Study On The Response Of Enhalus Acoroides To Ocean Acidification(OA) And Its Mechanism To Alleviate OA

The earth is in the Sixth Mass Extinction due to the increase of CO₂,and ocean acidification（OA）is causing more and more serious ecological damage.As one of the three major marine ecosystems,seagrass has great potential in reducing OA and increasing carbon sequestration.As a dominant species in the Indo-Pacific convergence zone,Enhalus acoroides has significant potential in alleviating OA and increasing carbon sequestration.Previous studies have simply explained one of the possible inorganic carbon（C_i）uptake modes in E.acoroides,but little is known about its ability and mechanism to absorb C_i,its response mechanism to OA and its ability to mitigate OA.This study completely explained the structural characteristics of carbon storage of E.acoroides,transition of the C_iabsorption mode during OA,and explained its response mechanism at the molecular level.The main contents and results are listed as follows:1.Through the analysis of the content of wet weight,dry weight,and the content of C and N in each part of the E.acoroides adult plant,it was found that the underground stem accounted for the most weight proportion,accounting for about half of the whole plant,and the underground stem had the highest content of C,accounting for 40.62%of dry weight and 8.21%of wet weight.The content of C in the underground tissue of E.acoroides was also higher than that in the aboveground tissue.This indicates that carbon in E.acoroides is mainly stored in underground tissues,especially in underground stems,suggesting that E.acoroides may be an efficient carbon sink for marine plants.2.Measuring the photosynthetic oxygen release rate of E.acoroides proved that,at 2.2 m M C_iconcentration,its CO₂fixation efficiency increased to 81.89 t CO₂/year/Ha under p H 6.5 from 27.59 t CO₂/year/Ha at p H 8.2,and even reached88.11 t CO₂/year/Ha at p H 6.5 with unlimited C_i,and the CO₂fixation efficiency increased with p H drops.These results indicated that OA was beneficial to increase the carbon sequestration and carbon sequestration capacity of E.acoroides,and E.acoroides had the potential to alleviate OA.3.The high carbon sequestration capacity of E.acoroides is made possible by three pathways for C_iutilization,which included absorbing CO₂directly,transforming HCO₃^-into CO₂through extracellular carbonic anhydrase,and absorbing HCO₃^-directly by anion-exchange protein then transforming it to CO₂through intracellular carbonic anhydrase,as verified by inhibitor addition experiments.In addition to the direct absorption of CO₂,HCO₃^-is absorbed through two pathways at p H 8.2,with the direct transport of HCO₃^-by anionic transporter being more active.With increasing acidification,the direct absorption of CO₂becomes predominant and the absorption of HCO₃^-no longer functions effectively.The effectiveness of the two HCO₃^-absorption pathways is about equal at p H 7.5,but only CO₂dissolved in water is used directly at p H 6.5.With the increase of C_icontent in water and the decrease of p H,the photosynthesis and C_iabsorption ability of E.acoroides actually improved by transitioning among the three C_iuptake modes,thus increasing total carbon sequestration and providing protection against OA.This suggests a greater survival adaptability and carbon sequestration capacity of E.acoroides due to the transition between three pathways for C_iutilization.4.The expressions of the four crucial enzymes,ribulose-1,5-bisphosphate carboxylase/oxygenase（Rubisco）,phosphoglyceratekinase（PGK）,glyceraldehyde-3-phosphate dehydrogenase（GAPDH）,and triose-phosphate isomerase（TIM）,as well asγ-carbonic anhydrase 1,were significantly up-regulated under OA,as determined by using Tandem mass tags（TMT）-LC-MS/MS and parallel reaction monitoring（PRM）techniques.At the same time,Lhcb1-3 and Psb O in photosystemⅡas well as Lhca1-3 in photosystemⅠwere down-regulated.In contrast,no significant changes in respiration were observed.These results indicate that,under OA conditions,respiration did not change,but the dark reactions were enhanced,including the recycling of inorganic carbon and its assimilation via the Calvin cycle,and as well,light energy absorption and utilization tended to be more balanced.The above results strongly suggest that tropical seagrass E.acoroides may play an important role in mitigating OA by increasing carbon sequestration through the conversion of CO₂to organic substances.In addition,OA increased the expression of peroxidase and ascorbate peroxidase of E.acoroides,thereby improving its ability to resist stress,resulting in stronger adaptability and vitality.5.Through simulated OA in the laboratory,the changes in metabolite content in E.acoroides seedlings were measured and analyzed by untargeted metabolomics technology.It was found that OA had a great effect on the metabolism of E.acoroides seedlings,and the different metabolites were mainly concentrated in the Citrate cycle,Sulfur metabolism,Fatty acid biosynthesis,Glyoxylate and dicarboxylate metabolism,Flavonoid biosynthesis,Carbon metabolism,Oxidative phosphorylation related pathways.6.Based on the combined analysis of TMT proteomics and non-targeted metabolomics,it was found that OA down-regulated the expression of malate dehydrogenase in the TCA cycle of E.acoroides,resulting in the accumulation of metabolites in the TCA cycle,which might promote the production of glutamate and the accumulation of acetyl Co A and ultimately resulted in the increase of terpenoids and fatty acids.It may also promote the synthesis of chlorophyll and cytochrome,thereby improving photosynthesis and the regulation of morphogenesis,as well as the enhancement of stress resistance.7.Through metabolomics analysis,it is found that OA increased the content of some disease resistance metabolites and metabolites with economic value in E.acoroides.Although the influence of these metabolites on the survival adaptability of E.acoroides is still unknown based on the current study,this can be one of the research directions in the future.In addition,it is also worth studying whether OA can be used to improve the economic value of E.acoroides,so as to turn OA into benefits to some extent.In conclusion,this study shows that E.acoroides has the possibility of being an efficient carbon sink due to its structural and carbon distribution characteristics,as well as through the transition of C_iabsorption mode,the regulation between dark and light reactions in photosynthesis,the regulation of antioxidant enzymes and antioxidant metabolites,and the regulation of TCA cycle.These results not only systematically explain the carbon sequestration capacity of E.acoroides and the specific response mechanism in response to OA,but also provide more evidence for using seagrass as an efficient carbon sink to increase carbon sequestration and mitigate OA.