Mechanism Of Reduced Glutathione Enhancing Resistance To Pb²⁺ Stress In Lolium Perenne L.

Heavy metal pollution has been the focus problem because of their persistent pollution endangering to the environment and human health.The combined use of exogenous growth regulators and phytoremediation has become the main measure to solve the heavy metal pollution problem in recent years by the advantages of low cost,easy operation and high efficiency.Lead（Pb）is one of the most common and harmful heavy metals in the environment.It is an urgent problem for researchers at present that how to lessen lead pollution.Reduced glutathione（GSH）plays an important role in uptake and transport of lead and growth in plants under heavy metal stress.L-buthionine-sulfoximine（BSO）,a GSH synthase inhibitor,can reduce GSH content in plants.Most studies focus on the mechanism of enhancing plant resistance to cadmium stress,while the physiological and molecular mechanism of GSH enhancing plant resistance to lead stress is still an open problem.Lolium perenne has the advantages of rapid growth,large biomass,well-developed root system and strong adaptability,which can be as an ideal material for the study of heavy metal pollution soil remediation,and can also be used to speed up the research process by hydroponics.In this paper,Lolium perenne‘cuttle’with 12 weeks old was selected as the experimental materials,the effects of lead stress on growth,ultrastructure,nutrient element were analyzed,the response of photosynthesis and antioxidant system to lead stress were defined,the key genes involved in GSH synthesis were identified by transcriptome analysis,and Lp GCS were cloned and functionally identified.which explored the regulatory mechanism of GSH in Lolium perenne under lead stress from morphological,physiological,biochemical and molecular levels.These results would lay a theoretical foundation for revealing the mechanism of resistance to heavy metal lead stress in plant,and provide a reference for breeding materials for remediation of lead contaminated soil.The main research results are as follows:（1）GSH protected the integrity of chloroplast,mitochondria and other ultrastructures of Lolium perenne under Pb²⁺stress,and alleviated the inhibition of lead on growth.On the contrary,BSO aggravated the damage of ultrastructures and the inhibition of growth under Pb²⁺stress.（2）GSH promoted the uptake and transport of lead in Lolium perenne under Pb²⁺stress,and increased the content of lead in cell wall and cytosol.BSO reduced the lead content in leaves and transport in plant,and reduced the content of lead in cell wall,but increased the lead content in cytosol.（3）GSH increased the content of Ca,Mg,N,S and K in roots,stems and leaves,but significantly decreased the content of Na.Spraying BSO reduced the content of Na in roots,stems and leaves and k content in roots,but significantly increased the content of P in roots and stems and K content in stems.（4）GSH application significantly increased chlorophyll content,photosynthetic parameters and chlorophyll fluorescence kinetic parameters.The content of carotenoids and photosynthetic parameters were decreased after BSO spraying.（5）GSH application increased the activity of superoxide dismutase（SOD）,glutathione peroxidase（GPX）and glutathione reductase（GR）,and significantly increased GSH/oxidized glutathione（GSSG）ratio,GSH,phytochelate（PCs）and sulfhydryl group（T-SH）content.Meanwhile,after treatment with GSH,the production of anion（O₂^-）and hydrogen peroxide（H₂O₂）,the relative permeability（EC）and the content of malondialdehyde（MDA）were decreased.However,BSO increased MDA content and EC values,decreased the activity of SOD in leaves,GPX,γ-GCS and GR,and decreased GSH/GSSG ratio and the contents of GSH,GSSG,MT,PCs and T-SH.（6）A total of 1246 differential genes were identified after GSH treatment,of which 587were up-regulated and 659 were down-regulated.A total of 135 GO terms were significantly enriched,with a high proportion of small molecule metabolism process,generation of precursor metabolites and energy,oxidoreductase activity,tetrapyrrole binding,cytoplasmic part,intracellular organelle part and organelle part.Twelve KEGG metabolic pathways were significantly enriched,including carbon fixation in photosynthetic organisms,photosynthesis-antenna proteins,glycolysis/fructose,fructose and mannose metabolism,photosynthesis,pentose phosphate pathway,glyoxylate and dicarboxylate metabolism,glutathione metabolism,nitrogen metabolism,porphyrin and chlorophyll metabolism,monoterpenoid biosynthesis and ascorbate and aldarate metabolism.A total of 526 differential genes were identified after BSO treatment,of which 344 were up-regulated and 182 were down-regulated.There was no significant enrichment term in GO database,only one KEGG metabolic pathway named plant-pathogen interaction was significantly enriched.（7）After GSH application,a total of 31 genes were identified in photosynthesis-antenna proteins pathway,photosynthesis and porphyrin and chlorophyll metabolism pathways,among which 30 genes were up-regulated,including PSⅠprotein（Lp PSAG,Lp PSAN）,PSⅡproteins（Lp PSBO,Lp PSBP,Lp PSBQ,Lp PSBW,Lp PSB27,Lp PSB28）,ATPase（Lp DELTA）,light-harvesting chlorophyll protein complexes（Lp LHCA1,Lp LHCA2,Lp LHCA4,Lp LHCB1,Lp LHCB6）,glutamyl-t RNA reductase（Lp HEMA）,magnesium protoporphyrin monoester cyclase gene（Lp MPEC）and red chlorophyll-decomposable metabolite reductase（Lp PCCR）.（8）After the application of GSH,the expression of glutathione S-transferase（Lp GST）and ascorbate peroxidase（Lp APX）were up-regulated and the expression of glutathione peroxidase（Lp GPX）was down-regulated in the glutathione metabolic pathway.The expression of glutathione S transferase（Lp GST）gene was upregulated after the application of BSO.（9）Lp ABCF4（ABC）and Lp ABCB1（ABC）were identified after GSH spraying,and both of them were down-regulated.The expression of Lp NRAMP2（NRAMP）was down-regulated.after the application of BSO,Lp ABCC10（ABC）gene was up-regulated and Lp NRAMP5（NRAMP）gene was up-regulated.（10）Overexpression of glutamylcysteine synthase（Lp GCS⁺）promoted the growth and development of tobacco.Together,GSH can regulate the gene expression of Lolium perenne,increase the absorption of nutrient elements and the differentiation distribution of lead,increase antioxidant capacity,alleviate lead stress,maintain ultrastructure integrity,enhance photosynthesis,and promote the growth and development of the plant.