The science of the stomata of plants: a continuously growing list of references, abstracts and illustrations, helping researchers to data on publications.
The moss Physcomitrella patens genome encodes orthologues of the basic helix loop helix (bHLH) transcription factors regulating stomatal development in flowering plants (a) Developing P. patens sporophyte, arrow indicating region of stomatal placement, and (b) excised sporophyte with stomata (orange/brown pores) forming a ring around the base. (c) Close-up of the sporophyte epidermis with single celled guard cells and central pores. (d and e) Bootstrapped Maximum Likelihood phylogenies of the SMF gene family comprising the FAMA, SPCH and MUTE subfamilies and the SCRM/ICE gene family in sequenced land plants. Internal node names in bold red indicate inferred subfamily ancestry. Internal nodes are coloured to indicate either duplication (red), speciation (green) or haplotype (blue) origin of the descendant nodes. Edge values represent bootstrap values. External node names comprise species abbreviations, original accession numbers of the protein sequences and accepted gene names of experimentally studied representatives in bold red. Species abbreviations in five-letter-code: Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, Sorghum bicolor, Selaginella moellendorffii and Physcomitrella patens. (f, g and h) Relative expression of PpSMF1, PpSMF2 and PpSCRM1 in the developing sporophyte (grey bars) and protonema tissue (black bars) analysed by qRT-PCR. Error bars indicate standard error of the mean. Three replicates per tissue type were used. The scale bar in a = 100μm, in b = 100μm, in c = 25μm.
Origin and function of stomata in the moss Physcomitrella patens
by 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., Decker E., Frank W., Gray J. E., Fleming A., Reski R., Beerling D. J. (2016)
PpSMF1 and PpSCRM1 are required for stomatal development in the moss Physcomitrella patens (a) Stacked UV fluorescence images (upper panel), scanning electron microscope images (middle panel) and bright field images (bottom panel) showing the spore capsule base and epidermal close-ups from P. patens wild-type, ΔPpSMF1, ΔPpSMF2 and ΔPpSCRM1 knock-out mutants, respectively. The top panel wild-type representative is from Villersexel K3 ecotype of P. patens, the middle panel wild-type representative is from the Gransden D12 ecotype and the bottom panel wild-type relates to the Gransden 2004 ecotype. There were no discernible differences between the sporophytes of the different background lines. For both of the ΔPpSCRM1 lines generated we observed one such instance of aborted stomata (see bottom right panel) in the 7 capsules of each line surveyed. (b) Number of stomata formed per sporophyte in two independent lines of each genotype versus wild-type controls. Error bars indicate one standard error of the mean. For ΔPpSMF1 and ΔPpSCRM1 and the corresponding wild-types, n = 7 capsules of each line were analysed. For ΔPpSMF2 and wild-type background, 5 capsules were surveyed. A One-way ANOVA was performed to test for differences between the wild-type and ΔPpSMF2 lines and no significant differences (denoted ns) were found. (c) RT-PCR to confirm loss of the respective transcript in each of the P. patens knock-out lines (top panel). A Rubisco (RBCS) control was run to verify the integrity of the produced cDNA (Bottom panel). For labelling purposes the wild-types Villersexel K3, Gransden D12 and Gransden 2004 are denoted Vx, GrD12 and Gr04. For PpSMF2 two bands were amplified in the control for which the smaller 239bp product represents the size expected for PpSMF2. Scale bars in a = 50 μm in the top and middle panels, in the bottom panel = 15 μm.
Abstract
Stomata are microscopic valves on plant surfaces that originated over 400 million years (Myr) ago and facilitated the greening of Earth’s continents by permitting efficient shoot-atmosphere gas exchange and plant hydration1.
However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood2-4 and their function has remained a matter of debate for a century5.
Here, we show that genes encoding the two basic helix-loop-helix proteins PpSMF1 (SPEECH, MUTE and FAMA-like) and PpSCREAM1 (SCRM1) in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss.
Targeted P. patens knockout mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lack stomata.
Protein-protein interaction assays reveal heterodimerization between PpSMF1 and PpSCRM1, which, together with moss-angiosperm gene complementations6, suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit is required to activate transcription for moss stomatal development, as in A. thaliana7.
Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land-plant sporophytes.
Phylogeny and expression profiles of stomatal patterning genes in Physcomitrella patens. (A,C,E) Phylogenetic trees constructed using amino acid sequences of selected Arabidopsis EPF1 (A), TMM (C) and ERECTA (E) gene family members based on Phytozome V11 (Goodstein et al., 2012), using the neighbour-joining method (Saitou and Nei, 1987; Takata et al., 2013) on MEGA6 (Tamura et al., 2013). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). Amino acid sequences from P. patens (Pp), Selaginella moellendorffii (Sm), Zea mays (Zm), Symphytum tuberosum (St), Medicago truncatula (Mt) and A. thaliana (At) were used to generate trees, except for ERECTA, for which S. moellendorffii and S. tuberosum gene family members were omitted, owing to the large overall number of genes in the ERECTA family. For complete analyses of all three gene families, see Fig. S1. (B,D,F) Expression profiles of PpEPF1 (B), PpTMM (D) and PpERECTA1 (F) based on microarray data taken from the P. patens eFP browser (Ortiz-Ramírez et al., 2016; Winter et al., 2007) for spore, protoplast, protonemal, gametophyte and sporophyte tissue. Red indicates a relatively high transcript level, with the arrows highlighting phases of sporophyte development when the respective genes appear to be relatively highly expressed. For the expression profiles of other PpERECTA gene family members, see Fig. S2.
Abstract
The patterning of stomata plays a vital role in plant development and has emerged as a paradigm for the role of peptide signals in the spatial control of cellular differentiation.
Research in Arabidopsis has identified a series of Epidermal Patterning Factors (EPFs) which interact with an array of membrane-localised receptors and associated proteins (encoded by ERECTA and TMMgenes) to control stomatal density and distribution.
However, although it is well established that stomata arose very early in the evolution of land plants, until now it has been unclear whether the established angiosperm stomatal patterning system represented by the EPF/TMM/ERECTA module reflects a conserved, universal mechanism in the plant kingdom.
Here, we use molecular genetics to show that the moss Physcomitrella patens has conserved homologues of angiosperm EPF, TMM and at least one ERECTA gene which function together to permit the correct patterning of stomata and that, moreover, elements of the module retain function when transferred to Arabidopsis.
Our data characterise the stomatal patterning system in an evolutionary distinct branch of plants and support the hypothesis that the EPF/TMM/ERECTA module represents an ancient patterning system.
PLANT STOMATA ENCYCLOPEDIA 
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