| Woody biomass is one of the most important renewable biological resources,which was widely used in paper making,construction industry and biofuels production.In China,the forest coverage rate is low and the contradiction between wood supply and demand is prominent.Poplar is one of the most widely planted fast-growing tree species around world,and also one of the major trees for plantation in China.With the completion of whole genome sequencing and the establishment of genetic transformation and regeneration system,poplar has become one of the model plants to study the growth and development of trees.Therefore,the regulation mechanism of wood formation in poplar is the key to forest breeding.The main component of wood is the cell wall.Lignin,as one major component of secondary cell walls,mainly exists in the cells of the transport tissues and lignified tissues.In plants,lignin synthesis mainly includes shikimic acid synthesis pathway,phenylpropionic acid synthesis pathway and specific lignin synthesis pathway.There are three monomers of lignin:syringyl lignin（S-lignin）and guajacyl lignin（G-lignin）hydroxy-phenyl lignin（H-lignin）.After the monomer is synthesized in the cytoplasm,it is transported to the cell wall through the transmembrane,and then the polymerized lignin is formed under the action of enzymes.The polymerization of lignin monomers is essential for the formation of lignin macromolecules.It is reportedthat Laccase（LAC）may be involved in this process,but its role in lignin synthesis is not clear.In order to explore the role of laccase for the synthesis of lignin in poplar,we cloned a laccase gene PtoLAC14 and investigated its biological functions in the regulation of the lignin synthesis in poplar.The main results are as follows:（1）To identify the genes related to wood formation in poplar trees,a FOX library containing multi-tissue full-length CDS of P.trichocarpa was constructed.Then the FOX library was genetically transformed into Arabidopsis thaliana,and the mutant with enhanced lignification in stem was screened.The gene was cloned and sequenced,and it was transferred into the poplar laccase gene PtrPLAC14.In order to further understand the role of this gene in wood formation in poplar,PtoLAC14 gene was cloned by using PtrPLAC14 gene as reference sequence.（2）PtoLAC14 encodes a protein composed of 558 amino acids.Multiple sequence alignment revealed that PtoLAC14 is highly similar to Arabidopsis laccase genes AtLAC4 and AtLAC17 in amino acid sequence,and the similarities between PtoLAC14 and AtLAC4,AtLAC17 are 75.85%and 55.88%,respectively.The amino acid sequences of these three genes contain four typical laccase copper ion binding domains,suggesting that these genes may be functionally conserved,which indicating that PtoLAC14 may also play an important role in lignin synthesis.（3）The results of real-time quantitative PCR detection and the use of the promoter to drive GUS reporter gene showed that PtoLAC14 was predominantly expressed in the xylem of young stem tissue,while the expression level was low or not expressed in other tissues,suggesting that PtoLAC14 may be involved in the lignin biosynthesis in the xylem of young plant tissues.（4）To further elucidate the biological functions of PtoLAC14,we obtained transgenic lines with overexpression of PtoLAC14 and knockout transgenic lines with targeted gene editing of CRISPR/Cas9.It was found that,compared with the wild type,overexpression of PtoLAC14 resulted in a phenotype of dwarfed transgenic poplar plants,reduced stem diameter,and reduced biomass,while the knockout mutants showed a phenotype of taller plants,slightly thicker stem diameters and increased biomass,indicating that PtoLAC14 was involved in the wood formation.（5）The stem tissue sections of transgenic plants and their corresponding wild-type plants were observed after toluidine blue staining.Compared with wild-type,there was no significant difference in the number of xylem cell layers in transgenic plants that overexpressed and knocked out PtoLAC14.Scanning electron microscopy results showed that,compared with the wild-type,overexpression of PtoLAC14 resulted in thickening of secondary wall thickness of xylem cells,inhibition the expansion of vascular cells and fiber cells.In contrast,the CRISPR/Cas9-generated mutation of PtoLAC14 showed the opposite results,that is,the secondary wall thickness of xylem decreased,with the xylem vascular cells and fiber cells enlarged.（6）The results phloroglucinol staining and chemical assay showed that,compared with the wild type,overexpression of PtoLAC14 would lead to an increased in lignin content in transgenic plants.Maüle staining and quantitative HPLC results showed that the ratio of S/G monomer in transgenic plants with overexpression of PtoLAC14 was decreased,that is,the content of G-type lignin was increased and S-type lignin was decreased.Inversely,transgenic PtoLAC14-KO lines displayed no significant difference intotal lignin content,but increased ratio of monomer S/G was,that is,the content of S-type lignin was increased and G-type lignin was decreased.The above results indicate that PtoLAC14 plays an important role in the synthesis of lignin and lignin monomer component in poplar.（7）In order to further elucidate the function of PtoLAC14 in lignin synthesis,the PtoLAC14protein was purified by prokaryotic induction and purification.The catalytic efficiency of PtoLAC14was analyzed with G-type and S-type lignin monomers as substrates,respectively.The Km value and michaelis constant reflect that PtoLAC14 has a higher affinity to G-type lignin monomers than S-type lignin monomers,indicating that PtoLAC14 has catalytic polymerization selectivity for different types of lignin monomers,consistent with the previous findings that the lignin monomer ratio was changed in the transgenic lines.（8）Since the proportion of lignin monomer significantly affects the biomass enzymatic saccharification.As mentioned above,the S-type and G-type lignin content ratios of PtoLAC14transgenic materials was significantly changed,we further determined the efficiency of biomass enzymatic saccharification.The results showed that the PtoLAC14-KO transgenic lines were more easily digested and their saccharification efficiency was higher than that of the wild type.In detail,the pretreatment with H2SO4 or NaOH could lead to hexose yields 6.61%-10.5%or 11.96%-16.31%increased from PtoLAC14-KO lines than WT.In summary,this study indicated that PtoLAC14 is involved in the synthesis of lignin in poplar and has substrate selectivity in the polymerization process of lignin monomer.The PtoLAC14-KO transgenic plants maintained a normal plant growth with slightly increased biomass yields.Importantly,the PtoLAC14-KO poplar showed significantly increased ligin S/G ratio,promoted the degradation of poplar,enhanced biomass enzymatic saccharification,and leading to remarkably improve the utilization rate of wood.Hence,this study provides a potent strategy for genetic improvement and molecular breeding of poplar and other plants. |
