Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the last steps of monolignol biosynthesis. In Arabidopsis, one CCR gene (CCR1, At1g15950) and two CAD genes (CAD C At3g19450 and CAD D At4g34230) are involved in this pathway. A triple cad c cad d ccr1 mutant, named ccc, was obtained. This mutant displays a severe dwarf phenotype and male sterility. The lignin content in ccc mature stems is reduced to 50% of the wild-type level. In addition, stem lignin structure is severely affected, as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde, sinapaldehyde, and ferulic acid. Male sterility is due to the lack of lignification in the anther endothecium, which causes the failure of anther dehiscence and of pollen release. The ccc hypolignified stems accumulate higher amounts of flavonol glycosides, sinapoyl malate and feruloyl malate, which suggests a redirection of the phenolic pathway. Therefore, the absence of CAD and CCR, key enzymes of the monolignol pathway, has more severe consequences on the phenotype than the individual absence of each of them. Induction of another CCR (CCR2, At1g80820) and another CAD (CAD1, At4g39330) does not compensate the absence of the main CCR and CAD activities. This lack of CCR and CAD activities not only impacts lignification, but also severely affects the development of the plants. These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

SummaryWe show here that the in vivo methylation-based tagging technique DamID (DNA adenine methyltransferase identification) can be used for studies of DNA-protein interactions or chromatin profiling in plants. We have demonstrated the feasibility, reproducibility and sensitivity of the method in Arabidopsis thaliana, using the well-known yeast GAL4 transcription factor, for which DNA-binding sites (UAS G ) were introduced into the plant genome. We monitored the methylation resulting from the activity of DNA adenine methyltransferase fused to the protein of interest, by combining digestion with methylation-sensitive restriction enzymes and quantitative PCR. We then used DamID to identify genomic targets of LHP1, a protein mostly associated with euchromatin. We showed that LHP1 was targeted to the promoter and transcribed regions of four genes: AG, AP3, FT and PI. Our data also demonstrate that LHP1, like its animal homologues, has a high binding affinity for A/T-rich regions, binding particularly strongly to the large regulatory introns of AG and PI. We identified three major characteristics of LHP1 binding, highlighting the similarities between plant LHP1 and animal HP1 proteins.

RÉSUMÉ figure I show the results obtained with the two strains. For L.P.I., the two strains gave the same results on F 2 2A, 7A, 1B, 2B, 4B, 1D, 2D, 3D ; only 7A and 2B gave a resistant reaction. The F 2 4A, 5B, 7B gave opposite results between the strains. With P. max, the same result for the resistance was given only with 3A, and opposite results with the F 2 2B, 3B, 5B. For LE. the resistance or susceptibility reactions were different with the strains. No concordance was found between characters of partial resistance and characters of tolerance, which were thus two different genetic systems. The relation between L.P.I. and P. max was only partial with strain 42. Statistical analysis showed interaction between F Z monosomic and strains for L.P.I. (table 2)

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