【作者】 谢彦杰；

【导师】 杨清； 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， 2011， 博士

【摘要】 动物中的研究表明,血红素加氧酶/一氧化碳(heme oxygenase/carbon monoxide, HO/CO)是一种内源信号系统,HO(EC 1.14.99.3)产生的CO起着生理信号分子或生物活性小分子的功能,影响着细胞的繁殖和各种生长因子的合成。在植物中,HO参与了光敏色素发色团的生物合成。先前我们通过研究发现,盐胁迫下小麦幼苗根部HO活性上升并导致内源CO含量增加,CO通过NO信号介导的上调根部抗氧化防护和维持离子稳态来提高小麦幼苗耐盐性,但还不清楚HO诱导与盐适应性之间的关系。我们进一步的研究发现,低浓度NaCl预处理(25 mM)不仅能激活小麦HO-1基因的表达和活性提高,还能诱导产生盐适应现象；200mM NaC1处理则能导致相反的结果。进一步探讨了HO-1上调在小麦幼苗获得性耐盐性(盐适应)中的作用,发现HO-1专一性抑制剂锌原卟啉(zinc protoporphyrin IX, ZnPP)能够逆转盐适应的产生,而200 mMNaCl产生的毒害效应能被外加HO-1诱导剂haemin部分逆转,这说明盐适应过程与HO-1上调有关。此外,CO(HO的催化产物之一)处理能模拟低浓度NaCl诱导产生的盐适应现象。CO预处理能重建活性氧(reactive oxygen species,ROS)稳态平衡,这一过程是通过上调超氧化物歧化酶(superoxide dismutase, SOD)、抗坏血酸过氧化物酶(ascorbate peroxidase, APX)和胞内过氧化物酶(peroxidase,POD)的转录本、总酶活和同工酶活性,下调NADPH氧化酶和细胞壁POD总酶活以及同工酶活性来实现的。同时也发现,过氧化氢(hydrogen peroxide, H2O2)稳态的维持是HO-1介导的盐适应所必需。上述结果表明,HO-1是通过维持ROS稳态来介导小麦幼苗盐适应性。在拟南芥中,共有四个基因编码HO,它们分别是HY1、HO2, HO3和HO4。为了阐明各HO基因在盐适应过程中的功能,进一步利用遗传突变体研究HO介导盐适应性产生中的具体机制。首先比较了拟南芥野生型和四个HO单突变体(hy1-100、ho2、ho3和ho4)在NaCl胁迫下的萌发率和主根生长差异。结果表明,hy1-100对NaCl最敏感,且没有盐适应表型；而HYl过表达株系(35S：HYl-1/2/3/4)对NaCl则更具抗性。进一步研究发现,低浓度NaCl能激活NADPH氧化酶D(respiratory burst oxidase homologues, RbohD)转录本,其产生的ROS呈双峰诱导(峰Ⅰ和峰Ⅱ),ROS介导了低盐胁迫下HY1的诱导表达和其产生的盐适应现象；虽然hy1-100突变体的HY1无法正常表达,但依然检测到了ROS峰Ⅰ存在,这说明ROS峰Ⅰ的产生不依赖于HY1。我们还研究了受haemin诱导的HY1表达与RbohD产生的ROS峰Ⅱ对盐适应产生的影响。在atrbohD突变体中,haemin预处理能诱导HYl的表达,但无法诱导ROS和盐适应的产生；在hy1-100突变体中,haemin预处理既不能诱导HY1基因表达,RbohD产生的ROS峰Ⅱ也无法形成。上述结果说明,HY1在盐适应信号中起着重要作用,其功能的发挥需要RbohD产生的ROS峰Ⅱ参与。由于渗透胁迫和盐胁迫存在诸多共性,因此进一步比较了拟南芥野生型Col-0、hy1-00突变体和HY1过表达突变体35s：HY1-3/1-4对渗透胁迫和盐胁迫的应答差异。结果表明,与野生型相比,hy1-100突变体的萌发受抑对mannitol和NaCl更敏感,而35s：HY1-3/1-4对mannitol和NaCl不敏感。HO-1活性诱导剂haemin和专一性抑制剂ZnPP能不同程度地缓解或更加抑制拟南芥野生型在渗透和盐胁迫下的鲜重累积和主根生长受抑；haemin不影响hy1-100突变体在胁迫下的表型,而ZnPP在一定程度上加剧了胁迫下hy1-100突变体的受抑程度。通过外源添加HO各催化产物发现,CO和胆红素(bilirubin, BR)是HY1调节拟南芥在胁迫下主根和侧根生长发育中的有效产物。与野生型相比,虽然hy1-100的生长受抑对渗透胁迫和盐胁迫均表现为超敏感,但hy1-100突变体在渗透胁迫均能存活,而盐胁迫下存活率较野生型则显著下降,这暗示着HY1在盐胁迫和渗透胁迫信号转导中功能可能存在差异。接着检测了渗透胁迫和盐胁迫下野生型和hy1-100突变体胁迫应答基因和脱落酸(abscisic acid, ABA)合成基因的表达,发现在渗透胁迫下,上述两类基因在hy1-100突变体中的诱导程度显著高于野生型的；而在盐胁迫下的结果则恰相反。同时,ABA能诱导HYl基因表达,hy1-100突变体的萌发受抑对外源ABA超敏感,35s：HYl-3/1-4过表达突变体则对ABA不敏感。自然干旱实验的结果表明,hy1-100突变体较野生型更耐旱,而35s：HY1-3过表达突变体不耐旱。进一步实验证明这可能是由于各材料气孔的开关对ABA响应的敏感性不同引起的,HY1可能通过调节气孔开放和关闭来改变耐旱性。一氧化氮(nitric oxide, NO)是植物逆境信号转导的重要信号分子,还不清楚HY1与NO信号在耐盐信号转导中的关系。进一步的实验从遗传学角度继续研究了盐胁迫下NO产生的来源,并初步探讨了HY1与硝酸还原酶(nitrate reductase, NR)途径产生的NO之间的关系。NR抑制剂钨酸钠(tungstate)和动物NOS抑制剂NG-硝基-L-精氨酸甲酯(NG-nitro-L-arginine methyl ester hydrochloride, L-NAME)均能抑制NaCl诱导的NO产生,同时NaCl引起的拟南芥主根生长减缓和鲜重累积下降更受抑制；NaCl诱导了拟南芥根部两个NR的编码基因NIA1和NIA2及一氧化氮相关蛋白1(nitric oxide associated 1, NOA1)转录水平的提高,这些结果说明NR和L-Arg途径对盐胁迫诱导的NO产生均有贡献。遗传学的证据还表明,NaCl无法诱导NR突变体nial-1/2-5、NOA1突变体noal和nial-1/2-5/noal突变体根部NO含量的提高,而外源NO供体硝普钠(sodium nitroprusside, SNP)能同时增加各突变体的NO含量,说明NaCl诱导拟南芥根部NO的产生与NIA1、NIA2和NOA1均有关。同时,与野生型相比,nial-1/2-5、noal和nial-1/2-5/noal突变体的生长抑制均对NaCl表现为超敏感。进一步还比较了盐胁迫下拟南芥野生型、hy1-100, ho2, ho3和ho4突变体根部NO产生的差异。结果显示,hy1-100突变体在盐胁迫下产生更多的NO；NO供体二乙胺/一氧化氮复合物(diethylamine/nitric oxide adduct, NONOate)无法缓解hy11-100突变体萌发对NaCl超敏的现象,而NO清除剂2-苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt, PTIO)逆转了35s：HYl-3更抗盐的表型；HO-1诱导剂haemin能缓解NaCl胁迫下拟南芥野生型的萌发和子叶张开绿化受抑,但haemin对noal突变体的缓解效应则明显趋弱,haemin无法缓解nial-1/2-5和nial-1/2-5/noal突变体生长抑制对NaCl的超敏现象。这些结果暗示,盐胁迫信号转导途径中HY1可能与NR产生的NO存在互作,它们都是耐盐信号转导途径所必需的组分。先前的研究表明,大豆血红素加氧酶响应紫外(ultraviolet, UV)辐射,HO的诱导能缓解UV-B胁迫引起的氧化伤害,且这一过程可能与ROS有关。由于缺乏遗传学证据,还不清楚HO调节紫外胁迫的具体机理。本论文从遗传学角度探讨了拟南芥HO各基因对UV-C的应答变化,并比较了UV-C辐射下野生型和hy1-100突变体类黄酮合成代谢途径和抗氧化防御系统的差异。结果发现,UV-C辐射胁迫下,拟南芥根部HY1、H02、HO3和H04均受诱导,其中以HYl的诱导最为剧烈。同时,与野生型相比,hy1-100突变体的生长抑制对UV-C辐射胁迫表现为超敏感。进一步实验结果表明这与其类黄酮代谢水平降低有关：与野生型相比,hy1-100突变体紫外吸收物质含量显著降低,类黄酮代谢途径4个重要代谢酶查尔酮合酶(chalcone synthase, CHS)、查尔酮异构酶(chalcone isomerase, CHI)、黄烷酮3-羟化酶(flavonoid 3-hydroxylase,F3H)和黄酮醇合成酶(flavonol synthase, FLS)的表达也有所降低。此外,UV-C辐射显著诱导了上述4个关键基因的表达；hy1-100突变体的CHI和FLS受诱导程度则部分受抑制,而CHS的表达则明显受抑制。hy1-100突变体上述基因诱导受抑是由其UV信号转导受阻引起的：UV-C辐射显著诱导了野生型根部类调控黄酮代谢的相关转录因子的表达；与之相对的是,HYl突变后,UV-C辐射对相关转录因子的诱导表达明显受到抑制。此外,抗氧化酶基因定量PCR的结果显示,hy-100突变体对UV-C辐射超敏感还与其抗氧化防护系统无法有效诱导有关。更多还原

【Abstract】 Recent studies suggested that the heme oxygenase/carbon monoxide (HO/CO) is an endogenous siganlling system in modulating inflammatory reactions, influencing cell proliferation and production of growth factors in animals. It was also confirmed that carbon monoxide (CO), mainly generated by heme oxygenase enzymes (HOs, EC 1.14.99.3), functions as a physiological messenger or bioactive molecule in animal cells.The major role of HO in phytochrome chromophore biosynthesis was discovered in plants. Our previous result showed that salt stress induced an increase in endogenous CO production and the activity of the CO synthetic enzyme HO in wheat seedling roots. CO might confer an increased tolerance to salinity stress by maintaining ion homeostasis and enhancing antioxidant system parameters in wheat seedling roots, both of which were partially mediated by NO signal. However, little was known about the relationship between HO induction and salt acclimation in plants. In this study, HO-1 up-regulation and its role in acquired salt tolerance (salinity acclimation) were also investigated in wheat plants. We discovered that pretreatment with a low concentration of NaCl (25 mM) followed by the transfer to 200 mM NaCl stress, not only led to the induction of wheat HO-1 protein and gene expression as well as the increased HO activity in seedling roots, but also induced salinity acclimatory response. Opposite phenomenon were observed in seedling plants upon 200 mM NaCl stress. The effect is specific for HO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blocked the above cytoprotective actions, and the cytotoxic responses conferred by 200 mM NaCl were reversed partially when HO-1 inducer haemin was added. Furthermore, the HO’s catalytic product CO aqueous solution pretreatment mimicked the low concentration of NaCl-induced salinity acclimatory responses. Meanwhile, the reestablishment of ROS homeostasis triggered by CO pretreatment followed by salinity stress was mainly guaranteed by the induction of total and isozymatic activities, or corresponding transcripts of superoxide dismutase (SOD), ascorbate peroxidase (APX), and cytosolic peroxidase (POD), as well as the down-regulation of NADPH oxidase expression and decreased cell-wall POD activity. A requirement of H2O2 homeostasis for HO-1-mediated salinity acclimation was also discovered. Taken together, the above results suggested that up-regulation of HO-1 expression was responsible for salinity acclimation through the regulation of ROS homeostasis.In Arabidopsis thaliana, a family of four genes(HYl, HO2, HO3 and HO4) encodes haem oxygenase (HO), and their major role in phytochrome chromophore biosynthesis was discovered. We further characterize the detailed mechasim of HY1 mediated salt acclimation by using mutant analyses. To characterise the contribution of different HO isoforms involved in salt acclimation, effects of NaCl on seed germination and primary root growth in Arabidopsis wild-type and four HO mutants(hy1-100, ho2, ho3 and ho4) were compared. Among the four HO mutants, hy1-100 displayed maximal sensitivity to salinity and provided no acclimatory response. Whereas, HY1-over-expressing plants (35S:HY1) displayed tolerance characteristics. Mild salt-stress stimulated a biphasic change in RbohD transcripts and a consequent increase in its derived reactive oxygen species (ROS, peak I and II) production in wild-type. Furthermore, ROS peak I-mediated HY1 induction and thereafter salt acclimation were observed, while only the ROS peak I appeared in hy1-100 mutant. In a subsequent test, haemin-induced HY1 expression and RbohD-derived ROS peak II formation, confirmed their causal relationship with salt acclimation. Meanwhile, in atrbohD mutants, haemin pretreatment resulted in the induction of HY1 expression, whereas no similar response appeared in hyl-100, and neither peak II of ROS nor any following salt acclimatory responses were observed. Together, the above findings suggest that HY1 plays an important role in salt acclimation signalling, which required participation of RbohD-derived ROS peak II.As both salt and osmotic stresses share common characterics, we further compared responses of Arabidopsis wild type Col-0, hyl-100 mutant and HY1 over-expressing lines 35s:HY1-3/1-4 to osmotic and salt stresses. Further results showed that, compared with wild type, hy1-100 mutant displayed hypersensitive phynotype, whereas 35s:HY1-3/1-4 was insensitive to exogenous NaCl and mannitol. Application of the HO-1 inducer haemin and potent inhibitor ZnPP can alleviate or aggravate the osmotic or salt, stress-induced inhibition of fresh weight and primary root growth. No significant response of hy1-100 mutant was obsereved when haemin was added upon stress conditions. Meanwhile, ZnPP slightly aggravates growth inhibition of hy1-100 mutant upon both osmotic and salt stresses. Further analysis discovered that both CO and BR were the effective by-product of HO modulating primary and latral root development when Arabidopsis seedlings were exposed to stresses. Interestingly, althought hy1-100 mutant exhibited hypersensitive phenotyes, different survive rates under osmotic and salt stresses were oberseved. By using real-time PCR analyses, we found that the inductions of both stress response and ABA metabolism genes expression were much higher in hy1-100 mutant than those of wild type under osmotic stress. Whereas opposite phenonmen were observed in salt stress condition. Additionally, ABA treatment results in the induction of HY1 expression, and the germination inhibition of hy1-100 was hypersensitive to exogenous ABA. Oppssite results were also found in 35s:HY1-3/1-4 plants. Finally, we also noticed that enhanced drought resistance of hy1-100 mutant plants was due to an efficient stomatal closure and inhibition of opening induced by ABA.Former results showed that HY1 is a key component of the Arabidopsis salt acclimation. hy1-100 mutant displayed hypersensitive to salt stress, whereas the HY1 over expression line (35S:HY1) showed salt tolerant phenotype. Nitric oxide (NO) has been demonstrated to play an important role in salt stress signaling. However, the relationship between HY1 and endogenous NO function under salt stress was still unknown. In this chapter, we studied the origin of NO under salt stress by combined using pharmacological and genetic approaches, and the relationship between HY1 and nitrate reductase (NR)-derived NO production were also discussed. The results showed that the both NR and L-argine (L-Arg) pathway were responsible for NaCl-triggered NO generation. NR inihibitor tungstate and mammalian nitric oxide synthase (NOS) inihibitor NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) were able to block the NO generation induced by NaCl; meanwhile, root growth inhibition and fresh weight accumulation of wild-type induced by NaCl were also aggravated. The transcription levels of NIA1, NIA2 and nitric oxide associated 1 (NOA1) in wild-type roots were also induced by NaCl treatment. By contrast, NR mutant nia1-1/2-5, NOA1 mutant noal and nia1-1/2-5/noal failed to accumulate NO under salt stress, whereas the application of NO donor sodium nitroprusside (SNP) could increase NO production. Above results indicated that NO generation in Arabidopsis roots induced by NaCl is related to NR and NOA1 up-regulation. Moreover, nia1-1/2-5, noa1 and nia1-1/2-5/noa1 are all hypersensitive to NaCl stress, which could be partially rescued by exogenous NO donor diethylamine/nitric oxide adduct (NONOate). We next compared the differences of NO generation between WT, hy1-100, ho2, ho3 mutant under salt stress. The results showed that more NO production could be observed in hy1-100 mutant seedling root under salt stress. Interestingly, NO donor NONOate failed to alleviate the hypersensitivity phenotype of hy1-100 mutant upon NaCl during germination period, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (PTIO) reversed the salt tolerance phenotype of 35S:HY1-3. HO-1 inducer haemin could alleviate germination and cotyledon opening inhibition of WT seeds under salt stress. Whereas weak alleviation can be observed in noal mutant seedlings, and haemin failed to alleviate hypersensitivity phenotype of nia1-1/2-5 and nia1-1/2-5/noa1 mutants under salt stress. These results indicate that a cross-talk might exist between NR-derived NO and HY1 function during salt stress signaling, both of which are necessary for Arabidopsis salt tolerance.Previous research demonstrated that the soybean HO can respond to ultraviolet (UV) radiation. The induction of HO can alleviate oxidative damage caused by UV-B radiation, the ROS may be involved in this process (Yannarelli et al.,2006). However, detailed genetic evidence is required for further investigating the mechanism of HO in regulating UV-B radiation response. In this chapter, we examined the responses of four HO genes under UV-C radiation in Arabidopsis. The difference between wild-type and hy1-100 mutant for flavonoid biosynthetic metabolism and antioxidant defence system in response to UV-C was also studied. The results showed that, compared with the wild type, the hypersensitivity of hy1-100 mutant in response to UV-C radiation appeared, and this was due to its low level of flavonoid biosynthetic metabolism. Compared with the wild-type, hy1-100 contained less UV-absorbing compounds, and the down-regulating of the transcripts of four key enzymes for the flavonoid biosynthetic metabolism (chalcone synthase (CHS), chalcone isomerase(CHI), flavonoid 3-hydroxylase (F3H), and flavonol synthase (FLS)) were also found, respectively. Moreover, four key genes in the Arabidopsis wild-type flavonoid biosynthetic metabolism were significantly induced after UV-C radiation, whereas in the hy1-100 mutant, the induction of CHS and FLS is partially blocked and CHS expression is reversed significantly. We further hypothesis that this suppression of hy1-100 mutant might due to its interruption of the UV signal perception. For example, the expression of transduction factor HY5, HYH (HY5 homolog), MYB11 and MYB12 in the root of wild-type can be markedly induced by UV-C radiation, whereas partially inhibited in hyl-100 mutant. Furthermore, the results of real-time PCR analysis indicated that the hyper-sensitivity of the hy1-100 mutant in response to UV-C radiation may also be contributed to the failure in induction of the antioxidant defence genes expression, such as CSD1 (Cu/Zn superoxide dismutase 1), CAT1 (catalasel), CAT2 (catalase2), FSDl (Fe superoxide dismutase), cAPXl (cytosolic ascorbate peroxidase 1), and cAPX2 (cytosolic ascorbate peroxidase 2).更多还原