H1 Histone in Developing and Aging Coleoptiles of Etiolated Wheat Seedlings

It has been established that the DNA and H1 histone contents in aged coleoptile of 8-day-old etiolated wheat seedling are about 40 and 30%, respectively, lower than those in young seedlings. H1 histone in wheat seedlings is represented as six electrophoretically different subfractions. The ratios of H1 histone subfractions in wheat coleoptile and initial leaf are similar. In contrast to some animal cells, apoptosis in wheat coleoptile is not accompanied by changes in the set and ratios of H1 histone subfractions. Aging of coleoptiles is associated with a progressive diminution of the H1 histone and DNA contents. H1 histone/DNA ratio in aged coleoptile is 1.5-2-fold higher than that in the young organs. Therefore, the content of H1 histone in chromatin of coleoptile decreases with age more slowly than DNA content.     

Apoptosis in the Initial Leaf of Etiolated Wheat Seedlings: Influence of the Antioxidant Ionol (BHT) and Peroxides

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27·10^–4 M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H₂O₂, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.     

外源H2O2对盐胁迫下小麦幼苗生理指标的影响

以‘郑麦-004’小麦幼苗为供试材料,采用Hoagland营养液培养方法,通过添加H2O2的清除剂过氧化氢酶(CAT)和抗坏血酸(ASA),研究0.05μmol/L外源H2O2处理对150mmol/L NaCl胁迫下小麦幼苗生长和抗氧化系统活性的影响,探讨低浓度外源H2O2对盐胁迫下小麦幼苗伤害的防护作用及其生理机制。结果显示:外源H2O2能缓解盐胁迫对小麦幼苗生长的抑制效应,降低丙二醛(MDA)含量和超氧自由基(O2.-)的产生速率,使小麦幼苗的株高、根长和干重均显著增加,并能提高超氧化物歧化酶(SOD)、过氧化物酶(POD)、CAT、抗坏血酸氧化酶(APX)等保护酶活性和抗氧化物质谷胱甘肽(GSH)的含量;而H2O2清除剂(CAT和AsA)能够逆转外源H2O2对盐胁迫下小麦幼苗生长的促进作用。研究表明,低浓度外源H2O2处理能促进小麦幼苗中的酶类和非酶类抗氧化剂的产生,减少脂质过氧化物的含量,提高小麦幼苗的耐盐性。     

Necessity of Superoxide Production for Development of Etiolated Wheat Seedlings

It was found that production of superoxide (O₂ ^{– ·}) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O₂ ^{– ·} production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O₂ ^{– ·} production was clearly observed in the period from two to four days of seedling development, then the rate of O₂ ^{– ·} production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O₂ ^{– ·} production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O₂ ^{– ·} production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O₂ ^{– ·} amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O₂ ^{– ·} controls plant growth by cell elongation at the early stages of seedling development but later O₂ ^{– ·} controls (induces) apoptotic DNA fragmentation and protein disintegration.     

Apoptosis in Etiolated Wheat Seedlings: 2. Effects of the Antioxidant Butylated Hydroxytoluene and Peroxides

The antioxidant butylated hydroxytoluene (BHT, 50 mg/l, 2.27 × 10^–4 M) was found to prevent the development of characteristic signs of senescence and apoptosis in the cells of etiolated wheat (Triticum aestivum L.) seedlings. In particular, BHT blocked the apoptotic and age-induced formation of specific cytoplasmic mitochondria-containing vesicles in the coleoptiles. In contrast, the oxidants (H₂O₂ and cumene hydroxyperoxide) accelerated apoptosis (DNA fragmentation) in the coleoptiles and induced it in the first leaves, while in the control leaves, there were no signs of apoptosis. Thus, the programmed developmental apoptosis is controlled by the reactive oxygen species (ROS), and anti- and prooxidants can actively affect this process. In the coleoptile, BHT induced substantial changes in the ultrastructure of all cell organelles (nucleus, mitochondria, plastids, Golgi apparatus, and endoplasmic reticulum). It also induced the formation of unusual membrane structures in the cytoplasm and impaired nucleus and cell divisions. As a result, giant multilobed nuclei and multinuclear cells appeared. The effects of the antioxidant were tissue-specific: BHT did not noticeably affect cell ultrastructure in the first leaf. In roots of etiolated seedlings, BHT stimulated unusual plastid differentiation that resulted in the formation of chloroplasts, which is a phenomenon abnormal for roots. The BHT effects on the plant are evidently related to its antioxidant properties. Indeed, its structural analog, 3,5-di-tert-butyltoluene, which does not exhibit antioxidant properties, was physiologically inert. The BHT-controlled ROS evidently triggered apoptosis and produced age-dependent structural rearrangements of the cytoplasm and the formation of specific mitochondria-containing vesicles, which actively synthesize mtDNA. ROS inactivation by BHT is evidently responsible for BHT-induced changes in the structure of all cell organelles. Therefore, we believe that ROS control cell division (including nucleus division and cell-wall formation) and affect the differentiation of plastids and Golgi apparatus. In such a way, ROS effectively control plant growth and development.     

Apoptosis in Etiolated Wheat Seedlings: 1. Ultrastructure of the Apoptotic Cell

We studied the process of apoptosis in etiolated wheat (Triticum aestivum L.) seedlings. As a result, an integral pattern of the apoptotic plant cell ultrastructure was established. In the apoptotic cells of the coleoptile, we observed chromatin condensation and margination, an increased density and specific cytoplasm fragmentation accompanied by the appearance of unusual cytoplasmic vesicles containing subcellular organelles, mitochondria in particular, in the vacuoles.     

Luminol-Dependent Chemiluminescence Analysis of Chitooligosaccharide-Induced Rapid Production of Hydrogen Peroxide by Intact Wheat Seedlings

The feasibility of a luminol-dependent chemiluminescence analysis of hydrogen peroxide production by intact wheat seedlings using a KhL-003 chemiluminometer was determined. It was shown that the minimal H₂O₂ concentration that can be detected in a 0.5-ml sample with this instrument is 0.125 µM. Analysis of biological activity of a mixture of chitooligosaccharides with molecular masses from 5 to 10 kD and acetylation degree of 65% demonstrated that, at a concentration of 1 µg/ml, they induce rapid overproduction of H₂O₂ in roots of 3-day-old wheat seedlings.     

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.     

Cd~(2+)胁迫对小麦幼苗生理生化特性的影响

以小麦品种'西旱2号'和'宁春4号'幼苗为材料,采用室内水培实验研究了不同浓度Cd(NO3)2处理对叶绿素含量、抗氧化酶活性及渗透性调节物含量等的影响.结果表明,两小麦品种幼苗的叶绿素a(Chl a)、叶绿素b(Chl b)及叶绿素总量在镉胁迫下均比对照降低,且高浓度镉对'西旱2号'幼苗叶片Chl b的破坏程度强于Chl a;两品种镉胁迫幼苗的过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性均比对照不同程度地升高;两品种镉胁迫幼苗的可溶性糖含量均与对照无显著差异,但'宁春4号'幼苗叶片的脯氨酸含量却比对照显著升高,且此效应具有浓度依赖性;镉胁迫下两品种小麦幼苗叶片的MDA含量变化与对照无显著差异.研究显示,两品种小麦幼苗叶片的光合色素在镉胁迫下均受到不同程度的破坏,但它们均能通过增加体内保护酶活性和渗透调节物质含量来缓解膜脂过氧化伤害,从而对镉胁迫均表现出较强的耐受能力.     

The Effects of Phytohormones and 5-Azacytidine on Apoptosis in Etiolated Wheat Seedlings

The development of etiolated wheat (Triticum aestivum L.) seedlings is necessarily accompanied by apoptosis in their coleoptiles and first leaves. Internucleosome DNA fragmentation, which is characteristic of apoptosis, was detected in the coleoptile as soon as six days after germination. After eight days of germination, DNA fragmentation was clearly expressed in the coleoptile and was noticeable in the apical part of the first-leaf blade. Growing of intact seedlings or incubation of their shoots in the presence of such phytohormones as benzyladenine, gibberellin A₃, fusicoccin C, and 2,4-D at the concentration of 10^–5 M did not essentially affect DNA fragmentation in the coleoptile. As distinct from antioxidants, none of the phytohormones used prevented apoptosis in wheat seedlings. In contrast, ABA (10^–5 M) and an ethylene producer, ethrel (2-chloroethylphosphonic acid, 10^–2–10^–3 M), stimulated sharply DNA fragmentation in the coleoptile. An inhibitor of DNA methylation, 5-azacytidine, was very efficient in the stimulation of DNA fragmentation in the coleoptiles of eight-day-old seedlings at its concentration of 100 g/ml. Thus, some phytohormones can regulate apoptosis, and DNA methylation is involved in this process. Our results indicate that apoptosis activation by some phytohormones may be mediated by their regulation of DNA methylation/demethylation, which is responsible for the induction of genes encoding apoptogenic proteins and/or the repression of antiapoptotic genes.     

Antioxidant responses of wheat plants under stress

Currently, food security depends on the increased production of cereals such as wheat (Triticum aestivum L.), which is an important source of calories and protein for humans. However, cells of the crop have suffered from the accumulation of reactive oxygen species (ROS), which can cause severe oxidative damage to the plants, due to environmental stresses. ROS are toxic molecules found in various subcellular compartments. The equilibrium between the production and detoxification of ROS is sustained by enzymatic and nonenzymatic antioxidants. In the present review, we offer a brief summary of antioxidant defense and hydrogen peroxide (H₂O₂) signaling in wheat plants. Wheat plants increase antioxidant defense mechanisms under abiotic stresses, such as drought, cold, heat, salinity and UV-B radiation, to alleviate oxidative damage. Moreover, H₂O₂ signaling is an important factor contributing to stress tolerance in cereals.     

Carbon Monoxide： A Novel Antioxidant Against Oxidative Stress in Wheat Seedling Leaves

Carbon monoxide （CO）, an endogenous signaling molecule in animals, also provides potent cytoprotective effects including attenuation of lung lipid peroxidation induced by oxidant in the mouse. Our recent work demonstrated that 0.01 μmol/L hematin （a CO donor） treatment of wheat plants alleviated salt-induced oxidative damage in seedling leaves. In this report, we further discovered that hematin pretreatment （≤ 0.1 μmol/L） could delay wheat leaf chlorophyll loss mediated by further treatment of H202 and paraquat, two reactive oxygen species （ROS） sources, in dose-and even time-dependent manners. Also, compared with the control samples, seedling leaves pretreated with 0.01 or 0.1 μmol/L hematin for 24 h exhibited lower levels of H2O2 and lipid peroxidation, as well as higher contents of chlorophyll and activities of antioxidant enzymes. Such beneficial effects exerted by hematin were mimicked by the pretreatment of antioxidant butylated hydroxytoluene （BHT）, and differentially reversed when CO scavenger hemoglobin （Hb）, or CO specific synthetic inhibitor ZnPPIX was added, respectively. Taken together, the results presented In this paper directly illustrate for the first time that CO is able to strongly protect plants from oxidative damage caused by the overproduction of ROS, and strengthens the evidence that CO is a potent antioxidant in various abiotic and biotic stresses, as similar results have been shown in animal tissues.     

不同处理条件下小麦胚芽鞘长的遗传分析

以小麦双单倍体(DH)群体(旱选10号×鲁麦14)的150个株系及其亲本为材料,对不同处理条件下的胚芽鞘长、幼苗株高和幼苗生物学产量进行遗传分析,以探讨胚芽鞘长、幼苗株高及幼苗生物学产量的遗传基础及其相关性.结果表明:(1)随着播种深度的增加,胚芽鞘长、幼苗株高显著增加,16.1%PEG-6000处理条件下的幼苗株高显著低于对照,干旱胁迫条件下的幼苗生物学产量显著低于正常供水对照;(2)胚芽鞘长、幼苗株高、幼苗生物学产量的遗传力分别为92.5%、90.3%和78.1%;(3)胚芽鞘长、幼苗株高及幼苗生物学产量均受多基因控制,决定各性状的基因估计数目分别为7、8和14对;(4)根据对性状偏度和峰度系数的估算结果,控制这3个性状的多基因之间均有互补作用;胚芽鞘长与幼苗株高、生物学产量及成熟期株高之间呈显著正相关.     

Involvement of Lysosomal Cysteine Proteases in Hydrogen Peroxide-induced Apoptosis in HL-60 Cells

Hydrogen peroxide is a well-known mediator of apoptosis. As a mechanism for H₂O₂-induced apoptosis, both a mitochondrial Cyt.c-dependent pathway and a lysosome-mediated pathway have been suggested. However, the relative roles of and the relation between these two pathways in H₂O₂-induced apoptosis remain to be discovered. In this study, to find the relative roles of the lysosomal and mitochondrial pathways, the effects of E-64-d, a cell-permeable inhibitor of lysosomal cysteine proteases, on apoptosis caused by H₂O₂ in HL-60 cells were investigated. It was found that the concentration of H₂O₂ strongly affected the inhibitory effect of E-64-d on the apoptosis in HL-60 cells: dose-dependent inhibition (up to 40%) of both DNA fragmentation and caspase-3 activation was observed when a high concentration of H₂O₂ (50 μM) was used to induce apoptosis, but no inhibitory effect was detected when a low concentration (10 μM) was used. Consistent with these observations, apparent lysosomal destabilization was observed only with 50 μM H₂O₂. The release of mitochondrial Cyt.c, in contrast, was observed at both 10 μM and 50 μM. These results indicated that the mitochondrial Cyt.c-mediated pathway predominates in the H₂O₂-induced apoptosis in HL-60 cells and the lysosomal mediated pathway is partially involved when high concentrations of H₂O₂ are used to induce apoptosis.     

核酸内切酶在细胞凋亡中的作用

核酸内切酶在形成细胞凋亡的典型特征——DNA片段化中,发挥着直接的重要作用.介绍了已知的参与细胞凋亡的二价金属离子依赖性和非依赖性核酸内切酶种类,其中二价金属离子依赖性主要有nuc18、DNaseⅠ、Ca^2＋/Mg^2＋核酸内切酶、Ca^2＋/Mn^2＋核酸内切酶、DNaseγ、nuc58和nuc40;二价金属离子非依赖型主要有DNaseⅡ及类似核酸内切酶.此外,还初步探讨了核酸内切酶降解染色质DNA的过程及其作用机制.     

小麦幼苗光呼吸与活性氧累积的关系

用Na2 S处理小麦幼苗 ,提高了光呼吸速率 ,减少了O- ·2 的含量 ;用草酸处理 ,降低了光呼吸速率 ,增加了O- ·2 的含量。光呼吸速率的变化与H2 O2 含量无关 ,却与膜脂过氧化水平呈负相关。体外试验表明 :Na2 S和草酸对O- ·2 和H2 O2 没有作用。小麦幼苗中 ,过氧化氢酶活性比乙醇酸氧化酶活性高 3个数量级 ,Na2 S和草酸对乙醇酸氧化酶活性分别有促进和抑制作用 ,却对过氧化氢酶活性没有影响。这些结果表明光呼吸是植物体内防止活性氧积累和伤害的一道防线。     

小麦叶片顺乌头酸酶对NO和H2 O2 的敏感性

外源一氧化氮（nitric oxide,NO)供体硝普钠（sodium nitroprusside,SNP)和过氧化氢（hydrogen peroxide,H2O2)处理抑制小麦（Triticu aestivum L.)叶片顺乌头酸酶活性,抑制呈明显的浓度及时间效应;同时外源NO衍生代谢物过氧亚硝酸阴离子（peroxynitrite,ONOO^-)的供体3－morpholinosydnonimine hydrochlloride(SIN-1)和水杨酸（salicylic acid,SA)对酶活性也具有抑制作用,而且小麦叶片线粒体顺乌头酸酶对H2O2和SIN－1更敏感。分别以SNP与过氧化氢酶（catalase,CAT)专一性抑制剂氨基三唑（3－amino-1,2,4-triazole,3-AT)处理离体小麦叶片,发现在其内源H2O2含量上升的同时,顺乌头酸酶活性均呈浓度与时间依赖性下降趋势。表明NO除直接抑制顺乌头酸酶活性外,还可能经H2O2介导间接对顺乌头酸酶产生抑制作用。