Phenylalanine ammonia lyase (PAL, EC 4.3.1.5)
There are four putative PAL (PAL1 – 4) genes in Arabidopsis, these being of at least 78% amino acid sequence similarity to each other. Although PAL is one of the most intensely studied enzymes in plant metabolism, very little is understood about the precise physiological roles of these multigene families in the various phenylpropanoid pathway derived metabolic networks in different plant species including Arabidopsis. Our overall strategy was to characterize fully each PAL isoenzyme, as well as identifying their precise roles in metabolism in Arabidopsis.
The available EST database yielded interesting, albeit preliminary, observations into possible expression patterns of each of the 4 cytosolic Arabidopsis PAL homologues in developing Arabidopsis (1). Relative to PAL1, the three other PAL homologues display 92.7, 79.9 and 85.3% similarity, and 90.3, 73.9 and 80.5% identity, respectively. All are cytosolic and lack any targeting peptide sequence. Of these, only three of the four PAL homologues were detected in the EST database (Fig. 2A), with PAL3 escaping detection perhaps due to low copy number, transient expression and/or being inducible only under certain conditions. Additionally, PAL1, 2 and 4 are differentially expressed in the various tissues examined, although with no indication of either distinct spatial and/or temporal expression patterns in, for example, particular cell types. PAL1 and PAL2 also apparently differ from PAL4 in that both are mainly expressed in above-ground (presumably lignified) organs, green siliques and roots, whereas PAL4 seems to be mainly present in just the roots and green siliques. Interestingly, the relative abundances of PAL1 and PAL2 ESTs are highest overall in root tissues, this perhaps being an indication that the ESTs for the above-ground organs were from Arabidopsis tissues which were not (very) actively lignifying at the time(s) of collection. Furthermore, the presence of PAL2 in essentially every tissue known to undergo lignification (including developing seeds) might suggest it to be the most likely candidate for an exclusive and/or dominant role in targeting carbon to the lignin pathway. PAL4 was also highly abundant in developing seed tissues whereas PAL1 was not, suggesting a different metabolic role for PAL4 within this tissue. Finally, no ESTs for any of the PAL isoforms were detected in flower buds.
There are thus three ongoing subprojects: (i) cloning of Arabidopsis PAL genes and characterization of recombinant PAL isoenzymes; (ii) analysis of PAL gene “knockouts” and (iii) identifying temporal and spatial patterns of expression of the various PAL promoters when fused to GUS/GFP reporter genes. Progress is shown in Tables 1 – 3.(2)