Physiological And Molecular Mechanisms Of Elevated CO₂ Concentration Alleviating Cadmium Stress Of Soybean（Glycine Max）

With the rapid development and growth of industrialization,the atmospheric CO₂concentration（a CO₂）has increased from 280 μmol.mol-1 to 416 μmol.mol-1 now.It is expected to keep rising at an average rate of2-3 μmol.mol-1 per year.Elevated CO₂concentration（e CO₂）can promote crop growth and regulate physiological processes in response to biotic and abiotic stresses.e CO₂ increases C3photosynthesis,regulates its growth and development,thus increasing biomass and yield.Cadmium（Cd）is the most common heavy metal pollutant in farmland.The World Health Organization has listed Cd as a key food pollutant,which poses a great threat to human health and food safety.As our daily food,soybean is a kind of crop that is extremely easy to accumulate a large amount of Cd.In this study,soybean Williams 82 was used as the material,we used physiological and biochemical methods together with transcriptomic comparison to explore the effects of e CO₂ on Cd-stressed soybean.The four different treatments: a CO₂（400 μmol.mol-1）,Cd Cl2（60 mmol.L-1）,e CO₂concentration（700 μmol.mol-1）,e CO₂ and Cd Cl2 interaction treatment were studied here.In this study,we used the physiological and biochemical methods,transcriptomics and molecular biology research,in order to explore the physiological and molecular mechanisms of e CO₂ in alleviating Cd stress.The main results are as follows:1.The e CO₂ alleviated the inhibition of Cd on soybean growth,significantly increased the dry weight of soybean leaves and roots.Elevated CO₂ can also significantly regulate the activities of superoxide dismutase（SOD）,catalase（CAT）,peroxidase（POD）,glutathione enzyme（GSH）,proline,soluble sugar and flavonoids.These defensive mechanisms reduced the contents of Cd2+,MDA,and H2O2 in soybean.2.In order to explore the possible mechanisms of that e CO₂ enhances the tolerance of soybean to Cd,transcriptome analysis of soybean leaves were conducted under AC,Cd Cl2,EC,and EC+Cd treatments.Transcriptome data showed that a total of 1158 differentially expressed genes（DEGs）were found under EC alone compared to the control group,including 342 genes that specifically responded to EC.Under Cd stress,a total of 7541 DEGs were identified,including 1339 genes that specifically responded to Cd.The total number of DEGs under the combined treatment of EC+Cd were 7076,among them 1816 genes were specifically response to EC+Cd.In addition,606 genes simultaneously responded to AC+Cd,EC,and EC+Cd.The results of KEGG enrichment analysis showed that the DEGs which respond to EC,Cd and EC+Cd were mainly enriched in plant hormone signal transduction,MAPK signal pathway,starch and sucrose metabolism,flavonoid synthesis,and photosynthesis.Based on physiological results,we analyzed the DEGs under different treatments,the main conclusions are as follows:（1）Under the treatment of Cd alone and EC+Cd,22 genes encoding POD enzymes were simultaneously changed.These genes were involved in phenylpropanoid synthesis,MAPK signaling,and keratinization processes.Compared with the control group,the expression levels of Glyma.10G222500（PER12）,Glyma.09G002500（PEX17）,and Glyma.16G055900（PEX55）were significantly upregulated under Cd treatment,but returned to normal levels under EC+Cd.（2）Heatmap analysis showed that 27 genes involved in regulating ROS synthesis significantly changed under Cd,EC and EC+Cd compared to AC,among them 17 genes related to EC+Cd were significantly upregulated compared to the treatment of Cd alone.（3）The expression of the proline dehydrogenase encoding gene Glyma.13G049700（PRODH2）,which inhibits proline degradation,was significantly upregulated under Cd treatment,but the expression of this gene decreased to the control level under EC+Cd.The Glyma.03G069400 encoding delta-1-pyrroline-5-carboxylate synthase（P5CS）was up-regulated under AC+Cd and further up-regulated under EC+Cd compared with AC.（4）Sixteen genes involved in glucose metabolism and 17 genes involved in glucose transport were selected in our study,all of which were related to carbohydrate transport and metabolic processes.（5）Thirty-nine genes encoding glutathione S-transferase（GST）were screened,and 26 of them were significantly upregulated under Cd and EC+Cd conditions.（6）We also identified 17 genes involved in the response to cadmium ions,cadmium ion transmembrane transport,and cadmium ion detoxification processes.The Glyma.15G262300 encoding metal tolerant protein 10（MTP10）,moreover,Glyma.11G208700 encoding OXS3-like were significantly downregulated under Cd,but returned to the control level under EC+Cd.（7）The expression of MAPK related genes MEK2,MEKK1,MEKK3,MEKK7,MPK3,and MPK4 b were significantly upregulated under Cd stress and EC+Cd conditions compared to the control.The heat map results showed that Glyma.04G199900（b HLH137）,Glyma.03G105700（b HLH18）,Glyma.08G294000（b HLH89）,Glyma.05G094400（MEKK17）,Glyma.02G228300（MEKK18）and Glyma.09G072000（ERF17）were down-regulated under Cd stress,but EC+Cd treatment significantly increased the expression of these genes compared to Cd alone.3.According to the analysis of transcriptome data,we obtained a target gene Gm MYB84 that simultaneously responded to EC,Cd stress,and EC+Cd treatment（it was reported that actively regulated the response of plants to drought and salt stress）.The homologous gene of Gm MYB84 in Arabidopsis is At MYB116.We found that myb116 Arabidopsis mutant was more sensitive to Cd stress compared to WT,while the overexpression of Gm MYB84 in Arabidopsis plant OE-Gm MYB84 significantly increased its tolerance to Cd stress.In details,the OE-Gm MYB84 Arabidopsis plants in response to Cd stress showed higher germination rates,root length,leaf dry weight,chlorophyll and flavonoid content,but lower MDA,and H2O2 content.Under Cd stress,the soybean Gm MYB84 knockout mutant constructed by CRISPR-Cas9 significantly inhibited its growth,and showed decreased the dry weight of leaves and roots,increased the content of H2O2,MDA,O2-.4.In previously study,Glyma.01G172700 and Glyma.02G042500 were proved to involve in lignin and chitin synthesis,which play an important role in plant development and defense to abiotic stresses.Our study showed that Gm MYB84 could combine with downstream genes Glyma.01G172700 and Glyma.02G042500.In summary,elevated CO₂ concentration may affect soybean response to Cd stress through multiple pathways,among them Gm MYB84 is one of the important genes that plays a positive role in alleviating Cd stress.In addition,Gm MYB84 as a transcription factor can bind to downstream genes Glyma.01G172700 and Glyma.02G042500,which may result in affecting the synthesis of lignin and chitin related to abiotic stress,thereby alleviating Cd stress.This study explored the key genes and possible signaling pathways that elevated CO₂ concentration alleviating cadmium stress,providing a theoretical basis for cultivating heavy metal resistant soybeans in the future.