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The initiation of stomatal development in the developing Arabidopsis epidermis is characterized by an asymmetric ‘entry’ division in which a small cell, known as a meristemoid, and a larger daughter cell is formed. The meristemoid may undergo further asymmetric divisions, regenerating a meristemoid each time, before differentiating into a guard mother cell which divides symmetrically to form a pair of guard cells surrounding a stomatal pore. Recently EPF2 and BASL have emerged as regulators of these asymmetric divisions and here we present results indicating that these two factors operate independently to control stomatal development
Several factors that regulate asymmetric cell divisions involved in the differentiation of specialized plant cell types have been identified from molecular genetic studies, including putative transcription factors, receptors, phosphatases, kinases and proteases.1 Recently, we and others reported putative peptide ligands for the receptor-like protein TMM which regulates both the number and spacing of stomata during epidermal development. 2,3 EPIDERMAL PATTERNING FACTORs 1 and 2 (EPF1 and EPF2) are members of a family of secretory peptides, all with previously unknown function.4–6 EPF1 and EPF2 are both specifically expressed in stomatal precursor cells. Knockdown mutants of epf1 exhibit pairs of adjacent stomata in their epidermis, and epf2 mutants are characterized by increased stomatal density and the presence of an abnormally large proliferation of arrested meristemoids. Thus EPF1 functions to orientate asymmetric divisions of meristemoids and prevent stomata from forming in contact with one another, whereas EPF2 acts to restrict the number of ‘entry’ and asymmetric divisions of meristemoids and to promote the differentiation of daughter cells into pavement cells.
A novel protein, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which accumulates in stomatal precursor cells has also recently been reported.7 Interestingly, BASL accumulates in a distinct polarized pattern inside stomatal precursor cells, marking the periphery of those cells about to undergo an asymmetric division. Knockdown basl mutants show extra symmetric divisions of meristemoids and BASL is therefore proposed to be necessary for promoting correctly orientated asymmetric divisions of stomatal precursors. Epigenetic studies have shown that double mutants epf1;basl and tmm;basl have additive phenotypes relative to the single mutants suggesting that BASL acts independently of these previously characterised stomatal development regulators. As EPF2 and BASL are both expressed in stomatal precursor cells and are both involved in regulating their asymmetric divisions, we investigated whether EPF2 and BASL could act together to regulate this step in stomatal development.
The basl-4;epf2-1 leaf epidermis contained large numbers of additional small cells which appeared to be the result of increases in both symmetric and asymmetric divisions of stomatal lineage cells. This resulted in a reduced density of mature tessellated epidermal pavement cells (Fig 1A–D). Stomatal density was not altered in fully expanded leaves of either basl-4 or basl-4;epf2-1 in comparison to the wild-type control (Col-0) but was significantly higher in epf2-1 (p < 0.003) than wild-type or basl-4;epf2-1 (p < 0.03) (Fig. 1E). In contrast to basl-1 and basl-2,7 basl-4 did not show increased stomatal density. This may be explained by the different tissue examined as Dong et al. examined cotyledons, whereas we used mature leaves. There were however clearly more clustered stomata in basl-4 than wild-type and the epf2-1 mutation caused a significant reduction in the number of paired or clustered stomata in basl-4;epf2-1 (p > 0.016) (Fig. 1F). Neither basl-4 nor epf2-1 showed any obvious effect on growth rate or leaf shape, but basl-4;epf2-1 plants developed narrow twisted leaves, perhaps because of their considerably reduced number of epidermal pavement cells.
To confirm that the additional small epidermal cells that we found in our mutants were the result of excessive asymmetric or symmetric divisions of meristemoids we expressed the pTMM:GUS-GFP gene construct which is specifically expressed in stomatal lineage cells, in each of our genetic backgrounds and observed GUS and GFP cellular expression patterns (Fig. 2C). GUS staining and GFP fluorescence directed by the TMM promoter indicted that the additional small epidermal cells that we observed in our single and double mutants were indeed arrested stomatal lineage cells.
The recently reported Arabidopsis gene products BASL and EPF2 are both involved in regulating the asymmetric cell division programthat leads to stomatal development. Lack of BASL expression leads to a large number of symmetric rather than asymmetric divisions of stomatal precursors, and lack of EPF2 leads to excessive numbers of asymmetric divisions. Our analysis of the double mutant basl-4;epf2-1 suggests however, that BASL and EPF2 operate independently to regulate asymmetric divisions during stomatal development.
Previously published online: www.landesbioscience.com/journals/psb/article/10704