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PIRIN2 (PRN2) was earlier reported to suppress syringyl (S)-type lignin accumulation of xylem vessels of Arabidopsis thaliana. In the present study, we report yeast two-hybrid results supporting the interaction of PRN2 with HISTONE MONOUBIQUITINATION2 (HUB2) in Arabidopsis. HUB2 has been previously implicated in several plant developmental processes, but not in lignification. Interaction between PRN2 and HUB2 was verified by β-galactosidase enzymatic and co-immunoprecipitation assays. HUB2 promoted the deposition of S-type lignin in the secondary cell walls of both stem and hypocotyl tissues, as analysed by pyrolysis-GC/MS. Chemical fingerprinting of individual xylem vessel cell walls by Raman and Fourier transform infrared microspectroscopy supported the function of HUB2 in lignin deposition. These results, together with a genetic analysis of the hub2 prn2 double mutant, support the antagonistic function of PRN2 and HUB2 in deposition of S-type lignin. Transcriptome analyses indicated the opposite regulation of the S-type lignin biosynthetic gene FERULATE-5-HYDROXYLASE1 by PRN2 and HUB2 as the underlying mechanism. PRN2 and HUB2 promoter activities co-localized in cells neighbouring the xylem vessel elements, suggesting that the S-type lignin-promoting function of HUB2 is antagonized by PRN2 for the benefit of the guaiacyl (G)-type lignin enrichment of the neighbouring xylem vessel elements.

Plants have developed defense mechanisms against viruses by using an RNA silencing-based process, which has many common features with the endogenous RNA silencing pathway used for regulating the level of transcripts derived from developmental genes. In the virus-induced gene silencing (VIGS) method, it is possible to take advantage of this mechanism by inserting a plant nucleic fragment within the viral genome to knock down the corresponding gene. This tool has been used in many species as a fast and easy reverse genetics technique in order to gain information on the role of genes with poorly understood functions. Here we describe in detail two Agrobacterium-mediated infection protocols in flax, based on a whole plant vacuum infiltration and a leaf syringe infiltration that systemically impact the transcript levels in the stem.