HSI2/VAL1 PHD-like domain promotes H3K27 trimethylation to repress the expression of seed maturation genes and complex transgenes in Arabidopsis seedlings.

BACKGROUND: The novel mutant allele hsi2-4 was isolated in a genetic screen to identify Arabidopsis mutants with constitutively elevated expression of a glutathione S-transferase F8::luciferase (GSTF8::LUC) reporter gene in Arabidopsis. The hsi2-4 mutant harbors a point mutation that affects the plant homeodomain (PHD)-like domain in HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2 (HSI2)/VIVIPAROUS1/ABI3-LIKE1 (VAL1). In hsi2-4 seedlings, expression of this LUC transgene and certain endogenous seed-maturation genes is constitutively enhanced. The parental reporter line (WT LUC ) that was used for mutagenesis harbors two independent transgene loci, Kan R and Kan S . Both loci express luciferase whereas only the Kan R locus confers resistance to kanamycin. RESULTS: Here we show that both transgene loci harbor multiple tandem insertions at single sites. Luciferase expression from these sites is regulated by the HSI2 PHD-like domain, which is required for the deposition of repressive histone methylation marks (H3K27me3) at both Kan R and Kan S loci. Expression of LUC and Neomycin Phosphotransferase II transgenes is associated with dynamic changes in H3K27me3 levels, and the activation marks H3K4me3 and H3K36me3 but does not appear to involve repressive H3K9me2 marks, DNA methylation or histone deacetylation. However, hsi2-2 and hsi2-4 mutants are partially resistant to growth inhibition associated with exposure to the DNA methylation inhibitor 5-aza-2'-deoxycytidine. HSI2 is also required for the repression of a subset of regulatory and structural seed maturation genes in vegetative tissues and H3K27me3 marks associated with most of these genes are also HSI2-dependent. CONCLUSIONS: These data implicate HSI2 PHD-like domain in the regulation of gene expression involving histone modifications and DNA methylation-mediated epigenetic mechanisms.

Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobacco PAL genes and active heterotetrameric enzymes.

PAL (L-phenylalanine ammonia-lyase), the first enzyme of phenylpropanoid biosynthesis, is often encoded by multigene families in plants. A PCR-based approach was used to isolate cDNA clones corresponding to the four PAL genes of tobacco (Nicotiana tabacum). By careful comparison of cDNA and genomic clones, a new PAL gene (PAL4) was defined. PCR amplification of PAL sequences from cDNA led to the generation of chimaeric clones between PAL1 and PAL4, and incorrect annotation of PAL4 ESTs (expressed sequence tags) as PAL1 in the EST database has given rise to a randomly shuffled tentative consensus sequence. The PAL2 previously described in the literature was shown, by domain swapping experiments with PAL1, to possess a single nucleotide substitution leading to an inactive enzyme. The altered amino acid resulting from this substitution maps to the base of the active site pocket in the three-dimensional structure of PAL. The inactive PAL2 allele could not be recovered from 13 different tobacco cultivars examined. PALs 1-4 were co-expressed in multiple plant organs, and were also co-induced following exposure of cell cultures to yeast elicitor or methyl jasmonate. All four tobacco PAL proteins expressed in Escherichia coli displayed normal Michaelis-Menten kinetics, with Km values between 36 and 60 muM. Co-expression of different PAL proteins in E. coli resulted in formation of heterotetramers, which possessed kinetic properties within the same range as those of the individual homotetramers. The potential physiological function of heterotetrameric PAL forms is discussed.