拟南芥GLP13基因在植物抗氧化胁迫响应中的作用

类萌发素蛋白（germin-like protein, GLPs）是一类与小麦萌发素序列相似性较高、位于胞外基质的可溶性糖蛋白, 在植物的生长发育阶段以及对生物和非生物胁迫的应答中起着重要的作用。为了研究GLP13基因的生理功能, 我们分离并鉴定了GLP13的敲减突变体glp13, 同时构建了其超表达植株35S：：GLP13。用甲基紫精（methyl viologen, MV）处理2种不同基因型和野生型（WT）植株, 结果发现, 与野生型相比, 突变体glp13子叶变绿率较低, 主根生长受抑制较明显; 而超表达植株35S：：GLP13子叶变绿率较高, 主根生长的受抑制程度较WT轻。用MV处理2周的35S：：GLP13植株, 其叶绿素荧光参数Fv/Fm 的下降较野生型对照缓慢。半定量RT-PCR分析结果表明, 与野生型相比, 经MV处理4小时后的35S：：GLP13中抗氧化酶系基因FSD1的表达上调, 而CAT1、CSD1和UGT71C1的表达水平在35S：：GLP13、glp13和野生型植株三者之间没有明显差异。以上结果表明GLP13基因在拟南芥抗氧化胁迫响应中起重要作用。     

AtERF49在拟南芥应答盐碱胁迫中的作用

盐碱胁迫是造成作物减产的主要逆境因素之一。植物AP2/ERF（APELATA2/ethylene response factors）转录因子在植物生长发育及其响应非生物逆境胁迫过程中发挥重要作用。探究AtERF49在拟南芥中对盐碱胁迫的应答,为深入解析AtERF49参与植物对盐碱胁迫的分子机理奠定基础。选取拟南芥野生型Col-0、过表达AtERF49转基因拟南芥和CRISPR/Cas9突变体erf49为试验材料,用150 mmol/L混合盐碱（摩尔比NaHCO₃∶Na₂CO₃=9∶1）溶液进行处理,使用荧光定量PCR技术对该基因的基本特性、盐碱胁迫及光合响应基因表达模式等进行分析。结果表明,盐碱胁迫处理后,突变体erf49叶片萎蔫并发生白化,而过表达AtERF49植株叶片稍有变黄。此外,在盐碱胁迫条件下,过量表达AtERF49上调盐碱胁迫响应基因（RD29A和RAB18）以及光合响应基因rbcL的表达。拟南芥叶片叶绿素荧光参数测定结果表明,过表达AtERF49植株的光系统Ⅱ实际量子产能Y（Ⅱ）、光化学淬灭系数（qP）显著高于Col-0,光损伤程度（NO）和非光化学淬灭系数（qN）显著低于Col-0,而突变体erf49与之相反。因此,AtERF49通过调控下游盐碱胁迫响应基因的表达以及植物的光合作用效率,改变参与植物对盐碱胁迫的应答。     

盐胁迫过程中抗坏血酸对植物的保护功能

以拟南芥抗坏血酸突变体(υtc-1)和野生型(ωt)为材料,研究了抗氧化系统对盐胁迫的响应机制．以揭示抗坏血酸(ASA)的抗氧化机理及对植物的保护功能。结果显示：100mmol／L NaCl处理12、24、48、72h,υtc-1和ωt体内MDA(丙二醛)及H2O2(过氧化氢)的含量均明显增加,但υtc-1增加的程度明显高于ωt,说明盐胁迫可能对υtc-1造成了更严重的氧化伤害。胁迫过程中,ωt体内的几种抗氧化酶[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)]活性均升高,而υtc-1体内SOD、CAT活性降低,APX活性在胁迫24h之前增加,24h之后降低;同时,υtc-1中总的抗坏血酸含量和还原型谷胱甘肽／氧化型谷胱甘肽(GSH／GSSG)的比值下降,而ωt与此相反。本研究表明：抗坏血酸参与活性氧(AOS)的代谢,减轻AOS对植物的伤害;并可能对植物细胞内的抗氧化酶具有调节作用,增强逆境胁迫下植物的抗逆能力,对植物有重要的生物学保护功能。     

基于光子学技术研究弱光胁迫早期拟南芥光合损伤机理

弱光限制植物的光合作用,降低了光合作用效率,造成农业产量下降.本文主要研究了弱光处理早期,拟南芥光合作用相关指标的变化.研究中发现在弱光处理的早期,植株生长表型和最大光化学效率（Fv/Fm）没有明显变化,实际光化学效率Y（Ⅱ）以及光系统电子传递效率（ETR）下降较明显.此外,弱光处理原生质体,利用2 ＇,7＇-二氯二氢荧光素二乙酯（dichlorofluorescin diacetate,H2DCF-DA）染色,共聚焦显微镜观察,发现细胞中有较明显的活性氧（ROS）合成,且定位于叶绿体.该研究结果为植物弱光耐受性的研究提供理论依据.     

过量表达GST基因对盐胁迫下转基因拟南芥氧化损伤的影响

利用模式生物拟南芥作为实验材料,通过测定谷胱甘肽-抗坏血酸代谢相关酶（GST、GPX、APX、GR、DHAR、MDHAR）的活性和GSH、ASA、MDA含量以及生物量等来研究过量表达具有过氧化物酶活性的盐地碱蓬谷胱甘肽转移酶基因（GST基因）对盐胁迫下转基因拟南芥氧化损伤的影响。结果显示,转基因拟南芥比野生型具有较高的GST、GPX以及MDHAR酶活性;前者还具有较多的还原型谷胱甘肽和抗坏血酸,并且谷胱甘肽库氧化水平较野生型高。盐胁迫不但部分抑制了野生型拟南芥的生长,同时也导致了大量脂质过氧化物的积累;而盐胁迫对转基因拟南芥的生长抑制不明显,也没有较多的脂质过氧化物的积累。结果表明,过量表达盐地碱蓬谷胱甘肽转移酶基因提高．广转基因拟南芥依赖于还原型谷胱甘肽的过氧化物清除途径,同时有可能改变了GSH和ASA的代谢途径,这两方面的作用导致了转基因拟南芥氧化损伤的降低,使转基因拟南芥在盐胁迫下保持较好的生长态势。     

转录因子WRKY6和PR1在拟南芥胁迫记忆中的表达模式

植物暴露在细菌或其它微生物病原体下,会形成全身防御,称为系统获得性抗性SAR（Systemic Acquired Resistance）,该系统可以在病原体二次侵染时有效抑制病原体对植物的伤害。其中,WRKY转录因子和病程相关蛋白PRs（Pathogenesis-related proteins）在植物抗病信号调控途径中起着重要作用。本研究以模式植物拟南芥为实验材料,对WRKY6和PR1（PATHOGENESIS RELATED）两个转录因子进行初步研究。首先,从拟南芥eFP数据库中获得WRKY6和PR1的基因表达数据,进行生物信息学分析,获得WRKY6和PR1基因在不同胁迫条件下的表达热图。其次,通过实时荧光定量PCR技术,比较了经过生物胁迫和非生物胁迫处理后WRKY6和PR1的基因表达水平。结果表明,拟南芥经过生物胁迫丁香假单胞菌[Pseudomonas syringae pv.tomato（P_st） DC3000]处理后,WRKY6和PR1的基因表达模式具有一定的相似性,然而经过非生物胁迫和机械损伤组合处理后,WRKY6和PR1基因又呈现出不同的表达模式。本研究初步探索了WRKY6和PR1基因的表达模式及其关系,为今后进一步研究系统性获得抗性应答机制提供了思路。     

盐胁迫对拟南芥AtPUB18基因的诱导表达及其启动子分析

用300mmol/L NaCl处理拟南芥幼苗,分别于处理后0、1、2、4、8、16、24、48h通过Northern Blot检测其AtPUB18基因的表达量。结果显示:拟南芥AtPUB18基因的表达量受高盐胁迫的诱导而升高,于处理后4h表达量达到最高,处理后16h表达量最低。采用PCR技术克隆AtPUB18的启动子,序列为1 974bp;序列分析发现启动子内含有大量与非生物胁迫相关的顺式作用元件,如HSE、LTR、MBS及ABRE;将启动子克隆到表达载体pCambia1300-221-GUS中,驱动报告基因GUS表达。组织化学染色结果表明,未经过高盐处理的幼苗中GUS基因表达水平很低;300mmol/L NaCl处理后GUS基因表达量显著升高。研究表明,AtPUB18的表达受高盐胁迫诱导,且AtPUB18基因的启动子是一个盐胁迫诱导型启动子。     

盐胁迫过程中抗坏血酸对植物的保护功能

以拟南芥抗坏血酸突变体 vtc- 1 和野生型 wt 为材料 ,研究了抗氧化系统对盐胁迫的响应机制 ,以揭示抗坏血酸 ASA 的抗氧化机理及对植物的保护功能 .结果显示 :10 0 mm ol/ L Na Cl处理 12、2 4、4 8、72 h,vtc- 1和 wt体内 MDA 丙二醛 及 H2 O2 过氧化氢 的含量均明显增加 ,但 vtc- 1增加的程度明显高于 wt,说明盐胁迫可能对vtc- 1造成了更严重的氧化伤害 .胁迫过程中 ,wt体内的几种抗氧化酶 [超氧化物歧化酶 SOD 、过氧化氢酶 CAT 、抗坏血酸过氧化物酶 APX ]活性均升高 ,而 vtc- 1体内 SOD、CAT活性降低 ,APX活性在胁迫 2 4 h之前增加 ,2 4 h之后降低 ;同时 ,vtc- 1中总的抗坏血酸含量和还原型谷胱甘肽 /氧化型谷胱甘肽 GSH/ GSSG 的比值下降 ,而 wt与此相反 .本研究表明 :抗坏血酸参与活性氧 AOS 的代谢 ,减轻 AOS对植物的伤害 ;并可能对植物细胞内的抗氧化酶具有调节作用 ,增强逆境胁迫下植物的抗逆能力 ,对植物有重要的生物学保护功能     

GRAS转录因子AtSCL4负调控拟南芥应答渗透胁迫

探讨拟南芥GRAS转录因子AtSCL4(Arabidopsis thaliana SCL4)在渗透胁迫中发挥的生物学功能,为GRAS蛋白在非生物胁迫中的功能研究奠定基础。以野生型和AtSCL4突变体拟南芥为试验材料,通过生理指标测定和qRT-PCR方法研究渗透胁迫下AtSCL4调控植物抗逆的生物学机制。研究发现AtSCL4受渗透胁迫诱导后显著上调表达,且AtSCL4突变体的抗渗能力强于野生型。在渗透胁迫下,AtSCL4可以负调节ATMYB6的表达,减小气孔开放度,降低叶片水分流失;AtSCL4通过负调控P5SC1和BADH的转录来提高植物体内脯氨酸和甜菜碱的含量;AtSCL4通过负调节AtSOD1和PER4的表达来增加抗氧化酶活性而降低活性氧含量。AtSCL4可负向调控抗逆基因表达和生理变化应答渗透胁迫。     

外源一氧化氮对镉胁迫下玉米幼苗根生长及氧化伤害的影响

以玉米幼苗为材料,通过在镉处理的同时补充外源一氧化氮(NO)供体硝普钠(SNP)及其类似物[K3Fe(CN)6]、以及NO消除剂,分析NO对植物耐镉性的影响,探讨NO在植物逆境胁迫响应中的作用及其机理。结果显示:添加20μmol·L-1 SNP能显著降低镉引发的玉米幼苗根生长抑制及根尖内源镉的积累,减少电解质的渗漏以及超氧化物自由基(O2.-)和过氧化氢(H2O2)的上升幅度,抑制超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性的增加,进一步提高镉胁迫下谷胱甘肽还原酶(GR)的活性。SNP的上述效应可被NO消除剂2-(4-羧基-2-苯基)-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)所逆转,而SNP类似物K3Fe(CN)6的应用对上述反应几乎无影响,说明该反应具有NO特异性。研究表明,外源NO能够显著缓解镉胁迫对玉米幼苗生长造成的伤害,该缓解作用主要是通过降低植株体内内源镉积累和减轻镉诱发的氧化伤害来实现的。     

拟南芥活性氧不敏感型突变体的筛选与特性分析

采用 EMS化学诱变方法与 H2 O2 氧化胁迫选择 ,以根在重力作用下的弯曲生长为指标 ,筛选得到拟南芥活性氧不敏感型突变体。对突变体杂交后代遗传分析表明 ,突变株对活性氧不敏感性状为隐性单基因突变所致 ;生理生化分析表明突变体对 H2 O2 有很强的抗性 ,表现为气孔开度对 H2 O2 不敏感和 H2 O2 胁迫时较低的膜脂过氧化水平。运用 L SCM技术并结合 H2 O2 荧光探针 H2 DCFDA检测外源 ABA诱导保卫细胞内产生 H2 O2 的情况 ,结果显示突变体体内荧光强度比对照低 ,暗示了突变体体内消除 H2 O2 的能力可能有所提高 ,增强了植株对氧化胁迫的抗性。拟南芥活性氧不敏感突变体的筛选 ,不仅为人们深入研究活性氧在细胞内的作用提供良好的实验材料 ,而且还将大大加深人们对信号转导途径的再认识     

Exogenous ethanol treatment alleviates oxidative damage of Arabidopsis thaliana under conditions of high-light stress

Abiotic stresses, such as high light and salinity, are major factors that limit crop productivity and sustainability worldwide. Chemical priming is a promising strategy for improving the abiotic stress tolerance of plants. Recently, we discovered that ethanol enhances high-salinity stress tolerance in Arabidopsis thaliana and rice by detoxifying reactive oxygen species (ROS). However, the effect of ethanol on other abiotic stress responses is unclear. Therefore, we investigated the effect of ethanol on the high-light stress response. Measurement of chlorophyll fluorescence showed that ethanol mitigates photoinhibition under high-light stress. Staining with 3,3′-diaminobenzidine (DAB) showed that the accumulation of hydrogen peroxide (H₂O₂) was inhibited by ethanol under high-light stress conditions in A. thaliana. We found that ethanol increased the gene expressions and enzymatic activities of antioxidative enzymes, including ASCORBATE PEROXIDASE1 (AtAPX1), Catalase (AtCAT1 and AtCAT2). Moreover, the expression of flavonoid biosynthetic genes and anthocyanin contents were upregulated by ethanol treatment during exposure to high-light stress. These results imply that ethanol alleviates oxidative damage from high-light stress in A. thaliana by suppressing ROS accumulation. Our findings support the hypothesis that ethanol improves tolerance to multiple stresses in field-grown crops.     

ITN1, a novel gene encoding an ankyrin-repeat protein that affects the ABA-mediated production of reactive oxygen species and is involved in salt-stress tolerance in Arabidopsis thaliana

Salt stress and abscisic acid (ABA) induce accumulation of reactive oxygen species (ROS) in plant cells. ROS not only act as second messengers for the activation of salt-stress responses, but also have deleterious effects on plant growth due to their cytotoxicity. Therefore, the timing and degree of activation of ROS-producing or ROS-scavenging enzymes must be tightly regulated under salt-stress conditions. We identified a novel locus of Arabidopsis, designated itn1 (increased tolerance to NaCl1), whose disruption leads to increased salt-stress tolerance in vegetative tissues. ITN1 encodes a transmembrane protein with an ankyrin-repeat motif that has been implicated in diverse cellular processes such as signal transduction. Comparative microarray analysis between wild-type and the itn1 mutant revealed that induction of genes encoding the ROS-producing NADPH oxidases (RBOHC and RBOHD) under salt-stress conditions was suppressed in the mutant. This suppression was accompanied by a corresponding reduction in ROS accumulation. The ABA-induced expression of RBOHC and RBOHD was also suppressed in the mutant, as was the case for RD29A, an ABA-inducible marker gene. However, the ABA-induced expression of another marker gene, RD22, was not impaired in the mutant. These results suggest that the itn1 mutation partially impairs ABA signaling pathways, possibly leading to the reduction in ROS accumulation under salt-stress conditions. We discuss the possible mechanisms underlying the salt-tolerant phenotype of the itn1 mutant.     

Production of reactive oxygen species,impairment of photosynthetic function and dynamic changes in mitochondria are early events in cadmium‐induced cell death in Arabidopsis thaliana

Background information. Cadmium (Cd) is a highly toxic heavy metal that causes changes in plant metabolism through inhibiting photosynthesis and respiration. The effects of Cd on the morphology and function of the chloroplast and mitochondria, as well as on the production and localization of ROS (reactive oxygen species), were studied at the single‐cell level in Arabidopsis. Results. The present study showed that the morphology of chloroplasts changed after Cd treatment, and the photochemical efficiency dramatically declined prior to obvious morphological distortion in the chloroplasts. A quick burst of ROS was detected after Cd treatment. The ROS appeared first in the mitochondria and subsequently in the chloroplast. Simultaneously, the mitochondria clumped irregularly around the chloroplasts or aggregated in the cytoplasm, and the movement of mitochondria was concomitantly blocked. Furthermore, the production of ROS was decreased after pre‐treatment with ascorbic acid or catalase, which prevented inhibition of photosynthesis, organelle changes and subsequent protoplast death. Our results suggest that the distribution and mobility of mitochondria, the morphology of chloroplasts and the accumulation of ROS play important roles in Cd‐induced cell death. The results are in good agreement with previous reports of many types of apoptotic‐like cell death. Conclusion. The changes in the distribution and mobility of mitochondria, and morphology of chloroplasts, as well as the accumulation of ROS, play important roles in Cd‐induced cell death.     

Abscisic acid-regulated responses of aba2-1 under osmotic stress: the abscisic acid-inducible antioxidant defence system and reactive oxygen species production

We investigated the interaction among abscisic acid (ABA), reactive oxygen species (ROS) and antioxidant defence system in the transduction of osmotic stress signalling using Arabidopsis thaliana WT (Columbia ecotype, WT) and an ABA-deficient mutant (aba2-1). For this, 50 μm ABA and osmotic stress, induced with 40% (w/v) polyethylene glycol (PEG8000; -0.7 MPa), were applied to WT and aba2-1 for 6, 12 or 24 h. Time course analysis was undertaken for determination of total/isoenzyme activity of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), NADPH oxidase (NOX; EC 1.6.3.1) activity; scavenging activity of the hydroxyl radical (OH˙), hydrogen peroxide (H(2) O(2) ); endogenous ABA and malondialdehyde (MDA). The highest H(2) O(2) and MDA content was found in PEG-treated groups of both genotypes, but with more in aba2-1. ABA treatment under stress reduced the accumulation of H(2) O(2) and MDA, while it promoted activity of SOD, CAT and APX. APX activity was higher than CAT activity in ABA-treated WT and aba2-1, indicating a protective role of APX rather than CAT during osmotic stress-induced oxidative damage. Treatment with ABA also significantly induced increased NOX activity. Oxidative damage was lower in ABA-treated seedlings of both genotypes, which was associated with greater activity of SOD (Mn-SOD1 and 2 and Fe-SOD isoenzymes), CAT and APX in these seedlings after 24 h of stress. These results suggest that osmotic stress effects were overcome by ABA treatment because of increased SOD, CAT, APX and NOX.     

Susceptibility of brown adipocytes to pro-inflammatory cytokine toxicity and reactive oxygen species

Brown adipose tissue (BAT) cells have a very high oxidative capacity. On the other hand, in obesity and obesity-related diabetes, levels of pro-inflammatory cytokines are elevated, which might promote BAT dysfunction and consequently impair carbohydrate metabolism and thereby exacerbate cellular dysfunction and promote diabetes progression. Therefore, the antioxidative enzyme status of a brown adipocyte cell line and its susceptibility towards pro-inflammatory cytokines, which participate in the pathogenesis of diabetes, and reactive oxygen species (ROS) were analysed. Mature brown adipocytes exhibited significantly higher levels of expression of mitochondrially and peroxisomally located antioxidative enzymes compared with non-differentiated brown adipocytes. Pro-inflammatory cytokines induced a significant decrease in the viability of differentiated brown adipocytes, which was accompanied by a massive ROS production and down-regulation of BAT-specific markers, such as uncoupling protein 1 (UCP-1) and β-Klotho. Taken together, the results strongly indicate that pro-inflammatory cytokines cause brown adipocyte dysfunction and death through suppression of BAT-specific proteins, especially of UCP-1 and β-Klotho, and consequently increased oxidative stress.     

Mitochondrial ATP synthase subunit d,a component of the peripheral stalk,is essential for growth and heat stress tolerance in Arabidopsis thaliana

As rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name: ATPQ, AT3G52300, referred to hereafter as ATPd), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants for ATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNA interference to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed that ATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide-ranging defects in energy-demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and support the importance of complex V in normal plant growth, and provide new information about its requirement for heat stress tolerance.     

The effects of dopamine on antioxidant enzymes activities and reactive oxygen species levels in soybean roots

In the current work, we investigated the effects of dopamine, an neurotransmitter found in several plant species on antioxidant enzyme activities and ROS in soybean (Glycine max L. Merrill) roots. The effects of dopamine on SOD, CAT and POD activities, as well as H₂O₂, O₂^•−, melanin contents and lipid peroxidation were evaluated. Three-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), without or with 0.1 to 1.0 mM dopamine, in a growth chamber (25°C, 12 h photoperiod, irradiance of 280 μmol m⁻² s⁻¹) for 24 h. Significant increases in melanin content were observed. The levels of ROS and lipid peroxidation decreased at all concentrations of dopamine tested. The SOD activity increased significantly under the action of dopamine, while CT activity was inhibited and POD activity was unaffected. The results suggest a close relationship between a possible antioxidant activity of dopamine and melanin and activation of SOD, reducing the levels of ROS and damage on membranes of soybean roots.