Abstract
BackgroundAlthough it is known that CLAVATA3 (CLV3) acts as 12- and/or 13-amino acid (AA) secreted peptides to regulate the number of stem cells in shoot apical meristems (SAMs), how functional CLV3 peptides are generated and if any particular sequences are required for the processing remain largely unknown.ResultsWe developed a mass spectrometry (MS)-based in vitro assay to monitor the cleavage of heterologously produced CLV3 fusion protein. Through co-cultivation of the fusion protein with Arabidopsis seedlings, we identified two cleavage sites: the previously reported one before Arg70 and a new one before Met39. Using synthetic peptides together with MALDI-Tof-MS analyses, we demonstrated that the non-conserved 5-AA motifs flanking N-termini of the CLV3 and its orthologous CLE1 peptides were critical for their cleavages and optimal activities in vitro. We also found that substitutions of Leu69 by Ala in fusion protein and in synthetic peptide of CLV3 compromised their cleavages, leading to significantly reduced activities in regulating the sizes of shoot and root meristems.ConclusionsThese results suggest that 5-AA residues flanking the N-terminus of CLV3 peptide are required for proper cleavages and optimal function in stem cell regulation.
Highlights
It is known that CLAVATA3 (CLV3) acts as 12- and/or 13-amino acid (AA) secreted peptides to regulate the number of stem cells in shoot apical meristems (SAMs), how functional CLV3 peptides are generated and if any particular sequences are required for the processing remain largely unknown
Produced CLV3 fusion protein is active in vitro For the production of the CLV3 proprotein, a construct was made in which tandem aligned Trx and His tags were fused to the Nterminus of CLV3
We observed that the incubation of clv3-2 seedlings with 1 μM TH-ProCLV3 for 8 days led to significant reduced sizes of SAMs (Figure 1B, H, and I), while no evident reduction was observed in SAMs of clv1-1 seedlings (Figure 1B, D, and E), suggesting that the TH-ProCLV3 fusion protein produced in E. coli is active in restricting SAMs in a CLV1-dependent manner
Summary
It is known that CLAVATA3 (CLV3) acts as 12- and/or 13-amino acid (AA) secreted peptides to regulate the number of stem cells in shoot apical meristems (SAMs), how functional CLV3 peptides are generated and if any particular sequences are required for the processing remain largely unknown. It has been known for a long time that small peptides act as endocrinal hormones and neurotransmitters to facilitate intercellular communications in animals [1,2,3,4]. These studies provide basic knowledge on the cleavage of CLE peptides, sequences involved in the cleavage recognition and how such cleavages contribute to the activity of these peptides remain elusive
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