| 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). |
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