The asymmetric cell divisions necessary for stomatal lineage initiation and progression in Arabidopsis () require the function of the basic helix-loop-helix (bHLH) transcription factor (). Mutants lacking do not produce stomata or lineages. Here, we isolated a new allele carrying a point mutation in the bHLH domain that displayed normal growth, but had an extremely low number of sometimes clustered stomata in the leaves, whereas the hypocotyls did not have any stomata. In vivo tracking of leaf epidermal cell divisions, combined with marker lines and genetic analysis, showed that the leaf phenotype is dosage dependent and results from the decreased ability to initiate and amplify lineages, defects in asymmetric cell fate allocation, and misorientation of asymmetric division planes. Notably, application of brassinosteroids (BRs) partly rescued the stomatal leaf phenotype of Transcriptomic analysis combining with BR treatments revealed that the expression of a set of SPCH target genes was restored by BRs. Our results also show that BR-dependent stomata formation and expression of some, but not all, SPCH target genes require the integrity of the bHLH domain of SPCH.

Summary Epidermal differentiation in Arabidopsis thaliana aerial organs involves stomatal lineage development. Lineages derive from meristemoids, which arise from asymmetric divisions of protodermal cells. Each meristemoid divides repeatedly in an inward spiral before it transits to a guard mother cell (GMC) that produces the stoma, leaving a trail of surrounding stomatal lineage ground cells (SLGCs) that eventually differentiate into endoreplicated pavement cells. MUTE is a bHLH transcription factor that is expressed in late meristemoids and drives their transition to GMCs. Loss‐of‐function mute mutants are stomata‐less dwarf plants with arrested lineages, in which stunted putative SLGCs surround a halted meristemoid. We analysed MUTE functions using a chemically inducible system for mute‐3 complementation based on conditional MUTE expression in its normal domain. Continuous induction from germination produced stomata‐bearing, normal‐sized plants with viable mute‐3 seeds. In 2‐week‐old mute‐3 cotyledons, meristemoids appeared to retain their identity and synchronously formed stomata in response to induced MUTE expression. However, arrested SLGCs were not complemented: many produced stomata, leading to stomatal clusters, and others remained unexpanded and diploid. In contrast, non‐lineage pavement cells, which are under‐endoreplicated in mute‐3, expanded and increased their ploidy level upon induction, showing that the lack of response of SLGCs is specific to this arrested cell type. Leaf phenotypic mosaics include wild‐type lineages and adjacent mute‐3 lineages, whose meristemoids and putative SLGCs remained arrested, indicating that the role of MUTE in SLGC fate is strictly lineage‐autonomous. These results show that timely MUTE expression is essential to prevent stomatal fate in SLGCs and to promote their differentiation as pavement cells.

Loss of function of the positive stomata development regulators SPCH or MUTE in Arabidopsis thaliana renders stomataless plants; spch-3 and mute-3 mutants are extreme dwarfs, but produce cotyledons and tiny leaves, providing a system to interrogate plant life in the absence of stomata. To this end, we compared their cotyledon transcriptomes with that of wild-type plants. K-means clustering of differentially expressed genes generated four clusters: clusters 1 and 2 grouped genes commonly regulated in the mutants, while clusters 3 and 4 contained genes distinctively regulated in mute-3. Classification in functional categories and metabolic pathways of genes in clusters 1 and 2 suggested that both mutants had depressed secondary, nitrogen and sulfur metabolisms, while only a few photosynthesis-related genes were down-regulated. In situ quenching analysis of chlorophyll fluorescence revealed limited inhibition of photosynthesis. This and other fluorescence measurements matched the mutant transcriptomic features. Differential transcriptomes of both mutants were enriched in growth-related genes, including known stomata development regulators, which paralleled their epidermal phenotypes. Analysis of cluster 3 was not informative for developmental aspects of mute-3. Cluster 4 comprised genes differentially up−regulated in mute−3, 35% of which were direct targets for SPCH and may relate to the unique cell types of mute−3. A screen of T-DNA insertion lines in genes differentially expressed in the mutants identified a gene putatively involved in stomata development. A collection of lines for conditional overexpression of transcription factors differentially expressed in the mutants rendered distinct epidermal phenotypes, suggesting that these proteins may be novel stomatal development regulators. Thus, our transcriptome analysis represents a useful source of new genes for the study of stomata development and for characterizing physiology and growth in the absence of stomata.