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

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A Correction to this article was published on 12 September 2021

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Abstract

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.

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Acknowledgements

We thank Jerry Cohen for his donation of several auxin conjugates that are no longer commercially available. We would also like to thank Lisa Campanella for her help in editing this manuscript and thank you Scott Kight for your common sense. This work was supported by a Margaret and Herman Sokol Fellow Award (#MHSFA07022), as well as a Wehner Foundation Grant (#WFG19001).

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Authors and Affiliations

  1. Department of Biology, Montclair State University, 1 Normal Ave, Montclair, New Jersey, 07043, USA

    Edgar A. Medina, Samuel Desind, Abdulkader Hallak, Abdullah Alhaddad & James J. Campanella

  2. Department of Biology and Horticulture, Bergen Community College, 400 Paramus Rd, Paramus, New Jersey, 07652, USA

    John V. Smalley

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  1. Edgar A. Medina
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  2. Samuel Desind
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  3. Abdulkader Hallak
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  6. James J. Campanella
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Contributions

J.J.C. conceived the project, performed phylogenetic analysis, 3D protein modeling, analyzed data, cloned and characterized PcILR2A238S, and wrote the article with the contributions of all the authors; E.A.M. cloned MvILR1, MtIL1, and PhILR1 and characterized them; S.D. cloned PcILR1 and PcILR2 and characterized them; A.H. designed and cloned MtILRG240S and MvILRG238S and characterized them; A.A. designed and cloned PhILRA238S and characterized it; J.V.S. performed the codon usage analysis and helped to analyze data.

Corresponding author

Correspondence to James J. Campanella.

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The original online version of this article was revised: “The Supplementary Table 1 was originally published with the wrong format; it has now been replaced with the correct file”.

Supplementary Information

Below is the link to the electronic supplementary material.

344_2021_10467_MOESM1_ESM.jpg

Supplementary file1 Principal coordinate analysis of codon usage to determine if hornwort hydrolases were bacterial contaminants acquired during genomic sequencing. The two-dimensional plot was created using ggplot2 (Wickham 2009) (jpg 86 kb)

344_2021_10467_MOESM2_ESM.jpg

Supplementary file2 Amino acid sequence similarity matrix of ILR1 orthologues from various Plantae species and several prokaryotes. The matrix was generated with MatGAT v1.1 using the default values for protein analysis. The color variation (green to red) indicates levels of similarity: green-shaded values have a higher similarity, whereas red-shaded values have a lower similarity (jpg 397 kb)

344_2021_10467_MOESM3_ESM.jpg

Supplementary file3 Amino acid alignment of orthologue amidohydrolases. Alignment was performed using CLUSTAL X v2.1. Red Letters indicate conserved amino acids (Cys137, His139, Glu173, His197, and His397). The red arrow indicates where the homologous Ser209 can be located. The blue bands designate the sequence of the hornwort amidohydrolases (jpg 736 kb)

344_2021_10467_MOESM4_ESM.jpg

Supplementary file4 3D-modelling analysis of the protein structures of A) AtILL2, B) PcILR1, C) PcILR2, D) PhILR, E) MtILR, and F) MvILR. The red amino acids, indicated by arrows, are conserved and found in the enzymes’ active site. The green bar indicates the alignment between the residue homologues of Ser209 (in AtILL2) and the conserved Phenylalanine across from it (Phe381 in AtILL2) (jpg 552 kb)

344_2021_10467_MOESM5_ESM.jpg

Supplementary file5 3D-modelling analysis of A) PcILR1 and B) PcILR2 examining further polymorphic structural differences (AA76, AA208, AA221). Close-up view of active-site around C) PcILR1 Ser221 and D) PcILR2 Gly221 to examine topologic changes. Red arrows indicate residues which are conserved between the two proteins. Black arrows indicate amino acid polymorphisms (jpg 405 kb)

Supplementary file6 (jpg 338 kb)

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Medina, E.A., Desind, S., Hallak, A. et al. Examination of the M20D Auxin Conjugate Peptidase Family from Hornwort and Implications on the Evolution of the Tracheophytes. J Plant Growth Regul 41, 2695–2706 (2022). https://doi.org/10.1007/s00344-021-10467-3

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