Preliminary Study On Flg22-induced Defense Responses In Saccharina Japonica (Phaeophyta)

In the long process of evolution, plants have developed a variety of defense responses to resist the infection of potential pathogens in the surrounding. Specially, some molecules from the pathogens, such as flagellin and lipopolysaccharide, are able to induce non-specific defense responses in plants. It has been confirmed that flagellin and flg22, which is the most conserved22-amino-acid peptide in the N-terminal part of flagellin, function as effective elicitors, which can trigger defense responses in higher plants. Algae and higher plants share some conserved characteristics on defense response pathways. Therefore, in this study, flg22-induced defense responses both in female gametophytes and sporophytes of Saccharina japonica are investigated. Our results would enrich the theory of plant molecular pathology. The results are as follows:1. Cell death observation in flg22-induced S. japonica.The final concentration of flg22is200μM for female gametophytes, and500μM for sporophytes.The result of Evans blue dye shows that cell death is not observed in female gametophytes within3to6h induced by flg22, while amounts of cells are stained blue between9to12h, indicating the occurrence of cell death. Cell death in sporophytes is not observed within2to6h after flg22induction, whereas large amounts of cell death are observed from8to10h.2. Ultrastructural changes in flg22-induced female gametophytes of S. japonica.Electron microscopy result shows that the ultrastructure deforms significantly in cells of female gametophytes after being induced by flg22. The ultrastructural changes are similar with those of hypersensitive programmed cell death (PCD) in higher plants, including the condensation of chloroplasts and thickened cell walls, as well as relatively stable structures of mitochondria and nucleus. Chloroplasts condense after being induced by flg22from2to4h, together with cell wall thickening. From6to8h, the cells appear smaller than those at earlier stage. In addition, chloroplasts condense seriously, and slight dissolution of nuclear membranes is observed. However, the structure of mitochondria remains relatively stable during the whole stage of induction.3. The observation of DNA degradation in flg22-induced sporophytes of S.japonica.By using TdT-mediated dUTP-biotin nick and labeling (TUNEL), DNA degradation in cells of sporophytes is observed immediately2h after being induced by flg22. Fragments with3’-OH groups of degraded nuclear DNA increase gradually with the induction time and spread from the induction site.4. The quantitative detection of H2O2in flg22-induced S.japonica.By using Luminol-dependent fluorescence detection method, rapid release of H2O2is detected in cells of female gametophytes and sporophytes after flg22induction, with a peak of about46μM at2h and20μM at3h respectively. Then both of the concentration of H2O2decline gradually and return to the initial level at5h and6h respectively.5. Histological detection of ROS in flg22-induced S.japonica.Radical oxygen species (ROS) production is observed histologically using the fluorescent dye2’,7’-dichlorofluorescein diacetate (DCFH-DA). The green DCF fluorescence is detected immediately in female gametophytes and sporophytes1h after being induced by flg22. Both of the green DCF fluorescence intensity increase gradually with the induction time, reaching the highest levels at2h and3h respectively, then decrease gradually. The histological observation shows a consistent result with that of quantitative analysis of H2O2.6. The detection of antioxidant enzyme activities in flg22-induced female gametophytes of S.japonica.The changes of antioxidant enzyme activities in cells of female gametophytes are detected by the method of visible spectrometry. CAT activity increases immediately and consistently within0to1h, reaches the maximum value of10.4U·ml-1at1h, then decreases to control value at5h. SOD activity shows a similar change with that of CAT, exhibiting a peak value of73.29U·ml-1at2h. Compared with activities of CAT and SOD, GSH-PX activity increases slowly, and exhibits the peak value of252.08U·ml-1at3h, then decreases after3h, while there is no recover to control value at5h.7. The detection of phenols content in flg22-induced female gametophytes of S. japonica.Phenols content in cells of female gametophytes is detected by using Folin-Denis reagent. It is showed that phenols content increases within0to3h after flg22induction and decreases from3to5h. Maximum content of phenols is14.22μg·ml-1at3h. At5h, phenols content is almost equal to that of control experiments.According to our data, flg22is an effective elicitor which could induce defense responses in S. japonica.