bHLH transcription factors are common to all eukaryotes. They are especially numerous in multicellular organisms where they often play a prominent role in development (Degnan et al. 2009
). Phylogenetic analysis indicates that independent expansions in the animal and plant lineages resulted in unique subfamilies in these two groups (Ledent and Vervoort 2001
). Subgroup Ia bHLHs are a broadly conserved, plant specific group of transcription factors with an essential role in the production of stomata. The paralogs SPCH, MUTE and FAMA have well defined biological roles and characteristic domain architectures that make them an excellent system to understand how a multistep division of labor may evolve in the context of gene family evolution.
The role of SPCH, MUTE and FAMA in Arabidopsis stomatal development has been well described (MacAlister et al. 2007
, Pillitteri et al. 2007
and Ohashi-Ito and Bergmann 2006
). Putative orthologues of all three are readily identifiable from the genomes of other angiosperms, suggesting conserved function. The available expression pattern data also support this possibility. Microarray data for the expression of the poplar SPCH and MUTE-like sequences show higher levels in developing leaves than in mature leaves (Wilkins et al. 2009
), a pattern consistent with a role in stomatal development. More direct evidence is provided by Liu, Ohashi-Ito and Bergmann (2009)
who showed that stomatal development in rice is dependent on OsFAMA function and is modulated by OsSPCH, a conservation of function that spans the more than 140 million years since the divergence of the monocots and dicots (Chaw et al. 2004
). While this conservation of stomatal bHLH sequence and function within the angiosperms is consistent with the group Ia class having a conserved role in stomatal development, stomata are produced in a much wider group of plant taxa. If the stomatal bHLHs are generally required for stomatal development they should be generally conserved in the plants that produce them. Although SPCH, MUTE or FAMA orthologues cannot be unambiguously identified, the Selaginella
genomes contain subgroup Ia bHLHs with the key Ia domains ( and fig. S4
). Subgroup IIIb is also conserved in Physcomitrella
, suggesting that the heterodimerization between Ia and IIIb bHLHs could have been established early in plant evolution, even before the duplications that produced SPCH, MUTE and FAMA.
The mosses diverged from the ancestors of the angiosperms approximately 400MYA (Wolfe et al. 1989
), yet a Physcomitrella
Ia member (PpSMF1) is capable of promoting multiple steps of the stomatal lineage in Arabidopsis
. This type of multi-functionality of a single protein suggests a possible mechanism for the evolution of the stomatal bHLHs (). The duplication of a multi-functional ancestor sequence with a domain architecture similar to that of the present day Physcomitrella sequences would allow for alteration of the domain architecture (including MUTE losing the N-terminus and SPCH acquiring the MPKTD) and specialization for discrete stomatal lineage stages and expression patterns. FAMA appears to be closest to this ancestral form. MUTE, with its truncated N-terminus may have arisen by disruption of the initial start codon forcing the use of a later start codon at the beginning of the bHLH domain, where there is a conserved methionine residue. SPCH’s acquisition of the MPKTD is somewhat more difficult to trace since it is not a domain that is found in other Arabidopsis proteins. Insertion of a sequence with an abundance of serine and proline residues may have coincidentally created MAPK phosphorylation sites (P-X-S/T-P) that were able to capitalize on the MAPK pathway for SPCH regulation. Over time the functionally significant residues were maintained as the SPCH orthologues diverged, producing the islands of high conservation we now observe in this region (fig. S3
). Functional data from Arabidopsis also supports this evolutionary scenario. For example, deletion of the N-terminal region of FAMA renders it capable of producing MUTE-like overexpression phenotypes (Ohashi-Ito and Bergmann, 2006
), and deletion of the MPKTD of SPCH also generates a protein whose function more closely resembles that of MUTE (Lampard et al. 2008
Model of the expansion of the developmental complexity of the stomatal lineage
How might a single transcription factor have functioned to promote stomatal development in the ancestral case? A shorter stomatal lineage (for example, one without amplifying divisions as in the extant mosses) would simplify the problem. In the ancestral land plant, at the minimum, FAMA and MUTE-like activity would be essential to produce paired guard cells. FAMA-like activity is needed to specify the guard cell identity and drive specific differentiation programs like stomatal pore formation, whereas MUTE-like activity ensures that two guard cells will coordinately form a stoma. This is achieved by MUTE promoting a specific precursor from which guard cells are derived (the GMC). The GMC has well-defined proliferative potential (limited to a single symmetric division). Only in this cell and its daughters is FAMA expressed, ensuring that only two guard cells are formed per stomatal unit, and that they are directly adjacent with a common wall in which the pore could form. The asymmetric divisions currently regulated by SPCH may be a relatively late addition to the stomatal lineage that allowed for stomatal patterning, flexibility and other fine control of epidermal development. In addition, the MAP kinase regulation of early stages of stomatal development via SPCH would also allow environmental stimuli to feed into control of stomatal density (Lampard et al. 2008
). Throughout the land plants, guard cells morphologies are remarkably similar—two kidney shaped cells--except in the grasses, where guard cells are dumb-bell shaped. The ability of grass stomata to rapidly change pore size is dependent on subsidiary cells, themselves formed by asymmetric division (Franks and Farquhar, 2006). Interestingly, the unique and arguably more complex stomatal development in grasses correlates with the duplication of the SPCH gene in this lineage (Liu et al. 2009
Our cross-species complementation results highlight that the similarity in guard cell development across different taxa may arise from an underlying molecular similarity: the ancient and widespread group Ia bHLHs. The subgroup Ia bHLHs in stomatal development may be an example of a gene class that has always been specifically tied to a particular cell type. From studies with Arabidopsis
and maize, however, it is clear that many of the other genes required for guard cell specification and function are used in other processes. For example, the group IIIb bHLHs ICE1/SCRM and SCRM2 also have a well described role in cold response (Chinnusamy et al. 2003
) whereas the MAPK signaling pathway that regulates SPCH activity also has various functions including establishment of polarity in the early embryo (Lukowitz et al. 2004
) and stress and pathogen response (Asai et al. 2002
and Kovtun et al. 2000
). Although it is impossible to know which function came first, the selective pressure for well functioning stomata could have driven the recruitment of these components for stomatal development and patterning as a secondary event.
Subgroup Ia bHLHs enjoy a unique function in the production of stomata. The evolution of this group of paralogs is informative not only for understanding how important plant structures came to be, but suggests that these genes are excellent targets for breeding or transgenic approaches to modifying stomatal production, and hence CO2 acquisition, temperature and drought tolerance, across a wide range of economically important plant species.