Species and Organ Diversity in the Effects of Hydrogen Peroxide on Superoxide Dismutase Activity In Vitro

Superoxlde dlsmutase （SOD） is ubiquitous in aerobic organisms and constitutes the first link In the enzyme scavenging system of reactive oxygen species. In the present study, species and organ diversity of SOD activity In a solution and In an in-gel assay system, as well as the effects of hydrogen peroxide （H202） on SOD activity, were Investigated. In a solution assay system, SOD activity of jackfruIt root, shoot, leaves, axes, and cotyledons, of maize embryos and endosperms, of mung bean leaves and seeds, of sacred lotus axes and cotyledons, and of rice and wheat leaves was Increased by 1-15 mmol/L H2O2. However, SOD activity In rice root and seeds, maize roots and leaves, mung bean roots and shoots, and wheat seeds was decreased by 1-15 mmol/L H2O2. The SOD activity of wheat root and soybean roots, leaves, axes, and cotyledons was Increased by 1-4 mmol/L H2O2, but was decreased by concentrations of H2O2 〉4 mmol/L. The SOD activity of soybean shoots was not affected by 1-15 mmol/L H2O2. The SOD activity In crude mltochondrla of jackfruIt, maize, and upas seeds, as well as In purified mitochondria of jackfruIt, was also Increased by 1-15 mmol/L H2O2. In the In-gel assay system, the SOD In jackfruIt cotyledons was comprised of Mn-SOD, Cu/Zn-SOD, and Fe-SOD, the crude mltochondria of jackfruit seeds and maizes embryo was comprised of Mn-SOD and Cu/ Zn-SOD, and the crude mltochondria of maize seeds was comprised of Mn-SOD only. In the present study, H2O2 markedly Inhibited Cu/Zn-SOD and Fe-SOD activity.     

Cytoplasmic HSP70 homologues of pea: differential expression in vegetative and embryonic organs

Eukaryotes express several cytoplasmic HSP70 genes, and their encoded proteins participate in diverse cellular processes. Three cDNAs encoding highly expressed cytoplasmic HSP70 homologues from Pisum sativum were cloned and characterized. They were designated PsHSP71.2, PsHSC71.0, and PsHSP70b. These HSP70 genes have different expression profiles in leaves: PsHSP71.2 is observed only in response to heat stress, PsHSC71.0 is present constitutively, and PsHSP70b is weakly constitutively expressed, but induced strongly in response to heat stress. In addition to being heat induced, the PsHSP71.2 mRNA is also expressed in zygotic, but not maternal organs of developing pea seeds, while PsHSC71.0 and PsHSP70b mRNAs are present in maternal and zygotic organs throughout seed development. Immunoblot analysis of parallel protein samples detects a 70 kDa polypeptide in all samples, and a 72 kDa polypeptide that corresponds to the PsHSP71.2 gene product is observed in cotyledons beginning at mid-maturation and in axes beginning between late maturation and desiccation. This polypeptide is not detected in the seed coat. The 72 kDa polypeptide remains abundant in both cotyledons and axes through germination, but declines substantially between 48 and 72 h after the onset of imbibition. Differential control of HSP70 expression during heat stress, seed maturation, and germination is consistent with the hypothesis that there are functional distinctions between cytoplasmic HSP70s.     

Synthesis and degradation of the major allergens in developing and germinating soybean seed

Gly m Bd 28K,Gly m Bd 30K and Gly m Bd 60K are the major soybean(Glycine max(L.)Merr.)allergens limiting the consumption of a good protein source for sensitive individuals.However,little is known about their temporal-spatial expression during seed development and upon germination.The present data shows that soy allergens accumulated in both the embryonic axes and cotyledon,but expression patterns differed depending on the specific allergen.Allergens accumulated sooner and to a greater level in cotyledons than in embryonic axes.Gly m Bd 28 began at 14 d after flowering,7 to 14 d earlier than Gly m Bd 30K and Gly m Bd 60K.Comparatively,their degradation was faster and more profound in embryonic axes than in cotyledons.Gly m Bd 60K began to decline at 36 h after imbibition and remained detectable up to 108 h in cotyledons.In contrast,the Glym Bd 60K protein was reduced at 24 h,and eventually disappeared at 96 h.In cotyledons Gly m Bd 28K first declined at 24 h,then increased from 36 h to 48 h,followed by its large reduction at 72 h after seed germination.These findings provide useful information on soy allergen biosynthesis and will help move forward towards developing a hypoallergenic soybean for safer food.     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

Evidence for accumulation of the β-subunit of β-conglycinin in soybean [Glycine max (L) Merr.] embryonic axes

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins from soybean cotyledons and embryonic axes revealed the presence of similar storage proteins in both organs. Results from Western blot analysis conducted with antibodies raised against the purified #-subunit of #-conglycinin demonstrated accumulation of the #-subunit in embryonic axes. This accumulation followed a temporal pattern similar to that shown by the cotyledons. Axis #-conglycinin was broken down during the initial stage(s) of seed germination and was completely mobilized within 3 days after imbibition. Subcellular fractions were isolated from developing embryonic axes using metrizamide density gradients and analyzed by Western blots. Storage proteins were enriched in the lighter fractions of the gradient as well as with immunoglobulin heavy-chain binding protein. Electron microscopy of the storage-protein-enriched gradient fraction revealed small vesicles and protein aggregates. Protein A-gold immunocytochemistry was used to localize the occurrence of the #-subunit of #-conglycinin within the protein aggregates present in the metrizamide gradient and in the protein bodies present in mature embryonic axes.     

Changes in soluble carbohydrates and related enzymes induced by low temperature during early developmental stages of quinoa (Chenopodium quinoa) seedlings

Low temperature represents one of the principal limitations in species distribution and crop productivity. Responses to chilling include the accumulation of simple carbohydrates and changes in enzymes involved in their metabolism. Soluble carbohydrate levels and invertase, sucrose synthase (SS), sucrose-6-phosphate synthase (SPS) and alpha-amylase activities were analysed in cotyledons and embryonic axes of quinoa seedlings grown at 5 degrees C and 25 degrees C in the dark. Significant differences in enzyme activities and carbohydrate levels were observed. Sucrose content in cotyledons was found to be similar in both treatments, while in embryonic axes there were differences. Invertase activity was the most sensitive to temperature in both organs; however, SS and SPS activities appear to be less stress-sensitive. Results suggest that 1) metabolism in germinating perispermic seeds would be different from endospermic seeds, 2) sucrose futile cycles would be operating in cotyledons, but not in embryonic axes of quinoa seedlings under our experimental conditions, 3) low temperature might induce different regulatory mechanisms on invertase, SS and SPS enzymes in both cotyledons and embryonic axes of quinoa seedlings, and 4) low temperature rather than water uptake would be mainly responsible for the changes observed in carbohydrate and related enzyme activities.     

Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds

Sacred lotus (Nelumbo nucifera Gaertn. ‘Tielian’) seed is long-lived and extremely tolerant of high temperature. Water content of lotus and maize seeds was 0.103 and 0.129 g H2O [g DW] ?1, respectively. Water content, germination percentage and fresh weight of seedlings produced by surviving seeds gradually decreased with increasing treatment time at 100℃. Germination percentage of maize (Zea mays L. ‘Huangbaogu’) seeds was zero after they were treated at 100℃ for 15 min and that of lotus seeds was 13.5% following the treatment at 100℃ for 24 h. The time in which 50% of lotus and maize seeds were killed by 100℃ was about 14.5 h and 6 min, respectively. With increasing treatment time at 100℃, relative electrolyte leakage of lotus axes increased significantly, and total chlorophyll content of lotus axes markedly decreased. When treatment time at 100℃ was less than 12 h, subcellular structure of lotus hypocotyls remained fully intact. When treatment time at 100℃ was more than 12 h, plasmoly-sis gradually occurred, endoplasmic reticulum became unclear, nuclei and nucleoli broke down, most of mitochondria swelled, lipid granules accumulated at the cell periphery, and organelles and plas-molemma collapsed. Malondialdehyde (MDA) content of lotus axes and cotyledons decreased during 0-12 h of the treatment at 100℃ and then increased. By contrast, the MDA content of maize embryos and endosperms increased during 5-10 min of the treatment at 100℃ and then decreased slightly. For lotus seeds: (1) activities of superoxide dismutase (SOD) and glutathione reductase (GR) of axes and cotyledons and of catalase (CAT) of axes increased during the early phase of treatment at 100℃ and then decreased; and (2) activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) of axes and cotyledons and of CAT of cotyledons gradually decreased with increasing treat-ment time at 100℃. For maize seeds: (1) activities of SOD and DHAR of embryos and endosperms and of GR of embryos increased during the early phase of the treatment at 100℃ and then decreased; and (2) activities of APX and CAT of embryos and endosperms and of GR of endosperms rapidly decreased with increasing treatment time at 100℃. With decrease in seed germination, activities of SOD, APX, CAT, GR and DHAR of axes and cotyledons of lotus seeds decreased slowly, and those of embryos and endosperms of maize seeds decreased rapidly.     

Respiratory changes with chilling injury of soybeans

The leakage of solutes from cotyledons of soybeans (cv. Chippewa 64) was markedly stimulated by a chilling treatment (1 to 4 C) during the 1st minute of imbibition, but chilling after even 1 minute of water uptake resulted in little or no leakage increase. The respiratory rate of soybean particles was reduced more than 60% if a chilling treatment (15 minutes at 1 to 4 C) was given during the first minutes of imbibition, and little or no reduction was obtained if the chilling treatment was begun at 5 to 15 minutes after the start of imbibition. Using KCN as an inhibitor of cytochrome oxidase pathway of respiration and salicylhydroxamic acid as an inhibitor of the alternative pathway, it was found that the chilling injury involved a major reduction in the cytochrome pathway in whole axes and cotyledons and an engagement of the alternative pathway of respiration in cotyledon tissue. The suggestion is made that the chilling injury involves lesions resulting from temperature stress during the reorganization of membranes with water entry, and that both the leakage and the respiratory effects are consequences of these membrane lesions.     

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.     

Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds

Sacred lotus (Nelumbo nucifera Gaertn. ‘Tielian’) seed is long-lived and extremely tolerant of high temperature. Water content of lotus and maize seeds was 0.103 and 0.129 g H₂O [g DW] ⁻¹, respectively. Water content, germination percentage and fresh weight of seedlings produced by surviving seeds gradually decreased with increasing treatment time at 100°C. Germination percentage of maize (Zea mays L. ‘Huangbaogu’) seeds was zero after they were treated at 100°Cfor 15 min and that of lotus seeds was 13.5% following the treatment at 100°C for 24 h. The time in which 50% of lotus and maize seeds were killed by 100°C was about 14.5 h and 6 min, respectively. With increasing treatment time at 100°C, relative electrolyte leakage of lotus axes increased significantly, and total chlorophyll content of lotus axes markedly decreased. When treatment time at 100°C was less than 12 h, subcellular structure of lotus hypocotyls remained fully intact. When treatment time at 100°C was more than 12 h, plasmolysis gradually occurred, endoplasmic reticulum became unclear, nuclei and nucleoli broke down, most of mitochondria swelled, lipid granules accumulated at the cell periphery, and organelles and plasmolemma collapsed. Malondialdehyde (MDA) content of lotus axes and cotyledons decreased during 0 −12 h of the treatment at 100°C and then increased. By contrast, the MDA content of maize embryos and endosperms increased during 5–10 min of the treatment at 100°C and then decreased slightly. For lotus seeds: (1) activities of superoxide dismutase (SOD) and glutathione reductase (GR) of axes and cotyledons and of catalase (CAT) of axes increased during the early phase of treatment at 100°C and then decreased; and (2) activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) of axes and cotyledons and of CAT of cotyledons gradually decreased with increasing treatment time at 100°C. For maize seeds: (1) activities of SOD and DHAR of embryos and endosperms and of GR of embryos increased during the early phase of the treatment at 100°C and then decreased; and (2) activities of APX and CAT of embryos and endosperms and of GR of endosperms rapidly decreased with increasing treatment time at 100°C. With decrease in seed germination, activities of SOD, APX, CAT, GR and DHAR of axes and cotyledons of lotus seeds decreased slowly, and those of embryos and endosperms of maize seeds decreased rapidly.     

板栗种子发育期间ABA等生理指标与脱水耐性的相关性研究

对板栗种子发育期间胚轴和子叶中部分生理指标的变化以及它们与板栗种子脱水耐性的相关性进行研究.结果表明:随着板栗种子不断发育,在花后80 d胚轴和子叶中ABA、淀粉、可溶性蛋白质量分数同步达到最大值,可溶性糖质量分数达到最低,此时板栗种子的脱水耐性最强,可确定为板栗的最佳采收期.另外,通过相关分析可知,板栗种子在发育期间...     

Ureide metabolism during seedling development in French bean (Phaseolus vulgaris)

French bean ( Phaseolus vulgaris ) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.     

A study of water relations in neem (Azadirachta indica) seed that is characterized by complex storage behaviour

Neem (Azadirachta indica) seed is reputed to have limited tolerance to desiccation, to be sensitive to chilling and imbibitional stress, and to display intermediate storage behaviour. To understand this behaviour the properties of water in seed tissues were studied. Water sorption isotherms showed that at similar relative humidity (RH), the water content was consistently higher in axes than in cotyledons, mainly due to the elevated lipid content (51%) in the cotyledons. Using differential scanning calorimetry, melting transitions of water were observed at water contents higher than 0.14 g H2O g-1 DW in the cotyledons and 0.23 g H2O g-1 DW in the axes. Beside melting transitions of lipid, as verified by infrared spectroscopy, changes in heat capacity were observed which shifted with water content, indicative of glass-to-liquid transitions. State diagrams are given on the basis of the water content of seed tissues, and also on the basis of the RH at 20 degrees C. Longevity was considerably improved, and the sensitivity to chilling/subzero temperatures was reduced when axis and cotyledons were dehydrated to moisture contents < or = of approximately 0.05 g H2O g-1 DW. However, longevity during storage at very low water contents was limited. A possible mechanism for the loss of sensitivity to chilling/subzero temperatures at low water contents is discussed. The results suggest that dry neem seeds in the glassy state have great potential for extended storability, also at subzero temperatures.