Studies on the relationship between cyanide-resistant respiration and expression of alternative oxidase in mung bean using antibodies prepared by synthetic polypeptide

Twelve peptides, including eight conservative amino acid residues in the amino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled with α-chymotrypsinogen, and the antibodies were obtained through immunizing domestic rabbit by injecting this complex. By using these antibodies, which were raised to immunoreact with total proteins of purified mitochondria from different organs of mung bean seedlings, we find that there are two hybridizable AOX bands in mitochondria. Their molecular weights are about 35 and 38 ku, respectively. Moreover, the respiratory parameters of hypocotyl, true leaf and cotyledon of mung bean seedlings show that true leaf has the highest total respiration (Vt), alternative pathway (AP) capacity (Valt) and the activity of AP (ρValt) among the three organs. Vt and ρValt of cotyledon ranked the second. Hypocotyl has the lowest V, and ρValt, but its Valt is higher than that of cotyledon. These result     

Studies on the relationship between cyanide-resistant respiration and expression of alternative oxidase in mung bean using antibodies prepared by synthetic polypeptide

Twelve peptides, including eight conservative amino acid residues in the amino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled with α-chymotrypsinogen, and the antibodies were obtained through immunizing domestic rabbit by injecting this complex. By using these antibodies, which were raised to immunoreact with total proteins of purified mitochondria from different organs of mung bean seedlings, we find that there are two hybridizable AOX bands in mitochondria. Their molecular weights are about 35 and 38 ku, respectively. Moreover, the respiratory parameters of hypocotyl, true leaf and cotyledon of mung bean seedlings show that true leaf has the highest total respiration (V_t), alternative pathway (AP) capacity (V_alt) and the activity of AP (ρV_alt) among the three organs. V_t andρV _alt of cotyledon ranked the second. Hypocotyl has the lowest V_t andρV _alt, but its V_alt is higher than that of cotyledon. These results are consistent with the analysis of Western blotting of expression of AOX. The highest V_t andρV _alt in true leaf are accompanied two hybridizable polypeptides of AOX protein, 35 ku and 38 ku respectively. The next is cotyledon V_t andρV _alt with only one 38 ku hybridizable polypeptide of AOX protein. HypocotylV _t andρV _alt is the lowest and its immunoblotting band is similar to that of cotyledon, but the expressive amount of 38 ku protein is less than that of cotyledon. The results suggest that the 35 ku AOX may contribute mainly to true leafρV _alt.     

Effects of Enhanced UV-B Radiation on the Activity and Expression of Alternative Oxidase in Red Kidney Bean Leaves

An increase in ultraviolet(UV)B radiation on the earth's surface is a feature of current global climate changes.It hasbeen reported that alternative oxidase(AOX)may have a protective role against oxidative stress induced by environmentalstresses,such as UV-B.To better understand the characteristic tolerance of plants to UV-B radiation,the effects ofenhanced UV-B radiation on the activity and expression of AOX in red kidney bean(Phaseolus vulgaris)leaves wereinvestigated in the present study.The results show that the total respiration rate and AOX activity in red kidney bean leavesincreased significantly during treatment with enhanced UV-B.However,cytochrome oxidase(COX)activity did not changesignificantly.The H_2O_2 content was also markedly increased and reached a maximum of 4.45 mmol.L~(-1)·g~(-1)DW(dry weight)at 24 h of UV-B treatment,before dropping rapidly.Both alternative pathway content and alternative pathway activity wereincreased in the presence of exogenous H_2O_2.Immunoblotting analysis with anti-AOX monoclonal antibody revealed thatexpression of the AOX protein increased in red kidney bean leaves under enhanced UV-B radiation,reaching a peak at 72h.In addition,AOX expression in red kidney bean leaves was induced by exogenous H_2O_2.These data indicate that theincrease in AOX activity in red kidney bean leaves under enhanced UV-B radiation was mainly due to H_2O_2-induced AOXexpression.     

Effects of Enhanced UV-B Radiation on the Activity and Expression of Alternative Oxidase in Red Kidney Bean Leaves

An increase in ultraviolet （UV） B radiation on the earth＇s surface is a feature of current global climate changes. It has been reported that alternative oxidase （AOX） may have a protective role against oxidative stress induced by environmental stresses, such as UV-B. To better understand the characteristic tolerance of plants to UV-B radiation, the effects of enhanced UV-B radiation on the activity and expression of AOX in red kidney bean （Phaseolus vulgaris） leaves were investigated in the present study. The results show that the total respiration rate and AOX activity in red kidney bean leaves increased significantly during treatment with enhanced UV-B. However, cytochrome oxidase （COX） activity did not change significantly. The H2O2 content was also markedly increased and reached a maximum of 4.45 mmol·L^-1·g^-1 DW （dry weight） at 24 h of UV-B treatment, before dropping rapidly. Both alternative pathway content and alternative pathway activity were increased in the presence of exogenous H2O2. Immunoblotting analysis with anti-AOX monoclonal antibody revealed that expression of the AOX protein increased in red kidney bean leaves under enhanced UV-B radiation, reaching a peak at 72 h. In addition, AOX expression in red kidney bean leaves was induced by exogenous H2O2. These data indicate that the increase in AOX activity in red kidney bean leaves under enhanced UV-B radiation was mainly due to H2O2-induced AOX expression.     

表油菜素内酯对绿豆上胚轴内源IAA及其氧化酶的影响

用0.5ppm表油菜素内酯处理绿豆幼苗,显著促进上胚轴伸长生长,若切除真叶则可抑制表油菜素内酯诱导的效应。三碘苯甲酸(TIBA)也可抑制表油菜素内酯促进的伸长生长。外源IAA能部分恢复TIBA的抑制效应。经处理的上胚轴内源IAA含量明显高于对照。暗示表油菜素内酯可能通过对内源IAA的调节来促进绿豆上胚轴的伸长生长。 表油菜素内酯处理的绿豆上胚轴组织中,与生长素降解密切相关的IAA氧化酶以及过氧化物酶活性均明显低于对照。     

交替氧化酶结构和功能研究进展

交替氧化酶(a lternative ox idase,AOX)是植物线粒体呼吸链中抗氰呼吸途径(cyan ide-res istan t resp irationpathw ay)的末端氧化酶,它广泛存在于高等植物及部分真菌和藻类中。交替氧化酶是一种双铁羧基蛋白(d i-ironcarboxy late prote in),它不仅具有其它双铁羧基蛋白共有的结构特点以及去除分子氧的功能,更重要的是它还可以通过改变自身结构等方式来主动调节抗氰呼吸途径的运行程度,进而调节细胞多方面的代谢和功能,以适应环境条件的改变,增强植物适应各种逆境的能力,调节植物生长速率,并与细胞凋亡和光合作用相关。本文主要对交替氧化酶的结构与其在植物体内功能的最新研究进展进行综述。     

Mutants of the Phytopathogenic FungusMagnaporthe griseaDeficient in Alternative, Cyanide-Resistant, Respiration

The phytopathogenic fungusMagnaporthe griseahas a cyanide-resistant respiratory pathway. The fungicide SSF-126 ((E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl) acetamide) blocks the cytochrome electron transport ofM. griseaand induces the alternative respiratory pathway. Twelve mutants ofM. griseamore susceptible to SSF-126 than wild type were identified afterN-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. Five mutants retained a reduced alternative respiration activity, and seven mutants lacked alternative pathway activity. A monoclonal antibody against the maize alternative oxidase cross-reacted against a 40-kDa mitochondrial protein ofM. grisea,indicating that the 40-kDa protein is an alternative oxidase. Immunoblot analysis indicated that the seven completely deficient mutants grouped into two classes: four mutants produced the 40-kDa proteins while the other three mutants failed to produce the functional protein.     

光质对绿豆幼苗叶片超微弱发光及叶绿素含量的影响

以绿豆幼苗为试材,测定其叶片超弱发光(UBE)及叶绿素含量在不同光质条件下的变化,并探讨两者之间的关系.结果表明,生长在不同光质下绿豆幼苗叶片的UBE及延迟发光衰减参数1/P都随着其生长不断增强,且生长在白光下绿豆幼苗的UBE是生长在其他光质(红、黄、蓝、绿)下幼苗的2倍以上,而红光、黄光和绿光处理之间无显著差异;生长在白光下的绿豆幼苗叶片叶绿素含量显著高于红、黄、蓝、绿光处理幼苗,而红光和黄光处理又显著高于蓝光和绿光处理.研究发现,光质对绿豆幼苗叶片超弱发光和叶绿素含量影响相似,绿豆幼苗叶片超弱发光可能与叶绿体的发育和光合作用有关.     

黄化水稻幼苗转绿期AOX1基因家族的表达与功能分析

完全黄化的水稻幼苗叶片在持续光照下总呼吸速率、交替途径的速率以及交替途径在总呼吸中的比值均上升,但以水稻AOX1基因家族3个成员的特异性片段为探针,仅观察到其中AOX1c转录本的增加。交替途径的专一性抑制剂SHAM可以降低水稻幼苗在持续光照过程中的相对光合放氧速率与叶绿素含量。同时,水稻黄化幼苗光照前黑暗处理时间越长,在恢复光照后交替途径能力的增加越显著,表现了转绿进程与交替途径之间的相关性。推测加强交替途径可能是植物缓和能量和物质需求矛盾的一个重要调控机制。     

The Alternative Oxidase of Yarrowia lipolytica Mitochondria Is Unable To Compete with the Cytochrome Pathway for Electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Yarrowia lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Intracellular cAMP Content and the Induction of Alternative Oxidase in the Yeast Yarrowia lipolytica

The effect of cyanide, antimycin A, ethanol, and acetate on the induction of alternative oxidase in the yeast Yarrowia lipolytica VKM Y-155 was studied. The aerobic incubation of logarithmic-phase cells, whose respiration is sensitive to cyanide, in the presence of the aforementioned compounds led to the development of cyanide-resistant respiration, which could be suppressed by benzohydroxamic acid, an inhibitor of alternative oxidases. The incubation of cells with cyanide, ethanol, or acetate raised the intracellular pool of cAMP, which attained maximal values after a 2- to 3-min incubation period, then rapidly decreased to the initial value and did not change over the next three hours of incubation. The possible role of cAMP in the induction of alternative oxidase in yeast cells is discussed.     

The Cyanide-resistant Respiration in Callus of Nicotiana rustica cv. Gansu Yellow Flower Under Low Temperature

The increasement of the operation (ρValt) and the capacity of the alternative pathway (Valt) in tobacco (Nicotiana rustica L. cv. Gansu Yellow Flower) callus that treated by low temperature (4 ℃) were observed. The two electron transport pathways were changed alternatively, the cyanide-resistant respiration was the main respiratory pathway from the 2nd day to the 5th day, while the cytochrome pathway was the main pathway consistently in control callus. Cycloheximide and chloramphenicol treatment which inhibited protein synthesis resulted in dramatic and partly inhibition of Valt induced by low temperature respectively. This further proved above result that low temperature induced Valt. The same result was found when two inhibitors were used together. The analysis of heat production of callus indicated that the operation of the alternative pathway could be regulated to improve the micro-environmental temperature of tobacco callus to adapt to low temperature. The possible participation of the alternative pathway in the response to low temperature and its physiological roles were discussed.     

单克隆抗体和抗合成多肽抗体检测烟草愈伤组织中交替氧化酶表达的比较

单克隆抗体和抗合成多肽抗体检测烟草愈伤组织中交替氧化酶表达的比较 晏婴才 林宏辉* 梁厚果 杜林方 （四川大学生命科学学院,成都610064I）     

番茄交替氧化酶基因的克隆和表达

利用简并PCR扩增产物做探针筛选番茄cDNA基因文库获得一个全长交替氧化酶cDNA基因LeAoxlau.经序列分析得出,该基因全长1 418bp,编码区序列长1 077 bp,编码约40 kD的前体蛋白.该蛋白在转运到线粒体时被加工成32kD的成熟蛋白.Southern印迹杂交分析结果显示该基因以单拷贝形式存在于番茄的基因组中RT-PCR显示,该基因在在番茄植株的根、茎、叶和子叶中表达.重组表达实验表明该基因能在大肠杆菌中表达.     

表油菜素内酯对绿豆上胚轴核酸代谢的影响

0.5～5 ppm放线菌素D和0.01～0.1ppm亚胺环己酮均能抑制epiBR促进的绿豆上胚轴的生长。DNA、RNA含量测定表明epi—BR处理促进了它们的含量增加。绿豆上胚轴经 epiBR处理48h后,可增强其RNA聚合酶活性(以~H—UTP掺入RNA的DPM计算);DNA和RNA水解酶的活性随着epiBR处理时间的延长而逐渐降低;TIBA则抑制epiBR的上述作用。     

NaCl胁迫下SA浸种绿豆幼苗的生长及生理特征

以'中绿一号'绿豆品种为材料,对不同浓度水杨酸(SA)浸种绿豆种子在NaCl胁迫条件下的萌发、幼苗生长及相关生理指标变化进行分析.结果显示:(1)与未胁迫对照相比,未浸种对照绿豆在100～500 mmol·L-1 NaCl处理下的发芽率、芽长、根长和叶片叶绿素含量显著降低,而幼苗叶片丙二醛(MDA)与脯氨酸(Pro)含量水平显著上升(P＜0.05),且其升降幅度随NaCl胁迫浓度提高而增加;(2)与未浸种对照相比,未胁迫对照种子的萌发和幼苗的生长在20 mg·L-1 SA处理中受到抑制,而在40～80 mg·L-1 SA处理条件下得到促进,至100 mg·L-1 SA时又受到显著抑制.(3)适当浓度的SA浸种能够显著提高盐胁迫下绿豆幼苗的芽长、根长、叶绿素的含量,降低了MDA和Pro含量;在NaCl胁迫浓度为100～300 mmol·L-1时的SA适宜浓度浸种为60 mg·L-1,而500 mmol·L-1 NaCl时为80 mg·L-1.研究表明,适当浓度的SA浸种能有效缓解盐胁迫对绿豆幼苗的伤害,提高其耐盐性.     

NaCl胁迫下交替呼吸途径对叶绿素含量及其荧光特性的影响

以菜豆幼苗作为试验材料,分析了NaCl胁迫下交替呼吸对叶绿素含量以及叶绿素荧光特性变化特征的影响,以探讨交替呼吸途径在逆境下的生理学作用以及植物在盐胁迫下光系统Ⅱ(PSⅡ)的调节作用机制。结果表明:(1)随着NaCl胁迫浓度(0、100、200、300mmol/L)的增高,菜豆幼苗叶片叶绿素含量显著下降,叶片光系统Ⅱ(PSⅡ)潜在最大光化学量子效率(Fv/Fm)、光适应下最大光化学效率(Fv′/Fm′)、PSⅡ光适应下实际光化学效率[Y(Ⅱ)]和光化学荧光猝灭(qP)与对照相比均显著性下降,而非光化学猝灭(NPQ)较对照组显著增加,同时交替呼吸容量在NaCl胁迫下也显著上升。(2)与单独NaCl胁迫相比,在NaCl胁迫下施加交替呼吸的抑制剂水杨基氧肟酸(SHAM)会导致菜豆幼苗叶片叶绿素含量、Fv/Fm、Fv′/Fm′、Y(Ⅱ)和qP进一步显著下降、NPQ进一步显著增加。研究认为,NaCl胁迫导致菜豆叶片光系统Ⅱ光化学效率下降和光能耗散增加,交替呼吸途径可有效缓解NaCl胁迫下菜豆叶绿素含量的减少以及光系统Ⅱ光化学反应效率的下降。