Sequence and function of basic helix-loop-helix proteins required for stomatal development in Arabidopsis are deeply conserved in land plants

Abstract

Stomata are a broadly conserved feature of land plants with a crucial role regulating transpiration and gas exchange between the plant and atmosphere. Stereotyped cell divisions within a specialized cell lineage of the epidermis generate stomata and define the pattern of their distribution. The behavior of the stomatal lineage varies in its detail among different plant groups, but general features include asymmetric cell divisions and an immediate precursor (the guard mother cell [GMC]) that divides symmetrically to form the pair of cells that will differentiate into the guard cells. In Arabidopsis, the closely related basic helix-loop-helix (bHLH) subgroup Ia transcription factors SPEECHLESS, MUTE, and FAMA promote asymmetric divisions, the acquisition of GMC identity and guard cell differentiation, respectively. Genome sequence data indicate that these key positive regulators of stomatal development are broadly conserved among land plants. While orthologies can be established among individual family members within the angiosperms, more distantly related groups contain subgroup Ia bHLHs of unclear affinity. We demonstrate group Ia members from the moss Physcomitrella patens can partially complement MUTE and FAMA and recapitulate gain of function phenotypes of group Ia genes in multiple steps in the stomatal lineage in Arabidopsis. Our data are consistent with a mechanism whereby a multifunctional transcription factor underwent duplication followed by specialization to provide the three (now nonoverlapping) functions of the angiosperm stomatal bHLHs.

Fig. 1. Stomata are an ancient innovation in plants

(a) Land plant phylogeny including major groups described in this report; the first appearance of stomata is marked by a star. (b-d) Scanning electron micrographs of stomata (indicated by white arrows) in representative plant lineages: (b) Physcomitrella patens stomata on base of spore capsule, (c) Lycophyte Selaginella moellendorffii stomata on leaf, (d) Eudicot Eschscholzia californica (California poppy) stomata on leaf. (e) Diagram of stages in Arabidopsis (dicot) stomatal lineages with the point of action of the transcription factors, SPCH, MUTE and FAMA marked. A protodermal cell, the meristemoid mother cell (MMC) divides asymmetrically to form a small meristemoid (light grey). The meristemoid may self-renew by further asymmetric divisions or transition to a guard mother cell (GMC) (dark grey) which divides symmetrically to form the two guard cells. In Arabidopsis, asymmetric divisions require SPCH activity; GMC identity requires MUTE and the differentiation of the GCs requires FAMA function. Scale bars =100μm

Fig. 2. Phylogenetic tree and domain architecture of SPCH, MUTE and FAMA

(a) Maximum parsimony phylogentic tree based on complete predicted protein coding sequences of putative orthologues of stomatal bHLHs. Bootstrap values for 1000 replicates are given in nodes as percents. At3g56980 (a member of subgroup Ib, Heim et al. 2003) is used as an outgroup. Zm=Zea mays (maize), Os=Oryza sativa (rice), Vv=Vitis vinifera (grape), Rc= Ricinus communis (castor bean), Pt=Populus trichocarpa (poplar), Cs= Cucumis sativus (cucumber), Pg= Picea glauca (white spruce), Sm= Selaginella moellendorffii and Pp= Physcomitrella patens. (b) Diagram of domain architecture of SPCH, MUTE and FAMA-like sequences from angiosperms and subgroup Ia members from Selaginella and Physcomitrella. Regions that are unique to FAMA, MUTE and SPCH are red, orange and yellow respectively. The bHLH is in light blue with the Ia characteristic N-terminal extension in purple and the C-terminal extension in dark blue. The conserved C-terminal SMF domain is green

Fig. 3. Functional conservation of Physcomitrella and Arabidopsis group Ia bHLHs

(a-d) Confocal image of 10dpg cotyledons demonstrating that overexpression of PpSMF1 promotes SPCH, MUTE and FAMA-like overexpression phenotypes. (a) Wild type (b) induced ESTpro::PpSMF1 with unpaired guard cells marked with white *, and aberrantly shaped stomata by indicated by white arrow. Cell outlines are visualized by propidium iodide staining (red), chloroplast autofluorescence is blue. (c) 35S::SPCH, extra cell divisions noted by yellow arrow (d) induced ESTpro::PpSMF1 with extra SPCH-like divisions indicated in brackets and unpaired guard cells marked with yellow * (e-j) DIC images of 8 dpg cotyledons showing that expression of PpSMF1 can substitute for MUTE and FAMA function. (e) wild type (f) mute and (h) fama mutants; mute and fama do not produce mature guard cells but arrest at the meristemoid (black arrowhead) and GMC stage (black bracket) respectively. (g) expression of PpSMF1 under the MUTE promoter can partially rescue the mute phenotype; a normal stoma is marked with a black ‘*’. PpSMF1 rescue of fama may result in normal stomata (j, black *) or stomata within a fama GMC cluster, (i, black bracket). Scale bars are 50μm. a-d and e-j are at the same magnification. (k) quantification of rescue of the mute and fama phenotype as mean number of mature guard cells produced per seedling plus or minus standard error in independent transgenic lines (n=20 sides for each line) * mark statistically significant deviation from the mutant background. … do summary for the notation for the lines… for 4 lines of PpSMF1 and 2 of PpSMF2…

Fig.4. Model of the expansion of the developmental complexity of the stomatal lineage

(a) A single, multifunctional Ia member drives both the specification of GMC identity and guard cell differentiation. (b) Duplication of the ancestral Ia member allows for specialization for GMC specification (MUTE-like activity) and guard cell differentiation (FAMA-like activity). (c) Another duplication produces a third Ia member and allows for the evolution SPCH and the current stomatal lineage, including amplifying divisions.