线粒体交替氧化酶呼吸途径对植物叶片的光破坏防御作用及其调控机制

发布时间：2018-06-19 22:21

本文选题：交替氧化酶呼吸 + 光破坏防御　；参考：《山东农业大学》2016年博士论文

【摘要】：交替氧化酶(AOX)呼吸途径是植物线粒体内除细胞色素氧化酶(COX)呼吸途径之外的电子传递途径,它可以直接将电子从质体醌传递到氧气生成水而不伴随跨膜质子梯度的产生。因此,AOX呼吸途径作为一种非磷酸途径可以快速有效地消耗还原力,减少呼吸电子传递链的过还原以及活性氧(ROS)的产生,而不受跨膜质子梯度或胞内ATP/ADP的限制。AOX作为一种耗能呼吸途径,其生理功能已经被广泛研究,它能够维持呼吸电子传递链和三羧酸循环、清除活性氧以抵御各种生物非生物胁迫。研究指出,AOX呼吸途径有重要的光破坏防御作用。目前,被广泛认可的AOX呼吸途径的光破坏防御机制是:逆境下AOX呼吸途径通过快速氧化由苹果酸苹果酸-草酰乙酸穿梭途径转运出的叶绿体内的过剩还原力(NADPH)以防止光合电子传递链的过度还原从而缓解光抑制。然而,迄今为止对AOX呼吸途径的光破坏防御作用的研究都只在C3植物中进行,而对C4植物却鲜有研究。此外,有不少现象都无法用AOX吸途径依赖于苹果酸-草酰乙酸穿梭的光破坏防御作用来解释,如AOX呼吸的耗氧速率仅是光合放氧速率的百分之二左右,AOX对还原力如此小的消耗能力为何能起到不可替代的光破坏防御作用?此外,逆境下苹果酸-草酰乙酸穿梭途径对于光破坏防御的作用可以被其他光破坏途径补偿或替代,为何依赖于苹果酸-草酰乙酸穿梭的AOX呼吸途径却有不可替代的光破坏防御作用?作为叶绿体外部的一个重要的光破坏防御机制,AOX呼吸途径在光破坏防御过程中的调控机制却不甚清楚。本文通过对比研究多种C3和C4植物并且利用AOX呼吸途径缺失的突变体对AOX呼吸途径的光破坏防御作用进行了研究,同时对光下AOX呼吸途径的上调机制也进行了详细的研究和讨论。主要结果如下:(1)强光下,SHAM抑制AOX呼吸途径后,导致C3植物(包括黄瓜(Cucumis sativus)、杂交酸模Rumex K-1和柳树(Salix babylonica)以及拟南芥)叶片的光抑制程度加重,而对C4植物(包括3种NADP-ME型的玉米(Zea mays)、高粱(Sorghum bicolor)和黍子(Euchlaena mexicana);1种NAD-ME型的马齿苋(Portulaca oleracea);1种PPCK型的鼠尾草(Salvia farinacea))叶片的光抑制程度却没有明显的影响。并且,随着照光时间的增加或SHAM浓度的增加,C3植物叶片的光抑制程度加重,而对C4植物,照光时间的增加会加重叶片的光抑制程度,但SHAM浓度的增加不会加重叶片的光抑制程度。这说明,AOX呼吸途径在C3植物中起不可替代的光破坏防御作用,而对C4植物则不起光破坏防御作用。(2)随着照光时间的增加,c3植物黄瓜叶片中呼吸速率显著增加,其中细胞色素氧化酶(cox)呼吸途径的呼吸速率却没有显著增加,而aox途径的呼吸速率随着照光时间的增加而显著增加。与aox呼吸途径的活性相类似,aox的基因表达以及aox的蛋白在照光后都显著增加。而在c4植物玉米叶片中,总呼吸速率、cox呼吸速率随着照光时间的增加而增加,aox呼吸速率却没有显著的增加。玉米叶片中aox相关基因的表达以及aox蛋白在照光处理后也没有显著增加。强光下,aox呼吸途径只在c3植物中上调,并且起到不可替代的光破坏防御作用,而在c4植物中,aox呼吸途径没有显著变化,同时对叶片的光破坏防御也没有贡献。在c4植物中,尤其是在nad-me和nadp-me这两种类型的c4植物中,苹果酸-草酰乙酸穿梭的能力远大于c3植物,但是,aox却在c4植物中不起光破坏防御作用。这表明,强光下,aox呼吸途径不仅仅通过直接消耗由苹果酸-草酰乙酸穿梭机制转运出叶绿体的过剩的还原力nadph来缓解光合作用光破坏。aox呼吸途径可能还存在其他的光破坏防御机制。(3)强光下,当用光呼吸抑制剂将c3植物黄瓜和rumexk-1叶片的光呼吸抑制后,再用sham抑制aox呼吸途径后,叶片的光抑制程度不再增加。在低co2浓度(50ppm)或者低o2浓度(2%)以及低o2和低co2共同条件下,c3植物黄瓜和rumexk-1叶片的光呼吸都受到抑制,此时,aox呼吸途径被sham抑制后,不再加重c3植物黄瓜和rumexk-1叶片的光抑制程度。而对于c4植物玉米叶片,不论是施用光呼吸抑制剂或在不同控气条件下,sham处理的玉米叶片与对照叶片相比,光抑制程度始终没有显著差异。这表明,当光呼吸受抑后,aox呼吸途径在c3植物光破坏防御作用也受到抑制。(4)照光后co2的爆发(pib)表示了线粒体内甘氨酸脱羧过程中co2的释放,通常用来反映光呼吸的大小,sham抑制aox呼吸途径后,c3植物黄瓜叶片的pib与对照相比显著下降;叶片光合速率的氧敏感性也可以反映光呼吸的大小,黄瓜叶片用sham预处理后,其叶片光合速率的氧敏感性与对照相比也显著下降。此外,与对照相比,sham处理的叶片中甘氨酸与丝氨酸比值增加,这表明sham处理导致叶片线粒体内甘氨酸脱羧向丝氨酸转变过程受到抑制。这些结果都表明,在c3植物中,aox呼吸途径受抑后,叶片的光呼吸也会受到抑制。而在c4植物玉米中,由于其光呼吸极低,因此,在各种处理下,玉米叶片的pib、甘氨酸与丝氨酸的比值以及叶片光合速率的氧敏感性与对照相比无显著性差异,并且测定值极小。以上结果表明,aox呼吸途径对维持光呼吸的正常运转有重要的作用。光呼吸能有效的维持强光下叶绿体内的氧化还原平衡状态,并且清除对光合机构内极具毒害的乙醇酸和乙醛酸,此外,光呼吸对于光系统ii中d1蛋白的修复有重要的作用,因此,强光下,aox呼吸途径的上调能够维持或增加光呼吸循环的运转,这对于减轻强光下光合电子传递链受体侧的过还原和乙醇酸的毒害并以此增加植物的光破坏防御作用有重要的意义。(5)强光下,aox1a(aox呼吸途径缺失突变体)拟南芥突变体的光抑制程度显著高于野生型,而当突变体和野生型拟南芥叶片的光呼吸被光呼吸抑制剂或低co2浓度(50ppm)或者低o2浓度(2%)以及低o2和co2共同条件抑制后,aox1a突变体和野生型在强光下的光抑制程度不再表现出显著性差异。此外,与野生型相比,aox1a突变体的pib、光合速率的氧敏感性都显著下降,而甘氨酸与丝氨酸的比值显著的增加。通过用aox呼吸途径缺失的突变体再次证明,强光下,aox呼吸途径上调,并且通过维持光呼吸的正常运转,来缓解叶片的光抑制。(6)虽然光可以诱导c3植物黄瓜和烟草叶片aox呼吸途径的上调,但用3-(3,4-二氯苯基)-1,1-二甲基脲(dcmu)抑制光下植物的光合电子传递链的电子从qa到qb的传递,导致叶绿体内电子外泄产生大量活性氧后,叶片aox呼吸途径并未因此上调,这表明叶绿体内产生的活性氧并不会直接上调aox呼吸途径。此外,与暗处相比,当用iodoaceticacid(ia)抑制光合碳同化,从而导致还原力nadph的消耗减少,间接导致叶绿体内nadph积累后,叶片aox呼吸途径并未上调,这表明叶绿体内产生的还原力并不会直接上调aox呼吸途径。这些结果表明,强光下,叶绿体内活性氧的积累和还原力nadph的积累都不会直接影响aox呼吸途径的活性。(7)照光后黄瓜和烟草叶片线粒体内光呼吸代谢产物甘氨酸含量显著增加。与对照相比,甘氨酸处理后,黄瓜和烟草叶片aox呼吸途径显著增加,氨基乙腈(ann)预处理抑制了甘氨酸向丝氨酸的转变后,甘氨酸处理依旧增加aox呼吸途径。此外,在没有功能性叶绿体结构的烟草by-2细胞中,外施甘氨酸可以上调aox呼吸途径,并且在ann预处理后再施加甘氨酸也可以上调aox呼吸途径。这表明,甘氨酸可以直接上调aox途径而不依赖光呼吸或光合作用其他代谢产物。c4植物玉米叶片照光后,aox呼吸途径不上调,但是,用c3植物光呼吸代谢产物甘氨酸处理后,玉米叶片aox呼吸途径也出现上调。这表明,强光下,C3植物叶片AOX呼吸途径受到光呼吸代谢产物甘氨酸的上调,而C4植物光呼吸极低,在强光下,不会积累甘氨酸从而不会上调AOX呼吸途径。
[Abstract]:The alternative oxidase (AOX) respiration pathway is an electron transfer pathway outside the respiratory pathway of cytochrome oxidase (COX) in plant mitochondria. It can directly transfer electrons from plastid quinones to oxygen to generate water without the generation of transmembrane proton gradient. Therefore, AOX breathing path can be consumed quickly and effectively as a non phosphoric pathway. Reducing power, reducing the over reduction of the respiratory electron transfer chain and the production of active oxygen (ROS), not by the proton gradient of the transmembrane or the restriction of the intracellular ATP/ADP as an energy dissipation breathing pathway, its physiological function has been widely studied. It can maintain the respiratory electron transfer chain and the three carboxylic acid cycle, scavenge reactive oxygen species to resist various biological non - Biological stress. The study indicates that the AOX respiration pathway has an important light damage defense. Currently, the widely recognized AOX respiratory pathway is the optical destruction defense mechanism: in adversity, the AOX respiration pathway can prevent the photosynthesis by rapid oxidation of the excess reductive power (NADPH) in the green body of the leaf green, which is transported by the malate malate acetoacetic acid (malic acid) route. However, so far, the study of the optical destruction of the AOX respiration pathway has been carried out only in the C3 plant, but there is little research on the C4 plants. In addition, there are many phenomena that can not be explained by the AOX absorption method depending on the light destruction defense of the malate acylacetic acid shuttle, such as AOX. The rate of oxygen consumption of respiration is only about two percent of the rate of photosynthetic oxygen release. Why can the AOX's low reducing power play an irreplaceable role in the light damage defense? In addition, the effect of the malate oxoacetic acid shuttle pathway on the light destruction defense can be compensated or replaced by other optical destruction pathways under adverse circumstances. However, the AOX respiration pathway of malic acid and acetoacetic acid has an irreplaceable light destruction defense. As an important light destruction defense mechanism outside the chloroplast, the regulation mechanism of AOX breathing pathway in the process of light destruction is not clear. In this paper, several kinds of C3 and C4 plants were compared and the deletion of AOX respiration pathway was used in this paper. The mutants of AOX respiration pathway were studied and the mechanism of up regulation of AOX respiration pathway was also studied and discussed in detail. The main results are as follows: (1) under strong light, C3 plants (including cucumber (Cucumis sativus), Rumex K-1 and willow (Salix baby) are caused by SHAM inhibition of AOX respiration. Lonica) and Arabidopsis thaliana (Arabidopsis thaliana) increased the degree of light inhibition, but for C4 plants (including 3 NADP-ME types of Maize (Zea mays), sorghum (Sorghum bicolor) and millet (Euchlaena mexicana), and 1 NAD-ME types of purslane (Portulaca oleracea), and 1 species of cauda cauda) leaves had no obvious effect on the light inhibition. Moreover, with the increase of light time or the increase of SHAM concentration, the light inhibition of C3 plant leaves increased, while the increase of light time for C4 plants would aggravate the degree of light inhibition in leaves, but the increase of SHAM concentration would not aggravate the degree of light inhibition in leaves. This indicates that the AOX approach is an irreplaceable light destruction defense in the C3 plant. (2) the respiration rate of C3 plant cucumber leaves increased significantly with the increase of light time, and the respiration rate of cytochrome oxidase (COX) respiration pathway was not significantly increased, while the respiration rate of AOX pathway increased significantly with the increase of light time. And the respiratory path of AOX was increased with AOX respiration. The activity phase of the diameter was similar, the gene expression of AOX and the protein of AOX increased significantly after illumination. In the leaves of C4 plant corn, the total respiration rate and the respiration rate of Cox increased with the increase of light time, but the rate of AOX respiration was not significantly increased. The expression of the AOX phase gene in the maize leaves and the AOX protein were also treated after light treatment. No significant increase. Under strong light, the AOX respiration pathway is only up-regulated in the C3 plant and plays an irreplaceable light destruction defense. In C4 plants, the AOX respiration pathway does not change significantly and does not contribute to the light destruction defense of the leaves. In C4 plants, especially in the C4 plants of the two types of nad-me and nadp-me, apples The ability of acid - oacetoacetic acid to shuttle much more than C3 plants, but AOX does not act as a light damage defense in C4 plants. This indicates that under strong light, the AOX breathing pathway may not only transfer the excess reducing force NADPH of the chloroplast through the direct consumption of the malate acetoacetic acid shuttle mechanism to alleviate the possible photosynthesis pathway of the.Aox respiration pathway. There are other optical damage defense mechanisms. (3) under strong light, when photorespiration of C3 plant cucumber and rumexk-1 leaves is suppressed with photorespiration inhibitors, the light inhibition degree of leaves is no longer increased after sham inhibition of AOX respiration. Under the condition of low CO2 concentration (50ppm) or low O2 concentration (2%) and low O2 and low CO2 common conditions, C3 plant cucumber The photorespiration of the rumexk-1 leaves was inhibited, and when the AOX respiration pathway was inhibited by sham, the photoinhibition degree of the cucumber and rumexk-1 leaves of the C3 plant was no longer aggravated. The light inhibition degree of the maize leaves in the C4 plant, whether it was Shi Yongguang respiration inhibitor or under the different control gas control conditions, was compared with the control leaves. There is no significant difference at all times. This shows that the AOX respiration pathway is also inhibited in the light destruction of C3 plants when photorespiration is suppressed. (4) the outbreak of CO2 after illumination (PIB) indicates the release of CO2 during the process of mitochondrial glycine decarboxylation, which is usually used to reflect the small photorespiration. Sham inhibits the AOX respiration pathway, and PIB of C3 plant cucumber leaves PIB Compared with the control, the oxygen sensitivity of leaf photosynthetic rate can also reflect the size of photorespiration. The oxygen sensitivity of leaf photosynthetic rate of cucumber leaves decreased significantly after sham pretreatment. In addition, the ratio of glycine to serine in the leaves of sham treated leaves increased, which indicated that the sham treatment led to the increase of the ratio of the leaf photosynthetic rate to the photo ratio. The glycine decarboxylation of glycine decarboxylation to the serine transformation process was inhibited. These results showed that in the C3 plant, AOX respiration was suppressed and the leaf photorespiration was inhibited. In the C4 plant corn, the PIB, the ratio of glycine to serine, and the ratio of PIB, glycine and serine in the leaves of Maize, as a result of the low photorespiration. The oxygen sensitivity of the leaf photosynthetic rate is not significantly different from the photographic ratio and is very small. The above results show that the AOX respiration pathway plays an important role in maintaining the normal operation of photorespiration. Photorespiration can effectively maintain the redox equilibrium state in the chloroplasts under strong light and remove the toxic B in the photosynthetic apparatus. Alkyd and glyoxylic acid, in addition, photorespiration plays an important role in the repair of D1 protein in the optical system II. Therefore, under strong light, the up-regulation of AOX respiration pathway can maintain or increase the operation of the photorespiratory cycle, which reduces the toxicity of the excess of the acceptor and glycolic acid on the receptor side of the photosynthetic electron transfer chain under strong light and increases the light destruction of the plant. (5) under strong light, the photoinhibition of Arabidopsis mutant with aox1a (AOX deletion mutant) was significantly higher than that of wild type, while the photorespiration of mutant and wild Arabidopsis leaves was inhibited by photorespiration inhibitors or low CO2 concentration (50ppm) or low O2 concentration (2%) and low O2 and CO2 conditions. 1A mutant and wild type no longer showed significant difference in light suppression under strong light. In addition, the PIB of the aox1a mutant was significantly lower than the wild type, while the ratio of glycine to serine increased significantly. The mutant of the AOX respiration pathway was again proved to be under strong light, AOX respiration. The pathway is up-regulated, and the light inhibition of leaves is alleviated by maintaining normal operation of photorespiration. (6) although light can induce the up-regulated respiration pathway of C3 plant cucumber and tobacco leaves AOX, 3- (3,4- two chlorophenyl) -1,1- two methylurea (DCMU) inhibits the transmission of the electron from QA to QB of the photosynthetic electron transfer chain of the plants of the plant, resulting in the chloroplast. When a large amount of active oxygen is produced in the internal electron, the AOX respiration pathway of leaves does not rise, which indicates that the reactive oxygen species produced in the chloroplast does not directly up-regulate the AOX respiration pathway. In addition, when compared with the dark place, the consumption of iodoaceticacid (IA) inhibits the assimilation of photosynthetic carbon, resulting in a decrease in the consumption of the original force NADPH, which indirectly leads to the NADPH product in the chloroplast. After fatigue, the AOX respiration pathway did not increase, which indicated that the reduction force produced in the chloroplast did not directly up-regulate the AOX respiration pathway. These results showed that the accumulation of reactive oxygen in the chloroplast and the accumulation of reducing force NADPH did not directly affect the activity of AOX respiration pathway under strong light. (7) the mitochondria of cucumber and tobacco leaves were called in the mitochondria after illumination. The glycine content of the metabolites increased significantly. Compared with the control, the AOX respiration pathway of cucumber and tobacco leaves increased significantly after glycine treatment. Amino acetonitrile (ANN) pretreatment inhibited the transformation of glycine to serine, and glycine treatment still increased the AOX respiration pathway. In addition, the tobacco BY-2 cells with no functional chloroplast structure were found. In addition, exogenous glycine can up-regulate the AOX respiration pathway, and the application of glycine after Ann pretreatment can also increase the AOX respiration pathway. This indicates that glycine can directly up-regulate the AOX pathway without dependence on light respiration or other metabolites of photosynthesis,.C4 plant maize leaves light, AOX respiration pathway does not rise, but C3 plant light is used. After glycine treatment, the AOX respiration pathway of maize leaves also rises. This indicates that under strong light, the AOX respiration pathway of C3 plant leaves is up regulated by the glycine of the photorespiration metabolite, while the C4 plant has a very low photorespiration. Under strong light, no glycine can be accumulated so that the AOX respiration pathway is not up.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945

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