Response Mechanism Of Lignin Synthesis In Rice Under Copper Stress And The Role Of Rice Laccase In Plants Tolerance To Heavy Metal

Soil heavy metal pollution has become a worldwide environmental problem.Excess heavy metals in soil not only affect crop yield and quality,but also can harm human health through the food chain.Improving the plants resistance to heavy metal can reduce the negative effects of pollution caused by heavy metals.At present,researches on plant heavy metal tolerance include the following contents,such as limiting heavy metals uptake,heavy metal efflux in root,sequestration of organic compounds,the effects of antioxidant system and heavy metal fixation in cell wall.Lignin is one component of the cell wall,it can be generated by peroxidase and laccase-catalyzed polymerization of monomers and participate in plant response to environmental stresses.In present study,we focused on the response mechanism of lignin in rice under copper(Cu)stress and overexpressed rice laccase genes in Arabidopsis to study their roles in plant tolerance to heavy metals,results are as follows.First,we analyzed the effect of Cu treatment of different concentrations and times on rice seedlings at the transcriptional and physiological levels.The results showed that lignin biosynthesis pathway genes were upregulated under Cu stress,lignin content of rice roots increased significantly with the increasing concentrations and durations of Cu treatment;root growth was significantly inhibited by Cu stress,while the S/G values had not obviously changes between control and Cu-treated rice roots.Meanwhile,Cu treatment increased the activities of G-POD,which in the soluble and ionically bound cell wall fraction and LAC,except for the activities of covalently G-POD;the activity of ionically NADH-POD,H2O2 and total soluble phenol contents in rice roots were increased significantly under Cu treatments,compared with the control.These results suggested that Cu stress could notably increase the level of lignin accumulation in rice roots by enhancing the lignin polymerization.Cu stress can induce accumulation of lignin and hydrogen peroxide in rice roots.Meanwhile hydrogen peroxide can be used as a co-substrate for peroxidase involved in the polymerization of lignin precursors,so we used an NADPH oxidase inhibitor(diphenyliodonium,DPI)and a H2O2 scavenger(dimethyl thiourea,DMTU)to examine the role of Cu-induced H2O2 in lignin biosynthesis.The results showed that,compared with Cu treatment alone,DPI and DMTU pretreatment significantly reduced the dry weight of rice shoots and roots,decreased the content of hydrogen peroxide and lignin in rice roots,while the expressions of some lignin biosynthetic pathway genes were also significantly down-regulated.The activities of ionically bound cell wall G-POD,NADH-POD and LAC were significantly reduced.Meanwhile the pretreatments of DPI and DMTU can significantly promote translocation of Cu from root to shoot in rice seedling.In contrast,exogenous H2O2 treatment increased endogenous levels of hydrogen peroxide in rice roots,it also can significantly increase the expressions of lignin biosynthesis pathway genes,and lignin content in rice roots;short-term hydrogen peroxide treatment also can significantly improve the activities of ionically bound cell wall G-POD,NADH-POD and LAC.These results suggested that Cu stress-induced H2O2 could be involved in the regulation of lignin polymerization and accumulation,which may affect the process of Cu translocation from root to shoot in rice seedlings.Cu stress can significantly increase the laccase activity of rice root,while it can participate in the polymerization of monomers.Cetyl trimethyl ammonium bromide(CTAB),a specific inhibitor of laccase,was used to examine the effect of laccase on lignin biosynthesis and Cu tolerance in rice seedling at the physiological level.The results showed that CTAB treatment induced a significant reduction of laccase activity,the dry weight,root elongation and lignin accumulation of rice seedling,excess Cu treatment increased the degree of inhibition;with or without Cu treatment,CTAB could significantly improve Cu transport.These results indicated that laccase may play an important role in the process of lignin accumulation in rice roots.In addition,bioinformatics tools were used to systematically summarize the classification of rice laccase family genes,proteins characteristic,function prediction and spatiotemporal and abiotic stress-inducible expression patterns of rice laccase genes,as well as the detection of changes in expression levels of rice laccase genes under Cu,Cd stress.Analysis found that rice laccases belong to the family members of copper-binding proteins,mainly located in the secretory pathway,it can be divided into five sub-families.31 rice laccase genes located in eight chromosomes and distributed in various parts of the chromosomes,mainly expressed in the early development of the endosperm,growing roots and stems.Laccase genes can be induced by hormones,salt and drought stress,Cu,Cd stress significantly increased expression levels of some OsLAC genes,for instance,the highest induced expression levels of OsLAC11 and OsLAC30 appeared at 12 h after 20 p,M Cu treatment and 6 h after 100?M Cd treatment,respectively.These results suggested that the catalytic site sequences of rice laccases were very conservative,there were some differences in the patterns of spatio-temporal expression among rice laccase gene family members;some laccase genes can participate in the response to environmental stresses,among them,OsLAC11 and Os LAC30 were two heavy metal(Cu and Cd)stress response genes.To further investigate the biological function of OsLAC11 and OsLAC30,prokaryotic expression vectors of OsLAC11 and OsLAC30 were constructed and transformed into E.coli,laccase activity and recombinant proteins expression levels in E.coli were detected.Then,plant overexpression vectors of OsLAC11 and OsLAC30 were constructed and transformed into Arabidopsis,lignin accumulation,heavy metal(Cu and Cd)tolerance and absorption in wild-type and transgenic Arabidopsis were tested.The results showed that laccase activities were increased significantly and fusion protein were expressed in transgenic E.coli after induction of IPTG.Compared with wild type,lignin accumulation in roots of Arabidopsis over-expressing OsLAC11 were increased,but no significant changes in roots of Arabidopsis over-expressing OsLAC30 were found.After growth on 1/2 MS medium containing Cu(50 ?M)for 7 days,root length of Arabidopsis over-expressing OsLAC11 were significantly longer than the wild type,but there were no significant difference between Arabidopsis over-expressing OsLAC30 and wild type.In contrast,after growth on 1/2 MS medium containing Cd(50 ?M)for 7 days,no significant difference in root length were observed between wild type and Arabidopsis over-expressing OsLAC11,but root length of Arabidopsis over-expressing OsLAC30 were significantly longer than the wild type.Under hydroponic conditions,after 5 ?M Cu treatment for 3 days,compared with the wild-type,Cu concentration significantly decreased in roots of Arabidopsis over-expressing OsLAC 11,but not in shoots.Compared with the wild type,there were no significant changes in Cd concentration in roots and shoots of Arabidopsis over-expressing OsLAC30 after 10 ?M Cd treatment for 3 days.These results indicated that OsLAC11 was likely involved in the lignin biosynthesis,OsLAC11 could enhance Cu tolerance of Arabidopsis,reduced uptake of Cu in Arabidopsis roots,while OsLAC30 could enhance Cd tolerance of Arabidopsis but did not affect the absorption of Cd in Arabidopsis.