Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants

Stomata play significant roles in plant evolution. A trio of closely related basic Helix-Loop-Helix (bHLH) subgroup Ia genes, SPCH, MUTE and FAMA, mediate sequential steps of stomatal development, and their functions may be conserved in land plants. However, the evolutionary history of the putative SPCH/MUTE/FAMA genes is still greatly controversial, especially the phylogenetic positions of the bHLH Ia members from basal land plants. To better understand the evolutionary pattern and functional diversity of the bHLH genes involved in stomatal development, we made a comprehensive evolutionary analysis of the homologous genes from 54 species representing the major lineages of green plants. The phylogenetic analysis indicated: (1) All bHLH Ia genes from the two basal land plants Physcomitrella and Selaginella were closely related to the FAMA genes of seed plants; and (2) the gymnosperm ‘SPCH’ genes were sister to a clade comprising the angiosperm SPCH and MUTE genes, while the FAMA genes of gymnosperms and angiosperms had a sister relationship. The revealed phylogenetic relationships are also supported by the distribution of gene structures and previous functional studies. Therefore, we deduce that the function of FAMA might be ancestral in the bHLH Ia subgroup. In addition, the gymnosperm “SPCH” genes may represent an ancestral state and have a dual function of SPCH and MUTE, two genes that could have originated from a duplication event in the common ancestor of angiosperms. Moreover, in angiosperms, SPCHs have experienced more duplications and harbor more copies than MUTEs and FAMAs, which, together with variation of the stomatal development in the entry division, implies that SPCH might have contributed greatly to the diversity of stomatal development. Based on the above, we proposed a model for the correlation between the evolution of stomatal development and the genes involved in this developmental process in land plants.     

bHLH proteins know when to make a stoma

In Arabidopsis thaliana, stomata develop through a stereotypical pattern of cell divisions. Three recent publications demonstrate that three bHLH proteins act successively in such lineages to drive the formation of stomata. SPEECHLES drives the division that initiates the stomatal-cell lineage. Then MUTE induces the formation of the immediate stomatal precursor cell. Finally, FAMA causes the stomatal precursor cell to divide into the two guard cells that surround each stomatal pore.     

Plant development: three steps for stomata

Three basic helix-loop-helix proteins regulate sequential steps in the formation of stomata: SPEECHLESS initiates entry into the stomatal lineage; MUTE controls asymmetric divisions of stomatal precursor cells; and FAMA promotes guard cell differentiation.     

ABA inhibits entry into stomatal‐lineage development in Arabidopsis leaves

The number and density of stomata are controlled by endogenous and environmental factors. Despite recent advances in our understanding of stomatal development, mechanisms which prevent stomatal‐lineage entry remain unclear. Here, we propose that abscisic acid (ABA), a phytohormone known to induce stomatal closure, limits initiation of stomatal development and induces enlargement of pavement cells in Arabidopsis cotyledons. An ABA‐deficient aba2‐2 mutant had an increased number/proportion of stomata within a smaller cotyledon, as well as reduced expansion of pavement cells. This tendency was reversed after ABA application or in an ABA over‐accumulating cyp707a1cyp707a3 doublemutant. Our time course analysis revealed that aba2‐2 shows prolonged formation of meristemoids and guard mother cells, both precursors of stoma. This finding is in accordance with prolonged gene expression of SPCH and MUTE, master regulators for stomatal formation, indicating that ABA acts upstream of these genes. Only aba2‐2 mute, but not aba2‐2 spch double mutant showed additive phenotypes and displayed inhibition of pavement cell enlargement with increased meristemoid number, indicating that ABA action on pavement cell expansion requires the presence of stomatal‐lineage cells.     

Roles of CONSTITUTIVE PHOTOMORPHOGENIC 10 in Arabidopsis stomata development

Stomata are epidermal bi-celled structures that differentiate within special cell lineages initiated by a subset of protodermal cells. Recently, we showed that the Arabidopsis photomorphogenic repressor COP10 controls specific cell-lineage and cell-signaling developmental mechanisms in stomatal lineages. Loss-of-function cop10-1 mutant cotyledons and leaves produced (in the light and in the dark) abundant stomatal clusters, but nonlineage epidermal cells were not affected. Here we examine COP10 role in hypocotyls, cylindrical organs displaying a distinct epidermal organization with alternate files of protruding and non-protruding cells, with the latter producing a limited number of stomata. COP10 prevents stomatal clusters and restricts stomata production in hypocotyls; these roles are specific to lineage cells as in cotyledons, since COP10 loss of function does not elicit stomatal fate in nonlineage cells; COP10 also sustains the directional cell expansion of all hypocotyl epidermal cell types, and seems necessary for the differentiation between protruding and non-protruding cell files.     

Stomatal development in Arabidopsis and grasses: differences and commonalities

Stomata, found on the epidermis of all terrestrial plants, consist of two specialized cells called guard cells, which surround a tiny pore. Major advances have been made in our understanding of the genetic control of stomatal development in Arabidopsis and grasses. In Arabidopsis, three basic-helix-loop-helix (bHLH) genes control the successive steps that lead to stomatal formation. SPEECHLESS (SPCH) drives the cell division that initiates the stomatal cell lineage, MUTE induces the formation of the immediate stomatal precursor cell, and FAMA causes the stomatal precursor cell to divide into the two guard cells. Recent results demonstrate that these genes share functions with their grass homologs, and that MUTE is expressed later in development than its grass counterparts. Other differences in stomatal development between these two plant groups are exemplified by the PANGLOSS1 (PAN1) gene of maize. PAN1, which encodes a leucine-rich repeat receptor-like kinase with an inactive kinase domain, promotes polarization of the subsidiary mother cell and orients its cell division plane. Because such events do not exist in Arabidopsis, it is likely that the PAN1-like genes of Arabidopsis and PAN1 are paralogs. Together, these results indicate that distinctions in the regulation of gene expression and protein function are both responsible for the divergence of stomatal development between Arabidopsis and grasses.     

BASL and EPF2 act independently to regulate asymmetric divisions during stomatal development

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 developmentKey words: stomata, development, meristemoids, asymmetric cell division, leaf epidermis, cell polarity, peptide signal