不同生境两种生态型芦苇叶片质膜H+-ATPase的比较

利用两相法化纯化质膜微囊,研究了分布西北沙地区的两种生态型芦苇(Phragmites communis trih.)水生芦苇和重度盐化草甸芦苇,分别简称为水芒和盐芦)叶片质膜H - ATPase的部分性质.结果显示,与水芦相比,盐芦质膜H -ATPase的ATP水解活性升高,Km值由1.27mmol\l降至Vmax没有显著差异.并且该酶活性对温度的敏感必玫PH谱型也发生了变化.以对硝基苯磷酸盐为底物,低浓度时盐芦的的质膜H -ATPase水解活性有差异.钡酸盐抑制实验表明,两种生态的质膜H -ATPase磷酸-酶区的催化性质不同.胰酶对质膜H -ATPase活性的活化谱型也存在差异,说明该酶C末端的结构或性质发生了变化.此外,与水芦相比,盐芦质膜H -ATPase的质子泵活性的耦联程度也升高了.以上结果明,当芦苇从水生环境向盐渍环境过渡时,质膜H -ATPase的催化性质发生了变化,这些变化可能是由酶结构的修饰和不同的同工酬酶谱引起的.H -ATPase催化性质的变化可能是对盐渍生境的适应性反应.     

不同生境两种生态型芦苇的抗氧化系统

以分布于甘肃临泽平川乡的两种芦苇生态型——水生芦苇(水芦)和重度盐化草甸芦苇(盐芦)叶片为材料,研究了其抗氧化系统的特征。结果表明,与水芦相比,盐芦中未出现活性氧和MDA(丙二醛)的积累,抗氧化酶SOD(超氧化物歧化酶)、CAT(过氧化氢酶)、POD(过氧化物酶)和APX(抗坏血酸过氧化物酶)的活性显著升高。总抗坏血酸和类胡萝卜素含量在两种生态型芦苇中没有差异,但还原型抗坏血酸和总谷胱甘肽含量在盐芦中显著升高。而且,盐芦的LOX(脂氧合酶)活性比水芦低。这些结果表明,盐芦中有效的抗氧化防御系统对抵抗盐渍胁迫起着重要的作用。此外,盐芦中高活性的Ca^2 -和Mg^2 -ATPase对细胞中过多离子的转运以及避免离子毒害起着重要的作用。     

盐胁迫对豌豆根液泡膜H^＋—ATPase活性及含量的影响

为了阐明液泡膜H^ -ATPase在盐胁迫下的作用和适应性调节机制,对豌豆（Pisum sativum L.）植株进行不同盐浓度和不同盐胁迫时间（1－3d）的处理后,分别测定液泡膜H^ -ATPase的H^ 转运活性、水解性和蛋白含量（A亚基）的变化。结果表明,100mmol/L和200mmol/L NaCl 处理1dH^ -ATPase的水解活性没有变化,而250mmol/L NaCl处理1d引起水解活性降低约25％。100mmol/L NaCl处理2d内水解活性没有变化,而第3天活性下降约20％。但是上述盐胁迫均能提高液泡膜H^ -ATPase的质子转运活性,说明盐胁迫后H^ -ATPase的水解活性和质子转运活性的变化不成比例,盐胁迫可能导致偶联比率的改变。Western blot研究发现,上述盐胁迫对液泡膜H^ -ATPase（A亚基）的含量基本无影响,仅100mmol/L NaCl处理3d后A亚基的量略有下降,这些结果证明,盐胁迫能刺激提高豌豆根液泡膜H^ -ATPase的H^ 泵效率,且泵效率的提高是源于偶联比率的改变,而不是由于ATP水解活性的提高和蛋白含量的增加。     

胡杨愈伤组织质膜的两相分离法及其H＋-ATPase的特性

以胡杨愈伤组织为材料,用PEG 3350/DextranT 500构成的两相系统提取质膜微囊,研究质膜H+-ATPase的特性.结果显示由6.3% PEG 3350、6.3% Dextran T500、KCl、磷酸缓冲液(pH 7.8)和蔗糖构成的两相系统提取膜微囊的H+-ATPase活性分别被Na3VO4、KNO3、NaN3抑制了约75%、2.6%和1.3%.方向性检测显示原位膜微囊占提取质膜微囊的90%,翻转膜微囊仅占10%.去垢剂对质膜H+-ATPase活性的影响说明0.015%的Triton X-100和0.01%～0.1%的Brij 58适用于测定质膜H+-ATPase活性.Lineweaver-Burk动力学分析该酶的Km值为0.65 mmol*L-1,Vmax为37.59 μmol Pi*mg-1 protein*h-1.研究结果表明两相法提取的质膜微囊主要是正向密闭的膜微囊;胡杨愈伤组织质膜H+-ATPase的最适pH为6.5,最适温度为37℃左右.     

两种生态型芦苇叶绿体的光合电子传递和抗氧保护体系

分布于甘肃省河西走廓的两种不同生态型芦苇－水生芦苇（水芦,生长在深约１ｍ的水滩里）和沙丘芦苇（沙芦,生长在高约５ｍ的沙丘上）在其离体叶绿体的光化学活性上表现为前者高于后者。其中,沙芦全链电子传递速率及光系统Ⅱ（ＰＳⅡ）电子传递速率明显低于水芦,而光系统Ⅰ（ＰＳⅠ）电子传递速率却与水芦接近。沙芦叶片和叶绿体中抗氧化酶活性及叶片抗坏血酸含量均高于水芦。综合结果表明,喜水植物芦苇登陆后,在自然选择压力下     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H^＋-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响。结果表明,0.1 mmol/L SNP处理显著缓解了150 mmol/L NaCl 胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性。进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜 H -ATPase和焦磷酸酶活性诱导有关。此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响。应用外源CaSO4 和 EGTA 处理也证实,Ca2 可能在NO诱导的质膜 H -ATPase和焦磷酸酶活性的提高过程中起信号作用。另外,分析盐胁迫下小麦幼苗根部 Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一。     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H+-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响.结果表明,0.1 mmol/L SNP处理显著缓解了1 50 mmol/L NaCl胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性.进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜H -ATPase和焦磷酸酶活性诱导有关.此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响.应用外源CaSO4和EGTA处理也证实,Ca2 可能在NO诱导的质膜H -ATPase和焦磷酸酶活性的提高过程中起信号作用.另外,分析盐胁迫下小麦幼苗根部Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一.     

NaCl胁迫下大麦根液泡膜H+-ATPase活性、离子吸收与钙的关系

NaCl胁迫2 d,耐盐大麦(Hordeum vulgare L.cv) ("滩引2号")根系液泡膜H+-ATPase活性增强,H+-PPase活性下降.以质膜Ca2+通道抑制剂La3+ (1 mmol/L)或Ca2+螯合剂EGTA (5 mmol/L)处理大麦幼苗,抑制了NaCl诱导的液泡膜H+-ATPase活性的增强,但提高了H+-PPase活性;用CaM拮抗剂三氟拉嗪(TFP,20 μmol/L)处理,也抑制了液泡膜H+-ATPase活性的增强.NaCl胁迫下,外加La3+,TFP或La3++TFP处理,使Na+吸收增加,K+和Ca2+吸收降低.结果表明,NaCl胁迫下,液泡膜H+-ATPase活性提高和离子吸收的变化可能与Ca-CaM系统有关.     

等渗盐胁迫对番茄抗氧化酶和ATP酶及焦磷酸酶活性的影响

用Ca(NO3)2 80 mmol/L和NaCl 120 mmol/L等渗溶液处理番茄幼苗后,细胞质和叶绿体中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)的活性升高,并且NaCl胁迫的作用明显高于Ca(NO3)2胁迫.Ca(NO3)2处理提高了线粒体中SOD、CAT、APX的活性,而NaCl处理降低了它们的活性.根系质膜H -ATPase、液泡膜H -ATPase、焦磷酸酶(H -PPase)的活性和叶片丙二醛(MDA)及脯氨酸含量在两种盐胁迫后明显增加.NaCl处理对植株生长的抑制程度明显高于Ca(NO3)2处理.     

胡杨愈伤组织质膜的两相分离法及其H~+-ATPase的特性

以胡杨愈伤组织为材料,用PEG3350/DextranT500构成的两相系统提取质膜微囊,研究质膜H+-AT-Pase的特性。结果显示:由6.3%PEG3350、6.3%DextranT500、KCl、磷酸缓冲液(pH7.8)和蔗糖构成的两相系统提取膜微囊的H+-ATPase活性分别被Na3VO4、KNO3、NaN3抑制了约75%、2.6%和1.3%。方向性检测显示原位膜微囊占提取质膜微囊的90%,翻转膜微囊仅占10%。去垢剂对质膜H+-ATPase活性的影响说明0.015%的TritonX-100和0.01%～0.1%的Brij58适用于测定质膜H+-ATPase活性。Lineweaver-Burk动力学分析该酶的Km值为0.65mmol·L-1,Vmax为37.59μmolPi·mg-1protein·h-1。研究结果表明:两相法提取的质膜微囊主要是正向密闭的膜微囊;胡杨愈伤组织质膜H+-ATPase的最适pH为6.5,最适温度为37℃左右。     

Comparison of Plasma Membrane H^＋-ATPase Activity in Two Ecotypes of Reed （Phragmites communis） Leaves from Different Habitats

Plasma membrane (PM) vesicles of the leaves of two ecotypes of reed (Phragrnites communis Trin.), swamp reed (SR) and heavy salt meadow reed (HSMR) growing in the desert region of Northwest China, were purified by two-phase partitioning and the properties of their PM H^ -ATPases (EC 3.6.1.35) were compared. The specific activity of this enzyme was greater in HSMR than in SR and the Km lower (1.27mmol/L in SR and 0.30mmol/L in HSMR), and the Vmax of ATP hydrolysis activity showed no significant difference between the two ecotypes. The PM H^ -ATPase was more sensitive to denaturing temperatures in HSMR than in SR, and the pH profile also showed a slight difference, suggesting that the catalytic mechanism of this enzyme was different in HSMR compared with that in SR. The p-nitrophenyl phosphate (PNPP) hydrolysis activity of H^ -ATPase was higher in HSMR than in SR at low concentrations of PNPP, but showed no difference at high PNPP concentration. The Km for PNPP hydrolysis was 3.61mmol/L and 1.92mmol/L in SR and HSMR, respectively. And the Vmax of PNPP hydrolysis showed no significant difference in the two reed ecotypes. An experiment with the inhibitor vanadate showed that the catalytic mechanisms of the phosphatase domain of the ATPase were different in the two ecotypes. The data obtained following trypsin treatment showed a difference in the enzyme activity pattern, suggesting that there existed a possible change in the C-terminus of the ATPase, either in the structure or in the property or both of them. In addition, compared with SR, the ATP-dependent H^ pumping activity of ATPase and the coupling between proton transport and ATP hydrolysis in HSMR were increased. These results indicated that the properties of PM H^ -ATPase were changed in HSMR with compared to those in SR, which might include enzyme modifications and different isoforms expressed. The alterations of the properties of this enzyme might be an adaptive response to the habitat.     

Regulation of the structure and catalytic properties of plasma membrane H<Superscript>+</Superscript>-ATPase involved in adaptation of two reed ecotypes to their different habitats

The properties and kinetics of ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activities of plasma membrane H⁺-ATPase from the two reed ecot ypes, swamp reed (SR) and dune reed (DR), were investigated. The pH optimum of the plasma membrane H⁺-ATPase in both reed ecotypes was similar but the sensitivity of the enzyme to the reaction medium pH seemed to be higher in DR than that in SR. Compared to SR, the DR exhibited a higher V_max value for ATP hydrolysis whereas the K_m value was almost similar in both reed ecotypes. The PNPP hydrolysis of the plasma membrane H⁺-ATPase was also studied in both reed ecotypes at increasing PNPP concentrations. K_m and V_max for PNPP hydrolysis showed great differences in the two reed ecotypes and in DR the K_m and V_max values were 2- and 10-fold, respectively, higher than those in SR. The ATP hydrolysis activity of the plasma membrane was markedly inhibited by hydroxylamine in both reed ecotypes, and the percentage inhibition of ATP hydrolysis rate seemed higher in DR than that in SR. In addition, the structure or property of the C-terminal end of the plasma membrane H⁺-ATPase were also different in the two reed ecotypes. These data suggest that different isoforms of the plasma membrane H⁺-ATPase might be developed and involved in the adaptation of the plant to the long-term drought-prone habitat.This research was supported by Natural Science Foundation of China (No. 30270238 & No. 30470274) and the National Key Basic Research Special Funds of China (G1999011705).     

磷饥饿时番茄幼苗根系质膜H+-ATP酶活性

磷饥饿提高了番茄幼苗质膜H+-ATP酶活性并促进了番茄幼苗根部的H+分泌。动力学分析表明,磷饥饿使番茄幼苗根部质膜H+-ATP酶的Km值明显降低,亦即提高了该酶对其底物的亲和力,但对该酶的Vmax影响不大。另外,磷饥饿并不改变ATP酶的最适pH值(最适pH值为6.5)。钒酸盐显著抑制番茄幼苗根部质膜ATP酶的活性以及H+分泌,也显著抑制番茄幼苗的Pi吸收。与对照相比,上述抑制作用在饥饿处理的植物中表现得更强。以上结果表明,磷饥饿时高亲和性Pi传递系统的诱导很可能包含质膜H+-ATP酶的参与。     

Effect of Lysophosphatidylcholine on ATP and ρ-Nitrophenyl Phosphate Hydrolysis by the Plasma Membrane H+-ATPase from Soybean Hypocotyls

The stimulatory effect of lysophosphatidylcholine (lyso-PC) on ATP and ρ-nitrophenyl phosphate (PNPP) hydrolysis by the plasma membrane H+-ATPase from soybean (Glycine max (L.) Merr.) hypocotyls was studied. Results showed that lyso-PC stimulated the hydrolysis of ATP; ATP hydrolysis was enhanced dramatically when lyso-PC was within 0-0.03%, and increased slightly when lyso-PC was higher than 0.03%. At the concentration of 0.03%, lyso-PC stimulated ATP hydrolysis by 80.5%. Kinetics analysis showed that V max increased from 0.46μmol Pi·mg-1 protein·min-1 to 0.87 μmol Pi·mg-1 protein·min-1 while Km increased from 0.88 mmol/L to 1.15 mmol/L under lyso-PC treatment. The optimum pH of ATP hydrolysis was shifted from 6.5 to 7.0. Moreover, it was found lyso-PC enhanced the inhibition of ATP hydrolysis by hydroxylamine. In the presence of 200 mmol/L hydroxylamine, ATP hydrolysis was inhibited by 74.4%, while it was inhibited by 84.4% when treated with lyso-PC. However, PNPP hydrolysis and the inhibitory effect of vanadate were not affected by lyso-PC. The above results indicated that the kinase domain might be an action site or regulatory region of the C-terminal autoinhibitory domain in the plant plasma membrane H+-ATPase.     

Antioxidant defense system in <Emphasis Type="Italic">Phragmites communis</Emphasis> Trin. ecotypes

The antioxidant defense system in three ecotypes of reed (Phragmites communis Trin.), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), from northwest China were investigated. The HSMR possessed the highest ratio of ascorbate (ASC)/dehydroascorbate (DHA) and activities of superoxide dismutase (SOD) and catalase among the three reed ecotypes, whereas, the DR exhibited the highest ratio of glutathione/glutathione disulfide and activities of ASC peroxidase (APX) and DHA reductase. Malondialdehyde and hydrogen peroxide contents were highest in HSMR, intermediate in SR, and lowest in DR. In addition, different isoenzymes of glutathion reductase, APX, SOD and DHA were also observed in three reed ecotypes.     

Differential expression of photosynthesis-related genes of reed ecotypes in response to drought and saline habitats

Three ecotypes of reed (Phragmites communis Trinius), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), growing in desert regions of northwest China were simultaneously investigated in their natural state for gas exchange patterns and the expression of three photosynthesis-related genes, cab (the gene for the light-harvesting chlorophyll a/b binding protein, LHC), psbA (the gene for the reaction centre D1 protein of photosystem 2, PS2), and 16S rDNA (the gene for plastid 16S rRNA). Stomatal conductance (gs) and intercellular CO2 concentration (ci) were markedly lower in the two terrestrial ecotypes (DR and HSMR) as compared to SR, paralleling a similar observed depression in net photosynthetic rate (PN). However, DR with the lowest measured gs and ci still exhibited a higher PN compared to HSMR. These results suggest that both stomatal and non-stomatal factors account for the comparatively low carbon assimilation in the terrestrial ecotypes. An increase in the expression of photosynthesis-related genes was observed in DR compared to SR, whereas the reverse situation was true in HSMR. The expression of photosynthesis-related genes may contribute to reed plants' photosynthetic capacity per leaf area under natural water deficits, but the levels of photosynthesis-related gene expression are not directly correlated with reed plants' general ability for survival and adaptation under water deficient conditions.     

Leaf anatomy and C4 photosynthetic enzymes in three reed ecotypes

Differences in leaf interveinal distances, chloroplasts distribution in bundle sheath cells (BSC) and activities of C₄ photosynthetic enzymes in the leaves of three ecotypes of Phragmites communis Trinius, namely swamp reed (SR), heavy salt meadow reed (HSMR) and dune reed (DR), occurring in the desert region of northwest China were investigated. The two terrestrial ecotypes, DR and HSMR, had denser vascular system, more and longer BSC chloroplasts and higher capacity of CO₂ concentrating mechanism of NAD-ME subtype as compared with the SR ecotype. The enhanced NADP-ME pathway in the HSMR might contribute to its adaptation to the salinity habitat.     

Nitric oxide functions as a signal in salt resistance in the calluses from two ecotypes of reed

Calluses from two ecotypes of reed (Phragmites communis Trin.) plant (dune reed [DR] and swamp reed [SR]), which show different sensitivity to salinity, were used to study plant adaptations to salt stress. Under 200 mm NaCl treatment, the sodium (Na) percentage decreased, but the calcium percentage and the potassium (K) to Na ratio increased in the DR callus, whereas an opposite changing pattern was observed in the SR callus. Application of sodium nitroprusside (SNP), as a nitric oxide (NO) donor, revealed that NO affected element ratios in both DR and SR calluses in a concentration-dependent manner. N(omega)-nitro-l-arginine (an NO synthase inhibitor) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (a specific NO scavenger) counteracted NO effect by increasing the Na percentage, decreasing the calcium percentage and the K to Na ratio. The increased activity of plasma membrane (PM) H(+)-ATPase caused by NaCl treatment in the DR callus was reversed by treatment with N(omega)-nitro-l-arginine and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde. Western-blot analysis demonstrated that NO stimulated the expression of PM H(+)-ATPase in both DR and SR calluses. These results indicate that NO serves as a signal in inducing salt resistance by increasing the K to Na ratio, which is dependent on the increased PM H(+)-ATPase activity.