Examination of the M20D Auxin Conjugate Peptidase Family from Hornwort and Implications on the Evolution of the Tracheophytes

Edgar A. Medina, Samuel Desind, Abdulkader Hallak, Abdullah Alhaddad, John V. Smalley, James J. Campanella

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We have identified and cloned five auxin conjugate amidohydrolases (M20D peptidases) in four different hornwort species (Phaeoceros carolinianus, Megaceros tosanus, Megaceros vincentianus, and Paraphymatoceros hallii). Sequence analysis suggests that all five enzymes have greater than 60% overall similarity to tracheophyte amidohydrolases. Phylogenetic analysis supports the hypothesis that the bryophyte and tracheophyte hydrolases are derived from a common ancestor. Enzyme studies of hornwort auxin amidohydrolases all demonstrate greater activity and substrate recognition than the more ancient liverwort hydrolase (MpILR1). The mean wild-type hornwort hydrolytic activity (23.2 ± 6.8 pmol auxin released/min/ml), although higher than the liverwort activity (1.3 ± 1.1 pmol auxin released/min/ml), was almost a magnitude lower than the average activity in tracheophyte hydrolases (186.2 ± 52.1 pmol auxin released/min/ml). Two hornwort orthologues from M. vincentianus and M. tosanus possess a Glycine238/240 replacing the tracheophytically conserved Serine209, while two from P. hallii and P. carolinianus have an Alanine238 at that homologous residue location. Further enzymatic studies and three-dimensional structural analyses of the hornwort enzymes present supporting evidence that the Ala238-line of hornworts is the likely clade from which tracheophytes arose.

Original languageEnglish
Pages (from-to)2695-2706
Number of pages12
JournalJournal of Plant Growth Regulation
Issue number7
StatePublished - Oct 2022


  • Auxin conjugate aminopeptidases
  • Auxin conjugates
  • Auxin regulation
  • Bryophyte
  • Hornwort
  • Molecular evolution
  • Tracheophyte


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