The Role of the Basic Helix Loop Helix Transcription Factor Speechless in the Initiation of the Stomatal Lineage.

Stomata are essential to the survival of plants due to their role in balancing gas exchange and transpiration. A series of stereotyped divisions within the expanding epidermis produce stomata beginning with the asymmetric division of a precursor cell, the meristemoid mother cell, to form a smaller cell, the meristemoid, and a larger sister cell. The meristemoid has limited stem cell like properties and can continue to divide asymmetrically to increase the total number of epidermal cells. Eventually it will convert to a rounded guard mother cell (GMC) which will divide symmetrically to form the paired guard cells. Changes in turgor pressure in the guard cells regulate the aperture of a pore between them. Working with Arabidopsis mutants with defects on stomatal production, I identified a gene, speechless (spch), specifically required for asymmetric meristemoid forming divisions. Using a map based strategy I cloned spch and found it to encode a bHLH type transcription factor. Translational reporter fusions show SPCH to be constitutively nuclear and expressed in small, non-differentiated epidermal cells and young meristemoids. Although the bHLH family in Arabidopsis has many members, spch is very closely related to fama which is required for the last step in the stomatal lineage, the differentiation of guard cells. I found another closely related gene, mute, to be required for the middle step in the stomatal lineage, the conversion of meristemoids to GMCs. Although these three are closely related, they function at distinct stages of the stomatal lineage and are not able to functionally substitute for one another. The formation of stomata was an early adaptation to life on land and they remain extremely well conserved in extant plants. SPCH, MUTE and FAMA are required for stomatal production in Arabidopsis, and clear orthologues of each are identifiable across a number of other angiosperms. Outside of the seed plants, orthologues cannot be unambiguously identified, but members of the same bHLH subgroup (subgroup Ia) are present. A subgroup Ia member from the moss Physcomitrella patens was found to be functional at multiple stomatal lineage stages in Arabidopsis, suggesting that a multifunctional ancestor may have given rise to the three stomatal bHLHs. In addition to domains SPCH, MUTE and FAMA share, SPCH contains a novel region with several consensus MAPK phosphorylation sites. Proper stomatal spacing is established through the activity of an inhibitory signaling network that prevents inappropriate meristemoid formation. This network includes a MAP kinase cascade. SPCH, as gatekeeper of entry into the stomatal lineage, is an ideal target for regulation. Working in collaboration with Dr. Greg Lampard, we have shown SPCH to be phosphorylated in vitro by the kinases implicated in stomatal patterning, that the phosphorylation domain negatively regulates SPCH function in planta and that SPCH interacts genetically with members of the inhibitory signaling pathway. Finally, I report the results of a screen for SPCH interacting proteins and transcriptional profiling of SPCH overexpressing lines to identify genes that may be effectors of SPCH activity and/or targets of SPCH transcriptional regulation. This thesis represents a significant advance in understanding the regulation of the stomatal lineage and provides new insights into how stomata are formed and patterned.