Characterization of the EPF family of signaling peptides controlling stomatal development in monocots

Stomata are pores on plant epidermis which control water and gas exchanges between plants and the atmosphere. These two processes are very crucial for adaptation against environmental changes and photosynthesis process which increases biomass of a plant. As such, proper density and distribution of stomata are critical for plant growth and survival. In Arabidopsis, several Epidermal Patterning Factor (EPF) family members of small cysteine-rich peptides have been discovered as important signaling molecules that possess diverse functions in controlling plant development, including stomatal patterning and differentiation. EPF1 and EPF2, released from stomatal precursor cells, enforces stomatal spacing divisions and inhibits initiation of stomatal cell lineage, respectively. They are known as negative regulators of stomata development. STOMAGEN/EPFL9, on the other hand, being a positive regulator promotes stomatal differentiation in the epidermis and expressed in the underlying mesophyll tissues. Although much is known about Dicots’ EPFs, how EPF peptides control different stomatal patterns and morphologies exist in grasses remains unclear. In this study, we searched for stomatal EPF homologs in other agriculturally important cereal crops using a bioinformatics followed by functional genomics studies. Our phylogenetic analysis revealed that there are genes encoding putative EPFs including stomatal EPFs, EPF1, EPF2, and STOMAGEN, in each of the grass species examined. To understand the roles of EPF peptides in grass stomatal development, we generated estradiol inducible overexpression lines for both wheat and Brachypodium EPF1, EPF2 and STOMAGEN homologous genes in Arabidopsis. It resulted in identification of antagonistic stomatal ligands, which are duplicated ortholog of Arabidopsis AtEPF2 and AtSTOMAGEN peptides, in the wheat and Brachypodium. We further investigated the behavior of AtEPF1/2-like genes from wheat and Brachypodium in the regulation of epidermal development by analyzing cross-species complementation studies followed by phenotypic effects of exogenously applied bioactive grass EPF peptides on Brachypodium wild type seedlings, Bd21-3. Application of bioactive BdEPF1/2 peptides inhibited the stomatal initiation process in Bd21-3, whereas BdSTOMAGEN application promoted stomatal development in Bd21-3, resulting in an epidermis with clustered stomata. Overall, the present study furthers our knowledge on conserved EPF peptides which controls different stomatal patterns that exist in nature and provides putative strategies to improve crop productivity by using plant-derived antagonistic peptides that optimize stomatal density on plant epidermis