Liverwort bHLH transcription factors and the origin of stomata in plants

Stomata are distributed in nearly all major groups of land plants, with the only exception being liverworts. Instead of having stomata on sporophytes, many complex thalloid liverworts possess air pores in their gametophytes. At present, whether stomata in land plants are derived from a common origin remains under debate. 1 Duckett J.G. Pressel S. The evolution of the stomatal apparatus: intercellular spaces and sporophyte water relations in bryophytes-two ignored dimensions. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2018; 373https://doi.org/10.1098/rstb.2016.0498 Crossref PubMed Scopus (41) Google Scholar ,2 Chater C.C.C. Caine R.S. Fleming A.J. Gray J.E. Origins and evolution of stomatal development. Plant Physiol. 2017; 174: 624-638https://doi.org/10.1104/pp.17.00183 Crossref PubMed Scopus (104) Google Scholar ,3 Harris B.J. Harrison C.J. Hetherington A.M. Williams T.A. Phylogenomic evidence for the monophyly of bryophytes and the reductive evolution of stomata. Curr. Biol. 2020; 30: 2001-2012.e2https://doi.org/10.1016/j.cub.2020.03.048 Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar In Arabidopsis thaliana, a core regulatory module for stomatal development comprises members of the bHLH transcription factor (TF) family, including AtSPCH, AtMUTE, and AtFAMA of subfamily Ia and AtSCRM1/2 of subfamily IIIb. Specifically, AtSPCH, AtMUTE, and AtFAMA each successively form heterodimers with AtSCRM1/2, which in turn regulate the entry, division, and differentiation of stomatal lineages. 4 Pillitteri L.J. Sloan D.B. Bogenschutz N.L. Torii K.U. Termination of asymmetric cell division and differentiation of stomata. Nature. 2007; 445: 501-505https://doi.org/10.1038/nature05467 Crossref PubMed Scopus (368) Google Scholar ,5 Ohashi-Ito K. Bergmann D.C. Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell. 2006; 18: 2493-2505https://doi.org/10.1105/tpc.106.046136 Crossref PubMed Scopus (343) Google Scholar ,6 MacAlister C.A. Ohashi-Ito K. Bergmann D.C. Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature. 2007; 445: 537-540https://doi.org/10.1038/nature05491 Crossref PubMed Scopus (401) Google Scholar ,7 Kanaoka M.M. Pillitteri L.J. Fujii H. Yoshida Y. Bogenschutz N.L. Takabayashi J. Zhu J.K. Torii K.U. SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation. Plant Cell. 2008; 20: 1775-1785https://doi.org/10.1105/tpc.108.060848 Crossref PubMed Scopus (380) Google Scholar In the moss Physcomitrium patens, two SMF (SPCH, MUTE and FAMA) orthologs have been characterized, one of which is functionally conserved in regulating stomatal development. 8 Chater C.C. Caine R.S. Tomek M. Wallace S. Kamisugi Y. Cuming A.C. Lang D. MacAlister C.A. Casson S. Bergmann D.C. et al. Origin and function of stomata in the moss Physcomitrella patens. Nat. Plants. 2016; 2: 16179https://doi.org/10.1038/nplants.2016.179 Crossref PubMed Scopus (95) Google Scholar ,9 MacAlister C.A. Bergmann D.C. Sequence and function of basic helix-loop-helix proteins required for stomatal development in Arabidopsis are deeply conserved in land plants. Evol. Dev. 2011; 13: 182-192https://doi.org/10.1111/j.1525-142X.2011.00468.x Crossref PubMed Scopus (73) Google Scholar We here provide experimental evidence that orthologous bHLH TFs in the liverwort Marchantia polymorpha affect air pore spacing as well as the development of the epidermis and gametangiophores. We found that the bHLH Ia and IIIb heterodimeric module is highly conserved in plants. Genetic complementation experiments showed that liverwort SCRM and SMF genes weakly restored a stomata phenotype in atscrm1, atmute, and atfama mutant backgrounds in A. thaliana. In addition, homologs of stomatal development regulators FLP and MYB88 also exist in liverworts and weakly rescued the stomatal phenotype of atflp/myb88 double mutant. These results provide evidence not only for a common origin of all stomata in extant plants but also for relatively simple stomata in the ancestral plant.