Characterization of the auxin conjugate amidohydrolase orthologue family from several hornwort species and implications for the evolution of M20D hydrolases in tracheophytes

We have previously characterized a liverwort auxin conjugate hydrolase (MpILR1) which appears to be the molecular ancestor for all tracheophyte M20D peptidases. Additional studies into mosses suggested that the amidohydrolase gene family has been lost in the moss clade. Since no expressing M20D class hydrolases are found in mosses, and a hydrolase of low activity has been detected in liverwort, we are now examining when this family of hydrolases became fully. We have refocused our studies on the most recently evolved bryophytes, i.e. hornworts which emerged about 25 million years after liverwort. We have detected five auxin conjugate amidohydrolases in four different species of hornwort (Phaeoceros carolinianus, Megaceros tosanus, Megaceros vincentianus, and Paraphymatoceros hallii). We have cloned and enzymatically characterized two enzymes from the species P. carolinianus (PcILR1,-2) and are beginning the characterization of a third from the species P. hallii (PcILR). Sequence analysis suggests that all five enzymes are plant hydrolases and have greater similarity to plant eucaryotic genes than procaryotic hydrolases. Phylogenetic analysis shows that the bryophyte clade of hydrolases clearly precedes the tracheophytes in evolution. PcILR1 and PcILR2 appear to be highly homologous to each other with a similarity of ~98%. We suspect a relatively recent gene copy event since the DNA sequence of the two P. carolinianus genes have not genetically drifted much from each other. Enzymatic analysis of PcILR1 and PcILR2 suggests a slight increase in activity compared to the liverwort, but no activity as high as in tracheophytes. We did observe a larger range of substrate recognition in the PcILR2 enzyme (IAA-IsoLeu, IAA-Leu, IAA-Phe, IAA-Gly, IBA-Ala, IPA-Ala). Limited analysis of the P. halli (PhILR) enzyme suggests only restricted activity so far (IAA-Leu was cleaved at low levels, but not IAA-Ala or IAA-Val). Continued studies will determine whether exaptation of M20D occurred after the divergence of the tracheophytes from the common hornwort/tracheophyte ancestor.