拟南芥转录因子Ethylene-insensitive3（EIN3）抑制花青素的合成

Ethylene-insensitive3（EIN3）和 EIN3-like1（EIL1）蛋白是乙烯信号转导途径中一类重要的核转录因子。花青素是植物体中的一类水溶性天然色素,在植物的许多生理过程中起重要作用。本研究以拟南芥双突变体ein3-1eil1-3为研究材料,通过RT-PCR技术确定了拟南芥双突变体ein3-1eil1-3中EIN3和EIL1基因均已被敲除,单突变体ein3-1中的EIN3基因被敲除。通过肉眼定性观察发现突变体ein3-1eil1-3的种子和叶片内均呈紫色。通过紫外分光光度计定量分析发现,花青素积累量也明显比突变体ein3-1和野生型多。通过GUS染色发现EIN3启动子主要在花、柱头、成熟花粉、种子胚和果荚等组织中有较强的表达。这与突变体ein3-1eil1-3的种子和叶片内均呈紫色并花青素含量增高一致。因此,拟南芥转录因子EIN3可能与EIL1共同参与抑制花青素的合成。     

拟南芥H_2O_2突变体的筛选及特性分析

通过化学诱变剂甲基磺酸乙酯(EMS)诱变模式植物拟南芥(Arabidopsis thaliana)获得突变体筛选群体.在5 mmol/L H2O2胁迫下,以叶片温度差异为筛选指标,利用远红外成像技术进行突变体的筛选,获得了对H2O2不敏感突变体hpi1(hydrogen peroxide-insensitive1)和敏感突变体hps1(hydrogen peroxide-sensitive1).进一步研究发现,两种突变均为单基因隐性突变,气孔密度同野生型一样,而叶片温度、气孔开度和叶片失水率则有明显的差异.种子萌发实验表明,hpi1对甘露醇(Man)和NaCl不敏感而对ABA敏感,hps1则对3种胁迫都表现出敏感特性.     

H2S位于H2O2下游参与乙烯诱导拟南芥气孔关闭过程

H2O2和H2S是植物体内重要的信号分子,二者均参与乙烯诱导的拟南芥气孔关闭过程。以拟南芥野生型及其突变体为材料研究了H2O2和H2S在乙烯诱导拟南芥气孔关闭过程中的相互关系。结果表明,乙烯能够诱导野生型拟南芥叶片H2S含量及L-/D-半胱氨酸脱巯基酶(L-/D-CDes)活性显著增加,促进气孔关闭,但对H2O2合成突变体AtrbohD、AtrbohF、Atpao2和Atpao4植株叶片无显著作用;乙烯亦可引起H2S合成突变体Atl-cdes和Atd-cdes气孔保卫细胞H2O2水平的显著增加,但对其气孔运动没有显著作用。此外,H2O2清除剂和合成抑制剂均能抑制乙烯诱导的拟南芥叶片H2S含量和L-/D-CDes活性的增加及气孔开度的减小;而H2S清除剂和合成抑制剂虽能抑制乙烯诱导的气孔关闭,却不能改变乙烯对拟南芥叶片气孔保卫细胞H2O2的作用效应。由此表明H2S位于H2O2下游介导乙烯诱导拟南芥气孔关闭过程。     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO2浓度升高可以诱导植物叶片气孔关闭,提高植物对高浓度CO2的适应性.但植物如何感知CO2浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚.利用高通量、非侵入的远红外成像技术,建立了拟南芥(Arabidopsis thaliana)气孔对CO2浓度变化反应相关的突变体筛选技术,筛选出对环境CO2浓度敏感的拟南芥突变体ecs1.遗传学分析表明,ecs1 为单基因隐性突变体,突变基因ECS1编码一个跨膜钙离子转运蛋白.与野生型拟南芥相比,360 μL·L-1CO2可引起ecs1突变体叶片温度上升和气孔关闭,ecs1突变体对900 μL·L-1CO2长时间处理具有较强的适应性.进一步的实验表明,360 μL·L-1CO2即可诱导ecs1突变体叶片积累较高浓度的H2O2,而900 μL·L-1CO2才能够诱导野生型拟南芥叶片积累H2O2.因此,ECS1可能参与调节高浓度CO2诱导的拟南芥气孔关闭和H2O2产生,H2O2可能作为第二信号分子介导CO2诱导拟南芥气孔关闭的反应.     

活性氧不敏感型拟南芥的突变体对H2O2的响应

检测拟南芥ros突变株对H2O2响应的结果表明,此种突变体对H2O2有较强的耐受性,表现为气孔开度对H2O2不敏感和H2O2胁迫时的膜脂过氧化水平较低。采用激光扫描共聚焦显微术(LSCM)并结合H2O2荧光探针H2DCFDA检测外源ABA诱导保卫细胞的结果显示,突变体内荧光强度比野生型拟南芥低,暗示此种突变体消除H2O2的能力可能有提高,从而可增强植株抗氧化胁迫的能力。     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。     

拟南芥神经酰胺酶基因对氧化胁迫的响应

以拟南芥哥伦比亚生态型（Col）和神经酰胺酶突变体为实验材料,通过对突变体的一系列生理生化指标的测定,来研究拟南芥神经酰胺酶基因（AtCER）对H2O2的响应。利用PCR和Northern blot获得了9个AtCER纯合单突变体。不同浓度H2O2处理野生型和突变体后,发现突变体对H2O2的反应比野生型更加敏感。H2O2处理后突变体叶片出现比野生型更严重的黄化现象和坏死斑点,总叶绿素含量也比野生型下降的更快,电导率测定也发现突变体比野生型的电导率增加得更多。抗氧化酶活性的分析结果发现H2O2处理后,突变体的抗氧化酶活性比野生型提高了1．5～3倍。上述研究结果说明AtCER参与了H2O2诱导的氧化胁迫反应。     

H_2O_2介导H_2S诱导的拟南芥气孔关闭

以拟南芥（Arabidopsis thaliana）为材料,研究了过氧化氢（H2O2）在硫化氢（H2S）调控气孔运动信号转导中的作用。结果表明,光下H2S的供体硫氢化钠（NaHS）能够诱导拟南芥气孔关闭;且能够显著提高叶片和保卫细胞胞质H2O2含量;H2O2的清除剂AsA和H2O2合成酶的抑制剂可不同程度地抑制NaHS诱导的拟南芥气孔关闭及叶片和保卫细胞胞质H2O2水平的升高;NaHS对AtrbohD、AtrbohF、Atpao2和Atpao4突变体气孔关闭、叶片和保卫细胞胞质H2O2水平升高的诱导作用要明显的小于野生型,但对AtPAO2和AtPAO4过表达株系叶片和保卫细胞H2O2水平的升高较野生型显著。据此推测,来源于NADPH氧化酶、细胞壁过氧化物酶和多胺氧化酶途径的H2O2参与H2S诱导的拟南芥气孔关闭。     

H2S位于SOS上游参与盐胁迫诱导的拟南芥气孔关闭

以拟南芥野生型、SOS突变体印tsosl、Atsos2和Atsos3）、H2S合成相关酶L-／D-半胱氨酸脱巯基酶（L-／D-CDes）基因缺失突变体（Atl-cdes和Atd-cdes）和过表达株系（OEL—CDes和OED-CDes）为材料研究了H,s和SOS信号转导途径在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片H,S含量、L-／D-CDes活性及其基因表达量显著升高,诱导野生型拟南芥和OEL—CDes和OED．CDes叶片气孔关闭,但对Atl-cdes和Atd-cdes气孔开度无显著影响;而H2S清除剂次牛磺酸（hypotaurine,HT）可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明H2S参与盐胁迫诱导的拟南芥气孔关闭过程。外源H2S诱导野生型拟南芥气孔关闭,但对SOS突变体气孔开度无显著影响;同时盐胁迫下Atsosl、Atsos2和Atsos3亦表现出H2S含量及L-／D-CDes活性显著升高,且与野生型相比,盐胁迫对Atl-cdes和Atd-cdes叶片AtSOS基因表达量无显著影响。表明盐胁迫诱导气孔关闭过程中H2S位于SOS上游。     

H2S位于SOS上游参与盐胁迫诱导的拟南芥气孔关闭

以拟南芥野生型、SOS突变体(Atsos1、Atsos2和Atsos3)、H2S合成相关酶L-/D-半胱氨酸脱巯基酶(L-/D-CDes)基因缺失突变体(Atl-cdes和Atd-cdes)和过表达株系(OEL-CDes和OED-CDes)为材料研究了H2S和SOS信号转导途径在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片H2S含量、L-/D-CDes活性及其基因表达量显著升高,诱导野生型拟南芥和OEL-CDes和OED-CDes叶片气孔关闭,但对Atl-cdes和Atd-cdes气孔开度无显著影响;而H2S清除剂次牛磺酸(hypotaurine,HT)可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明H2S参与盐胁迫诱导的拟南芥气孔关闭过程。外源H2S诱导野生型拟南芥气孔关闭,但对SOS突变体气孔开度无显著影响;同时盐胁迫下Atsos1、Atsos2和At-sos3亦表现出H2S含量及L-/D-CDes活性显著升高,且与野生型相比,盐胁迫对Atl-cdes和Atd-cdes叶片AtSOS基因表达量无显著影响。表明盐胁迫诱导气孔关闭过程中H2S位于SOS上游。     

Arabidopsis UVH3 gene is a homolog of the Saccharomyces cerevisiae RAD2 and human XPG DNA repair genes

To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype. The uvh3 locus was mapped within chromosome III to the GL1 locus. A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants. Cosmid N9 was found to confer UV-resistance, H2O2-resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation. Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV-damage. The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product. In humans, the homologous XPG protein is also involved in removal of oxygen-damaged nucleotides by base excision repair. We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues. Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues.     

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     

Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens

Reactive oxygen species (ROS) are responsible for mediating cellular defense responses in plants. Controversy has existed over the origin of ROS in plant defense. We have isolated a novel extracellular peroxidase gene, CaPO2, from pepper (Capsicum annuum). Local or systemic expression of CaPO2 is induced in pepper by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection. We examined the function of the CaPO2 gene in plant defense using the virus-induced gene silencing technique and gain-of-function transgenic plants. CaPO2-silenced pepper plants were highly susceptible to Xcv infection. Virus-induced gene silencing of the CaPO2 gene also compromised hydrogen peroxide (H(2)O(2)) accumulation and hypersensitive cell death in leaves, both locally and systemically, during avirulent Xcv infection. In contrast, overexpression of CaPO2 in Arabidopsis (Arabidopsis thaliana) conferred enhanced disease resistance accompanied by cell death, H(2)O(2) accumulation, and PR gene induction. In CaPO2-overexpression Arabidopsis leaves infected by Pseudomonas syringae pv tomato, H(2)O(2) generation was sensitive to potassium cyanide (a peroxidase inhibitor) but insensitive to diphenylene iodonium (an NADPH oxidase inhibitor), suggesting that H(2)O(2) generation depends on peroxidase in Arabidopsis. Together, these results indicate that the CaPO2 peroxidase is involved in ROS generation, both locally and systemically, to activate cell death and PR gene induction during the defense response to pathogen invasion.     

Carbonylation and Loss-of-Function Analyses of SBPase Reveal Its Metabolic Interface Role in Oxidative Stress, Carbon Assimilation, and Multiple Aspects of Growth and Development in Arabidopsis

Sedoheptulose-1,7-bisphosphatase (SBPase) is a Calvin cycle enzyme and functions in photosynthetic carbon fixation. We found that SBPase was rapidly carbonylated in response to methyl viologen (MV) treatments in detached leaves of Arabidopsis plants. In vitro activity analysis of the purified recombinant SBPase showed that SBPase was carbonylated by hydroxyl radicals, which led to enzyme inactivation in an H(2)O(2) dose-dependent manner. To determine the conformity with carbonylation-caused loss in enzymatic activity in response to stresses, we isolated a loss-of-function mutant sbp, which is deficient in SBPase-dependent carbon assimilation and starch biosynthesis. sbp mutant exhibited a severe growth retardation phenotype, especially for the developmental defects in leaves and flowers where SBPASE is highly expressed. The mutation of SBPASE caused growth retardation mainly through inhibition of cell division and expansion, which can be partially rescued by exogenous application of sucrose. Our findings demonstrate that ROS-induced oxidative damage to SBPase affects growth, development, and chloroplast biogenesis in Arabidopsis through inhibiting carbon assimilation efficiency. The data presented here provide a case study that such inactivation of SBPase caused by carbonyl modification may be a kind of adaptation for plants to restrict the operation of the reductive pentose phosphate pathway under stress conditions.     

Rice tillering dwarf mutant dwarf3 has increased leaf longevity during darkness-induced senescence or hydrogen peroxide-induced cell death

Senescence or cell death in plant leaves is known to be inducible by darkness or H(2)O(2). When the Arabidopsis gene MAX2/ORE9 is disrupted, leaf senescence or cell death in response to the above stimuli is delayed. Because the rice (Oryza sativa L.) gene DWARF3 (D3) is orthologous to MAX2/ORE9, we wished to know whether disruption of D3 also results in increased longevity in leaves. We found that darkness-induced senescence or H(2)O(2)-induced cell death in the third leaf [as measured by chlorophyll degradation, membrane ion leakage and expression of senescence-associated genes (SAGs)] in a d3 rice mutant was delayed by 1-3 d compared to that in its reference line Shiokari. Moreover, the mRNA levels of D3, HTD1 and D10, which are orthologs of Arabidopsis MAX2/ORE9, MAX3 and MAX4, respectively, increased during cell death. These results suggest that D3 protein in rice, like MAX2/ORE9 in Arabidopsis, is involved in leaf senescence or cell death.