Plant Biology 2023, Braga, Portugal, 9 - 12 July 2023, pp.284
In Arabidopsis thaliana, stomatal development is positively regulated by three related bHLH transcription
factors: SPCH, MUTE, and FAMA. They orchestrate the transitions between three consecutive cell stages
during stomatal lineage development: meristemoid initiation and self‐renewal, meristemoid‐to‐guard
mother cell transit, and stoma differentiation. Loss‐of‐function mutations in any of these proteins result
in a seedling‐lethal phenotype and the complete absence of stomata, hindering the study of their
functions at later developmental stages. Our previous work described hypomorphic mutations in SPCH
(spch‐5, SPCHPPP, and spch‐2), which produced viable and fertile plants and allowed further inspection
of SPCH functions.
By taking advantage of the sequence conservation between SPCH and MUTE, we constructed three
synthetic MUTE alleles by introducing the SPCH mutations into the MUTE coding sequence as proteins
fused to GFP to study the MUTE‐leaded transition from meristemoid to the guard mother cell. Under the
control of the MUTE promoter, we used them to complement the stomata‐deficient mute‐3 mutant. The
synthetic alleles or MUTE versions, MUTEv5, MUTEPPP, and MUTEv2, partially complemented the mute‐
3 phenotype and formed stomata. However, they did not fully restore the normal functions of MUTE.
Qualitative and quantitative characterization of the lines carrying the MUTE versions revealed that they
produce distinct phenotypes with varying proportions of arrested lineages and clustered stomata.
An RNAseq‐based transcriptome analysis was performed to describe the molecular phenotypes
underlying these epidermal defects. Transcripts for many genes associated with stomatal development
and direct MUTE targets were correctly expressed in lines carrying the various versions, as expected by
their ability to produce stomata. However, key markers for specific cell stages were misregulated,
providing a basis to explain the abnormal stomatal development. We are using a dual‐luciferase trans‐
activation system in N. benthamiana to test the capacity of the different MUTE versions to regulate the
transcription of MUTE target genes, which will provide information on the functional basis for MUTE