Phenylalanine ammonia-lyases and 4-coumaric acid coenzyme A ligases in Chara braunii, Marchantia polymorpha, and Physcomitrium patens as extant model organisms for plant terrestrialization.

ABSTRACT

The conquest of land posed severe problems to plants which they had to cope with by adapting biosynthetic capacities. Adaptations to respond to UV irradiation, water loss, pathogen and herbivore defense, and the earth's pull were essential. Chemical compounds alleviating these problems can be synthesized by the phenylpropanoid pathway, the core of which are three enzymes phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase, and 4-coumaric acid coenzyme A-ligase (4CL). The genomes of model organisms, Chara braunii as aquatic alga and the two bryophytes Physcomitrium patens and Marchantia polymorpha, were searched for sequences encoding PAL and 4CL and selected sequences heterologously expressed in Escherichia coli for biochemical characterization. Several possible isoforms were identified for both enzymes in Marchantia polymorpha and Physcomitrium patens, while only one or two isoforms could be retrieved for Chara braunii. Active forms of both enzymes were found in all three organisms, although the catalytic efficiencies varied in a wide range. l-Phenylalanine was accepted as best substrate by all PAL-like enzymes, despite annotations in some cases suggesting different activities. The substrate spectrum of 4CLs was more diverse, but caffeic and/or 4-coumaric acids generally were the best-accepted substrates. Our investigations show that PAL and 4CL, important enzymes for the formation of phenolic compounds, are present and active in extant charophytes and bryophytes as model organisms for the conquest of land.