Molecular Cloning And Function Analysis Of Two Cinnamyl Alcohol Dehydrogenases From Plagiochasma Appendiculatum

The bryophytes are key taxa in the evolutionary adaptation of plants from an aqueous to a non-aquatic environment. They are rich in secondary metabolites such as terpenoids, bibenzyls, flavonoids, lignans and lignins, which have a wide range of bioactivities. Moreover, these secondary metabolites play a vital role in plant adaptation to terrestrial environments. And lignins provide plants with mechanical strength, hydrophobicity and stress tolerance. However, the genes involved in lignin biosynthesis in liverworts remain unidentified because of the lack of genetic information. Cinnamyl alcohol dehydrogenase (CAD), a key enzyme in lignin biosynthesis, catalyses the reduction of the cinnamyl aldehydes to cinnamyl alcohols; this is the last step in the synthesis of monolignols. Almost all CAD members of land plants could be divided into three classes according to the phylogenetic analysis, together with gene structure and function.In the present investigation, two cDNAs encoding CADs were obtained from a Chinese liverwort Plagiochasma appendiculatum thallus library and were designated as PaCADl and PaCAD2. They were ligated into prokaryotic expression vector, expressed in BL21and purified. Purified proteins were used for enzyme characterization. Moreover, p-coumaryl, caffeyl and5-hydroxyconiferyl aldehydes and corresponding alcohols were chemically synthetized. The recombinant PaCAD proteins displayed high levels activity using P-coumaryl.caffeyl,coniferyl,5-hydroxyconiferyl and sinapyl aldehydes as substrates to form corresponding alcohols.The enzyme kinetics results showed that PaCAD1and PaCAD2used caffeyl and coniferyl aldehyde as the favorite substrate respectively and showed high catalytic efficiency toward P-coumaryl aldehyde but lower catalytic efficiency towards5-hydroxyconiferaldehyde and sinapyl aldehyde. Sequence alignment and phylogenetic analysis showed that PaCAD1and PaCAD2belong to a clade in Class II together with some proteins which have a conserved VTG(X)2G(X)9L(X)5motif. In addition, total lignin content was analyzed in leaves and callus.In accord with the higher lignin content in the leaves than in the callus, the expression level of PaCAD2was also higher in leaves than in the callus. The expression of PaCAD1and PaCAD2was induced by Methyl jasmonic acid (MeJA) treatment. This suggested that these two PaCADs played important roles in lignin biosynthesis and the defense of abiotic stress in P. appendiculatum. This is the first time that the CADs in liverworts have been functionally characterized. This study provided two candidate genes to improve the lignin content through metabolic engineering, and it also would contribute to understand the evolution and origin of lignin.