Comparison of lignin deposition in three ectopic lignification mutants.

The Arabidopsis thaliana mutants de-etiolated3 (det3), pom-pom1 (pom1) and ectopic lignification1 (eli1) all deposit lignins in cells where these polymers would not normally be found. Comparison of these mutants provides an opportunity to determine if the shared mutant phenotype arose by perturbing a common regulatory mechanism in each of the mutants. The mutants were compared using a combination of genetics, histochemistry, chemical profiling, transcript profiling using both Northern blots and microarrays, and bioinformatics. The subset of cells that ectopically lignified was shared between all three mutants, but clear differences in cell wall chemistry were evident between the mutants. Northern blot analysis of lignin biosynthetic genes over diurnal and circadian cycles revealed that transcript abundance of several key genes was clearly altered in all three mutants. Microarray analysis suggests that changes in the expression of specific members of the R2R3-MYB and Dof transcription factor families may contribute to the ectopic lignification phenotypes. This comparative analysis provides a suite of hypotheses that can be tested to examine the control of lignin biosynthesis.

Light, the circadian clock, and sugar perception in the control of lignin biosynthesis.

Experiments were undertaken to investigate some of the mechanisms that may function to regulate lignin biosynthesis (lignification) in Arabidopsis thaliana. Northern blot analyses revealed that several genes encoding enzymes involved in the synthesis of lignin monomers displayed significant changes in transcript abundance over a diurnal cycle. Northern blot analysis also suggested that some of the changes in diurnal transcript abundance were likely to be attributable to circadian regulation, whereas others were likely to be attributable to light perception. Comparison of circadian changes in transcript abundance of lignin biosynthetic genes between wild-type plants and the sex1 mutant, which is impaired in starch turnover, suggested that carbon availability related to starch turnover might determine the capacity to synthesize lignins. This hypothesis was supported by the observation that the sex1 mutant accumulated fewer lignins than wild-type plants. Consistent with the relationship between carbon availability and lignin accumulation, analysis of dark-grown wild-type A. thaliana seedlings uncovered a role for sugars in the regulation of lignin biosynthesis. Analysis of lignin accumulation, as determined by qualitative changes in phloroglucinol staining, suggested that metabolizable sugars positively influence the abundance of lignins. Transcriptome analysis supports the hypothesis that sugars are not merely a source of carbon skeletons for lignification, but they also function as a signal to enhance the capacity to synthesize lignins.

Characterisation of a pine MYB that regulates lignification.

A member of the R2R3-MYB family of transcription factors was cloned from a cDNA library constructed from RNA isolated from differentiating pine xylem. This MYB, Pinus taeda MYB4 (PtMYB4), is expressed in cells undergoing lignification, as revealed by in situ RT-PCR. Electrophoretic mobility shift assays (EMSAs) showed that recombinant PtMYB4 protein is able to bind to DNA motifs known as AC elements. AC elements are ubiquitous in the promoters of genes encoding lignin biosynthetic enzymes. Transcriptional activation assays using yeast showed that PtMYB4 could activate transcription in an AC-element-dependent fashion. Overexpression of PtMYB4 in transgenic tobacco plants altered the accumulation of transcripts corresponding to genes encoding lignin biosynthetic enzymes. Lignin deposition increased in transgenic tobacco plants that overexpressed PtMYB4, and extended to cell types that do not normally lignify. Taken together, these findings are consistent with the hypothesis that PtMYB4 is sufficient to induce lignification, and that it may play this role during wood formation in pine.