The NMR structure of stomagen reveals the basis of stomatal density regulation by plant peptide hormones

Abstract

stomatal development in plants is regulated by defensin-like secretory epidermal patterning factor (EPF) peptide hormones. only one of these, stomagen, is a positive regulator, whereas EPF1, EPF2, and possibly others are negative regulators. Here we explore the structure-function relationships of EPFs, by integrating nmR and semi-in vitro stomagen experiments. We show that stomagen is composed of a loop and a scaffold containing three disulphide bonds. A mutant composed of the stomagen loop and the EPF2 scaffold positively regulates the stomatal density on Arabidopsis cotyledons. The reciprocal mutant composed of the EPF2 loop and the stomagen scaffold acts negatively. Deletion of the disulphide bond introduces unfolding and inactivity. our results suggest that the loop confers the functional specificity of EPFs and that the scaffold is structurally required for their activity. This structural decomposition approach to elucidating the functional site could be adapted for the analysis of other cysteine-rich peptide families.

A Tale of Two Systems: Peptide Ligand-Receptor Pairs in Plant Development

“…The nuclear magnetic resonance (NMR) structure of the EPF family peptide EPFL9/Stomagen revealed a conserved peptide backbone composed of an antiparallel b sheet supported by three disulfide bonds, which is intercepted by a less conserved loop domain (Ohki et al 2011). Domain-swap experiments between EPFL9/ Stomagen and EPF2, which are positive and negative regulators of stomatal development, respectively, revealed that the divergent loop domain is likely to determine the functional specificity of each EPF peptide (Ohki et al 2011).…”

“…Domain-swap experiments between EPFL9/ Stomagen and EPF2, which are positive and negative regulators of stomatal development, respectively, revealed that the divergent loop domain is likely to determine the functional specificity of each EPF peptide (Ohki et al 2011). It would be enlightening to investigate further the relationship between structure and biological function by analyzing the loop region and its binding affinity to the receptor among EPF family members known to have different and redundant biological functions.…”

“…Misfolded and unfolded EPFL9/Stomagen generated via removal of conserved cysteine residues resulted in inactive peptide because of its improper structural conformation. This strongly suggests that correct disulfide formation is critical for EPF family members, including EPFL9, to exhibit proper folding and activity (Ohki et al 2011). …”

“…There are 11 EPF family members in Arabidopsis and they all have a characteristic, carboxy-terminal domain containing six or eight cysteine residues that are essential for the formation of intramolecular disulfide bonds (Rychel et al 2010). Recent biochemical and structural studies on EPF-LIKE9 (EPFL9)/Stomagen, a member of the EPF family of peptides, revealed that the mature form of EPFL9 is 45 amino acids, which is relatively large compared with CLE peptides that have three intramolecular disulfide bonds derived from its carboxy-terminal end Ohki et al 2011). Misfolded and unfolded EPFL9/Stomagen generated via removal of conserved cysteine residues resulted in inactive peptide because of its improper structural conformation.…”

Signaling Peptides in Plants

“…In this context as well, signaling peptides have a significant impact on development [44,[72][73][74]. Multiple cysteine-rich signaling peptides that belong to the EPF family, in particular EPF1, EPF2, EPF-LIKE6/CHALLAH (EPFL6/CHAL) and EPFL9/ STOMAGEN, are associated with the regulation of stomatal density and positioning [18,19,44,72,[75][76][77][78]. Overexpression of EPF1 and EPF2 reduces stomatal density.…”