Mutation Of Osccoaomt Genes Causes Brittle Phenotype And Changes Copper Tolerance In Oryza.sativa

Copper(Cu)is an essential nutrient element,which is involved in electron transfer,redox reaction and cell wall synthesis in plants.It is also an important environmental pollutant.However,copper in excess will inhibit the growth of plants and affect the normal metabolism process.Cell wall has been demonstrated to play an indispensable role in plant resistance to copper stress,the mechanism of ligninwork in copper toxicity is very little known.This study chose Caffeoyl CoznzymeA-O methyl transferase(CCoAOMT)as the research object,which was a key enzyme in the process of lignin biosynthesis.The aim is to change the accumulation and distribution of heavy metals in plants by regulating the biosynthesis of cell wall components and lignin monomers,which can improve the plant tolerance.Follows are the results:1.According to multiple alignment,the ORF of OsCCoAOMT were highly conservative except the 5'end.OsCCoAOMT1 shared high homology with many CCoAOMTs from other plant species.While OsCCoAOMT20 was from the other group,which might be specific in rice.Both of OsCCoAOMT1 and OsCCoAOMT20 were abundantly expressed in leaf sheathes,moderately expressed in roots and expressed at low levels in leave.Subcellular localization indicated that they localized in the cell wall and plasmalemma.2.After copper treatment,OsCCoAOMTs had a transient response.To investigate whether the expression of CCoAOMT is regulated by Cu-induced H2O2,we applied the pretreatment with DMTU which is a H2O2 scavenger,the results showed no significant difference.The CCoAOMT genes also inducible by oxidative stress after 0.2mM H2O2 treatment,but the low level Cu-induced H2O2 cannot induced CCoAOMT upregulation.The results indicated the CCoAOMT genes may play important roles in stress defense.3.Meanwhile,we reported two T-DNA inserted mutants osccoaomtl and osccoaomt20,which were characterized by brittle culms that can be easily broken by bending.At the seedling stage,the mutant showed dwarfism with drooping leaves,and most of osccoaomtl showed etiolation.At the mature stage,the plant height of both mutants were reduced.Chemical analysis(acetyl bromide and thioacidolysis revealed that the Syringyl/Guaiacyl(G/C)lignin monomer ratio and total lignin content were modified in two down-regulated lines.More strikingly,the cellulose contents of osccoaomtl and osccoaomt20 mutants in both shoots and roots decreased dramatically compared to the wild-type plants,while the content of hemicelluose were a little higher.4.After Cu treatment,the Cu content in the mutant roots was 20-35%more than that in the wild-type.In the leaf sheathes,the Cu content in the mutants was not as higher as before.In the upper part,the Cu content in mutant leaves was almost comparable to that in the wild-type,even a little lower.However,the accumulation of copper in the mutant cellulose was significantly less than wild types,and the copper content in the hemicellulose was higher low concentration treatment.In conclusion,the OsCCoAOMT genes were involved in lignin synthesis and cell wall formation,may binding the Cu on cell wall to prevent it from entering the root cells and transporting to the upper part of the plant,thereby protecting the plant under copper stress.