Modification of Lignin Biosynthesis in Forest Trees
Two full length complementary DNAs from aspen (Populus tremuloides) were cloned and sequenced. Northern blot analysis of aspen RNA showed high Pt4CL1 mRNA levels in secondary developing xylem tissues and lower internodes, moderate levels in top internodes, and low level in leaves. Pt4CL2 mRNA was not detected in developing secondary xylem tissues and was barely noticeable in lower internodes. A high level of Pt4CL2 mRNA was observed in top internodes, and a weak expression of Pt4CL2 mRNA was found in leaves. Pt4CL2 mRNA was not detected in developing xylem tissues but was evident in epidermal layers, indicating that Pt4CL2 is epidermis-specific in aspen stem. Escherichia coli-expressed Pt4CL1 and Pt4CL2 proteins were purified, and the purified recombinant proteins were characterized for their catalytic activities with various hydroxycinnamic acid derivatives. Unlike Pt4CL2, Pt4CL1 showed high affinity for lignin-specific substrates, such as ferulic and 5-hydroxyferulic acids, suggesting the involvement of this protein in lignin biosynthesis. To test whether lignin biosynthesis can be controlled by Pt4CL1, transgenic aspen trees with suppressed Pt4CL1 expression were produced. The trees had a 40-45% reduction in lignin quantity and a 9-15% increase in cellulose content, resulting in a cellulose:lignin ratio of 4:0 compared to 2:0 in the wild-type aspen. The severe lignin reduction did not adversely affected growth and development in transgenic trees, but resulted in the formation of thicker stems, larger leaves, and longer internodes than the control. The results indicate that lignin can be genetically reduced without compromising cellulose biosynthesis and tree growth.