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. 2015 Jul 23;11(7):e1005374. doi: 10.1371/journal.pgen.1005374

Fig 1. Molecular framework of the SPCH•SCRM positive feedback for stomatal-lineage specification.

Fig 1

(A) SPCH and SCRM are mutually required for initiating the entry asymmetric division of stomatal cell lineages. Shown are false-colored confocal microscopy images of abaxial rosette leaf epidermis from 10–12 day-old seedlings. Wild type (left) epidermis gives rise to stomatal lineage cells: Cyan, early meristemoids; light green, late meristemoids and guard mother cells; green, immature and mature guard cells; white, stomatal-lineage ground cells or pavement cells. spch or scrm scrm2 mutant epidermis is solely composed of pavement cells (white). Scale bars, 20 μm. (B) Expression heat map of SPCH, SCRM, and SCMR2 from a microarray study [13] in wild type and mutants enriched in specific epidermal cells: scrm-D mute (M: meristemoids); scrm-D (GC: stomatal guard cells); spch (PC: pavement cells). (C) Promoter GFP reporter expression patterns of SPCHpro::nucGFP (top), SCRMpro::nucGFP (middle), and SCRM2pro::nucGFP (bottom) in early protoderm of 11-day-old wild-type (left), spch (middle), and scrm scrm2 (right) seedlings. SPCH does not require itself or SCRMs for its own promoter activity. In contrast, SCRMs require SPCH and themselves, indicating that SCRMs form a positive feedback loop essential for pattern formation. Scale bar, 20 μm. (D, E) ChIP assays on SCRM (D) and SCRM2 (E) promoter regions using anti-GFP antibody on control wild type or transgenic seedlings expressing functional SPCH-GFP in scrm-D, GFP-SCRM, GFP-scrm-D, or GFP-SCRM2. Each amplicon is indicated in a red letter. Shown as a graph are mean ± SEM of fold enrichment over wild-type Col from three biological replicates. Line, intergenic region or intron; arrow, transcription start site; filled rectangle, coding region. (F) Transactivation assays in N. benthamiana. Reporter luciferase expression driven by SCRM promoter is strongly induced when both SPCH and SCRM are present. Reporter firefly luciferase activity was normalized against constitutively expressed Renilla luciferase, and the values are normalized against controls without effector proteins. Bars indicate means of three biological replicates; error bars, S.E.M.