Processing and Formation of Bioactive CLE40 Peptide Are Controlled by Posttranslational Proline Hydroxylation
Nils Stührwohldt1, Alexandra Ehinger2, Kerstin Thellmann2
1Department of Plant Physiology and Biochemistry, Institute of Biology, University of Hohenheim, 70593 Stuttgart, Germany nils.stuehrwohldt@uni-hohenheim.de.
Plant Physiology
|September 10, 2020
Summary
Researchers identified subtilases that process the CLE40 peptide, a regulator of stem cell differentiation in Arabidopsis roots. Pro hydroxylation prevents secondary cleavage, ensuring bioactive CLE40 formation and activity.
Area of Science:
- Plant molecular biology
- Peptide signaling
- Developmental biology
Background:
- Small signaling peptides are crucial regulators of plant development.
- Posttranslational modifications (PTMs) are essential for peptide biogenesis and activity, but processing proteases and modification roles remain largely unknown.
- The Clavata3/Endosperm Surrounding Region (CLE) peptide family, including CLE40, regulates stem cell differentiation in Arabidopsis roots.
Purpose of the Study:
- To identify proteases involved in CLE40 precursor processing.
- To elucidate the role of PTMs, specifically Pro hydroxylation, in CLE40 biogenesis and activity.
- To understand the regulation of stem cell differentiation by CLE40 in Arabidopsis roots.
Main Methods:
- Yeast two-hybrid screening to identify interacting proteins.
- In vitro protease assays using recombinant subtilases and CLE40 precursor.
- In planta analysis of CLE40 processing and activity in wild-type and mutant Arabidopsis.
- Mass spectrometry to confirm peptide modifications.
Main Results:
- Three subtilases (SBT1.4, SBT1.7, and SBT4.13) were identified as redundant proteases cleaving the CLE40 precursor at two sites.
- C-terminal cleavage is essential for mature CLE40 signal biogenesis.
- Cleavage at a second site within the mature peptide attenuates CLE40 activity.
- Pro hydroxylation of the CLE40 precursor prevents this secondary cleavage, leading to the formation of mature, bioactive CLE40.
- SBT-mediated processing and Pro hydroxylation are critical for regulating CLE40 activity in planta.
Conclusions:
- Subtilases redundantly process the CLE40 precursor, releasing the mature peptide and regulating its activity.
- Pro hydroxylation acts as a crucial PTM that prevents secondary cleavage, thereby ensuring the biogenesis and bioactivity of CLE40.
- This study reveals a novel regulatory mechanism for peptide hormone formation and function in plant development, highlighting the interplay between proteolytic processing and PTMs.
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