Stomatal Density is Controlled by a Mesophyll-Derived Signaling Molecule

Abstract

Stomata are composed of a pair of guard cells and a pore between them, and their density and positions are regulated by developmental and environmental signals. In a screen in which we overexpressed many genes coding for putative secretory proteins one by one in Arabidopsis, we identified a gene named STOMAGEN, which increases stomatal density when overexpressed. The STOMAGEN gene encodes a small peptide with a putative secretory signal sequence at its N-terminus and is expressed preferentially in mesophyll cells. This peptide belongs to the EPIDERMAL PATTERNING FACTOR (EPF) family of the cysteine-rich peptides superfamily. The mature form was a 45-amino-acid peptide (stomagen) with three intramolecular disulfide bonds. Stomagen treatment at very low concentrations, as low as 10 nM, increased the stomatal density of wild-type Arabidopsis plants. We propose that stomagen is a mesophyll-to-epidermis signaling molecule that positively regulates stomatal density. We also suggest that stomagen increases stomatal density by competing with negative regulators EPF1 and EPF2 for the receptor-like protein TOO MANY MOUTHS.

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

“…The anti-parallel β-sheet is packed with the amino-terminal region, which contains a one-turn 3 10 -helix (residues 10-13), supported by three disulphide bonds, the locations of which (Cys8-Cys41, Cys13-Cys20, and Cys16-Cys43) were identical to those previously obtained by enzymatic digestion and mass spectrometry (MS) 11 . This stomagen core, consisting of the β-sheet and a one-turn 3 10 -helix, does not correspond to a single continuous stretch in the aminoacid sequence; it is separated into N-and carboxy-terminal regions (residues 8-24 and 39-44).…”

“…The functions of the other members remain unclear. Interestingly, it is currently known that stomagen (EPFL9) acts as a positive regulator [10][11][12] . One of the most intriguing questions in this area is how and why peptides from the same family can produce opposite effects on stomatal density.…”

Generation of Signaling Specificity in Arabidopsis by Spatially Restricted Buffering of Ligand–Receptor Interactions

“…However, the CHALf proteins are similar in sequence to stomatal regulators EPF1, EPF2, and STOMAGEN Kondo et al, 2009), and CHAL itself was originally identified in a stomatal screen (Abrash and Bergmann, 2010). In overexpression assays, CHAL confers distinct, quantifiable stomatal phenotypes (Abrash and Bergmann, 2010), and Hara et al (2009) report in an EFPL family survey that CLL1 and CLL2 can similarly repress stomatal production.…”

“…The EPFLs are small proteins predicted (and in one case, demonstrated) to be processed into ;45-to 75-amino acid long secreted peptides with compact Cys knot structures (Kondo et al, 2009;Sugano et al, 2009). Three members of the gene family, EPF1, EPF2, and EPFL9/STOMAGEN, have clear roles in stomatal development.…”

“…Three members of the gene family, EPF1, EPF2, and EPFL9/STOMAGEN, have clear roles in stomatal development. EPF1 and EPF2 are expressed specifically within subsets of stomatal lineage cells, and STO-MAGEN is expressed in leaf mesophyll; all three depend largely upon TMM for their ability to affect stomatal development (Hara et al, 2007Hunt and Gray, 2009;Kondo et al, 2009;Sugano et al, 2009). A fourth member of the family, EPFL6/ CHALLAH (CHAL), however, exhibits different behaviors.…”