The core stomatal proteins SPCH, MUTE and FAMA associate with both types of ICE proteins, VrICE1 and VrICE4, in Vitis riparia

SPEECHLESS (SPCH), MUTE and FAMA drive stomatal development in Arabidopsis. They do so in association with SCREAM/INDUCER OF CBF EXPRESSION (SCRM/ICE) proteins. Orthologous proteins have also been reported for other plants, including now for Vitis, suggesting that a similar process occurs during stomata formation also in other plants. However, the details of this process likely vary because the Vitis protein sequences are most similar to those of dicots other than Arabidopsis. We recently reported a 2nd type of ICE protein in angiosperms, VrICE4L, with unique sequences in its putative protein-interacting bHLH and ACTL domains. This brings up the question whether this second ICE protein is also involved in stomatal formation. Overexpression of any one of the Vitis riparia SPCH, MUTE, FAMA or ICE proteins was found to affect stomata numbers in tobacco leaves. Localization, bimolecular fluorescence complementation (BiFC) and pull-down experiments pointed that VrSPCH, VrMUTE and VrFAMA can interact with both types of ICE proteins present in angiosperms, represented by VrICE1 and VrICE4. Taken together, these findings suggest that stomatal development in Vitis involves SPCH/ICE, MUTE/ICE and FAMA/ICE complexes with functions that are less similar to those in Arabidopsis than to those in other dicots.