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.     

Possible Involvement of Reactive Oxygen Species Scavenging Enzymes in Desiccation Sensitivity of Antiaris toxicaria Seeds and Axes

The relationships among desiccation sensitivities of Antiaris toxicaria seeds and axes, changes in activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase, (TBA)-reactive substance were studied. Desiccation tolerance of seeds and axes decreased with dehydration. Desiccation tolerance of axes was higher than that of seeds, and that of epicotyls was higher than radicles. Activities of SOD, CAT and DHAR of seeds increased during the initial phase of dehydration, and then decreased with further dehydration, whereas activities of APX and GR decreased with dehydration. These five enzyme activities of axes, however, increased during the initial phase of dehydration, and then decreased with further dehydration. The rate of superoxide radical production, and the contents of H2O2 and TBA-reactive products of seeds and axes gradually increased with dehydration. These results show that the A. toxicaria seed is a typical recalcitrant seed. Loss of desiccation tolerance in seeds and axes was correlated with activities of seeds and axes.     

黄皮种子线粒体呼吸速率和活性氧清除酶活性对脱水的响应及其生态学意义

顽拗性种子脱落时具有较高的含水量和代谢活性, 对脱水高度敏感; 但顽拗性种子脱水敏感性的机理至今仍然不清楚。该文以顽拗性黄皮(Clausena lansium)种子为材料, 研究了种子和胚轴对水分丧失的响应, 在脱水过程中胚轴和子叶的呼吸速率, 胚轴和子叶线粒体的细胞色素c氧化酶(CCO)活性、外膜完整性、CCO和交替氧化酶(AOX)途径以及线粒体活性氧清除酶活性的变化。结果表明, 随着水分的丧失, 种子和胚轴的存活率逐渐下降, 种子的脱水敏感性大于胚轴; 胚轴和子叶的呼吸速率以及线粒体外膜的完整性降低。胚轴和子叶线粒体的CCO途径以及胚轴AOX途径的呼吸速率在脱水初期增加, 随着继续脱水下降, 胚轴线粒体AOX途径的呼吸速率则随着脱水显著下降。胚轴线粒体的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性和子叶线粒体的APX活性随着脱水迅速下降; 胚轴线粒体的脱氢抗坏血酸还原酶(DHAR)活性和子叶线粒体的SOD、DHAR和GR活性在脱水初期增加, 然后下降。这些数据表明黄皮种子的脱水敏感性与线粒体的呼吸速率和活性氧清除酶的活性降低密切相关, 也与长期适应热带/亚热带的生境有关。     

酶促和非酶促抗氧化系统在玉米胚脱水耐性获得中的作用

以发育中的玉米胚为材料,研究了玉米胚脱水耐性的发育变化及其与抗氧化系统之间的关系。结果表明,授粉后18d的胚获得萌发能力,但不耐脱水;授粉后36d的胚开始获得耐脱水能力,并随着发育逐渐增加。随着发育,胚的超氧化物歧化酶（SOD）、抗坏血酸过氧化物酶（APX）、谷胱甘肽还原酶（GR）和脱氢抗坏血酸还原酶（DHAR）的活性逐渐降低,过氧化氢酶（CAT）活性逐渐增加。授粉后16～22d的玉米胚中检测不到抗坏血酸,24d后胚中抗坏血酸的含量显著增加;还原性谷胱甘肽含量在整个发育过程中逐渐增加。脱水胚的SOD、APX和DHAR的活性比对照（未脱水）胚低,而GR和CAT活性在发育早期比对照胚低,在发育中、后期高于对照胚。脱水胚的抗坏血酸和还原性谷胱甘肽含量明显低于对照胚。胚中丙二醛的含量随着发育逐渐下降,脱水胚的丙二醛含量显著高于对照。这些结果说明CAT活性和谷胱甘肽含量的增加以及脂质过氧化产物丙二醛含量的下降与玉米胚脱水耐性的获得密切相关。     

Trichiliadregeana胚轴的脱水敏感性与抗坏血酸的抗氧化作用

以顽拗性TrichiliadregeanaSond.种子为材料,研究其胚轴的脱水敏感性与抗坏血酸的抗氧化作用。T.dregeana胚轴的脱水耐性随着脱水进程逐渐下降,50%的胚轴被脱水致死的含水量(W50)大约为0.16gH2O/gDW。在脱水过程中,胚轴的电解质渗漏速率逐渐增加,超氧物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、谷胱苷肽还原酶(GR)和脱氢抗坏血酸还原酶(DHAR)的活性下降,硫代巴比妥酸(TBA)-活性产物的含量增加。2.5~10.0mmol/L抗坏血酸处理能有效地增加胚轴的脱水耐性和SOD、APX、CAT和GR的活性,降低电解质渗漏速率和TBA活性产物的含量。结果表明,T.dregeana胚轴的脱水耐性与抗氧化酶的活性增加和脂质过氧化作用的降低密切相关。     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

Response of Chinese Wampee Axes and Maize Embryos to Dehydration at Different Rates

Survival of wampee (Clausena lansium Sksels) axes and maize (Zea mays L.) embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutsse (SOD), ascorbate peroxidase (APX) and catalase (CAT) of wampee axes markedly increased during the sady phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the eady phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.     

乙烯与NO互作对镉胁迫下荷花的抗坏血酸-谷胱甘肽循环的影响

以荷花‘微山湖红莲’实生苗为试验材料,研究镉(Cd,50 μmol·L^-1)胁迫下,外源乙烯前体1-氨基环丙烷羧酸(ACC,100 μmol·L^-1)、ACC与一氧化氮合酶(NOS)抑制剂N-硝基-L-精氨酸(L-NNA,200 μmol·L^-1)、ACC与硝酸还原酶(NR)抑制剂钨酸钠(Tu,1 mmol·L^-1),ACC与一氧化氮(NO)清除剂2-苯基-4,4,5,5-四甲基咪唑啉-3-氧代-1-氧(PTIO,200 μmol·L^-1),外源NO供体硝普钠(SNP,500 μmol·L^-1)、SNP与乙烯信号转导抑制剂硫代硫酸银(STS,100 μmol·L^-1)处理下荷花幼苗叶片的受害程度及抗坏血酸(AsA)-谷胱甘肽(GSH)循环的变化情况.结果表明: Cd胁迫下,荷花叶片受害症状明显,其相对电导率、丙二醛(MDA)、AsA和GSH含量显著上升,抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)、单脱氢抗坏血酸还原酶(MDHAR)和脱氢抗坏血酸还原酶(DHAR)活性明显降低;ACC的添加进一步增加了Cd对荷花叶片的毒害症状,并加剧了4种抗氧化酶活性的降低,但增加了抗氧化剂的含量;SNP的添加对荷花叶片的伤害起到加重作用,并导致GR和MDHAR活性降低以及AsA和GSH含量的升高;PTIO可显著提高Cd和ACC复合处理下荷花叶片APX、GR、MDHAR和DHAR的活性并降低AsA和GSH的含量,而L-NNA和Tu效果不如PTIO明显;STS可显著缓解Cd和SNP复合处理下荷花叶片的毒害症状,并提高4种抗氧化酶的活性、降低AsA和GSH的含量.由此说明,乙烯和NO在AsA-GSH循环中存在互作,二者相互促进,共同调控AsA-GSH循环,进而参与调控荷花对Cd胁迫的响应.     

Ascorbate and glutathione metabolism during development and desiccation of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The ascorbate-glutathione system was studied during development and maturation of beech (Fagus sylvatica L.) seeds, the classification of which in the orthodox category is controversial. This study revealed an increase in glutathione content after acquisition of desiccation tolerance, which was more intensive in embryonic axes than in cotyledons. During seed maturation, the redox status of glutathione markedly changed toward the more reducing state, especially in cotyledons. Ascorbic acid content decreased during maturation, mostly in cotyledons. Activities of the enzymes of the ascorbate-glutathione cycle—ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2)—were markedly higher in embryonic axes than in cotyledons throughout the study period. In the course of seed maturation, the activities of these enzymes decreased. Importance of the ascorbate-glutathione cycle in desiccation tolerance of beech seeds was discussed in relation to results for typical orthodox and recalcitrant seeds of other broadleaved species.     

Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds

Lipid peroxidation resulting from loss of free radical scavenging is thought to be involved in deterioration of sunflower (Helianthus annuus L.) seeds during accelerated ageing. In other respects, presoaking of seeds in a solution of low water potential (osmopriming) has been demonstrated to reinvigorate aged seeds. The aim of the present work was to study the effect of osmopriming on the germination of aged sunflower seeds and to investigate whether this effect was associated with the restoration of antioxidant defence systems. Seeds were aged for 5 days at 45°C and 100% relative humidity and then primed for various durations up to 7 days at 15°C in a solution of polyethylene glycol 6000 at ?2 MPa. Lipid peroxidation was estimated by measuring malondialdehyde (MDA) and conjugated diene contents, and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were measured throughout the treatments. Accelerated ageing resulted in a marked decrease in the germination rate, and was associated with an increase in the levels of MDA and conjugated dienes, thus indicating lipid peroxidation. Ageing was also characterized by a decrease in the activities of CAT and GR. The activities of SOD and DHAR were much less altered. No APX activity was detected whatever the seed treatment. Priming of aged seeds progressively restored the initial germinative ability and resulted in a marked decrease in the levels of MDA and conjugated dienes, indicating a fall in lipid peroxidation processes. These effects of priming were also well correlated to the recovery of SOD, CAT and GR activities. Priming treatment for 7 days led to full restoration of the cell detoxifying mechanisms which were strongly altered during ageing. Glutathione content showed the same changes as GR activity. There existed a clear-cut relationship between seed germinative energy, expressed as the germination rate, and the efficiency of free radical scavenging systems, in particular CAT and GR activities and glutathione content. The results suggest that the antioxidant defence systems might play a key role in seed vigour.     

A comparative study of water distribution, free radical production and activation of antioxidative metabolism in germinating pea seeds

The aim of this study was to investigate whether there is a relationship between hydration of the embryo axes and cotyledons and the resumption of the oxidative metabolism in both organs of germinating seeds of pea (Pisum sativum L. cv. Piast). Nuclear magnetic resonance (¹H-NMR) spectroscopy and imaging were used to study temporal and spatial water uptake and distribution in pea seeds. The observations revealed that water penetrates into the seed through the hilum, micropyle and embryo axes, and cotyledons hydrate to different extents. Thus, inhomogeneous water distribution may influence the resumption of oxidative metabolism. Electron paramagnetic resonance (EPR) measurements showed that seed germination was accompanied by the generation of free radicals with g₁ and g₂ values of 2.0032 and 2.0052, respectively. The values of spectroscopic splitting coefficients suggest that they are quinone radicals. The highest content of free radicals was observed in embryo axes immediately after emergence of the radicle. Glutathione content decreased during the entire germination period in both embryo axes and cotyledons. A different profile was observed for ascorbate, with significant increases in embryo axes, coinciding with radicle protrusion. Electrophoretic analysis showed that superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) were present in dry seeds and were activated later during germination, especially in embryo axes. The presence of all antioxidative enzymes as well as low molecular antioxidants in dry seeds allowed the antioxidative machinery to be active as soon as the enzymes were reactivated by seed imbibition. The observed changes in free radical levels, antioxidant contents and enzymatic activities in embryo axes and cotyledons appear to be more closely related to metabolic and developmental processes associated with preparation for germination, and do not correspond directly to the hydration of the tissues.     

Cytological and physiological changes in orthodox maize embryos during cryopreservation

Cytological and physiological changes during cryopreservation were studied in maize embryos at 35 days after pollination (DAP). Both dehydration and freezing caused cytological damage, such as plasmolysis, swelled mitochondria, increased heterochromatin, and nuclear shrinkage. Dehydration alone slightly impaired plasma membrane integrity while a drastic increase in electrolyte leakage was observed after freezing of embryos with moisture content above 23%. Damage to cellular ultrastructure and plasmalemma integrity was negatively related to moisture content in unfrozen embryos and positively related in frozen embryos. The pattern of changes in activity of antioxidant enzymes differed from one another during dehydration and/or freezing–thawing treatment. Dehydration increased activity of ascorbate peroxidase (APX) and glutathione reductase (GR) but decreased activity of superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR). Freezing further decreased GR and SOD activity and resulted in extremely low DHAR activity. Embryos at intermediate moisture contents had low catalase (CAT) activity before freezing but highest CAT activity after freeze–thaw. Both dehydration and freezing promoted membrane lipid peroxidation which resulted in an approximately threefold increase at most in the malondialdehyde content in postthaw embryos. Changes in viability of postthaw embryos can be closely related to damage in cellular ultrastructure and plasmalemma integrity but directly related neither to antioxidants nor lipid peroxidation levels.     

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.     

Kinetics of the Anti-oxidant Response to Salinity in the Halophyte Cakile maritima

The effects of NaCl stress on the activity of anti-oxidant enzymes (superoxide dismutase, catalase (CAT),peroxidase (POD),ascorbate peroxidase (APX), monodehydroascorbate reductase, dehydroascorbate reductase (DHAR), and glutathionereductase (GR)), anti-oxidant molecules (ascorbate and glutathione), and parameters of oxidative stress (malondialdehyde(MDA), electrolyte leakage, and H_2O_2 concentrations) were investigated in Cakile maritima, a halophyte frequent along theTunisian seashore. Seedlings were grown in the presence of salt (100, 200, and 400 mmol/L NaCI). Plants were harvestedperiodically over 20 days. Growth was maximal in the presence of 0-100 mmol/L NaCl. At 400 mmol/L NaCl, growthdecreased significantly. The salt tolerance of C. maritima, at moderate salinities, was associated with the lowest values ofthe parameters indicative of oxidative stress, namely the highest activities of POD, CAT, APX, DHAR, and GR and high tissuecontent of ascorbate and glutathione. However, prolonged exposure to high salinity resulted in a decrease in anti-oxidantactivities and high MDA content, electrolyte leakage, and H_2O_2 concentrations. These results suggest that anti-oxidantsystems participate in the tolerance of C. maritima to moderate salinities.     

Dehydroascorbate reductase and glutathione reductase play an important role in scavenging hydrogen peroxide during natural and artificial dehydration of Jatropha curcas seeds

Changes in H₂O₂ and the main antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), in endospermic and embryonic tissues were studied in developing and artificially dried Jatropha curcas seeds. Immature seeds were desiccation-tolerant at 80 days after flowering, as they were able to germinate fully after artificial drying on silica gel had reduced their water content to 10–12% of fresh weight. In both endospermic and embryonic tissues, H₂O₂ level and, consequently, lipid peroxide content, decreased during seed development as well as after artificial dehydration of developing seeds. All examined antioxidant enzymes except DHAR showed a decrease in total activity in mature stages as compared with early stages. Expression analysis of SOD genes revealed that the decrease in total SOD activities was related to the decrease in Cu/Zn-SOD expression, while the continuous activity of SOD during maturation was related to an increase in Mn-SOD expression. Artificial drying resulted in increased SOD and DHAR activity, irrespective of the developmental stage. Our results revealed weak participation of CAT and APX in H₂O₂ scavenging, as well as no significant alterations in GR activities either during maturation or after artificial drying. Changes in SOD and GR isoenzyme patterns occurred during maturation-related drying, but not after artificial drying. These results highlight the role of ascorbate-glutathione cycle enzymes (DHAR and GR) in H₂O₂ scavenging during maturation or after artificial drying of developing J. curcas seeds.     

Kinetics of the Anti-oxidant Response to Salinity in the Halophyte Cakile maritima

The effects of NaCl stress on the activity of anti-oxidant enzymes （superoxide dismutase, catalase （CAT）, peroxidase （POD）, ascorbate peroxidase （APX）, monodehydroascorbate reductase, dehydroascorbate reductase （DHAR）, and glutathione reductase （GR））, anti-oxidant molecules （ascorbate and glutathione）, and parameters of oxidative stress （malondialdehyde （MDA）, electrolyte leakage, and H2O2 concentrations） were investigated in Cakile maritima, a halophyte frequent along the Tunisian seashore. Seedlings were grown in the presence of salt （100, 200, and 400 mmol/L NaCl）. Plants were harvested periodically over 20 days. Growth was maximal in the presence of 0-100 mmol/L NaCl. At 400 mmol/L NaCl, growth decreased significantly. The salt tolerance of C. maritima, at moderate salinities, was associated with the lowest values of the parameters indicative of oxidative stress, namely the highest activities of POD, CAT, APX, DHAR, and GR and high tissue content of ascorbate and glutathione. However, prolonged exposure to high salinity resulted in a decrease in anti-oxidant activities and high MDA content, electrolyte leakage, and H2O2 concentrations. These results suggest that anti-oxidant systems participate in the tolerance of C. maritima to moderate salinities.     

Oxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss) seeds

The seeds of Azadirachta indica were successfully cryopreserved for 12 months with 45% survival following drying to 0.16gH(2)Og(-1) dry mass (DM). Highest survival (94-96%) was recorded during the first month of cryostorage. Subsequent cryopreservation up to 12 months resulted in decreasing germination. Post-thawing pre-heat treatment enhanced the recovery marginally in seeds cryopreserved from 3 to 12 months. Viability of cryostored seeds was negatively correlated with leachate conductivity and accumulation of thiobarbituric acid reactive substances (TBRS) estimated in cotyledons and axes. Leachate conductivity of imbibed seeds was low during the first month of cryostorage but increased gradually with the duration of cryostorage to a maximum after 12 months. TBRS accumulation was gradual throughout cryostorage. Relatively low amounts of active oxygen species (AOS) detected during the first month of cryostorage were closely associated with very high activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in seeds (cotyledons and axes). Marked accumulation of AOS from 3 to 12 months was associated with decrease in antioxidant enzyme activity.     

The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt‐tolerant relative Lycopersicon pennellii

The possible involvement of the antioxidative system in the tolerance to salt stress was studied in the cultivated tomato Lycopersicon esculentum Mill. cv. M82 (M82) and its wild salt‐tolerant relative L. pennellii (Corn) D'Arcy accession Atico (Lpa). All analyses, except that of monodehydroascorbate reductase (MDHAR), were performed of the youngest fully‐expanded leaf of control and salt (100 m M NaCl) stressed plants, 4, 7, 10, 14, 18 and 22 days after completing the stress treatment. In Lpa, constitutive level of lipid peroxidation and activities of catalase (CAT) and glutathione reductase (GR) were lower while the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were inherently higher than in M82. Relative to M82, lipid peroxidation was much lower and the activities of SOD, CAT and APX were higher in Lpa at 100 m M NaCl. The activity of DHAR decreased more in Lpa than in M82 under salt stress, and the activity of MDHAR, which was lower in Lpa than in M82 under control conditions, increased much more and to a higher level in salt‐treated Lpa plants. GR activity decreased similarly in the two species under salt stress. The results of these analyses suggest that the wild salt‐tolerant Lpa plants are better protected against active oxygen species (AOS), inherently and under salt stress, than the relatively sensitive plants of the cultivated species.     

Antioxidative response of ascorbate-glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer saccharinum seeds

Ascorbate–glutathione systems were studied during desiccation of recalcitrant seeds of the silver maple (Acer saccharinum L.). The desiccated seeds gradually lost their germination capacity and this was strongly correlated with an increase in electrolyte leakage from seeds. Simultaneously the increase of reactive oxygen species (ROS) (superoxide radical – O₂⁻ and hydrogen peroxide – H₂O₂) production was observed. The results indicate that remarkable changes in the concentrations and redox status of ascorbate and glutathione occur in embryo axes and cotyledons. After shedding, concentrations of ascorbic acid (ASA) and the reduced form of glutathione (GSH) are higher in embryo axes than in cotyledons and their redox status is high in both embryo parts. Cotyledons in freshly shed seeds are devoid of GSH. At the first stages of desiccation, up to a level of 43% of moisture content, ASA content in embryo axes and GSH content in cotyledons increased. Below this level of moisture content, the antioxidant contents as well as their redox status rapidly decreased. The enzymes of the ascorbate–glutathione pathway: ascorbate peroxidase (APX) (EC 1.11.1.11), monodehydroascorbate reductase (MR) (EC 1.6.5.4), dehydroascorbate reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) increased their activity during desiccation, but mainly in embryonic axes. The changes are probably required for counteracting the production of ROS during desiccation. The relationship between ascorbate and glutathione metabolism and their relevance during desiccation of recalcitrant Acer saccharinum seeds is discussed.     

Cytological and physiological changes in recalcitrant Chinese fan palm (<Emphasis Type="Italic">Livistona chinensis</Emphasis>) embryos during cryopreservation

Cytological and physiological changes during cryopreservation were investigated in Livistona chinensis embryos excised 42 weeks after flowering. Both dehydration and freezing caused numerous cellular ultrastructural alterations. Dehydration seriously impaired plasma membrane integrity, while freezing caused a further increase in electrolyte leakage. Damage to cellular ultrastructure and plasmalemma integrity had an inverse relationship with water content in unfrozen embryos and a positive relationship in frozen embryos. Changes in activity of antioxidant enzymes differed during cryopreservation. Dehydration and freezing had little effect on superoxide dismutase activity, although these treatments greatly reduced embryo viability. Activity of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) changed only slightly during dehydration, but dehydration markedly decreased activity of ascorbate peroxidase (APX) and catalase (CAT). Freezing further decreased APX and GR activity but increased CAT activity in dehydrated samples. A novel DHAR isozyme was induced during the freeze–thaw cycle. Membrane lipid peroxidation was detected in the control embryos, and was promoted by both dehydration and freezing. The malondialdehyde (MDA) content in post-thaw embryos increased by a maximum of 30%. Thus, changes in viability of embryos were closely related to damage to cellular ultrastructure and plasmalemma integrity, but were not directly related to antioxidant activity nor MDA accumulation.