本文選題：交替氧化酶呼吸 + 光破壞防御��； 參考：《山東農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：交替氧化酶(AOX)呼吸途徑是植物線粒體內(nèi)除細(xì)胞色素氧化酶(COX)呼吸途徑之外的電子傳遞途徑,它可以直接將電子從質(zhì)體醌傳遞到氧氣生成水而不伴隨跨膜質(zhì)子梯度的產(chǎn)生。因此,AOX呼吸途徑作為一種非磷酸途徑可以快速有效地消耗還原力,減少呼吸電子傳遞鏈的過還原以及活性氧(ROS)的產(chǎn)生,而不受跨膜質(zhì)子梯度或胞內(nèi)ATP/ADP的限制。AOX作為一種耗能呼吸途徑,其生理功能已經(jīng)被廣泛研究,它能夠維持呼吸電子傳遞鏈和三羧酸循環(huán)、清除活性氧以抵御各種生物非生物脅迫。研究指出,AOX呼吸途徑有重要的光破壞防御作用。目前,被廣泛認(rèn)可的AOX呼吸途徑的光破壞防御機(jī)制是:逆境下AOX呼吸途徑通過快速氧化由蘋果酸蘋果酸-草酰乙酸穿梭途徑轉(zhuǎn)運出的葉綠體內(nèi)的過剩還原力(NADPH)以防止光合電子傳遞鏈的過度還原從而緩解光抑制。然而,迄今為止對AOX呼吸途徑的光破壞防御作用的研究都只在C3植物中進(jìn)行,而對C4植物卻鮮有研究。此外,有不少現(xiàn)象都無法用AOX吸途徑依賴于蘋果酸-草酰乙酸穿梭的光破壞防御作用來解釋,如AOX呼吸的耗氧速率僅是光合放氧速率的百分之二左右,AOX對還原力如此小的消耗能力為何能起到不可替代的光破壞防御作用?此外,逆境下蘋果酸-草酰乙酸穿梭途徑對于光破壞防御的作用可以被其他光破壞途徑補(bǔ)償或替代,為何依賴于蘋果酸-草酰乙酸穿梭的AOX呼吸途徑卻有不可替代的光破壞防御作用?作為葉綠體外部的一個重要的光破壞防御機(jī)制,AOX呼吸途徑在光破壞防御過程中的調(diào)控機(jī)制卻不甚清楚。本文通過對比研究多種C3和C4植物并且利用AOX呼吸途徑缺失的突變體對AOX呼吸途徑的光破壞防御作用進(jìn)行了研究,同時對光下AOX呼吸途徑的上調(diào)機(jī)制也進(jìn)行了詳細(xì)的研究和討論。主要結(jié)果如下:(1)強(qiáng)光下,SHAM抑制AOX呼吸途徑后,導(dǎo)致C3植物(包括黃瓜(Cucumis sativus)、雜交酸模Rumex K-1和柳樹(Salix babylonica)以及擬南芥)葉片的光抑制程度加重,而對C4植物(包括3種NADP-ME型的玉米(Zea mays)、高粱(Sorghum bicolor)和黍子(Euchlaena mexicana);1種NAD-ME型的馬齒莧(Portulaca oleracea);1種PPCK型的鼠尾草(Salvia farinacea))葉片的光抑制程度卻沒有明顯的影響。并且,隨著照光時間的增加或SHAM濃度的增加,C3植物葉片的光抑制程度加重,而對C4植物,照光時間的增加會加重葉片的光抑制程度,但SHAM濃度的增加不會加重葉片的光抑制程度。這說明,AOX呼吸途徑在C3植物中起不可替代的光破壞防御作用,而對C4植物則不起光破壞防御作用。(2)隨著照光時間的增加,c3植物黃瓜葉片中呼吸速率顯著增加,其中細(xì)胞色素氧化酶(cox)呼吸途徑的呼吸速率卻沒有顯著增加,而aox途徑的呼吸速率隨著照光時間的增加而顯著增加。與aox呼吸途徑的活性相類似,aox的基因表達(dá)以及aox的蛋白在照光后都顯著增加。而在c4植物玉米葉片中,總呼吸速率、cox呼吸速率隨著照光時間的增加而增加,aox呼吸速率卻沒有顯著的增加。玉米葉片中aox相關(guān)基因的表達(dá)以及aox蛋白在照光處理后也沒有顯著增加。強(qiáng)光下,aox呼吸途徑只在c3植物中上調(diào),并且起到不可替代的光破壞防御作用,而在c4植物中,aox呼吸途徑?jīng)]有顯著變化,同時對葉片的光破壞防御也沒有貢獻(xiàn)。在c4植物中,尤其是在nad-me和nadp-me這兩種類型的c4植物中,蘋果酸-草酰乙酸穿梭的能力遠(yuǎn)大于c3植物,但是,aox卻在c4植物中不起光破壞防御作用。這表明,強(qiáng)光下,aox呼吸途徑不僅僅通過直接消耗由蘋果酸-草酰乙酸穿梭機(jī)制轉(zhuǎn)運出葉綠體的過剩的還原力nadph來緩解光合作用光破壞。aox呼吸途徑可能還存在其他的光破壞防御機(jī)制。(3)強(qiáng)光下,當(dāng)用光呼吸抑制劑將c3植物黃瓜和rumexk-1葉片的光呼吸抑制后,再用sham抑制aox呼吸途徑后,葉片的光抑制程度不再增加。在低co2濃度(50ppm)或者低o2濃度(2%)以及低o2和低co2共同條件下,c3植物黃瓜和rumexk-1葉片的光呼吸都受到抑制,此時,aox呼吸途徑被sham抑制后,不再加重c3植物黃瓜和rumexk-1葉片的光抑制程度。而對于c4植物玉米葉片,不論是施用光呼吸抑制劑或在不同控氣條件下,sham處理的玉米葉片與對照葉片相比,光抑制程度始終沒有顯著差異。這表明,當(dāng)光呼吸受抑后,aox呼吸途徑在c3植物光破壞防御作用也受到抑制。(4)照光后co2的爆發(fā)(pib)表示了線粒體內(nèi)甘氨酸脫羧過程中co2的釋放,通常用來反映光呼吸的大小,sham抑制aox呼吸途徑后,c3植物黃瓜葉片的pib與對照相比顯著下降;葉片光合速率的氧敏感性也可以反映光呼吸的大小,黃瓜葉片用sham預(yù)處理后,其葉片光合速率的氧敏感性與對照相比也顯著下降。此外,與對照相比,sham處理的葉片中甘氨酸與絲氨酸比值增加,這表明sham處理導(dǎo)致葉片線粒體內(nèi)甘氨酸脫羧向絲氨酸轉(zhuǎn)變過程受到抑制。這些結(jié)果都表明,在c3植物中,aox呼吸途徑受抑后,葉片的光呼吸也會受到抑制。而在c4植物玉米中,由于其光呼吸極低,因此,在各種處理下,玉米葉片的pib、甘氨酸與絲氨酸的比值以及葉片光合速率的氧敏感性與對照相比無顯著性差異,并且測定值極小。以上結(jié)果表明,aox呼吸途徑對維持光呼吸的正常運轉(zhuǎn)有重要的作用。光呼吸能有效的維持強(qiáng)光下葉綠體內(nèi)的氧化還原平衡狀態(tài),并且清除對光合機(jī)構(gòu)內(nèi)極具毒害的乙醇酸和乙醛酸,此外,光呼吸對于光系統(tǒng)ii中d1蛋白的修復(fù)有重要的作用,因此,強(qiáng)光下,aox呼吸途徑的上調(diào)能夠維持或增加光呼吸循環(huán)的運轉(zhuǎn),這對于減輕強(qiáng)光下光合電子傳遞鏈?zhǔn)荏w側(cè)的過還原和乙醇酸的毒害并以此增加植物的光破壞防御作用有重要的意義。(5)強(qiáng)光下,aox1a(aox呼吸途徑缺失突變體)擬南芥突變體的光抑制程度顯著高于野生型,而當(dāng)突變體和野生型擬南芥葉片的光呼吸被光呼吸抑制劑或低co2濃度(50ppm)或者低o2濃度(2%)以及低o2和co2共同條件抑制后,aox1a突變體和野生型在強(qiáng)光下的光抑制程度不再表現(xiàn)出顯著性差異。此外,與野生型相比,aox1a突變體的pib、光合速率的氧敏感性都顯著下降,而甘氨酸與絲氨酸的比值顯著的增加。通過用aox呼吸途徑缺失的突變體再次證明,強(qiáng)光下,aox呼吸途徑上調(diào),并且通過維持光呼吸的正常運轉(zhuǎn),來緩解葉片的光抑制。(6)雖然光可以誘導(dǎo)c3植物黃瓜和煙草葉片aox呼吸途徑的上調(diào),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電子傳遞鏈的電子從qa到qb的傳遞,導(dǎo)致葉綠體內(nèi)電子外泄產(chǎn)生大量活性氧后,葉片aox呼吸途徑并未因此上調(diào),這表明葉綠體內(nèi)產(chǎn)生的活性氧并不會直接上調(diào)aox呼吸途徑。此外,與暗處相比,當(dāng)用iodoaceticacid(ia)抑制光合碳同化,從而導(dǎo)致還原力nadph的消耗減少,間接導(dǎo)致葉綠體內(nèi)nadph積累后,葉片aox呼吸途徑并未上調(diào),這表明葉綠體內(nèi)產(chǎn)生的還原力并不會直接上調(diào)aox呼吸途徑。這些結(jié)果表明,強(qiáng)光下,葉綠體內(nèi)活性氧的積累和還原力nadph的積累都不會直接影響aox呼吸途徑的活性。(7)照光后黃瓜和煙草葉片線粒體內(nèi)光呼吸代謝產(chǎn)物甘氨酸含量顯著增加。與對照相比,甘氨酸處理后,黃瓜和煙草葉片aox呼吸途徑顯著增加,氨基乙腈(ann)預(yù)處理抑制了甘氨酸向絲氨酸的轉(zhuǎn)變后,甘氨酸處理依舊增加aox呼吸途徑。此外,在沒有功能性葉綠體結(jié)構(gòu)的煙草by-2細(xì)胞中,外施甘氨酸可以上調(diào)aox呼吸途徑,并且在ann預(yù)處理后再施加甘氨酸也可以上調(diào)aox呼吸途徑。這表明,甘氨酸可以直接上調(diào)aox途徑而不依賴光呼吸或光合作用其他代謝產(chǎn)物。c4植物玉米葉片照光后,aox呼吸途徑不上調(diào),但是,用c3植物光呼吸代謝產(chǎn)物甘氨酸處理后,玉米葉片aox呼吸途徑也出現(xiàn)上調(diào)。這表明,強(qiáng)光下,C3植物葉片AOX呼吸途徑受到光呼吸代謝產(chǎn)物甘氨酸的上調(diào),而C4植物光呼吸極低,在強(qiáng)光下,不會積累甘氨酸從而不會上調(diào)AOX呼吸途徑。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q945

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 晏嬰才,林宏輝,梁厚果,張年輝;不同低溫脅迫對煙草愈傷組織抗氰交替途徑誘導(dǎo)和交替氧化酶表達(dá)影響的比較(英文)[J];植物學(xué)通報;2004年03期

2 郭連旺,許大全,沈允鋼;棉花葉片光合作用的光抑制和光呼吸的關(guān)系[J];科學(xué)通報;1995年20期

，

本文編號：2041556