Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity
Monday, 2016/07/11 | 07:38:23
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Michael T. Raissig, Emily Abrash, Akhila Bettadapur, John P. Vogel, and Dominique C. Bergmann SignificancePlants both control and are controlled by the global climate. Grasses in natural and agricultural systems participate in the exchange of atmospheric CO2 for biosphere-derived oxygen and water vapor via microscopic epidermal valves (stomata), but how these stomata are made in grasses is unknown. Using genetic screens and targeted genome editing, we identify and characterize master transcriptional regulators of stomatal initiation in the wheat relative Brachypodium. Surprisingly, the unique stomatal form and pattern of grasses is regulated by orthologs of Arabidopsis stomatal basic helix–loop–helix (bHLH) transcription factors, although the function of individual genes and regulation of their protein products have diverged. This finding suggests that the stomatal core bHLH transcription factors are excellent breeding targets to enhance performance in grasses. Abstract Stomata, epidermal valves facilitating plant–atmosphere gas exchange, represent a powerful model for understanding cell fate and pattern in plants. Core basic helix–loop–helix (bHLH) transcription factors regulating stomatal development were identified in Arabidopsis, but this dicot’s developmental pattern and stomatal morphology represent only one of many possibilities in nature. Here, using unbiased forward genetic screens, followed by analysis of reporters and engineered mutants, we show that stomatal initiation in the grass Brachypodium distachyon uses orthologs of stomatal regulators known from Arabidopsis but that the function and behavior of individual genes, the relationships among genes, and the regulation of their protein products have diverged. Our results highlight ways in which a kernel of conserved genes may be alternatively wired to produce diversity in patterning and morphology and suggest that the stomatal transcription factor module is a prime target for breeding or genome modification to improve plant productivity. See: http://www.pnas.org/content/early/2016/07/01/1606728113.full PNAS July 2016; Michael T. Raissig, doi: 10.1073/pnas.1606728113
Fig. 1. Stomatal development in Brachypodium requires BdICE1. (A) Stomatal production in eudicot leaves where stomatal stem cell populations are dispersed throughout the epidermis and stomata are randomly oriented (Left) and in grass leaves that exhibit a longitudinal gradient of development with divisions restricted to the leaf base and differentiation toward tip (Right). Grass stomata are restricted to specific files and all have the same orientation. Boxes highlight typical eudicot stomata consisting of two GCs (green) surrounding a pore (Left Box) and the typical four-celled grass stomata of two GCs (green) flanked by two SCs (yellow) (Right Box). (B–F) Diagrams and confocal images of Brachypodium stomatal development; these and all subsequent images represent the five main developmental stages, with the youngest at the left and cells toward the base of the leaf at the bottom. Stomatal cell files are established early and proliferate to make smaller cells (light purple) (B) and divide asymmetrically to produce GMCs (dark purple) (C). GMCs mature (blue) and recruit SCs (yellow) (D). GMCs divide symmetrically once (E) and differentiate to GCs (green) (F). Cell walls are stained with PI in confocal images. (G) DIC image of cleared WT (Bd21-3) epidermis. (H) stl epidermis. (I) Epidermis of stl complemented with Ubipro:YFP-BdICE1. Black arrowheads indicate stomata. All DIC images display abaxial first leaves, 6 dpg. (J) Quantification of stomatal density per field of view in WT (Bd21-3), stl, and stl; Ubipro:YFP-BdICE1 (lines #1 and #2). We analyzed the abaxial first leaf of 6-dpg T1 plants (progeny of initial transformed regenerants). Six individuals were analyzed per group, except for YFP− line #2, which had only one T1 and was excluded from statistical analysis. In the boxplots, the black horizontal line indicates the median; upper and lower edges of the box are the upper and lower quartiles; whiskers extend to the largest observation within 1.5 interquartile ranges of the box; circles indicate outliers. (Scale bars: 50 µm.) *P < 0.05, **P < 0.01. |
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