Abscisic Acid(ABA)-mediated Antioxidant Mechanism In Pyropia Yezoensis And Its Application In Production

Pyropia yezoensis（Py.yezoensis）grows in the intertidal zone.With the tide,it periodically undergoes the habitat conversion of dehydration and rehydration.Therefore,it was evolved a unique stress resistance mechanism,including antioxidative system.Additionally,abscisic acid（ABA）plays an important role in responding to stress.Therefore,it has a high scientific research value for the analysis of algae stress resistance mechanism.Consequently,we used the Py.yezoensis thallus as the experimental material,the changes of photosynthetic parameters,ABA content and related antioxidant enzyme activities was measured under different treatment conditions,and explored the specific regulatory mechanism of stress hormone ABA and antioxidant system under high salinity stress in Py.yezoensis.At the same time,transcriptome data were analyzed and verified the specific regulatory mechanism of ABA on major antioxidant enzymes at the molecular level.On the other hand,combined with the actual production situation,explored the mechanism of rotten diseases induced by low salinity and high temperature and the role of ABA in Py.yezoensis thallus.Finally,we found a key gene Py MPV17 related to the removal of reactive oxygen species（ROS）in transcriptome data.And constructed the overexpression and silencing of Py MPV17 by transgenic technology to verify the related stress resistant metabolic pathway,it laid a theoretical foundation for the cultivation of stress resistant strains of Py.yezoensis.The main results are as follows:1.Under high salinity stress,ABA has a positive regulation on antioxidant enzymes in Py.yezoensis.The activities of different antioxidant enzymes and the change of H₂O₂ content were determined by adding ABA synthesis inhibitors and exogenous ABA under hypersaline.The results show that the activities of related antioxidant enzymes（CAT,SOD,APX,GR）of Py.yezoensis treated with ABA inhibitors（Clo,Mev）had a downward trend,but the inhibition was weakened or even significantly increased after adding ABA.Transcriptome data showed that the expression of antioxidant enzyme genes were significantly increased after exogenous ABA treatment,which was consistent with the enzyme activity data.At the same time,we measured the change of H₂O₂ content.The results showed that ABA reduced the content of H₂O₂,It may be relevant that antioxidant enzymes play a significant role in transforming H₂O₂ to H₂O under ABA treatment,thus reducing the content of H₂O₂ in Py.yezoensis.2.The precursor IPP of ABA may be synthesized by MVA pathway and MEP pathway simultaneously in Py.yezoensis,but mainly by MEP pathway.The specific synthesis pathway of ABA may be similar to higher plants,mainly by carotenoid pathway,also called indirect synthesis pathway.We selected inhibitors of the two pathways of IPP to treat the algae at the same time,and measured the ABA content.The results showed that the content of ABA decreased significantly.Meanwhile,transcriptome data showed that seven genes in MEP pathway were annotated,including DXS、DXR、MCT、CMK、MECPS、HDS、HDR.While only AATC gene was annotated in MVA pathway.In addition,only DXS gene expression in MEP pathway was significantly decreased in algae treated with inhibitors.Therefore,we speculated that the synthesis of ABA precursor IPP in algae was mainly through MEP pathway,but there was MVA pathway.At the same time,our transcriptome data annotated a number of ABA carotenoid synthesis pathways,including FPS,ZEP,PDS,SDR.Among them,SDR is a differentially expressed gene,and the addition of inhibitors can significantly reduce the expression of this gene.Therefore,ABA is mainly synthesized through the carotenoid pathway in Py.yezoensis.3.The ABA-mediated antioxidant enzymes and their transcriptional level under the high temperature stress and low salinity conditions were studied.The content of ABA and the activity and expression of antioxidant enzymes were determined under different temperature（15℃,20℃）and salinity（30,25,20）.Here,the photosynthesis parameters,the contents of hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）were determined in different treatment conditions.The results showed that the photosynthesis system might be damaged under the conditions of low salinity of 20 at20℃temperature.Although the accumulation of H₂O₂was not detected,the MDA was significantly up-regulated in the samples with low salinity.At salinity of 20,the SOD and POD activities of the samples were up-regulated when they were stressed with high temperature of 20℃,which indicated that the oxygen radical generation and scavenging system in algal cells were active.However,all antioxidant enzymes and the related genes determined here were down-regulated under the conditions of salinity 20 and20℃.Therefore,we speculated that in the cultivation and production of Py.yezoensis in the sea area,the disease rot induced by high temperature is likely to be directly related to the decrease of ABA content and the inhibition of antioxidant enzyme system,which may be one of the physiological basis of the high temperature disease rot of Py.yezoensis in the production process.4.We found the Py MPV17 gene is differentially expressed under different treatment conditions in transcriptome data.Based on the possible mechanism of ABA mediated up regulation of antioxidant enzymes and functional studies under various stress conditions,the key gene Py MPV17 was screened and overexpressed or silenced strains were obtained by gene editing method,and verified at DNA and RNA levels.In the next step,we will study the stress resistance phenotype and related gene expression to verify the function of the target gene and its application value in the cultivation of new strains of Py.yezoensis.