Shunji Omori

Structural Characterization of Modified Lignin in Transgenic Tobacco Plants in Which the Activity of 4-Coumarate:Coenzyme A Ligase Is Depressed.

Transgenic tobacco (Nicotiana tabacum L.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the level of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.

Immunological characterization of transgenic tobacco plants with a chimeric gene for 4-coumarate:CoA ligase that have altered lignin in their xylem tissue

Transgenic (CS) plants that harbored a chimeric sense gene for 4-coumarate:CoA ligase (4CL), in which the activities of the enzyme were depressed and the structure of lignin was altered, were analyzed by histochemical and immunological methods. Antisera raised against recombinant forms of phenylalanine ammonia-lyase, caffeate O-methyltransferase, and cinnamyl alcohol dehydrogenase, in addition to antiserum against 4CL, were used for Western blotting and for localization of the corresponding polypeptides in stem tissues. Levels of 4CL protein in both upper and lower stem tissues of the CS plants with depressed 4CL activity were apparently lower than those in the control plants. The respective levels of the other enzyme proteins were almost the same in the CS and control plants. Immunohistochemical analysis revealed that suppression of the expression of the endogenous gene for 4CL by the introduced chimeric gene was not uniform throughout the secondary xylem and phloem since strongly stained and weakly stained areas were observed in the analysis. Collapsed vessel cells were seen specifically in the weakly stained regions of the xylem tissue. The xylem that was weakly stained with the antiserum against 4CL was also only weakly stained by acid-phloroglucinol, which stains the cinnamaldehyde units of lignin.