Synthetic alleles to study MUTE-dependent molecular transitions in stomatal development.

Stomatal abundance sets plants' potential for gas exchange, impacting photosynthesis and transpiration and, thus, plant survival and growth. Stomata originate from cell lineages initiated by asymmetric divisions of protodermal cells, producing meristemoids that develop into guard cell pairs. The transcription factors SPEECHLESS, MUTE, and FAMA are essential for stomatal lineage development, sequentially driving cell division and differentiation events. Their absence produces stomataless epidermis, hindering analysis of their roles during lineage development. MUTE drives the transition from proliferating meristemoids to guard mother cells, committed to stomatal fate. We aim to explore the molecular mechanisms underlying MUTE activity, using partial loss-of-function alleles predicted to impair DNA-binding and to potentially alter MUTE transcriptional activity. We engineered mutant allele coding sequences, generated Arabidopsis lines carrying them and analyzed their epidermal and transcriptional phenotypes using microscopy and RNA-seq. Synthetic alleles driven by the MUTE promoter rescued the stomata less phenotype of the seedling-lethal mute-3 mutant, enabling stomata differentiation and resulting in viable, fertile plants. Further examination of the developmental consequences of MUTE partial loss-of-function revealed arrested lineages, reduced stomatal abundance and altered stomatal spacing. Transcriptomic analysis of very young cotyledons from complemented lines indicated that only some MUTE targets require an intact MUTE bHLH domain. Comparison with existing lineage cell-specific transcriptional profiles showed that lineage development in the mutant lines was delayed compared to the wild-type but followed similar gene networks. These synthetic alleles provide new insight into MUTE ability to accurately and timely specify stomata formation.