Aging of Soybean Seeds in Relation to Metabolism at Different Relative Humidities

The present study was carried out to clarify the relationshipsbetween respiration, moisture content and the aging of dry soybean(Glycine max) seeds under different storage conditions. Ethanolicfermentation increased with increasing relative humidity (RH)during storage. However, at 75% RH which corresponded to a watercontent of 15.2% in seeds, the production of ethanol ceasedafter one month of storage. The ethanol that had accumulatedwithin the container was re-absorbed by the seeds themselvesand disappeared entirely within 3 months, unless the seeds wereexposed to HCN gas. Similar changes were also found for methanol.The activity of mitochondria increased only in seeds storedat 75% RH, it reached a maximum after the third month of storageand then rapidly declined. The amounts of both CoA and acetylCoA in seeds that had been stored for 4 months were far lowerat 75% RH than at 53% RH, and the viability of soybean seedsstarted to decrease after the fourth month at 75% RH. Therefore,a major cause of the rapid deterioration of seeds at 75% RHat room temperature seems to be the rapid development of mitochondrialrespiration, which is accompanied by the consumption of CoAand its acetyl derivatives. By contrast, the slow deteriorationof seeds during long-term storage at 33% to 68% RH and/or atsubzero temperatures seems to be due to the toxic action ofacetaldehyde, an intermediate in ethanolic fermentation, whichcan slowly occur. (Received April 10, 1995; Accepted July 10, 1995)     

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.     

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.     

Changes in the Content of Phytochrome I and II Apoproteins in Embryonic Axes of Pea Seeds during Imbibition

The contents of phytochrome I and II in crude extracts fromembryonic axes of Pisum sativum cv. Alaska seeds were immunochemicallydetermined using purified pea phytochrome I and II as standards.We have produced and used three different types of mouse monoclonalanti-pea phytochrome antibodies (mAP) such as one reacting preferentiallywith phytochrome I, one with phytochrome II, and one with bothI and II. Phytochrome II was separated from I in the samplesusing immobilized column chromatography with mAPl. The amountsof two phytochrome species were quantitatively measured withwestern blotting and ELISA. Ca. 0.2 µg /axis of phytochromeI and ca. 0.05 µg /axis of phytochrome II were detectedby ELISA after imbibition for 12 h in the dark, though smallamounts of both were detected in dry axes. Ca. 0.05 µg/axis each of phytochrome I and II were detected by ELISA afterimbibition for 12 h in the light, and the results were confirmedby western blotting. This study showed that phytochrome II isnot green-tissue-specific, being also found in dark-imbibedembryonic axes, and that although light significantly lowersthe content of phytochrome I in the axis, it does not significantlyaffect that of phytochrome II. (Received June 10, 1987; Accepted August 27, 1987)     

大豆种子老化过程中生理特性变化的研究

以两个大豆种子为试材,采用58℃±1℃热水老化法,进行了大豆种子不同老化水平生活力及生理特性的变化研究。结果表明:大豆种子衰老过程中,种子浸出液电导率值、丙二醛(MDA)、可溶性糖含量逐渐上升,种子活力与其呈负相关关系,与过氧化物酶(POD)活性呈正相关关系;随着种子老化程度加重,POD同工酶酶带颜色变浅并发生丢失,酶促防御系统解体。说明大豆种质资源更新标准不宜过低,中期库大豆资源更新发芽率应确定为70%~74%之间,70%的发芽率应作为大豆种质资源更新的下限值。     

Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition

Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O₂ uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 × 10⁻¹⁴ counts per O₂ molecule) was used to estimate the O₂ uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O₂ uptake (1.2 microliters O₂ per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues.     

Changes in Individual Nucleotides of Developing Soybean Seeds

Perchloric acid extracts of soybean seeds were separated by column chromatography into 17 peaks. Thirteen nucleotides were identified in 11 of these peaks (NAD, CMP, CDP, CTP, AMP, ADP, ATP, GMP, GDP, GTP, UMP, UDP, and UTP). Two of the peaks were identified as ascorbic acid and dehydroascorhic acid. Four peaks remain unidentified although one of these appears to be protein. Monophosphates decreased and triphosphates increased as development proceeded. Diphosphates were initially low and showed no statistically significant difference with maturity. The accumulation of triphosphates in late stages of development is discussed in relation to synthetic activities of the developing seeds.     

玉米种子老化过程中EST同工酶变化与染色体畸变的研究

对玉米自交系Mo17种子分别进行58℃热水和45℃恒温老化处理并对种子老化过程中种子发芽率、EST同工酶酶谱变化与染色体畸变规律进行了研究。结果显示：Mo17玉米种子萌发一天的种子胚EST酶谱共表现为14条清晰条带,58℃热水处理30min后,有四条酶带(Rf值为0．44、0．47、0．77、0．80)消失,酶带号为1、2、6、7、9的五条酶带着色变浅。由一、二级酶带变为二、三级酶带。45℃恒温老化处理60d后,Mo17种子EST酶谱中编号为2和5的两条酶带(Rf值为0．33、0．39)消失。处理70d后,种子发芽率降至39％,醇带号为11、12、13、14的四条酶带消失。随着老化时间延长。老化程度不断加重,发芽率低的玉米种子其根尖染色体畸变率相对较高,单桥、双桥、断片、落后及其它染色体畸变类型均被观察到。58℃热水老化其发芽率降至5％时,染色体畸变率上升为12．22％。     

Soybean Aphid Infestation Induces Changes in Fatty Acid Metabolism in Soybean

The soybean aphid (Aphis glycines Matsumura) is one of the most important insect pests of soybeans in the North-central region of the US. It has been hypothesized that aphids avoid effective defenses by inhibition of jasmonate-regulated plant responses. Given the role fatty acids play in jasmonate-induced plant defenses, we analyzed the fatty acid profile of soybean leaves and seeds from aphid-infested plants. Aphid infestation reduced levels of polyunsaturated fatty acids in leaves with a concomitant increase in palmitic acid. In seeds, a reduction in polyunsaturated fatty acids was associated with an increase in stearic acid and oleic acid. Soybean plants challenged with the brown stem rot fungus or with soybean cyst nematodes did not present changes in fatty acid levels in leaves or seeds, indicating that the changes induced by aphids are not a general response to pests. One of the polyunsaturated fatty acids, linolenic acid, is the precursor of jasmonate; thus, these changes in fatty acid metabolism may be examples of “metabolic hijacking” by the aphid to avoid the induction of effective defenses. Based on the changes in fatty acid levels observed in seeds and leaves, we hypothesize that aphids potentially induce interference in the fatty acid desaturation pathway, likely reducing FAD2 and FAD6 activity that leads to a reduction in polyunsaturated fatty acids. Our data support the idea that aphids block jasmonate-dependent defenses by reduction of the hormone precursor.     

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.     

甜高梁种子人工老化过程中活性氧清除酶活性的变化

研究甜高粱品种'意达利'种子人工老化过程中种子活力与活性氧清除酶活性之间关系的结果表明:随着老化处理(100%RH,43℃)的时间进程,种子的萌发率、活力指数以及由存活种子形成的幼苗鲜重下降,相对渗漏率增加,丙二醛含量下降.超氧物歧化酶、抗坏血酸过氧化物酶、过氧化氢酶、谷胱甘肽还原酶和脱氢抗坏血酸还原酶的活性在人工老化初期增加,而后下降.据此认为活性氧清除酶活性下降可能是甜高粱种子活力丧失的原因之一.     

Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds

In the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: α-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.     

Changes in Respiratory Metabolism during Callus Growth and Adventitious Root Formation in Jerusalem Artichoke Tuber Tissues

The CN-resistant alternative oxidase pathway was examined inrelation to the formation of callus and adventitious roots ofJerusalem artichoke tuber tissues. During the early stage ofincubation at 28?C, respiration was significantly activatedand the O₂-uptake rate via the Cyt pathway increased 2.5–2.6times by the second day of culture regardless of the presenceof 2,4-D. However, further increases and preservation of highlevels of Cyt pathway activities were observed only in the callusformingtissues. The capacity of the alternative pathway also increased,but the actually operating fraction was very small (0–4%of total respiration). On the other hand, during the later stagesof culture in which adventitious roots emerged from the callus,activation of the alternative pathway was observed. These resultssuggested that the alternative pathway was not involved in callusgrowth but was related to root formation in callus tissues ofJerusalem artichoke tubers. (Received October 29, 1986; Accepted April 20, 1987)     

Changes in Neutral Sugar Compositions of Cell Wall Polysaccharides during Redifferentiation of Cultured Carrot Cells and during Maturation of Soybean Seeds

Changes in the neutral sugar compositions of cell walls werestudied during regeneration of shoots and roots from culturedcarrot cells and during maturation of soybean seeds. There weremore arabinan and arabinose-rich acidic polysaccharides thangalactose-rich polysaccharides in the pectic fractions of thecell walls from cultured carrot cells and more galactan, arabinogalactanor both than the arabinose-rich polysaccharides in the samefractions from their mother tissue, i.e. root phloem tissue. The arabinose content of the cell walls decreased and the galactosecontent increased during root and shoot formation until galactoseexceeded arabinose in the cell walls of fully developed shootsand roots from cultured cells. The cell wall arabinose contentalso was higher than that of galactose in cotyledons and embryonicaxes of immature soybean seeds, and change in the neutral sugarcomposition of the cell wall during seed maturation was similarto that during the redifTerentiation of cultured carrot cells.During the very late stage of maturation, galactose in the cellwalls exceeded the content of arabinose. Results suggest that the redifferentiation of roots and shootsfrom cultured cells goes through a process of cell wall formationsimilar to that of embryogenesis or seed development in themother plants. Results also indicate that the predominant arabinanand arabinose-rich acidic polysaccharides have important functionsin cell walls during embryogenesis and in the eraly stages ofseed maturation and that galactan, arabinogalactan, or bothreplace these arabinose-rich polysaccharides after seed maturation. ²Present address: Department of Botany, the University of BritishColumbia, # 3529-6270 University Blvd.,Vancouver, B.C. V6T 2B1Canada (Received October 28, 1982; Accepted April 8, 1983)     

Oxylipin Metabolism in Soybean Seeds Containing Different Sets of Lipoxygenase Isozymes after Homogenization

The oxylipin metabolism was analyzed in soybean homogenates containing different sets of lipoxygenase isozymes (L-1, -2, and -3); namely, Suzuyutaka (containing L-1, -2, and -3), Yumeyutaka (containing only L-1), Kanto102 (containing L-2), Kyushu119 (containing L-3), and Ichihime (lacking all three isozymes). The amount of oxidized fatty acids in the esterified form was higher than that in the free form with every cultivar. Kanto102 formed the highest amount of oxidized lipids, and Yumeyutaka and Ichihime formed the lowest. With Kanto102 and Kyushu119, high amounts of keto fatty acids were formed, while they were undetectable with Yumeyutaka and Ichihime. Due to the lack of lipoxygenases in Ichihime, an accumulation of free fatty acids was expected; however, their amount in Yumeyutaka was significantly lower than was expected. It is suggested that a pathway existed to form C6-volatiles through hydroperoxides in the esterified form.     

Biogenesis of Protein Bodies in Embryonic Axes of Soybean Seeds (Glycine max. cv. Enrei)

Protein bodies in embryonic axes of soybean seeds have inclusion structures containing phytin globoids. Biogenesis of the protein bodies during seed development was examined by transmission electron microscopy. Protein bodies in embryonic axes originated from central vacuoles. The central vacuole in embryonic axes subdivided into smaller vacuoles with internal membranous structure. Then the subdivided vacuoles were directly associated with rough endoplasmic reticulum (rER), and were filled with proteinaceous matrix from the peripheral region. The increase of matrix was simultaneous with accumulation of β-conglycinin estimated by SDS-polyacrylamide gel electrophoresis. Glycinin-rich granules that had been found in developing cotyledons were not observed in embryonic axes. After proteinaceous matrix filled the protein bodies, electron-transparent regions presumably surrounded by a single membrane appeared in the matrix. Phytin globoids were constructed in this internal structures of protein bodies as the final step of protein body formation.     

Developmental Regulation of beta-Conglycinin in Soybean Axes and Cotyledons

Analysis of the expression of genes encoding the β-conglycinin seed storage proteins in soybean has been used to extend our understanding of developmental gene expression in plants. The α, α′, and β subunits of β-conglycinin are encoded by a multigene family which is organ-specific in its expression. In this study we report the differentially programmed accumulation of the α, α′, and β subunits of β-conglycinin. Multiple isomeric forms of each subunit are present in the dry seed, but the timing of their accumulation is unique for each subunit. The previously reported variation in amount of α′ and α subunits in axis and cotyledons is also reflected in the amount of subunit specific mRNA which is present in each tissue. The β subunit, previously undetected in soybean axes, is found to be synthesized but rapidly degraded. These differences in β-conglycinin protein accumulation may be reflected by the morphological differences observed in protein bodies between these two tissues.     

Alkaloid Changes in Tobacco Seeds during Germination

Nicotine, nornicotine, anabasine, and anatabine, normally found in growing and mature tobacco (Nicotiana tabacum L.) plants, were extracted and quantified from mature tobacco seeds and young tobacco seedlings. The rate of net alkaloid disappearance and accumulation in tobacco seedlings was related to phases of germination.