Evidence for the Early Evolutionary Loss of the M20D Auxin Amidohydrolase Family from Mosses and Horizontal Gene Transfer from Soil Bacteria of Cryptic Hydrolase Orthologues to Physcomitrella patens

  • James J. CampanellaEmail author
  • Stephanie Kurdach
  • Richard Skibitski
  • John V. Smalley
  • Samuel Desind
  • Jutta Ludwig-Müller


Inactive auxin conjugates are accumulated in plants and hydrolyzed to recover phytohormone action. A family of metallopeptidase orthologues has been conserved in Plantae to help regulate auxin homeostatic levels during growth and development. This hydrolase family was recently traced back to liverwort, the most ancient extant land plant lineage. Liverwort’s auxin hydrolase has little activity against auxin conjugate substrates and does not appear to actively regulate auxin. This finding, along with data that shows moss can synthesize auxin conjugates, led to examining another bryophyte lineage, Physcomitrella patens. We have identified and isolated three M20D hydrolase paralogues from moss. The isolated enzymes strongly recognize and cleave a variety of auxin conjugates, including those of indole butyric and indole propionic acids. These P. patens hydrolases not only appear to be “cryptic”, but they are likely to have derived from soil bacteria through Horizontal Gene Transfer. Additionally, support is presented that the plant-type M20D peptidase family may have been universally lost from mosses after divergence from the common ancestor with liverwort.


Physcomitrella patens Horizontal gene transfer Auxin conjugate regulation Amidohydrolase Cryptic genes Moss evolution 



The authors would like to thank Dirk Vanderklein and Scott Kight for their advice and encouragement. We would also like to thank Lisa Campanella for the help in revising this manuscript. The technical help of Adam Parker of MSU and Sabine Rößler, TU Dresden, is gratefully acknowledged. This work was supported by a Margaret and Herman Sokol Fellow Award, #07A. The authors of this article have no conflicts of interest.

Supplementary material

344_2019_9945_MOESM1_ESM.jpg (153 kb)
Supplementary Figure S1 Electrophoretic gel analysis of PCR products for specific, non-overlapping genomic regions of PpIAR31, PpIAR32, PpIAR33, and PpIAR34. Lanes 1 & 6: HiLo size marker, Lanes 2 & 4: PCR products for each hydrolase gene, Lanes 3 & 5: 18S PCR control. PCR was performed in duplicate for each reaction set. 2% agarose gel electrophoresed ~35 min at constant 150 volts and stained with ethidium bromide for visualization. The circles on the PpIAR31 panel indicate the lack of expected amplification for this hydrolase gene. (JPG 152 KB)
344_2019_9945_MOESM2_ESM.jpg (625 kb)
Supplementary Figure S2 A partial readout of the mRNA expression analysis of PpIAR32, PpIAR33, and PpIAR34, as performed by Lang et al. (2018). Data from Phytozome Web site P. patens v3.3. (JPG 624 KB)
344_2019_9945_MOESM3_ESM.jpg (116 kb)
Supplementary Figure S3 Electrophoretic gel analysis of RT-PCR products for specific, non-overlapping mRNA regions of PpIAR32, PpIAR33, and PpIAR34. Lanes 1 & 6: HiLo size marker, Lane 2: PpIAR32 RT-PCR product, Lane 3: PpIAR33, Lane 4: PpIAR34, Lane 5: 18S expression control. 1.5% agarose gel electrophoresed ~30 min at constant 150 volts and stained with ethidium bromide for visualization. (JPG 115 KB)
344_2019_9945_MOESM4_ESM.doc (23 kb)
Supplementary Table 1 (DOC 23 KB)


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Authors and Affiliations

  1. 1.Department of BiologyMontclair State UniversityMontclairUSA
  2. 2.Department of Biology and HorticultureBergen Community CollegeParamusUSA
  3. 3.Institute of BotanyTechnische Universität DresdenDresdenGermany

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