Tomato PAL genes: highly redundant but strongly underutilized in leaves
Phenylalanine ammonia-lyase (PAL) catalyzes the deamination of ' L'-phenyl-alanine to yield 'trans'-cinnamic acid, the initial committed step of the multi branched phenylpropanoid pathway in higher plants. As the first step in phenylpropanoid synthesis PAL is an important enzyme both in plant development and defense. To further characterize the number of PAL genes in tomato and their DNA sequences, PAL genes were PCR-amplified from tomato nuclear genomic DNA and cloned. DNA sequence analyses of two PCR-amplified protein-coding regions revealed that there are at least eighteen different PAL DNA sequences differing in deletions, insertions and nucleotide substitutions when compared with the previously characterized PALS gene. Based on the DNA sequence data, copy number of PAL genes was estimated at 24 to 25 copies per diploid tomato genome, a number consistent with earlier observations in our laboratory (Lee, 1992; Lim, unpublished). PCR amplification and cloning also was used to characterize the actual mRNA transcripts. Comparisons of cDNA sequences indicate that this very redundant gene family of at least eighteen different sequences is largely silenced with only a single sequence being expressed in tomato leaves. To explore the reason, the upstream sequences were determined together with patterns of DNA methylation. Cloning of upstream sequences amplified by an inverse PCR amplification strategy identified three major groups, a first previously characterized PAL5 sequence, a second group consisting of six highly homologous sequences and a third more distinct group. Analyses of methylation patterns by a bisulfite conversion sequencing strategy revealed that all sequences of the second group contained methylcytosines. This was in strong contrast to the complete absence of methylcytosines in the first group and suggested that the expression of a limited number of PAL genes could be directly related to this. To further investigate this relationship, ' in vivo', the actively expressed PAL promoter sequence was fused with a [beta]-glucuronidase coding sequence and introduced to tomato plants. The results provide evidence that methylation in CG dinucleotides is directly related to the observed transcriptional silencing. The significance of these observations with respect to sequence duplication and gene silencing are discussed.