Protein modification by Amadori and Maillard reactions during seed storage: roles of sugar hydrolysis and lipid peroxidation

The non-enzymatic modifications of proteins through Amadori and Maillard reactions play an important role in the loss of seed viability during storage. In the present study, the contribution of sugar hydrolysis and lipid peroxidation to Amadori and Maillard reactions, and to seed deterioration was investigated in mung-bean (Vigna radiata Wilczek). The contents of glucose and lipid peroxidation products in seed axes increased significantly during storage. The accumulation of Amadori products in seed axes was correlated to the lipid peroxidation, whereas the accumulation of Maillard products was closely correlated to sugar hydrolysis. The rate of accumulation of Maillard products was not well correlated to the content of Amadori products in both seed axes and protein/glucose model system, reflecting the complex nature of Amadori and Maillard reactions. The content of Amadori products in seed axes increased during the early stages of seed ageing, whereas the content of Maillard products increased steadily during the entire period of storage. The accumulation of Maillard products in seed axes was associated with the decline of seed vigour. These data suggest that, during seed ageing, sugar hydrolysis and lipid peroxidation are coupled with non-enzymatic protein modification through Amadori and Maillard reactions.     

Ethanol and acetaldehyde in imbibing soybean seeds in relation to deterioration

Deterioration as evidenced by decline in germination or seedling growth of soybean (cv. Essex) seeds during accelerated aging treatments at 41 C and 100% relative humidity is accompanied by increased levels of acetaldehyde and ethanol in imbibing embryonic axes and seeds. These increases become more pronounced with duration of the aging treatment. A similar inverse relationship between levels of acetaldehyde and ethanol and deterioration was observed when seeds were “naturally” aged for several years. During imbibition of low-vigor, accelerated-aged seeds at 25 C, acetaldehyde and ethanol increased from near trace amounts in dry tissues to maximum levels at 4 hours. Increases in acetaldehyde and ethanol during imbibition were less in high- than in low-vigor seeds. Increases were also less pronounced in low-vigor seeds when water uptake injury was avoided by osmotically decreasing water uptake rate with 30% polyethylene glycol. Embryonic axes from deteriorated seeds were characterized by low rates of O₂ uptake and high respiratory quotients relative to the unaged controls. Anaerobic conditions and respiratory inhibitors, such as sodium azide, increased acetaldehyde and ethanol in unaged seeds to levels similar to those in accelerated-aged seeds after 2 hours imbibition. It is suggested that, during aging, an imbalance between tricarboxylic and glycolytic activities, present during early imbibition to some degree even in vigorous unaged seeds, becomes more pronounced and leads to accumulation of ethanol and acetaldehyde.     

Quantitative Trait Locus Mapping of Seed Vigor in Soybean under −20 °C Storage and Accelerated Aging Conditions via RAD Sequencing

Due to its fast deterioration, soybean (Glycine max L.) has an inherently poor seed vigor. Vigor loss occurring during storage is one of the main obstacles to soybean production in the tropics. To analyze the genetic background of seed vigor, soybean seeds of a recombinant inbred line (RIL) population derived from the cross between Zhonghuang24 (ZH24, low vigor cultivar) and Huaxia3hao (HX3, vigorous cultivar) were utilized to identify the quantitative trait loci (QTLs) underlying the seed vigor under −20 °C conservation and accelerated aging conditions. According to the linkage analysis, multiple seed vigor-related QTLs were identified under both −20 °C and accelerated aging storage. Two major QTLs and eight QTL hotspots localized on chromosomes 3, 6, 9, 11, 15, 16, 17, and 19 were detected that were associated with seed vigor across two storage conditions. The indicators of seed vigor did not correlate well between the two aging treatments, and no common QTLs were detected in RIL populations stored in two conditions. These results indicated that deterioration under accelerated aging conditions was not reflective of natural aging at −20 °C. Additionally, we suggest 15 promising candidate genes that could possibly determine the seed vigor in soybeans, which would help explore the mechanisms responsible for maintaining high seed vigor.     

Prevention of imbibitional injury in low vigor soybean embryonic axes by osmotic control of water uptake

Deterioration of soybean [ Glycine max (L.) Merr. cv. Essex] seeds during accelerated aging at 41°C and 100% relative humidity predisposes the embryonic axis to injury during the initial period of imbibition. This injury was prevented or greatly reduced in severity when excised axes were imbibed on blotters containing 30% polyethylene glycol which slowed the rate of water uptake and when axes were pre-equilibrated to a high moisture level. Rates of water uptake by "high"(no treatment) and "low vigor"(accelerated aged) excised axes were identical. However, high vigor axes tolerated rapid water uptake during early imbition, whereas low vigor axes did not. Leakage of electrolytes during early imbibition was nearly six times greater in low than in high vigor axes. Polyethylene glycol significantly reduced the leakage of electrolytes from both low and high vigor axes. The data are in agreement with the hypothesis that seed deterioration in soybeans involves membrane changes which may predispose embryonic tissues to injury during imbibition. Reduction of the rate of water uptake during the initial period of imbibition would allow extra time for membrane repair or rearrangement, thus permitting the tissues to develop in a more orderly manner. The data indicate that deterioration in soybean seeds involves, at least in part, a decrease in ability of seed axes to tolerate rapid water uptake at the start of imbibition and that this weakness may be compensated by osmotic control of water uptake.     

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.     

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.     

Control of galactosyl-sugar metabolism in relation to rate of germination

The storage sugars stachyose and raffinose (galactosyl derivatives of sucrose) are metabolized early during germination of soybean [ Glycine max (L.) Merr.] seeds. The activities of four enzymes involved in the catabolism of these sugars were monitored in soybean cotyledons and embryonic axes during a 7-day germination period. An increase in enzyme activities correlated with a decline in galactosyl sugars. In embryonic axes, uridine diphosphate glucose (UDPglc)-hexose-l-P uridyltransferase (EC 2.7.7.12), an enzyme characteristic of the Leloir pathway, predominated over galactose-1-phosphate uridyltransferase (EC 2.7.7.10), an enzyme characteristic of the pyrophosphorylase pathway; whereas in cotyledons, the situation was reversed. There were differences between two cultivars. Ransom and Amsoy, in the levels of UDPglc-4-epimerase (EC 5.1.3.2); but not in glucose-1-phosphate uridyltransferase (EC 2 7.7.9). An accelerated aging treatment had a significant effect on the development of embryonic axes, as measured by dry weight. In vitro aging of seeds reduced the rate of growth and resulted in higher levels of galactose-containing sugars and significantly lower levels of UDPglc-hexose-l-P uridyltransferase. Thus, reduced development may be related to inability to mobilize or utilize stored carbon reserves. However, it has not been proved that the reduced enzyme activity is responsible for the effects of accelerated aging on growth and sugar metabolism.     

Production and characterization of antibodies to advanced glycation products on proteins

Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.     

Tocopherol and Organic Free Radical Levels in Soybean Seeds during Natural and Accelerated Aging

Soybean seeds which had aged in long-term storage (“natural aging”) or by exposure to high temperature and humidity (“accelerated aging”) were analyzed for their tocopherol and organic free radical contents. Tocopherol levels remained unchanged during both types of aging. Three principal tocopherol homologues (α, γ, δ) were present in fairly constant proportions throughout. Organic free radical levels were also remarkably stable, presumably due to the relatively immobile environment of the dry seed. These results, taken in conjunction with previous data on the stability of unsaturated fatty acids in soybean seeds, indicate that it is improbable that lipid peroxidation need play a significant role during natural or accelerated aging in this species.     

Maturation proteins and sugars in desiccation tolerance of developing soybean seeds

The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 μg/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 μg/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance.     

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.     

Lipid changes during natural aging of soybean seeds

When soybean seeds [ Glycine max (L.) Merr. cv. Wayne] were stored at approximately 4°C and low humidity for 44 months ("natural aging") there was a marked decline in vigor and viability which was associated with a decrease in the proportion of the polyunsaturated fatty acids. Other lipid parameters showed little change. Seeds subjected to high humidity at 40°C for several days ("accelerated aging") experience a comparable decline in vigor and viability, but without the change in fatty acid polyunsaturation. These results indicate that accelerated aging may cause loss of vigor in a manner quite different from natural aging. The accelerated aging treatment is therefore of limited usefulness in investigations of the mechanism of natural seed aging.     

Influence of accelerated and natural aging on free radical levels in soybean seeds

Embryonic axes and cotyledons of three soybean ( Glycine max L. cv. Gudzon) seed lots designated as "normal", "naturally aged" and "acceleratedly aged" were analyzed for their organic free radical contents. No signals of free radicals were found in ESR spectra from cotyledonary material of the three samples investigated. High levels of organic free radicals were observed in the embryonic axes. There were significant differences in the free radical contents of the embryonic axes: the relative values of free radicals were 100, 190 and 170% for samples aged normally, naturally and in an accelerated manner, respectively. These results point to the physiological role of the embryonic axes during natural or accelerated aging of seeds, notwithstanding that the axis is a small part as compared to the cotyledons. It is suggested that lipid peroxidation in embryonic axes may play an important role in the seed deterioration during aging.     

Dormancy as exaptation to protect mimetic seeds against deterioration before dispersal

Background and Aims

Mimetic seeds simulate the appearance of fleshy fruits and arilled seeds without producing nutritive tissues as a reward for seed dispersers. In this strategy of seed dispersal, seeds may remain attached to the mother plant for long periods after maturity, increasing their availability to naïve seed dispersers. The hypothesis that seed coat impermeability in many tropical Fabaceae with mimetic seeds serves as an exaptation to protect the seeds from deterioration and rotting while awaiting dispersal was investigated.

Methods

Seed coat impermeability was evaluated in five mimetic-seeded species of tropical Fabaceae in south-eastern Brazil (Abarema langsdorffii, Abrus precatorius, Adenanthera pavonina, Erythrina velutina and Ormosia arborea) and in Erythrina speciosa, a ‘basal’ species in its genus, which has monochromatic brown seeds and no mimetic displays. Seed hardness was evaluated as a defence against accelerated ageing (humid chamber at 41 °C for 144 h). Seed development and physiological potential of O. arborea was evaluated and the effect of holding mature seeds in pods on the mother plant in the field for a period of 1 year under humid tropical conditions was compared with seeds stored under controlled conditions (15 °C and 40 % relative air humidity).

Key Results

All five mimetic-seeded species, and E. speciosa, showed strong coat impermeability, which protected the seeds against deterioration in accelerated ageing. Most O. arborea seeds only became dormant 2 months after pod dehiscence. Germination of seeds after 1 year on the plant in a humid tropical climate was 56 %, compared with 80 % for seeds stored in controlled conditions (15 °C, 45 % relative humidity). Seedling shoot length after 1 year did not differ between seed sources.

Conclusions

Dormancy acts in mimetic-seeded species as an exaptation to reduce seed deterioration, allowing an increase in their effective dispersal period and mitigating the losses incurred by low removal rates by naïve avian frugivores.     

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.     

Lipid peroxidation associated with accelerated aging of soybean axes

Soybean seeds age rapidly during storage at high temperature and high relative humidity. The axes of such aged seeds contain high levels of malondialdehyde, a product of the peroxidation of unsaturated fatty acids. The levels of linoleic (18:2) and linolenic (18:3) acids in a polar lipid (phospholipid) fraction decrease during aging and more dramatically during postaging deterioration. None of these changes occurred in seeds that have been stored at high temperature but low relative humidity. No superoxide dismutase activity was detected in any nonimbibed seed. In viable seeds, activity was detectable 1.5 hours after the onset of imbibition, but none was found in aged seeds up to 5 hours. It is suggested that aging leads to peroxidative changes to lipids and that these could contribute to loss of viability.     

Scavenging of Active Oxygen Species by Glycated Proteins

Scavenging of active oxygen species by glycated proteins was investigated. Glycated proteins were prepared from bovine serum albumin (BSA), insulin, and lysozyme incubated with glucose. Glycated BSA at concentration of 0.5% scavenged 34% of hydroxyl radicals by ESR experiments using DMPO as a spin-trapping reagent. The ability to scavenge hydroxyl radicals by glycated BSA was higher than that by BSA. Hydrogen peroxides also were largely scavenged with an increase in the concentration of glycated proteins. However, the ability to scavenge superoxides by glycated BSA was lower than that by BSA because glycated proteins produced superoxides. Experiments using model compounds such as Amadori compound and caproyl pyrraline suggested that the scavenging ability of glycated proteins against hydroxyl radicals depends on Maillard reaction products in the advanced stage, while the ability against hydrogen peroxides is dependent upon Maillard reaction products in the early stage and brown pigments.     

Absence of Lipid Oxidation during Accelerated Aging of Soybean Seeds

When seeds of soybean were subjected to accelerated aging, the amount of total lipid which was extracted from the whole seed increased with “age,” whereas the extractable phospholipid decreased slightly. This small decline primarily reflected changes in the amounts of phosphatidylcholine and phosphatidylethanolamine. The levels of unsaturated fatty acids in the whole seed and in the seed axis showed no decline during aging. Similarly, the fatty acids in a polar lipid extract from the whole seed showed little change in unsaturation. These results suggest that oxidation of seed lipids may be unrelated to the process of seed aging.     

Protein Changes during the Stratification of Malus domestica Borkh. Seed

Apple seeds (Malus domestica Borkh. cv Golden Delicious) were stratified at 5 and 15°C for various lengths, weighed, and soluble protein of axis and cotyledon tissue was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only seeds treated at 5°C germinated; seeds treated at 15°C did not germinate. Optimal germination required 63 days of stratification. Excised embryos required less stratification time for germination than intact seeds. When stratification was less than 35 days, the resulting seedlings from 5°C stratified embryos were dwarfed and epinastic. After 63 days of stratification, axes from 5 and 15°C treated intact seeds had increased in fresh weight by 72 and 28% (w/w), respectively. The dry weights of the axes did not change significantly and both fresh and dry weights of cotyledons remained unchanged during stratification. Total soluble protein in axes and cotyledons changed very little during stratification. However, axis polypeptide profiles changed. Most obvious was the occurrence of a new polypeptide and the increase of four other clearly identifiable polypeptides during 5°C treatment. The levels of the five most predominant axis proteins decreased at the same time. We observed no changes in the profiles of soluble cotyledon proteins. Control seeds kept at −10°C showed none of the reported changes.