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Mobile MUTE specifies subsidiary cells to build physiologically improved grass stomata

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Science  17 Mar 2017:
Vol. 355, Issue 6330, pp. 1215-1218
DOI: 10.1126/science.aal3254
  • Fig. 1 BdMUTE is required for subsidiary cell formation.

    (A) Stomatal development in Brachypodium; in a stomatal file (1) the smaller cell of an asymmetric division (2) becomes a GMC (purple) and laterally induces SMC fate (yellow) (3). SMCs divide asymmetrically (4) before GMCs divide symmetrically (5) and the complex matures (6). (B to D) Differential interference contrast (DIC) images of the epidermis in WT (Bd21-3) (B), sid (C), and sid complemented with BdMUTEp:BdMUTE (D) [first leaf, 7 days after germination (dag)]. H, hair cells. Color key is at bottom right. (E) Stomatal density of four-celled (red), two-celled (green), aborted (blue), and three-celled (purple) complexes in WT (Bd21-3), sid, and complemented sid lines (n = 6 individuals per genotype, first leaf, 7 dag). (F) Gene model of BdMUTE with position and nature of sid/bdmute-1 and CRISPR-Cas9–induced (bdmute-2, bdmute-3, and bdmute-4) mutations indicated. Scale bars, 10 μm.

  • Fig. 2 BdMUTE is expressed during subsidiary cell recruitment and overexpression induces subsidiary cell–like divisions.

    (A) BdMUTEp:YFP-BdMUTE expression in sid during stomatal development. YFP-BdMUTE starts to be expressed in young GMCs and shows strong signal in mature GMCs and weak signal in SMCs. Expression is maintained until after GMC division in both GCs and SCs and disappears during complex maturation. All images from second leaf, 6 to 7 dag, T1 generation. (B) Epidermal development in Ubip:YFP-BdMUTE shows ectopic SMC-like divisions before, during, and after GMC division (arrows), resulting in stomata with multiple rows of SCs (see box in third panel) and SCs around hair cells (rightmost panel; H, hair cells). All images from about the sixth leaf, T0. Cell walls stained with propidium iodide (PI, purple). Scale bars, 10 μm.

  • Fig. 3 BdMUTE is a mobile transcription factor.

    (A) BdMUTEp:YFP-BdMUTE signal is detected in SMCs and SCs (arrows) in addition to GMCs before and after SC recruitment. (B) BdMUTEp:3xYFPnls signal is detected only in GMCs and GCs before and after SC recruitment. (C) BdMUTEp:BdSPCH1-YFP is detected only in GMCs and GCs before and after SC recruitment. (D) BdSPCH2p:YFP-BdMUTE is detected in SMCs and SCs (arrows) in addition to GMCs before and after SC recruitment. (E) Aniline-blue (AB) staining of callose indicates that secondary plasmodesmata connect nonsister GMCs and SMCs. (F) BdMUTEp:AtMUTE-YFP does not rescue SCs or GMC divisions in sid (arrowhead) nor does it move. (G) AtMUTEp:AtMUTE-YFP is observed exclusively in Arabidopsis GMCs. (H) AtMUTEp:YFP-BdMUTE is observed in GMCs and neighboring cells (asterisk) in Arabidopsis. Brachypodium images from second or third leaves of T1 individuals, 6 to 7 dag or 11 to 12 dag, respectively, except in (F) (about the fifth leaf, T0). Arabidopsis images from T2 cotyledons, 3.5 dag. Cell walls stained with PI (purple). Scale bars, 10 μm.

  • Fig. 4 Subsidiary cells enhance stomatal physiology.

    (A) WT and SC-less sid stomata close and open upon incubation with 50 μM ABA and 4 μM fusicoccin, respectively. Cell walls stained with PI. (B) Stomatal aperture (μm2) is significantly reduced in SC-less complexes (sid) upon fusicoccin treatment (n = 84 for WT; n = 123 for sid). (C) Maximum stomatal conductance (gsmax) is significantly reduced in sid (n = 8 per genotype, corrected for reduced density of functional stomata in sid). (D) Fresh weight of 5-week-old sid plants is reduced compared with WT (n = 10 per genotype). (E) Stomatal conductance (gs) in response to changing light conditions [acclimation at 1200 units of photosynthetically active radiation (PAR), dropped to 300 PAR, increased to 1200 PAR, dropped to 0 PAR]. The magnitude and speed of response was significantly greater in WT (left side, black dots) compared with sid individuals (right side, red dots; corrected for reduced density of functional stomata). Vertical dotted lines indicate light intensity changes. Each point equals the mean value of five independent experiments; error bars represent SEM.

Supplementary Materials

  • Mobile MUTE specifies subsidiary cells to build physiologically improved grass stomata

    Michael T. Raissig, Juliana L.Matos, M. Ximena Anleu Gil, Ari Kornfeld, Akhila Bettadapur, Emily Abrash, Hannah R. Allison, Grayson Badgley, John P. Vogel, Joseph A. Berry, Dominique C. Bergmann

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S7
    • Table S1
    • References