Analysis of Roles of Laccases in Lignin Biosynthesis in Arabidopsis thaliana
Laccases (benzenediol: oxygen oxidoreductase; EC 188.8.131.52) are multi-copper oxidases that have a loose substrate definition, capable of oxidizing a wide range of phenolic compounds, and transferring electrons to molecular oxygen to generate water. Previous genetic studies identified roles for Arabidopsis Lac 4 and 17 in lignin deposition, but did not address the mechanism of their actions. In this study, I further characterized T-DNA insertion lines in the lac4 and lac17 genes. I reproduced the xylem collapsed phenotype and the reduced lignin content from lac 4-2/17 double knock out mutant. I also complemented the double mutant by using a native promoter of Lac4 or Lac17 to drive the expression of Lac4 or Lac17 coding DNA sequence. I showed that the transgene restored the Mäule stain phenotype of lac4-2/17, making them become indistinguishable from wild type plants. Lignin from lac4-2/17 double mutant in Arabidopsis thaliana was further characterized using 2D NMR spectroscopy. My collaborator and I found that the Guaiacyl (G) unit was reduced, and the phenylcoumaran inter-unit linkages almost disappeared in lignin samples of the double mutant compared to that of the wild type. I then tested whether changing the ratio of guaiacyl (G) and sinapyl (S) subunit of lignin up-stream of laccase could rescue the lignin content of the laccase double mutant. The lac4-2/17/fah1-2/C4H-F5H quadruple hybrid was generated by transforming the lac4-2/17/fah1-2 triple mutant with the C4H-F5H construct. The lignin of the quadruple hybrid lines was rich in S and very low in G unit. However, the lignin content was not restored to the wild type level, which indicated that the two laccases, 4 and 17, were not specific to either S or G. Efforts to express and purify Lac4 from heterologous hosts to study the enzyme property were not successful. I further investigated whether other laccases were interchangeable with Lac4 and 17 by attempting to complement the Mäule stain phenotype of 4-2/17 plants with one of the other laccases from A. thaliana, Rhus vernicifera, or Trametes versicolor. I found that Lac10, 11 and 17 fully restored the phenotype of 4-2/17, while Lac 1, 2, 5, 12 restored the phenotype only partially. Lac 3, 15, and Laccases from R. vernicifera, T. versicolor did not complement. The results indicated that not all laccases in A. thaliana were functionally equivalent.