Study On Physiological Mechanism Of γ-Aminobutyric Acid Alleviating Cadmium Stress In Oilseed Rape

With the rapid development of urbanization and industry,the problem of Cadmium（Cd）pollution is becoming increasingly serious.Cd pollution is a common heavy metal pollution in farmland soils in China.It not only affects plant growth,but also harms animal and human health through food chain enrichment.γ-Aminobutyric acid（GABA）is a four-carbon non-protein amino acid that widely exists in organisms.In mammals,GABA is an inhibitory neurotransmitter in the central nervous system.When plants are under stress,GABA will rapidly accumulate and trigger a series of signal regulatory networks.Oilseed rape is a major oil crop in China,with well-developed roots,large biomass,and a certain concentration of Cd,making it an ideal phytoremediation material.In this study,Brassica napus L.,which has high yield,wide planting area,and relatively low Cd resistance,and Brassica juncea L.,which has low yield,small planting area,and strong Cd resistance,were used as materials.The effects of exogenous GABA of 5 mmol/L on Cd uptake and tolerance in oilseed rape under Cd stress were studied,and the physiological mechanism of GABA inducing Cd resistance in oilseed rape was explored,provide a theoretical basis for GABA to regulate plant growth and enhance heavy metal resistance mechanisms,and explore new candidate genes for GABA signal regulatory networks.The main research results are as follows:（1）GABA affects the growth of oilseed rape under Cd stress50μmol/L Cd inhibited the growth of Brassica napus and Brassica juncea,resulting in a decrease in aboveground and root biomass,as well as a decrease in total root length,root surface area,root volume,and average diameter;Photosynthesis was inhibited,and net photosynthetic rate（Pn）,stomatal conductance（Gs）,intercellular CO₂ concentration（Ci）and transpiration rate（Tr）decreased.Adding 5 mmol/L GABA externally increased the biomass and root development of the two oilseed rape varieties compared to Cd alone;The damage of Cd to the photosystem of oilseed rape was alleviated.Pn,Gs,and Tr in Brassica napus and Brassica juncea increased,chlorophyll and carotenoid contents increased,and the photochemical efficiency（Fv/Fm）of photosystem II（PSII）increased by 22.4%and 21.7%,respectively.This indicates that GABA can alleviate the physiological toxicity of Cd stress on oilseed rape growth.（2）GABA affects Cd absorption in oilseed rapeCd stress increased the content of Cd in the shoot and root of two kinds of oilseed rape,and the accumulation of Cd in Brassica juncea was greater than that in Brassica napus.In Brassica napus,Cd is mainly distributed in cell wall and cell fluid,and the subcellular distribution of Cd is as follows:cell wall>cell fluid>organelle;In Brassica juncea,Cd is more distributed in the cell fluid,and the subcellular distribution of Cd is as follows:cell fluid>cell wall>organelle.After GABA+Cd treatment,compared with Cd stress alone,the Cd content in shoot and root of Brassica napus decreased by 30.4%and 16.2%respectively,and that in shoot and root of Brassica juncea decreased by 25.6%and 17.8%respectively;The content of Cd in the cell wall and organelle in the shoot of Brassica napus decreased,but the distribution of Cd content in the root did not change significantly;The content of Cd in cell sap of shoot and root of Brassica juncea increased.Exogenous GABA can reduce the content of Cd in the two kinds of oilseed rape,help to reduce the accumulation of Cd in the organelles,and alleviate the toxicity of Cd stress on oilseed rape.（3）GABA alleviates oxidative damage and activates antioxidant system of oilseed rape under Cd stressCd stress caused oxidative damage,increased relative electrical conductivity and malondialdehyde content,and accumulation of active oxygen species（superoxide anion and hydrogen peroxide）in both oilseed rape varieties.After GABA+Cd treatment,compared to Cd stress alone,the oxidative damage degree of the two oilseed rape varieties was restored to a certain extent,and the contents of malondialdehyde,superoxide anion,and hydrogen peroxide were significantly decreased;The content of starch and sugar increased significantly;The content of ascorbic acid（ASA）in the shoot and root of Brassica napus increased by 17.2%and23.3%,respectively,while that of Brassica juncea increased by 15.6%and 9.2%,respectively;The content of glutathione（GSH）in the shoot of Brassica napus and Brassica juncea increased by 20.6%and 24.8%,respectively.The results of reactive oxygen species staining showed that GABA decreased the content of superoxide anion and hydrogen peroxide in oilseed rape leaves under Cd stress.The staining of roots with propidium iodide（PI）showed that GABA reduced cell death induced by Cd stress.GABA can alleviate the oxidative damage of two oilseed rape varieties under Cd stress,and resist the harm of Cd by activating their antioxidant systems.（4）Transcriptome sequencing analysis of oilseed rape under Cd stressTranscriptome analysis shows that there are 4659 differentially expressed genes in Brassica napus treated with GABA+Cd compared to the control,of which 2502 genes are upregulated and 2157 genes are downregulated;In Brassica juncea,there are 711 differentially expressed genes,352 upregulated genes,and 359 downregulated genes.The GO（Gene Ontology Consortium）annotation and KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway enrichment analysis of differentially expressed genes indicate that these genes are mainly related to photosynthesis,glutathione metabolism,starch and sucrose metabolism.GABA+Cd treatment upregulates the expression of glutathione S-transferase（GST）related genes in the glutathione metabolism pathway in oilseed rape.GST catalyzes GSH to enhance the scavenging ability of reactive oxygen species,alleviate oxidative damage in oilseed rape under stress,and improve the tolerance of oilseed rape to Cd stress.Three differentially expressed genes related to glutathione metabolism are screened（Bna A03g26140D,Bna C03g30880D,Bna A09g29540D）.These genes regulate GST activity in cells to alleviate the toxicity of Cd,but the specific molecular signaling pathway needs further study.To sum up,exogenous GABA can reduce the oxidative damage of oilseed rape under Cd stress,activate the antioxidant system,reduce the accumulation of Cd in oilseed rape,up-regulate the expression of GST-related genes,alleviate the toxicity of Cd to the growth and development of oilseed rape,enhance the tolerance of oilseed rape to Cd stress.Using GABA to alleviate Cd stress in oilseed rape provides a theoretical basis for improving the efficiency of oilseed rape in repairing Cd pollution in agricultural production.