ReportsBOTANY

Mobile MUTE specifies subsidiary cells to build physiologically improved grass stomata

See allHide authors and affiliations

Science  17 Mar 2017:
Vol. 355, Issue 6330, pp. 1215-1218
DOI: 10.1126/science.aal3254

Making more of your stomata

Stomata on grasses are made up of two guard cells and two subsidiary cells, and they perform better than stomata on broad-leaved plants, which are made up only of two guard cells. Raissig et al. found that the MUTE transcription factor in the wheat-like grass Brachypodium is a little bigger than the equivalent protein in the model broad-leaved plant Arabidopsis. The extension in the grass protein promotes its movement into adjacent cells, prompting them to become subsidiary cells. Mutant Brachypodium whose MUTE protein could not move between cells lacked stomatal subsidiary cells and grew poorly.

Science, this issue p. 1215

Abstract

Plants optimize carbon assimilation while limiting water loss by adjusting stomatal aperture. In grasses, a developmental innovation—the addition of subsidiary cells (SCs) flanking two dumbbell-shaped guard cells (GCs)—is linked to improved stomatal physiology. Here, we identify a transcription factor necessary and sufficient for SC formation in the wheat relative Brachypodium distachyon. Unexpectedly, the transcription factor is an ortholog of the stomatal regulator AtMUTE, which defines GC precursor fate in Arabidopsis. The novel role of BdMUTE in specifying lateral SCs appears linked to its acquisition of cell-to-cell mobility in Brachypodium. Physiological analyses on SC-less plants experimentally support classic hypotheses that SCs permit greater stomatal responsiveness and larger range of pore apertures. Manipulation of SC formation and function in crops, therefore, may be an effective approach to enhance plant performance.

When plants colonized land and formed a cuticle to prevent desiccation, the evolution of adjustable pores—stomata—in the epidermis allowed plants to balance carbon dioxide uptake with water loss. Stomata in today’s dicots resemble those of ~400 million years ago, consisting of two kidney-shaped guard cells (GCs) (1, 2). Grasses, however, have four-celled stomatal complexes with dumb-bell shaped GCs flanked by two paracytic (lineally unrelated) subsidiary cells (SCs) (Fig. 1, A and B) (14). This cellular organization may enable fast adjustments of pore aperture at a low energetic cost, while allowing higher gas-exchange capacity (1, 2, 5), and may have contributed to the successful diversification and dispersal of the grass family during global aridification 30 to 45 million years ago (1, 2, 6).

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.

Stomatal formation is limited to certain cell files in the grass leaf epidermis (Fig. 1A, stage 1) (3, 7). In Brachypodium distachyon, a stomatal initiation module—consisting of three basic helix-loop-helix (bHLH) transcription factors, BdICE1, BdSPEECHLESS1 (BdSPCH1), and BdSPCH2—guides the smaller daughters of asymmetric divisions toward guard mother cell (GMC) identity (Fig. 1A, stage 2) (7). The GMC then induces its lateral neighbor cells to acquire subsidiary mother cell (SMC) identity (Fig. 1A, stage 3) and to divide asymmetrically to form the SCs (Fig. 1A, stage 4). Finally, the GMC divides symmetrically (stage 5) and the complex matures (Fig. 1A, stage 6). Several aspects of stomatal development are conserved: BdICE1 and BdSPCH1/2 are orthologs of stomatal initiation factors in Arabidopsis (8, 9), and formation of mature GCs in rice requires the stomatal maturation bHLH OsFAMA (10). Recruitment of lateral SCs, however, is unique to grasses, and despite use of these cells to study polarity establishment (11, 12), factors defining SMC and SC identity are unknown. Here, we identify a locus responsible for the formation of SCs in Brachypodium and confirm that this morphological innovation improves physiological performance.

A forward genetic screen in Brachypodium targeting genes responsible for the unique morphology of grass stomata yielded the subsidiary cell identity defective (sid) mutant, which fails to recruit SCs and instead produces dicot-like two-celled stomata (Fig. 1, C and E). sid plants also exhibit some misoriented GMC divisions and aborted GCs (31.1% ± 7.0%; n = 599) (Fig. 1, C and E) but are viable and fertile. Bulk segregant mapping combined with whole-genome sequencing of sid uncovered a 5–base pair (bp) deletion in BdMUTE, an ortholog of Arabidopsis MUTE, which encodes a bHLH transcription factor associated with GMC identity (Fig. 1F and fig. S1) (13). Three independent clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) mutations within the BdMUTE gene recapitulated the sid phenotype (Fig. 1F and fig. S2).

We complemented sid with BdMUTEp:BdMUTE (Fig. 1, D and E) and a fusion of BdMUTE and yellow fluorescent protein (BdMUTEp:YFP-BdMUTE) (Fig. 1E) and confirmed that BdMUTE is required for SC formation and proper GMC division. BdMUTEp:YFP-BdMUTE is detected in GC and SC precursors, as well as in young GCs and SCs (Fig. 2A). Overexpressing BdMUTE induced polarized SMC-like divisions in epidermal pavement cells, resulting in multiple SC layers around stomata and SCs around hair cells (Fig. 2B), which suggested that BdMUTE is sufficient to specify SMC identity throughout the leaf.

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.

It was mysterious how BdMUTE acquired the ability to specify SMC identity, whereas its Arabidopsis ortholog specifies GMCs. Hints to the mechanism came from detecting YFP-BdMUTE signal not only in GMCs and early GCs but also in the neighboring SMCs and early SCs (Figs. 2A and 3A). All other described Brachypodium stomatal bHLH reporters are expressed only within stomatal files (7). Either the BdMUTE promoter is active in SMCs or the BdMUTE peptide moves from GMCs to neighboring cell files. In support of the latter scenario, YFP expression from a transcriptional reporter (BdMUTEp:3xYFPnls) was restricted to GMCs and GCs (Fig. 3B). Similarly, protein encoded by BdSPCH1-YFP (7) expressed with the BdMUTE promoter was restricted to GMCs (Fig. 3C), whereas YFP-BdMUTE expressed by the BdSPCH2 promoter [active only in the stomatal lineage (7)] appeared in both GMCs and SMCs (Fig. 3D); this indicated that BdMUTE protein is mobile. Punctate patterns of callose staining—a marker for plasmodesmata (14)—around GMCs (Fig. 3E) suggest that secondary plasmodesmata connecting the nonsister GMCs and SMCs are an available route of transport. As BdMUTE is small (237 amino acids, ~25.5 kDa), either those symplastic connections allow for its passive transport or, alternatively, domains within BdMUTE define its mobility. We took advantage of evidence that the even smaller AtMUTE (202 amino acids, ~22.9 kDa) is nonmobile in Arabidopsis (13) and cloned interspecific reporter hybrids to test whether movement capacity was an intrinsic feature of BdMUTE or whether it reflected differences between Brachypodium and Arabidopsis cells. BdMUTEp:AtMUTE-YFP fluorescence was restricted to Brachypodium GMCs and GCs, and although this version of AtMUTE-YFP is functional in Arabidopsis, it failed to rescue the sid SC and GMC defects (Fig. 3F). In the reciprocal experiment, YFP-BdMUTE expressed in Arabidopsis with the GMC-specific AtMUTE promoter (13) was visible in the neighbor cells, unlike GMC-only expression of AtMUTE-YFP (Fig. 3, G and H, and fig. S3), and BdMUTE-YFP signal in neighbors is not just a consequence of differential protein stability (fig. S3E). Together, our data suggest that BdMUTE is indeed mobile and that mobility is a protein-intrinsic feature of BdMUTE (fig. S4).

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.

The recruitment of lateral SCs in grass stomata is hypothesized to permit enhanced gas exchange capacity (gsmax) and stomatal responsiveness (dgs/dt) relative to dicots (5). SCs might serve as ion source and sink, facilitating rapid opening and closing, and could mechanically accommodate and restrict GC movement or morphology (1, 2, 5). Before the identification of SC-less sid, however, it was not possible to test the contribution of SCs to stomatal physiology directly. sid GCs can be forced open by the toxin fusicoccin (15) or forced closed by the hormone abscisic acid (ABA) (16), but maximal pore area is only half that of GCs from intact complexes (Fig. 4, A and B). Similarly, gsmax of sid stomata—when measured under conditions favoring maximum stomatal opening [low [CO2], high humidity, and saturating light intensity (fig. S5)] and corrected for lower density of functional stomata in sid (fig. S6)—is also only half of wild type (WT) (sid, 0.20 ± 0.01 mol H2O m–2 s–1; WT, 0.38 ± 0.03 mol H2O m–2 s–1) (Fig. 4C). Consequently, 5-week-old sid plants produced less biomass (Fig. 4D). Lifetime stomatal diffusion capacity can be assessed using the tissue–carbon isotope ratio (δ13C), because diffusion-limited leaves will incorporate a larger proportion of heavy isotopes (17). δ13C in sid mutants is higher than in WT (–28.57‰ ± 0.59‰ and –32.99‰ ± 0.47‰, respectively; P = 1.67–6) confirming that these mutants are diffusion limited (fig. S7).

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.

Finally, we assessed stomatal responsiveness to changing light conditions in WT and sid by acclimating the plants at near-saturating light, then simulating shade, increasing light again, and finally mimicking sudden nightfall (Fig. 4E). WT stomata quickly responded to changing light conditions, whereas both reaction times and amplitudes of the response were dampened in sid (Fig. 4E). In summary, sid stomata lacking SCs cannot open as wide (gsmax) (Fig. 4C), do not close as tightly in darkness (Fig. 4E), and are slow to respond to changing light (Fig. 4E).

The developmental innovation of SC-containing stomatal complexes is accompanied by changes in functional aspects and mobility of one of the core stomatal identity bHLH transcription factors. Whereas AtMUTE halts the asymmetric stem cell–like divisions and specifies GMC identity in Arabidopsis (13), BdMUTE is mobile, promotes divisions, and defines SMC identity in Brachypodium. Mobile transcription factors in plants maintain stem cell niches, pattern root hairs and trichomes, define the root endodermis, and even regulate flowering (18). Transcription factor transport, however, seems to be mostly associated with developmental plasticity, where different degrees of movement of orthologs fine-tune organ size (1921). In the stomatal context, we find species-specific mobility for BdMUTE associated with a developmental innovation. BdMUTE mobility might be an elegant mechanism to coordinate the development of GCs and SCs to enable formation and function of intimately connected four-celled complexes. Analysis of leaf-level gas exchange, stable isotopes, and biomass indicate that the presence of SCs correlates with improved stomatal responsiveness and greater aperture range, which enhance plant performance particularly when water is limited and/or temperatures are high. Therefore, engineering SC properties might allow for tuning of stomatal responsiveness and, thus, boost carbon assimilation and water use efficiency in crops.

Supplementary Materials

www.sciencemag.org/content/355/6330/1215/suppl/DC1

Materials and Methods

Figs. S1 to S7

Table S1

References (2232)

References and Notes

Acknowledgments: This work is supported by the Swiss National Science Foundation (P2ZHP3_151598 to M.T.R.) and through a grant from The Gordon and Betty Moore Foundation (GMBF2550.05) to the Life Science Research Foundation (to M.T.R.). The work conducted by the U.S. Department of Energy (DOE) Joint Genome Institute is supported by the Office of Science of the DOE under contract no. DE-AC02-05CH1123. E.A. was a NSF graduate research fellow and D.C.B. is a GBMF Investigator of the HHMI. Supplement contains additional data.
View Abstract

Subjects

Navigate This Article