A Race for Survival: Can Bromus tectorum Seeds Escape Pyrenophora semeniperda-caused Mortality by Germinating Quickly?

BACKGROUND AND AIMS: Pathogen-seed interactions may involve a race for seed resources, so that seeds that germinate more quickly, mobilizing reserves, will be more likely to escape seed death than slow-germinating seeds. This race-for-survival hypothesis was tested for the North American seed pathogen Pyrenophora semeniperda on seeds of the annual grass Bromus tectorum, an invasive plant in North America. In this species, the seed germination rate varies as a function of dormancy status; dormant seeds germinate slowly if at all, whereas non-dormant seeds germinate quickly. METHODS: Three experimental approaches were utilized: (a) artificial inoculations of mature seeds that varied in primary dormancy status and wounding treatment; (b) naturally inoculated undispersed seeds that varied in primary dormancy status; and (c) naturally inoculated seeds from the carry-over seed bank that varied in degree of secondary dormancy, habitat of origin and seed age. KEY RESULTS: In all three approaches, seeds that germinated slowly were usually killed by the pathogen, whereas seeds that germinated quickly frequently escaped. Pyrenophora semeniperda reduced B. tectorum seed banks. Populations in drier habitats sustained 50 times more seed mortality than a population in a mesic habitat. Older carry-over seeds experienced 30 % more mortality than younger seeds. CONCLUSIONS: Given the dramatic levels of seed death and the ability of this pathogen to reduce seed carry-over, it is intriguing to consider whether P. semeniperda could be used to control B. tectorum through direct reduction of its seed bank.     

Cytogenetic activity of the herbicide sodium trichloroacetate

It is established that sodium trichloracetate does not induce chromosomal aberrations in bone marrow cells of mice, does not modify mutagenic effect induced by rubomicin, but increases chromosome aberration frequency in the culture of human peripheral lymphocytes and in germs of Crepis tectorum and Allium cepa seeds.     

Artificial ageing of Vicia faba L. seeds caused by prolongation of G-1 phase

Broad bean (Vicia faba L. “Inovec”) seeds were artificially aged by means of storage at 30 %, resp. 25 % water content at 25 °C for 7-days to study the consequences on germination, root length and frequency of chromosomal aberrations. Under these conditions, significant changes in all parameters were observed. An increase of frequency of chromosomal aberrations in ana-telophase cells was confirmed by evaluation of c-metaphase cells. Synergic effect of artificial seed ageing was studied on different harvests of old seeds. Possible principles of this effect on cell level are discussed.     

皂荚天然群体间种实表型特性及种子萌发的差异分析

对位于湖北京山、湖南城步、贵州兴义、重庆秀山、四川成都、广西桂林、山东费县和甘肃天水的8个皂荚（Gleditsia sinensis Lam．）天然群体间果实和种子表型特性及种子萌发率的差异进行了研究,在此基础上,对种子特性与萌发率的相关性进行了分析。结果表明：不同皂荚群体间荚果的长度、宽度、厚度和质量以及种子长度、宽度、厚度和百粒质量均存在极显著差异（P〈O．01）;在低纬度、高降雨量和干扰程度相对大的区域产生的荚果及种子均较大。在不同温度（昼／夜温度35℃／20℃、30℃／15℃、25℃／10oC和15℃／5℃）条件下,8个皂荚群体间种子萌发率无显著差异,平均萌发率为7．70％;种皮经人为损伤后种子萌发率总体上有所提高且在不同温度条件下差异显著,其中在昼／夜温度15℃／5℃的条件下种子萌发率低于其他处理温度。在不同温度条件下,种子的长度、宽度、厚度和百粒质量与萌发率均呈正相关,但总体上相关性不显著。研究结果说明：不同皂荚群体的荚果和种子特性变异较大,且在降雨量高的地区其果实和种子较大;低温对皂荚种子的萌发有一定抑制作用;种皮损伤处理可解除皂荚种子的物理性休眠,但皂荚种子还可能存在生理休眠现象;一定程度的种子复合休眠可能是皂荚群体适应不同生境的重要生存策略之一。     

粗茎秦艽种子粗提物与破眠方法研究

为了探究影响粗茎秦艽（Gentiana crassicaulis Duthie ex Burk.）种子休眠的因素,破除休眠,寻找其种子快速萌发的方法,以干燥的粗茎秦艽种子为材料,测定种子吸水率及粗提物的活性,使用不同浓度的赤霉素（GA₃）、高锰酸钾（KMnO₄）、聚乙二醇（PEG6000）和过氧化氢（H₂O₂）溶液进行浸种处理,比较不同处理条件对粗茎秦艽种子萌发的影响。结果显示,粗茎秦艽种皮对种子吸水没有明显阻碍作用;不同浓度的种子粗提物对白菜、小麦的萌发和生长均表现出抑制作用;不同浓度的粗提物对粗茎秦艽种子自身的萌发也表现出一定的抑制作用,当粗提物浓度达到0.1 g/mL时,抑制作用最显著（P < 0.05）;高锰酸钾处理可提高粗茎秦艽种子的萌发率,浓度为1.5%时效果较显著（P < 0.05）,而过氧化氢处理对粗茎秦艽种子的萌发效果不如前者,此外,用500 mg/L的赤霉素浸种和300 mg/L的聚乙二醇预处理也可显著打破粗茎秦艽种子休眠（P < 0.01）。研究结果表明粗茎秦艽种子的内源抑制物是影响其休眠的因素之一;种皮的机械阻碍也在一定程度上影响了种子萌发;粗茎秦艽种子具有综合性休眠特性。高锰酸钾预处理、赤霉素浸种和聚乙二醇引发均可打破种子休眠、缩短种子出芽时间,提高种子的发芽能力。     

赤霉素与脱落酸对番茄种子萌发中细胞周期的调控

利用细胞流检仪检测番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．） ＧＡ－缺陷型、ＡＢＡ－缺陷型和相应的正常品种（野生型）成熟种子胚根尖细胞倍性水平时发现：ＧＡ－缺陷型和野生型种子绝大多数细胞ＤＮＡ 水平为２Ｃ,而ＡＢＡ－缺陷型种子则含有较多的４Ｃ细胞。在标准发芽条件下,ＡＢＡ－缺陷型和野生型种子浸种１ ｄ 后胚根尖细胞ＤＮＡ 开始复制,随后胚根突破种皮而发芽。然而ＧＡ－缺陷型种子除非加入外源ＧＡ,否则既不发生细胞ＤＮＡ 复制,也不发芽。这说明内源ＧＡ 是启动番茄种子胚根尖细胞ＤＮＡ 复制的关键因素,同时也说明番茄根尖细胞ＤＮＡ 复制是种子发芽的必要条件。实验证明：ＡＢＡ 不抑制细胞ＤＮＡ 合成,但阻止Ｇ２ 细胞进入到Ｍ 期。外源ＡＢＡ处理野生型种子与渗控处理结果相似,可以大幅度提高胚根尖４Ｃ／２Ｃ细胞的比例,但抑制种子的最终发芽     

入侵植物小花山桃草种群构件生物量结构及种子萌发特征

通过野外设置样方调查和室内萌发试验,研究小花山桃草种群各构件生物量的结构特征和它们之间的关系模型、繁殖分配以及种子萌发特点。结果表明:(1)小花山桃草根、茎、叶、花(果)序生物量与植株高度之间以及各构件生物量之间均呈正相关关系,可用幂函数模型或线性函数模型较好地表达;(2)各构件生物量在个体生物量中所占的比率表现为茎>花序>叶>根;(3)小花山桃草的繁殖投入和繁殖分配都随植株个体的增大而增加;(4)小花山桃草个体大小和繁殖投入之间为线性关系,而个体大小和繁殖分配之间为幂函数关系;(5)小花山桃草存在一个较小的繁殖阈值(0.6043g);(6)小花山桃草种子在有光照(12h)和黑暗条件下发芽率均可达到85%以上;未经贮藏的种子不萌发,低温沙藏(1～2℃)和室温干藏(14～32℃)一个半月种子萌发率分别可达92.5%和79%;低温沙藏时种子即可发芽,且发芽率可达61%。在研究地区,小花山桃草几乎整个生长季都可萌发,甚至初冬还有幼苗产生。小花山桃草构件生物量结构和繁殖分配特征、种子萌发特点等都有助于其入侵能力的提高,是其成功入侵我国的重要原因。     

beta]-Tubulin Accumulation and DNA Replication in Imbibing Tomato Seeds

The activation of the cell cycle in embryo root tips of imbibing tomato (Lycopersicon esculentum Mill. cv Lerica) seeds was studied by flow cytometric analyses of the nuclear DNA content and by immunodelection of [beta]-tubulin. With dry seeds, flow cytometric profiles indicated that the majority of the cells were arrested at the G1 phase of the cell cycle. In addition, [beta]-tubulin was not detectable on western blots. Upon imbibition of water, the number of cells in G2 started to increase after 24 h, and a 55-kD [beta]-tubulin signal was detected between 24 and 48 h. Two-dimensional immunoblots revealed at least three different [beta]-tubulin isotypes. Thus, [beta]-tubulin accumulation and DNA replication were induced during osmotic priming. These processes, as well as seed germination rate, were enhanced upon subsequent imbibition of water, compared with control seeds that imbibed but were not primed. By contrast, when aged seeds imbibed, DNA replication, [beta]-tubulin accumulation, and germination were delayed. In all cases studied, both DNA replication and [beta]-tubulin accumulation preceded visible germination. We suggest that activation of these cell-cycle-related processes is a prerequisite for tomato seed germination. Furthermore, [beta]-tubulin expression can be used as a parameter for following the initial processes that are activated during seed imbibition.     

Proteomic Profiling of the Aleurone Layer of Mature Arabidopsis thaliana Seed

The aleurone layer of mature Arabidopsis thaliana seed plays important roles in seed germination and dormancy. However, the proteomic profile of this cell layer is unknown partly because it is difficult to separate this thin cell layer from the mature seeds. In this study, we have used a simple technique to separate the aleurone layer along with the seed coat following germination of seeds and determined for the first time the putative protein composition of this cell layer. By subjecting the total proteins extracted from the seed coat to 2D gel electrophoresis followed by liquid chromatography/tandem mass spectrometry, we identified four AGI loci, AT4G28520, AT5G44120, AT1G03880, and AT1G03890; all of which belong to the seed storage family of proteins. Because in Arabidopsis the diploid aleurone cells of the seed coat perform protein storage functions similar to that of triploid endosperm of other plant species, it is assumed that the above AGI loci are associated with the aleurone layer of the seed coat.     

Dynamic distribution and the role of abscisic acid during seed development of a lady’s slipper orchid,Cypripedium formosanum

Background and Aims Although abscisic acid (ABA) is commonly recognized as a primary cause of seed dormancy, there is a lack of information on the role of ABA during orchid seed development. In order to address this issue, the localization and quantification of ABA were determined in developing seeds of Cypripedium formosanum.Methods The endogenous ABA profile of seeds was measured by enzyme-linked immunosorbent assay (ELISA). Temporal and spatial distributions of ABA in developing seeds were visualized by immunohistochemical staining with monoclonal ABA antibodies. Fluoridone was applied to test the causal relationship between ABA content and seed germinability.Key Results ABA content was low at the proembryo stage, then increased rapidly from 120 to 150 days after pollination (DAP), accompanied by a progressive decrease in water content and seed germination. Immunofluorescence signals indicated an increase in fluorescence over time from the proembryo stage to seed maturation. From immunogold labelling, gold particles could be seen within the cytoplasm of embryo-proper cells during the early stages of seed development. As seeds approached maturity, increased localization of gold particles was observed in the periplasmic space, the plasmalemma between embryo-proper cells, the surface wall of the embryo proper, and the inner walls of inner seed-coat cells. At maturity, gold particles were found mainly in the apoplast, such as the surface wall of the embryo proper, and the shrivelled inner and outer seed coats. Injection of fluoridone into capsules resulted in enhanced germination of mature seeds.Conclusions The results indicate that ABA is the key inhibitor of germination in C. formosanum. The distinct accumulation pattern of ABA suggests that it is synthesized in the cytosol of embryo cells during the early stages of seed development, and then exported to the apoplastic region of the cells for subsequent regulatory processes as seeds approach maturity.     

Discovering the type of seed dormancy and temperature requirements for seed germination of Gentiana lutea L. subsp. lutea (Gentianaceae)

Aims There are a number of mechanisms that regulate germination; among these, seed dormancy, one of the most important, is an adaptative mechanism in plants to promote survival by dispersing germination in space and time until environmental conditions are favourable for germination. The main goals of this study were to determine the temperature requirements for seed dormancy release and germination of Gentiana lutea subsp. lutea, to identify the class and level of seed dormancy and to suggest an optimal germination protocol.Methods Seeds belonging to two different localities were subjected to various pre-treatments, including cold stratification (0 and 5°C), warm stratification (25/10°C) and different combinations of these, and then incubated at a range of constant temperatures (5–25°C) and 25/10°C. Embryo growth during pre-treatments and incubation conditions were assessed at different times by measuring the embryo to seed length ratio (E:S ratio). The final germination percentage (FGP) and the germination rate (t 50) were calculated.Important findings Fleshy mature seeds of G. lutea subsp. lutea have linear underdeveloped embryos. Cold stratification at 0°C was effective in overcoming the physiological dormancy (PD) and promoted embryo growth and subsequent germination. After cold stratification at 0°C, both the root and the shoot emerged readily under a wide range of temperatures. G. lutea subsp. lutea seeds showed an intermediate complex morphophysiological dormancy (MPD). As regards the optimal germination protocol for this taxon, we suggest a period of cold stratification at ca. 0°C followed by seed incubation at 10–20°C. The optimal germination temperatures found for seeds of this taxon, as well as its pre-chilling requirement at 0°C, suggest that it is well adapted to a temperate climate; this behavior highlights an increasing threat from global warming for G. lutea, which could reduce the level of natural emergence in the field, prejudicing also the long-term persistence of the natural populations in Sardinia.     

Dormancy Breaking and Storage Behavior of Garcinia cowa Roxb. (Guttiferae) Seeds: Implications for Ecological Function and Germplasm Conservation

The dormancy breaking and storage behavior of Garcinia cowa Roxb. seeds were investigated.The seeds of G. cowa had 8-11 months dormancy in their natural habitat. Seeds were matured and dispersed at the end of the rainy season (mid-late August to late September) and were scatter-hoarded by rodents as food for winter after the seeds had fallen to the ground. Seedlings often emerged in the forest during the rainy season (May to August) the following year. Intact seeds of G. cowa failed to germinate after being sown at 30 ℃ for 120 d and the mean germination time (MGT) of seeds cultured in a shade (50% sunlight)nursery was 252 d. The most effective method of breaking dormancy was to remove the seed coat totally,which reduced the MGT to 13 d at 30 ℃. Germination was also promoted by partial removal of the seed coat (excising the hilum and exposing the radicle) and chemical scarification (immersion in 1% H2O2 for 1 d).Unscarified seeds take up water rapidly in the first 96 h, but water was absorbed by the outside seed coat,without penetrating through it. The moisture content (MC) of G. cowa seeds was high (50% in fresh weight)at shedding. The seeds could tolerate desiccation to some extent, until the MC reached approximately 40%;below that, the viability decreases rapidly and all seeds died at approximately 17% of MC. Seed viability decreased rapidly when seeds were chilled at 4 ℃; germination was 2% after storage for 1 week. Even stored at 10 ℃, seeds began to be damaged after 4 weeks. Seed storage for 1 yr revealed that in both dry (relative humidity (35 ± 5)%) and moist (wet sand) storage conditions, seed viability declined, but germination percentages for seeds stored under moist conditions are better than for seed stored under dry conditions.Because of their low tolerance to desiccation, marked chilling sensitivity and relatively short lifespan, G.cowa seeds should be classified into the tropical recalcitrant category. The ecological implications of dormant recalcitrant seeds and cues on storing recalcitrant seeds were discussed.     

Effect of stimulating maize germination on cell cycle proteins

The germination process can be accelerated if seeds are stimulated either by adding cytokinins or by osmopriming. Under these conditions, cells in maize ( Zea mays ) embryo axes shorten the time at which the first round of DNA replication and mitosis takes place, thus advancing the cell cycle. Using heterologous antibodies against different cell cycle proteins, we have followed the behaviour of several markers for G1 phase (cyclin D, E2F and p53) and a marker of G2 phase (cyclin B) under either control or "accelerated" germination conditions. The results showed two classes of behaviour: either there was no variation in the amount of the protein present under control or accelerated germination conditions, represented by cyclin Band E2F‐type proteins, or the amount of the proteins was drastically reduced, more rapidly under accelerated germination, as was the case for cyclin D‐ and p53‐type proteins. Although the cyclin D‐type protein was synthesized de novo during germination, the balance was towards degradation so that there was no cyclin D detected 15 h after germination in benzyladenine‐treated and osmoprimed seeds. A Cdk4‐type protein seemed to be present in cyclin D immunoprecipitates and its kinase activity paralleled the fluctuations of the cyclin amount during germination. These data are discussed in the context of early seed germination.     

A Role for the Surrounding Fruit Tissues in Preventing the Germination of Tomato (Lycopersicon esculentum) Seeds : A Consideration of the Osmotic Environment and Abscisic Acid

During tomato seed development the endogenous abscisic acid (ABA) concentration peaks at about 50 d after pollination (DAP) and then declines at later stages (60-70 DAP) of maturation. The ABA concentration in the sheath tissue immediately surrounding the seed increases with time of development, whereas that of the locule declines. The water contents of the seed and fruit tissues are similar during early development (20-30 DAP), but decline in the seed tissues between 30 and 40 DAP. The water potential and the osmotic potential of the embryo are lower than that of the locular tissue after 35 DAP also. Seeds removed from the fruit at 30, 35, and 60 DAP and placed ex situ on 35 and 60 DAP sheath and locular tissue are prevented from germinating. Development of 30 DAP seeds is maintained or promoted by the ex situ fruit tissue with which they are in contact. Their germination is inhibited until subsequent transfer to water, and germination is normal, i.e. by radicle protrusion, and viable seedlings are produced, compared with 30 DAP seeds transferred directly to water; more of these seeds germinate, but by hypocotyl extension, and seedling viability is very poor. Isolated seeds at 35 and 60 DAP re-placed in contact with fruit tissues only germinate when transferred to water after 7 d. At 30 DAP, isolated seeds are insensitive to ABA at physiological concentrations in that they germinate as if on water, albeit by hypocotyl extension. At higher concentrations germination occurs by radicle protrusion. Osmoticum prevents germination, but there is some recovery upon subsequent transfer to water. Seeds at 35 DAP are very sensitive to ABA and exhibit little or no germination, even upon transfer to water. The response of the isolated seeds to osmoticum more closely approximates that to incubation on the ex situ fruit tissues than does their response to ABA. This is also the case for isolated 60 DAP seeds, whose germination is not prevented by ABA, but only by the osmoticum; these seeds are inhibited when in contact with ex situ fruit tissues also. It is proposed that the osmotic environment within the tissues of the tomato fruit plays a greater role than endogenous ABA in preventing precocious germination of the developing seeds.     

Growth dynamics of Salmonella enterica strains on alfalfa sprouts and in waste seed irrigation water

Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis. The source of these epidemics appears to have been low-level contamination of seeds by Salmonella bacteria that developed into clinically significant populations during the seed germination process. To test the possibility that Salmonella enterica strains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds. An S. enterica serovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process. Germinating alfalfa seeds supported the multiplication of S. enterica cells prior to the emergence of the root radicle at 72 h. Thereafter, much lower rates of multiplication were apparent. The ability of S. enterica to grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain. Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools. Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds. The S. enterica strains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source. S. enterica strains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination. The ability to detect S. enterica in the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations.     

The effects of time of seed production on the germination response of Spergularia marina

Germination studies were carried out with seeds of Spergularia marina L. Griseb produced over an interval of six months (June-November). The response of the seeds to light and dark, various constant and alternating temperature regimes, and salinity were determined. In addition, the effects of soil moisture status at the time of seed production on the subsequent germination response of seeds were also determined. Light was an absolute requirement for germination. While a constant temperature regime did not generally favour germination of seed of any month, alternating temperature greatly enhanced germination with an optimum at 5/15°C in all seeds. When imbibed in solutions of different salinities, seeds collected in July and October behaved like true halophyte seeds whereas those collected in June. August, September and November behaved like glycophyte seeds.
High concentration of gibberellic acid (3 000 μ M ) stimulated dark germination in the June and November seed lots, but in light, low GA3 concentration (300 μ M ) stimulated germination most. The addition of kinetin (30 μ M ) plus gibberellic acid enhanced germination in the dark in contrast to GA3 alone; kinetin alone stimulated a very low percentage germination.
The moisture status of the soil at the time seeds were produced did not affect the germination response of an early seed crop (July) but affected that of the later seeds (August).
Judging from the different germination responses, it appears that the seeds belong to at least two physiological groups, one which appears to need either a dark-wet or cold-wet pretreatmem for high germination to occur; and the other group which does not need pretreatmem. The ecological significance of these varied responses is discussed in relation to the survival of the species in its habitat.     

GA and ABA Regulation on the Cell Cycle in Germination of Tomato Seeds

Flow cytometric determination of ploidy levels in embryos of GA-deficient, ABA-deficient mutant and isogenic wild type tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds revealed that, large amount of 2C DNA signals existed both in wild type and GA-deficient mutant seeds, showing that most cells had arrested in the cell cycle at presynthesis Gl, whereas a relative amount of 4C proportion which is a sign of seed germination was found in ABA-deficient mutant seeds, indicating that endogenous ABA play a role in regulating the switch from development to germination in seeds. DNA replication was stimulated 1 d after the seed was imbibed in water and a visible germination occurred subsequently either in wild type GA-deficient mutant seeds. But it was not the case for ABA-deficient mutant seeds unless an exogenous GA was supplemented. This demonstrated that DNA replication in embryo root tips cells was subjected to be a compulsory factor for seed germination, whereas endogenous GA triggered DNA synthesis. It was evident that exogenous ABA could inhibit seed germination not by suppressing DNA synthesis but by bloking the route leading to mitosis since a great amount of 4C proportion was found in the germinating wild type and GA-deficient mutant seeds in the ABA solution when visible ger mination did not occur. Finally a simple mode of hormonal regulation on cell cycle in high plants was hypothesized.     

Control of macaw palm seed germination by the gibberellin/abscisic acid balance

The hormonal mechanisms involved in palm seed germination are not fully understood. To better understand how germination is regulated in Arecaceae, we used macaw palm (Acrocomia aculeata (Jacq.) Lodd. Ex Mart.) seed as a model. Endogenous hormone concentrations, tocopherol and tocotrienol and lipid peroxidation during germination were studied separately in the embryo and endosperm. Evaluations were performed in dry (D), imbibed (I), germinated (G) and non‐germinated (NG) seeds treated (+GA₃) or not treated (control) with gibberellins (GA). With GA₃ treatment, seeds germinated faster and to a higher percentage than control seeds. The +GA₃ treatment increased total bioactive GA in the embryo during germination relative to the control. Abscisic acid (ABA) concentrations decreased gradually from D to G in both tissues. Embryos of G seeds had a lower ABA content than NG seeds in both treatments. The GA/ABA ratio in the embryo was significantly higher in G than NG seeds. The +GA₃ treatment did not significantly affect the GA/ABA ratio in either treatment. Cytokinin content increased from dry to germinated seeds. Jasmonic acid (JA) increased and 1‐aminocyclopropane‐1‐carboylic acid (ACC) decreased after imbibition. In addition, α‐tocopherol and α‐tocotrienol decreased, while lipid peroxidation increased in the embryo during germination. We conclude that germination in macaw palm seed involves reductions in ABA content and, consequently, increased GA/ABA in the embryo. Furthermore, the imbibition process generates oxidative stress (as observed by changes in vitamin E and MDA).