Funcution Of ABCG36 Transporter Involved In Lignin Biosynthesis In Arabidopsis

Lignin is the second most abundant plant biopolymer in nature.Lignin is mainly deposited in secondary cell walls of vascular plants.It is essential for physiological processes such as nutrient transport,mechanical support and plant pathogen defense.At the same time,lignin is also widely used in industrial and agricultural production.Lignin is mainly produced by oxidative coupling of p-coumaryl alcohol(H-type monolignol),coniferyl alcohol(G-type monolignol)and sinapyl alcohol(S-type monolignol),which are synthesized through phenylpropane metabolic pathway and eventually polymerized in cell wall by peroxidase and laccase.However,after the synthesis of monolignols,the mechanism of transport from cytosol to cell wall remains unclear.Therefore,clarifying the transmembrane transport process of monolignols is of great significance to the study of lignin biosynthesis and secondary cell wall regulation.It has been confirmed that the transmembrane transport of monolignols requires energy from ATP hydrolysis,which implies that ABC transporters may be involved in this process.Previous studies have confirmed that H-type monolignol in Arabidopsis is transported by AtABCG29 protein.However,it is still not clear how the other two important monolignols(G and S)are transported.In this study,we found that root growth of Arabidopsis abcg36 mutants were significantly inhibited by coniferyl alcohol and sinapyl alcohol,suggesting that ABCG36 protein may be involved in the transmembrane transport of G-type and S-type monolignols.In order to elucidate the biological function of ABCG36 in monolignols transport,this paper carried out an in-depth study,with specific results as follows:1.The expression of AtABCG36 gene was significantly induced by G-type and S-type monolignols stress in wild-type Arabidopsis,suggesting that AtABCG36 gene may be involved in the transport of G-type and S-type monolignols.AtABCG36 gene was cloned from Arabidopsis thaliana,and T-DNA insertion mutants abcg36-1 and abcg36-2 were screened and identified.2.Arabidopsis abcg36-1 and abcg36-2 mutants were treated with exogenous G-type and S-type monolignols.Compared with wild-type control,abcg36-1 and abcg36-2 mutants were more sensitive to G-type and S-type monolignols,plant growth was inhibited and root length was shorter.3.Tissue expression profile and in situ hybridization analysis showed that AtABCG36 was highly expressed in root and stem of Arabidopsis,and mostly accumulated in xylem of stem.Subcellular localization analysis confirmed that AtABCG36 protein was located on the cell membranes.4.Tissue section analysis showed that the cell wall thickness of abcg36-1 and abcg36-2 mutant xylem cells was thinner than that of wild type.The lignin contents of mutants abcg36-1 and abcg36-2 decreased significantly,and the monolignols ratio also changed significantly.qRT-PCR analysis revealed that the expression levels of key enzymes CCR1,CAD1 and CCoAOMT1 in abcg36-1 and abcg36-2 mutants were down-regulated in varying degrees compared with wild type.5.The expression of AtABCG36 gene in yeast can significantly increase its tolerance to G and S-type monolignols stress.At the same time,AtABCG36 transporter also enhanced the ability of yeast cells to excrete G-type and S-type monolignols.6.The homologous protein PtrABCG36 of AtABCG36 in Populus was isolated by bioinformatics analysis.The overexpression of PtrABCG36 gene in Arabidopsis made stronger tolerance to G-type and S-type monolignols stress than that in wild-type Arabidopsis,and the overexpression of PtrABCG36 promoted the lignin synthesis in the stem of transgenic Arabidopsi.In yeast cells,PtrABCG36 also promotes the excretion of G and S monolignols in vitro.In conclusion,AtABCG36,an important transporter gene in the lignin biosynthesis pathway of Arabidopsis,has been cloned in this study.Genetic and biochemical experiments have proved that AtABCG36 has the ability to transporting G-type and S-type monolignols across the membrane,thus affecting the development of lignin in the stem of Arabidopsis.Similarly,the homologous protein PtrABCG36 of AtABCG36 in poplar also has similar monolignols transport function.This study will help to elucidate the transport mechanism of lignin in plants and lay a foundation for the analysis of the regulation mechanism of wood development.