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

Stomatal abundance sets the plant potential for gas exchange, impacting photosynthesis and transpiration, and ultimately, 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 stomata commitment. We aim to explore the molecular mechanisms underlying MUTE activity, using partial loss-of-function alleles predicted to impair DNA-binding and potentially alter the expression of MUTE transcriptional targets. We engineered mutant allele coding sequences, generated Arabidopsis lines carrying these alleles and analyzed their epidermal and transcriptional phenotypes using microscopy and RNAseq. Synthetic alleles driven by the MUTE promoter rescued the stomataless 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 some MUTE targets, but not others, 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. To explore the molecular mechanisms underlying MUTE activity we designed two MUTE variants carriyng specific mutations in the bHLH domain: MUTEv5 (substitution Arg10>Trp10) and MUTEPPP (substitutions Hys3>Pro3, Glu7>Pro7 and Arg11>Pro11 ). These synthetic alleles and the wild-type allele driven by the MUTE promoter were mobilized into the mute-3 background to generate homozygous MUTEv5 and MUTEPPP lines, with MUTE lines used as the reference. For gene expression profiling we selected lines representative of the distinct quantitative epidermal phenotypes exhibited by the synthetic alleles. For MUTEv5, we chose line #2 with a moderate clustered stomata phenotype, and line #5 that showed a severe phenotype of arrested lineages. As a representative of the MUTEPPP lines, line #3 was chosen. As a control, MUTE line #1 was used. RNA-seq data were obtained from three independent biological replicates, each consisting of 50 seedlings collected at 2 days post-germination. Comparative gene expresión profiling análisis of RNA-seq data for lines MUTE#1, MUTEv5#2, MUTEv5#5 and MUTEPPP#3.