Induction of the mRNA that encodes for phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), which catalyzes the first reaction in the biosynthesis of a wide variety of phenylpropanoid natural products from phenylalanine, was investigated in. wounded tuber tissues of the potato (Solanum tuberosumL. cv. Irish Cobbler). Northern blot analysis showed that hybridizable RNA was not present in unwounded tissue, but the amount of hybridizable PAL-specific mRNA increased rapidly in the polysomal RNA fraction with a sharp, high peak at the early stage (0 h to 6 h) and two broad lower peaks at the later stage (6 h to 48 h) of the wound response. Addition of actinomycin D to the tissue prevented the appearance of hybridizable mRNA in the total RNA fraction, confirming that the increase resulted from synthesis of PAL mRNA de novo. Levels of translatable PAL mRNA activity in vitro increased in the polysomal RNA fraction in parallel with the changes in levels of hybridizable mRNA, with a subsequent increase in levels of PAL subunit polypeptides and enzymatic activity in wounded tissues. PAL subunits synthesized both in vivo and in vitro had the same molecular masses, of about 79 kDa, on SDS-polyacrylamide gel electrophoresis, but isoelectric focusing revealed the presence of isoforms of the native tetrameric enzyme with different pI values and changes in the relative levels of the isoforms after wounding. Furthermore, two-dimensional gel electrophoresis of PAL subunits synthesized in vitro showed that at least eight mRNAs that encoded subunit isoforms with different pI values were expressed sequentially after wounding.
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