Functional Analysis Of An Arabidopsis Hemoglobin

Hemoglobin (Hb) occurs in all the kingdoms of living organisms. In plants there are at least three distinct types of hemoglobin that have been categorized as symbiotic, nonsymbiotic and truncated hemoglobin (Dordas et al., 2003b). Symbiotic Hbs found in legumes and other nitrogen-fixing plants. The distributing of nonsymbiotic Hbs was more extensive, existing not only in the plants that had symbiotic Hbs, but also in other plants. Nonsymbiotic Hbs could be divided into two species: class 1 and class 2. Class 2 Hbs had similar affinities and dissociation kinetics of oxygen to symbiotic Hbs. Class 1 Hbs had different affinities and dissociation kinetics of oxygen and was called stress-induced hemoglobin, which could be induced by hypoxic stress and excessive nitrogenous nutrition (Dordas et al., 2003) . Class 1 Hbs almost found in whole plant kingdom and maybe the evolutional ancestor of symbiotic Hbs. Therefore, class 1 Hbs might play an important role in plant metabolism. But so far, the function of class 1 Hbs was unclear.The aim of our study is to investigate the function of class 1 Hbs of Arabidopsis (AtGLB1). We gained Arabidopsis lines with different hemoglobin levels using overexpression and RNAi technology. After northern gel blot analysis, we choose D1 (the overexpressing line) and R11 (the underexpressing line) from the transgenic lines for following research. The main results are as follows.After hypoxic stress treatments, the concentration of hydrogen peroxide and the activities of ADH increased in three lines (WT, D1, R11). But the extent of theincrease was different in three lines. The line overexpressing AtGLBl accumulated relatively little H2O2, and has enhanced tolerance to hypoxia. In contrast, the lines in which AtGLBl expression was suppressed by RNAi technique accumulated more H2O2, and were more sensitive to hypoxia. AtGLBl may play certain role in modulation of hydrogen peroxide level during hypoxic stress. Therefore, we detected the tolerance of protoplast and leaves with different hemoglobin 1 levels to exogenous hydrogen peroxide. The result showed that the line with high hemoglobin 1 level were more tolerant, while the line with low hemoglobin 1 level were more sensitive to exogenous hydrogen peroxide. AtGLBl might alleviate the hydrogen peroxide stress.All aerobic organisms have evolved a complex antioxidant system involved in scavenging H2O2, composed of nonenzymatic scavengers (such as ascorbate, glutathione, and hydrophobic molecules) and detoxifying enzymes. The latter include catalases (CAT; EC 1.11.1.6) and ascorbate peroxidases (APX; EC 1.11.1.11). Antioxidant system involved in scavenging H2O2 was also analyzed in our experiment. Before hypoxic stress treatment, the antioxidant system was not different in three lines. After hypoxic stress treatment, the activities of CAT and APX and ascorbate content were, indeed, increased in all three lines examined during the stress treatment, while only Dl can retain these relative high levels up to 48 h of the treatment, suggesting that antioxidant system may play an important role for the low H2O2 level of Arabidopsis over-expressing AtGLBl. However, how this Arabidopsis maintain the high level of these antioxidants for longer time compared with control lines remains unknown currently.The analyses of rapid-scan stopped-flow spectrophotometry showed that recombinant oxygenated AtGLBl could react with H2O2 and lead to the decrease of H2O2 level. The recombinant oxygenated AtGLBl with different concentrations could react with l.OxlO"4 M hydrogen peroxide. The higher the concentration of recombinant oxygenated AtGLBl was, the faster the reaction was. Therefore, AtGLBl might react with H2O2 and scavenge H2O2 directly during hypoxic stress. Alternatively, a recent report indicated that AtGLBl exhibited peroxidase-like activity (Sakamoto et al., 2004). Based on these results, we may also explain our observation for the enhanced tolerance of Arabidopsis over-expressing AtGLBl to hypoxia that hemoglobin 1 may contribute to scavenging H2O2, thus decreasing the H2O2 level and increasing the H2O2 tolerance. This needs to be further tested experimentally.AtGLBl play an important role during hypoxic stress. It is reported that function of a protein is be closely bound up to its location. We studied the location of AtGLB 1 through the instantaneous expression of the recombinant protein of AtGLBl and GFP in onion epidermis. The result showed that AtGLBl located in cytoplasm and nucleolus maily.