6 daging could be observed. Proteinase is the only enzyme foundso far that increases during accelerated aging. It is postulatedthat this enzyme may be the cause of the drop in all other enzymeactivities and therefore may play an important role in the lossof seed vigour. Examination of the components released from seeds into water,and of the rates of their leakage, led to the conclusion thatthe efflux reflects severely damaged seeds rather than vigourlessones.     

Dynamics of protein synthesis and degradation through the cell cycle

Protein expression levels depend on the balance between their synthesis and degradation rates. Even quiescent (G₀) cells display a continuous turnover of proteins, despite protein levels remaining largely constant over time. In cycling cells, global protein levels need to be precisely doubled at each cell division in order to maintain cellular homeostasis, but we still lack a quantitative understanding of how this is achieved. Recent studies have shed light on cell cycle-dependent changes in protein synthesis and degradation rates. Here we discuss current population-based and single cell approaches used to assess protein synthesis and degradation, and review the insights they have provided into the dynamics of protein turnover in different cell cycle phases.     

Molecular Marker Analysis of Differentially Aged Seeds of Soybean and Safflower

Storage of seeds for extended periods causes a number of degradative changes related to the aging process such as decreased seedling vigor and reduced germination. In this study, molecular markers were used to study the aging process in seeds of two different plants species. Seeds of three differentially aged seed groups, including control (un-aged), naturally aged, and accelerated aging, from soybean (Glycine max) and safflower (Carthamus tinctorius) were evaluated for genetic variability using random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) markers. For both plant species, naturally aged and accelerated aged groups clustered together with RAPD markers, whereas control and naturally aged seeds showed similarity in both AFLP and SSR profiles. Based on these findings, it can be concluded that observed changes in DNA profiles of seeds from different aged groups did not contribute to accumulation of genetic variations of the same magnitude. Therefore, seed of similar viability must be selected for molecular marker analysis for plant variety protection, among other comparative studies.     

Extracellular signal-regulated kinase 2 mRNA expression in the rat brain during aging

Aging is accompanied by the loss of memory and cognitive functions. The extracellular signal-regulated kinase (ERK) pathway has been shown to play an essential role in synaptic plasticity and memory. Although a reduction in basal ERK1/2 activity has been found in the cerebral cortex in aged rats, changes in ERK1/2 mRNA expression during aging have not been described. In this study, we investigated age differences in the mRNA expression of ERK2 in different brain regions of male Fisher 344 rats (three age-groups) using quantitative in situ hybridization. No age-dependent changes in ERK2 mRNA were detected in the cerebellum or cortical areas. However, in the hippocampus, a 20% decline in mRNA levels was observed in the CA3 region in the 12-month-old group as compared to the 3-month-old group. These results suggest that the impairment in ERK1/2 activity observed during aging is probably not regulated at the gene expression level.     

The Maillard reaction and oxidative stress during aging of soybean seeds

The chemical reactions that may lead to the loss of seed viability were investigated both during the accelerated aging and natural aging of soybeans ( Glycine max Merrill cv. Chippewa 64). Under conditions of accelerated aging (36°C and 75% RH), fluorescence of soluble proteins accumulated, which was closely correlated with the loss of seed germinability and vigor. We were able to show this correlation by using partially purified proteins for the assay. Fluorescence also increased in seeds under good storage conditions (5°C for up to 21 years), although there was a less significant correlation between seed viability and the accumulation of fluorescent products during the time of natural aging. The rise in protein fluorescence is interpreted as an increase of Maillard products. The carbonyl content of soluble proteins (a measure of the oxidative damage) did not change significantly during either accelerated aging or natural aging: however the elimination of carbonyls during germination seemed to be hindered in seeds that had poor germination. The Maillard reaction may be a consequence of the formation of reducing sugars through a gradual hydrolysis of oligosaccharides during aging. Preliminary evidence from the natural aging study showed that, when seeds were in the glassy state, the sugar hydrolysis was inhibited. These results suggest that the Maillard reaction and oxidative reaction may play an important role in seed deterioration.     

Aging and Loss of Germination in Rye Seeds Is Accompanied by a Decreased Fragmentation of Nuclear DNA at Loop Domain Boundaries

To investigate the processes that occur in the embryo cell nuclei in the course of natural and accelerated aging of rye seeds, nuclear DNA structural organization into chromatin loop domains was studied. The loss of germination was shown to be accompanied by a decreased excision of chromatin loop domains. The study of chromatin accessibility to DNase I did not reveal any considerable changes in chromatin architecture that would explain the decreased DNA fragmentation at matrix attachment regions. A soluble nuclear protein of ca. 31 kD was found to manifest nuclease activity, which declined with the loss of germination. The study of DNA fragmentation in histone-depleted nuclei (nucleoids) disclosed a nuclease activity resistant to 2 M NaCl extraction and sensitive to the specific inhibitors of DNA topoisomerase II; the latter activity also declined with aging. The authors conclude that the changes in DNA fragmentation patterns in aging seeds were primarily caused by a decreased activity of the enzymes accounting for the excision of chromatin loop domains.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts

The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR.     

Biochemical changes induced by accelerated aging in sunflower seeds. I. Lipid peroxidation and membrane damage

When stored at 42°C and 100% relative humidity for 1 to 8 days, sunflower seeds (Helianthus annuus L. cv. Rodeo) aged prematurely and lost 25% of their initial viability. A ten-fold increase in conjugated dienes as well as a decrease of unsaturated fatty acids in diacylglycerol and polar lipids fractions were observed after 8 days of accelerated aging, demonstrating the occurrence of lipid peroxidation in prematurely aged sunflower seeds. However, the viability remained relatively high. The absence of membrane damage in seeds and of lipid peroxidation in isolated microsqmes suggested that lipid peroxidation concerned mainly lipid reserves. These results suggest that, at least within the first 8 days of treatment, the lipid reserve in sunflower seeds might act as a detoxifying trap, protecting membranes from excessive damage.     

Protein Synthesis in Dormant and Non-Dormant Cocklebur Seed Segments

Using the axial and cotyledonary segments of lower cocklebur (Xanthium pensylvanicum Wallr.) seeds, protein synthesis as shown by incorporation of radioactive leucine was examined in relation to their dormant status. During the first 9 h of water imbibition, the protein synthesis was higher in the dormant axes than in the non-dormant, after- ripened ones. When imbibed for more than 12 h non-dormant axes had a higher activity than dormant ones. This was also the case with the cotyledonary segments. Cyctoheximide, an inhibitor of protein synthesis, blocked protein synthesis in the axial tissue regardless of its dormant status, and thereby inhibited germination of the non-dormant seeds. In the dormant seeds, however, cycloheximide at 3 mM slightly stimulated germination without stimulating the C₂H₄ production. Based on these results, it is suggested that in cocklebur seeds there may be some proteinaceous system which is involved in the maintenance of dormancy.