【作者】 武明珠；

【导师】 沈文飚；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 已经知道谷类糊粉层细胞可以作为一个独立的系统研究各种外加信号。由于其对GA和ABA响应的差异,糊粉层细胞已经成为研究植物激素调控细胞程序性死亡(programmed cell death, PCD)以及相关基因表达的一个模式系统。血红素加氧酶(heme oxygenase, HO, EC1.14.99.3)催化血红素(hemin)产生一氧化碳(carbon monoxide, CO).最近的研究发现HO/CO和一氧化氮(nitric oxide, NO)作用相类似,具有调控植物生长发育和抗逆的特性。在动物中的研究还发现氢气(hydrogen, H2)可以作为一种潜在的抗氧化物质,可以减轻活性氧(reactive oxygen species, ROS)造成的氧化伤害,但在植物中的生理作用不明。因此,本论文探讨了HO/CO和H2调控小麦糊粉层细胞α-淀粉酶基因表达以及PCD的分子机理。主要研究结果如下：1.以小麦糊粉层细胞的α-Amy2/54基因为分子探针研究一氧化氮(NO)／环鸟苷酸(cGMP)和血红素加氧酶-1(HO-1)的相互作用。研究发现释放NO的化合物sodium nitroprusside (SNP)和spermine NONOate (NONOate)均能以GA依赖的方式上调a-Amy2/54基因表达和α-淀粉酶的活性。HO-1的诱导剂氯化血红素(hemin)以及HO代谢产物CO也具有和NO相类似的作用。SNP对a-Amy2/54基因表达和酶活性的诱导作用能被cGMP的类似物8-Br-cGMP (8-Br)模拟；上述SNP/8-Br的作用被HO-1的专一性抑制剂ZnPPIX部分阻断,CO则可以逆转ZnPPIX作用。SNP和8-Br能上调HO-1的基因表达和酶活性,其中SNP的诱导作用则能被NO清除剂cPTIO和鸟苷酸环化酶的抑制剂LY83583抑制。分子生物学的证据表明GA和SNP、8-Br或cO共处理能上调GA诱导的GAMYB的基因表达和下调ABA引发的PKABA1的基因表达；GA和cPTIO、LY83583或ZnPPIX共处理则具有相反的效果。上述结果暗示在GA处理的小麦糊粉层中,HO-1可能参与NO/cGMP对a-Amy2/54基因表达的诱导作用。2.已经知道,HO-1在动植物中具有抵御各种胁迫和氧化伤害的作用。以小麦糊粉层细胞为实验材料,我们首先证明GA促进糊粉层细胞PCD,而ABA则具有抑制效应。GA处理后期的小麦糊粉层细胞HO活性和HO-1蛋白表达量均低于ABA处理。HO-1抑制剂ZnPPIX的预处理不仅能够降低HO的活性,同时也能加速GA诱导的小麦糊粉层细胞PCD。HO-1诱导物hematin以及HO产物CO水溶液和GA组合处理,不仅能上调HO的表达,还能延迟GA诱导的PCD；但是上述效应能够被ZnPPIX所逆转,说明HO上调表达能够延迟小麦糊粉层细胞的PCD。过氧化氢(H202)的含量和HO活性以及基因表达则呈现相反的效应,药理学实验证明清除或者提高H202含量可以延迟或者加速小麦糊粉层细胞的PCD. CO和BR能够上调CAT和APX的酶活性和基因表达,这可能与H202的含量降低相关。三种已知的抗氧化剂BHT、DTT和AsC能够延迟PCD,并且能够模拟hematin和CO的作用,包括上调HO的表达。总之,HO的上调表达延迟PCD的发生可能与H202的含量下降有关。3.在小麦的糊粉层细胞中,我们的研究发现动物中HO的诱导物CoCl2和CoPPIX预处理能够延缓GA诱导的PCD; ZnPPIX预处理不仅能够降低HO的活性,还能够加速糊粉层细胞的PCD. HO的代谢产物BR和CO的水溶液预处理则具有与CoCl2和CoPPIX相类似的效果,CO预处理也能上调HO的活性。CoCl2、CoPPIX、CO和BR预处理,都能下调GA诱导的NADPH氧化酶的基因表达和超氧阴离子(O2·-)的含量,上调SOD、CAT和APX的基因表达和酶活性,同时降低O2·-产生速率和H202的含量。因此我们提出：CoCl2和CoPPIX预处理诱导的HO的上调表达以及延迟的小麦糊粉层细胞的PCD可能与ROS含量下降有关。4.小麦糊粉层细胞中内源H2的含量随着GA处理的时间延长程呈现不断下降的趋势；应用外源H2水溶液和GA共处理则能延缓小麦糊粉层细胞的PCD。H2处理降低ABA和GA处理的糊粉层细胞原生质体中ROS的含量,以及小麦糊粉层细胞中超氧阴离子(O2·-)、H2O2和TBARS的含量。上述作用可能与H2能提高APX、CAT和SOD的基因表达和酶活性以及降低NADPH氧化酶的基因表达有关。研究还发现,H2对ROS中氧化性最强的羟自由基(·OH)具有直接的清除作用,从而提高糊粉层细胞的存活率。另外H2对亚硝酸阴离子(ONOO-)诱导的小麦糊粉层细胞PCD也具有明显的延缓作用。总之,我们推测H2延缓糊粉层细胞PCD的发生可能与其作为一种有效的抗氧化剂,通过迅速的扩散并通过细胞膜,从而直接清除·OH或通过调控抗氧化系统降低02‘-和H202的含量有关。更多还原

【Abstract】 Cereal aleurone cells can be used as a stand-alone system to study various applied signals. Because aleurone cells have different response to GA and ABA, it has become as a model system for the study of phytohormone-regulated PCD as well as corresponding gene expression. Heme oxygenase (HO, EC1.14.99.3) degrade heme to produce carbon monoxide (carbon monoxicide, CO) and HO/CO have the similar effects like nitric oxide (NO) in regulation of plant growth and development as well as resistence to biotic and abiotic stress. Recent study shows that in animals hydrogen (H2) act as an effective anti-oxidant in alleviating ROS caused damage. However, the effects of HO/CO on the a-Amylase gene expression, as well as HO/CO and H2on the PCD is not well understood. So this study investigated the molecular mechanisms of HO/CO and H2in regulation of a-amylase gene expression and PCD.The main results are shown as follows:1. a-Amy2/54gene expression was used as a molecular probe to investigate the interrelationship between nitric oxide (NO)/cyclic GMP (cGMP) and heme oxygenase-1(HO-1) in GA-treated wheat aleurone layers. The inducible expressions of a-Amy2/54and a-amylase activity were respectively amplified by two NO-releasing compounds, sodium nitroprusside (SNP) and spermine NONOate, in a GA-dependent fashion. Similar responses were observed when an inducer of HO-1, hemin-or one of its catalytic products, carbon monoxide (CO) in aqueous solution-was respectively added. The SNP-induced responses, mimicked by8-bromoguanosine3’,5’-cyclic monophosphate (8-Br-cGMP), a cGMP derivative, were NO-dependent. This conclusion was supported by the fact that endogenous NO overproduction was rapidly induced by SNP, and thereafter induction of a-Amy2/54gene expression and increased a-amylase activity were sensitive to the NO scavenger. We further observed that the above induction triggered by SNP/8-Br-cGMP was partially prevented by the addition of zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1. These blocking effects were clearly reversed by CO, confirming that the above response was HO-1-specific. Further analyses showed that both SNP and8-Br-cGMP rapidly up-regulated HO-1gene expression and increased HO activity, and SNP responses were sensitive to cPTIO and the guanylate cyclase inhibitor6-anilino-5,8-quinolinedione (LY83583). Molecular evidence confirmed that GA-induced GAMYB and ABA-triggered PKABA1transcripts were up-regulated or down-regulated by SNP,8-Br-cGMP or CO cotreated with GA. Contrasting changes were observed when cPTIO, LY83583or ZnPPIX was added. Together, our results suggested that HO-1might be involved in NO/cGMP-induced a-Amy2/54gene expression in GA-treated aleurone layers.2. Heme oxygenase-1(HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, we confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1protein expression exhibited lower levels in GA-treated layers, whereas hydrogen peroxide (H2O2) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H2O2either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with HO-1specific inhibitor zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of HO-1inducer hematin and the enzymatic reaction product of HO carbon monoxide (CO) aqueous solution, both of which brought about noticeable induction of HO expression, prevented GA-induced PCD substantially. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by hematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in the decrease of H2O2production and delay of PCD. Additionally, antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able to not only delay PCD, but also mimic the effects of hematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H2O2production. 3. In our experiments, we found that pretreatment with CoCl2and CoPPⅨ, which usually used as the HO inducer in animals, can delay GA-induced PCD in wheat aleurone layer. Further observation showed that ZnPPⅨ pretreatment not only decreases HO activity, but also accelerates PCD in the aleurone layers. Application of HO-1enzymatic reaction products BR and CO aqueous solution have the similar effects like CoCl2and CoPPⅨ in delaying GA-induced PCD, and it was also found that CO could upregulate HO activity. Further study showed that CoCl2, CoPPⅨ, CO, and BR pretreatment can down-regulate the gene expression of NADPH oxidase, and up-regulate SOD, CAT and APX gene expression and enzyme activities, while down-reculation superoxide anion (O2’) production and H2O2content. Overall, the above results suggested that up-regulation of HO expression in delaying of PCD by pretreatment with CoCl2or CoPPⅨ may be assiciated with the down-regulation of ROS accumulation.4. In our experiments, we found that endogenous H2content show a declining trend in GA treated wheat aleurone layer. H2aqueous solution cotreated with GA can delay the GA-induced PCD. Further research showed that H2can reduce the ROS content in ABA and GA treated aleurone protoplasts. It was also found that H2was able to alleviate superoxide anion (O2·-) and hydrogen peroxide (H2O2) accumulation and TBARS content. Above effects may associate with the up-regulation of APX, CAT, SOD gene expression and enzyme activity; and the down-regulation of NADPH oxidase gene expression by cotreatment with H2. H2could also delay the strongest oxidizing ROS-hydroxyl radical (·OH)-induced PCD, because H2can directly react with-OH. H2also delay ONOO--induced PCD in wheat aleurone layer. In short, as an effective anti-oxidant, H2can rapidly diffuse through the cell membrane, and could directly react with·OH or through regulating antioxidant enzyme down-regulation O2·-and H2O2content, thereby delaying the occurrence of aleurone cell PCD.更多还原