Arabidopsis Stomatal Initiation Is Controlled by MAPK-Mediated Regulation of the bHLH SPEECHLESS

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Science  14 Nov 2008:
Vol. 322, Issue 5904, pp. 1113-1116
DOI: 10.1126/science.1162263


Stomata, epidermal structures that modulate gas exchange between plants and the atmosphere, play critical roles in primary productivity and the global climate. Positively acting transcription factors and negatively acting mitogen-activated protein kinase (MAPK) signaling control stomatal development in Arabidopsis; however, it is not known how the opposing activities of these regulators are integrated. We found that a unique domain in a basic helix-loop-helix (bHLH) stomatal initiating factor, SPEECHLESS, renders it a MAPK phosphorylation target in vitro and modulates its function in vivo. MAPK cascades modulate a diverse set of activities including development, cell proliferation, and response to external stresses. The coupling of MAPK signaling to SPEECHLESS activity provides cell type specificity for MAPK output while allowing the integration of multiple developmental and environmental signals into the production and spacing of stomata.

In Arabidopsis, stomatal fate and pattern are regulated by three closely related basic helix-loop-helix (bHLH) transcription factors—SPEECHLESS (SPCH), MUTE, and FAMA—that, in partnership with the more distantly related bHLHs ICE1/SCREAM and SCREAM2, control initiation of asymmetric divisions, proliferation of transient precursor cells, and differentiation of stomatal guard cells, respectively (fig. S1) (14). Acting in opposition to these stomatal promoting factors are signaling systems that limit stomatal density and establish pattern. These negative regulators include the ERECTA (ER) family of leucine-rich repeat (LRR) receptor–like kinases (5, 6) and the LRR receptor–like protein TOO MANY MOUTHS (TMM) (7) that both may work in concert with the putative ligand EPF1 (8). A subtilisin protease, SDD1, also negatively regulates stomatal production but may act independently of this receptor-ligand module (8, 9).

Genetic evidence places a mitogen-activated protein kinase (MAPK) signaling cascade downstream of the receptors in stomatal development (10, 11). In all eukaryotes, MAPK cascades control a diverse array of activities, including the regulation of cell division and differentiation and the coordination of responses to environmental inputs (12, 13). The MAPK components implicated in stomatal development (YODA, MKK4/5, and MPK3/6) are broadly expressed (11, 14) and are involved in multiple activities. For example, YODA is required for asymmetric cell divisions in the embryo (15), and MKK4/5 and MPK3/6 were initially characterized by their roles in stress and pathogen responses (16, 17). Because of these multiple roles, a major challenge in MAPK signaling is to understand how common signaling elements evoke specific responses. Spatially or temporally restricted expression of MAPK substrates could provide this specificity. Developmental MAPK substrates have not been previously described in plants; however, the cell type–specific expression and activities of SPCH, MUTE, and FAMA make these proteins attractive candidates for such specificity factors. Here we show that SPCH is a substrate of MPK3 and MPK6 in vitro, that specific phosphorylation sites on SPCH regulate its activity in vivo, and that known components of the stomatal development signaling network modulate SPCH behavior.

The SPCH loss-of-function phenotype is strikingly similar to that caused by constitutive activation of the MAPK pathway components YODA and MKK4/5 (10, 11), and spch is epistatic to yoda (2); these results are consistent with SPCH being a downstream target of MAPKs in the epidermis. To test whether SPCH is regulated by MAPK activity, we examined the expression of transcriptional (SPCHpro::GFP) and translational (SPCHpro::SPCH-GFP) reporters (GFP, green fluorescent protein) in plants expressing constitutively active (CA) YODA (YODApro::CA-YODA) (11). At 5 days post-germination (dpg), both wild-type plants (20 of 20) and YODApro::CA-YODA plants (19 of 20) expressed SPCHpro::GFP (fig. S2, A and B). Wild-type plants also expressed SPCHpro::SPCH-GFP (20 of 20), but YODApro::CA-YODA plants did not (0 of 20; fig. S2, C and D); this result suggests that YODA does not prevent transcription of SPCH, but rather limits the production or abundance of SPCH protein.

Phosphorylation of transcription factors can modulate their levels and activities (18, 19). We tested whether SPCH was an in vitro substrate for phosphorylation by the MAPKs previously implicated in stomatal development (MPK3 and MPK6) (10). SPCH, but not its paralogs FAMA and MUTE, could be phosphorylated by both MPK3 and MPK6 (Fig. 1B and fig. S3A). Alignment of SPCH, MUTE, and FAMA proteins reveals high sequence conservation in their bHLH domains and C termini (Fig. 1A) (2). However, SPCH also has a unique 93–amino acid domain [herein referred to as the MAPK target domain (MPKTD)] that contains 10 consensus MAPK phosphorylation target sites. Five of these sites contain a Pro-X-Ser/Thr-Pro (P-X-S/T-P) motif, marking them as high-stringency sites (20) (Fig. 1A). Because SPCH differs from the nonsubstrate MUTE and FAMA proteins primarily in the MPKTD, we performed phosphorylation assays with a version of SPCH lacking the MPKTD and with the MPKTD alone. All MPK3 and MPK6 in vitro phosphorylation target sites appear to be contained within the SPCH MPKTD (Fig. 1B and fig. S3).

Fig. 1.

SPCH is a stomatal regulator that contains a unique MAPK phosphorylation target domain. (A) Protein alignment of SPCH, MUTE, and FAMA. Highest conservation is in the bHLH domain (black) and C terminus (dark gray). White lines within these domains indicate nonidentical residues. The sequence of the MPKTD is shown with beginning and ending amino acid residue positions. Deletions are denoted as a solid underline for Δ93, a long-dashed underline for Δ49, and a short-dashed underline for Δ31. Italics denote the predicted PEST sequence. MAPK target sites are in bold. High-stringency sites are denoted 1 (Ser193), 2 (Ser211), 3 (Thr214), 4 (Ser219), and 5 (Ser255). Abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; G, Gly; H, His; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; Y, Tyr. (B) In vitro activity of recombinant MPK3 (top) and MPK6 (bottom) on bacterially expressed SPCH variants. Lane labels indicate specific SPCH variant tested. Arrows correspond to phosphorylated SPCH and autophosphorylation of MPKs.

We then tested the functional importance of the SPCH MPKTD by using both the strong, broadly expressed 35S promoter and the endogenous SPCH promoter to express full-length SPCH and SPCHΔMPKTD variants in plants. Expression of 35S::SPCH was previously reported to induce divisions in pavement cells (2) and to produce extra stomatal lineage cells (3). 35S::SPCH expression in wild-type plants resulted in additional divisions in pavement cells and a modest increase in total epidermal cell number (Fig. 2B), whereas expression of SPCHpro::SPCH in the wild type resulted in no significant phenotypic effects (Fig. 3). In contrast to these results with full-length SPCH, SPCHΔMPKTD variants markedly affected epidermal development. When the entire domain was deleted (35S::SPCHΔ93 or SPCHpro::SPCHΔ93), the epidermis of transformed plants exhibited large clusters of stomata (Fig. 2C and fig. S4), a phenotype similar to that produced by 35S::MUTE (2, 3). The SPCHΔ93 results are unsurprising given the strong similarity of SPCH and MUTE—particularly when the MPKTD is removed (Fig. 1A)—and in light of previous reports that overexpression of FAMA deletion variants mimics 35S::MUTE (1).

Fig. 2.

Confocal images of phenotypes of SPCH variants expressed in plants. (A to D and G to J) Confocal images of 7-dpg abaxial cotyledons. Specific genotypes are noted in upper right corner of images. (E and F) Expression of TMMpro::TMM-GFP (green) in 35S::SPCHΔ49 (F) compared to wild-type (WT) control (E). (K and L) Comparison of expression pattern for full-length SPCH (K) and SPCH variant with four S/T → A substitutions (L) in abaxial leaves 1 and 2 at 11 dpg. SPCH expression in nuclei is shown in green. Note additional SPCH-expressing cells and persistence of SPCH in young guard cells (white arrow) in (L). Images in (A) to (J) are at the same magnification. Scale bars, 50 μm.

Fig. 3.

Production of divisions and stomata by SPCH variants in the wild type. Shown are average numbers of stomata and nonstomatal cells (pavement, meristemoid, and small dividing cells) in 0.25-mm2 sections of 7-dpg abaxial Col cotyledons expressing the indicated SPCH variant with the SPCH promoter. Asterisk indicates significant difference from SPCHpro::SPCH phenotype [joint confidence coefficient P = 99% (21)]. Error bars are ±SE.

More informative were the phenotypes induced by expressing a smaller deletion that eliminates eight target sites (four of five high-stringency sites) but leaves the fifth high-stringency site (Ser255) intact (SPCHΔ49, Fig. 1A) or the complementary deletion that removes the remaining target sites (SPCHΔ31, Fig. 1A). Expression of each deletion variant produced excessive numbers of asymmetric cell divisions in the epidermis, with SPCHΔ49 producing a stronger but qualitatively similar phenotype to that of SPCHΔ31 when expressed with the same promoter (Fig. 2D and fig. S4, B to D). The divisions induced by SPCHΔ49 and SPCHΔ31 were physically asymmetric and created cells with meristemoid morphology, much like the stomatal lineage–establishing divisions that SPCH promotes during normal development. To better characterize the cells produced by ectopic divisions, we monitored the expression of cell fate markers. Nearly all small, ectopic cells expressed TMMpro::TMM-GFP, a general marker of cells in the stomatal lineage (Fig. 2, E and F) (7). A smaller fraction expressed MUTEpro::GFP, a marker that is normally expressed in meristemoids just before their transition to guard mother cells (GMCs) (2). Thus, the population evidently consists of both meristemoids and other stomatal lineage cells (fig. S4H).

The division-promoting behavior of both SPCHΔ49 and SPCHΔ31 suggests that multiple residues within the MPKTD are functionally important. To define the specific residues, we repeated the in vitro and in vivo assays with SPCH variants in which the phosphorylatable S/Ts of the five high-stringency phosphorylation sites were substituted with nonphosphorylatable alanines. Each of these S/T → A variants was made as a fusion protein with yellow fluorescent protein (YFP) at the C terminus and was expressed with the SPCH promoter (21). Converting all five high-stringency MAPK target residues to alanines (SPCHpro::SPCH1-5 S/T>A) resulted in a protein that created ectopic stomata like those created by SPCHpro::SPCHΔ93 (Fig. 2G). Converting the first four sites to alanines (SPCHpro::SPCH1-4 S/T>A) resulted in ectopic division phenotypes similar to those seen with SPCHpro::SPCHΔ49 (Fig. 2H). The effect of SPCHpro::SPCH5 S/T>A, however, was much weaker than that of SPCHpro::SPCHΔ31 (Fig. 3 and fig. S4, D and E).

To test whether specific S/T residues or the overall number of S/T sites were important for SPCH regulation, we made additional combinations of S/T → A changes and assayed their ability to induce additional cell divisions. In representative lines from each variant (21), the ability to promote excess asymmetric cell division increased as more sites were eliminated (Fig. 3 and fig. S4). These results strongly suggest that multiple P-X-S/T-P sites are biologically important sites for SPCH regulation. Using mass spectrometry, we found evidence of phosphorylation at several of these functionally critical sites (fig. S5).

Elimination of MAPK target sites generated SPCH variants with greater activity, consistent with phosphorylation of the MPKTD having a repressive role. If the MPKTD is solely a negative regulatory domain, then each of the variants should still rescue spch mutant phenotypes. We assayed rescue of spch-3 by MPKTD deletion and S/T → A variants (fig. S6); in the course of this experiment, we found it necessary to refine our criteria for rescue to include not only the production of stomata (the ultimate result of SPCH activity) but also the generation of physically asymmetric cell divisions (the immediate consequence of SPCH activity), because multiple SPCH variants appeared to separate these two processes. For example, SPCHpro::SPCH1-4 S/T>A, SPCHpro::SPCHΔ49, and 35S::SPCHΔ49 did not produce stomata but did induce additional asymmetric divisions (figs. S4I and S6). It was possible to trace the failure to rescue spch to a single mutation (Ser193 → Ala in SPCHpro::SPCH1 S>A) (fig. S6), indicating a positive role for phosphorylation in the MPKTD in addition to negative regulatory elements.

Results with the S/T → A SPCH variants suggested that negative regulation by phosphorylation can be a mode of SPCH regulation. If the known stomatal regulators and MAPK components are endogenous regulators, then SPCH activity should be enhanced when these regulators are eliminated by mutation. Our results support this hypothesis; SPCHpro::SPCH-GFP expression in a tmm-1 background yielded a massive overproduction of stomata instead of a subtle increase in epidermal cell divisions (2) (Fig. 4, B and C). This stomatal overproliferation phenotype could be suppressed by expressing CA-YODA in SPCHpro::SPCH-GFP;tmm-1 plants (Fig. 4D). We further tested the effects of known stomatal regulators on SPCH behavior by expressing SPCHpro::SPCH and SPCHpro::SPCHΔ49 in backgrounds with mutations in either MPK3 or MPK6 and in a double mutant of the putative upstream receptor-like kinases ERL1 and ERL2. These backgrounds were specifically chosen because none has a stomatal patterning defect on its own (10, 22). In each MAPK and receptor mutant background, the phenotypic effect of SPCH expression was significantly enhanced relative to the wild type, consistent with these proteins being endogenous upstream regulators (Fig. 4, E to G, and fig. S7). As a control for whether the effect on SPCH activity was specific to MAPK-related stomatal regulators, we also expressed the variants in sdd1. SDD1 is also a negative regulator of stomatal development but was recently shown to act independently of YODA, TMM, and ER in perception of EPF1 (8). There was no statistically significant increase in stomatal production or clustering when SPCHpro::SPCH or SPCHpro::SPCHΔ49 were expressed in sdd1 (Fig. 4, H to J, and fig. S7, A and B). Taken together, the behavior of SPCH in these mutant backgrounds suggests that members of the established stomatal receptor/MAPK signaling system modulate SPCH activity in vivo. These results do not rule out additional regulators or MAPK pathway members being involved in SPCH regulation. Furthermore, although these results are consistent with TMM, ER-family receptors, and the MAPKs controlling SPCH activity itself, it is also possible that these proteins regulate the behavior of cells produced by SPCH activity.

Fig. 4.

Effects of endogenous stomatal regulators on SPCH function. (A) Scheme of known stomatal regulatory pathway (P, phosphorylation). (B to D) Suppression of tmm-1–mediated enhancement of SPCHpro::SPCH phenotypes by CA-YODA. (B) Baseline of tmm-1 clustered stomata. (C) Enhanced clusters in SPCHpro::SPCH; tmm-1. (D) Block in excess stomatal production by CA-YODA in SPCHpro::SPCH; tmm-1. (E to G) Enhancement of SPCH activity in erl1;erl2 mutant background. (E) erl1;erl2 with no stomatal clusters, (F) SPCHpro::SPCH; erl1;erl2, and (G) SPCHpro::SPCHΔ49; erl1;erl2 all result in a statistically significant increase in the stomatal density and fraction of stomata in clusters. (H to J) Lack of enhancement of SPCH by sdd1. (H) sdd1 mutants exhibit pairing of stomata and increased density. Expression of SPCHpro::SPCH (I) or SPCHpro::SPCHΔ49 (J) in sdd1 does not enhance the sdd1 stomatal overproduction phenotype.

Eliminating MAPK target sites affects SPCH function and subsequent stomatal development; however, these experiments do not address the mechanism by which phosphorylation affects SPCH. Substrates of MAPK phosphorylation are often associated with changes in localization, stability, or interaction partners (23, 24). All SPCH variants are constitutively nuclear (2) (Fig. 2, K and L), which suggests that SPCH phosphorylation does not alter its subcellular localization; however, it is possible that SPCH phosphorylation alters SPCH persistence. A structural hint of this mechanism is the presence of a predicted PEST domain [PESTfind score +7.63 (21)] in the SPCH MPKTD (fig. S5). Functionally, elimination of MPKTD phosphorylation sites results in excess SPCH protein as visualized by YFP expression. Typically, early in leaf development, SPCHpro:: SPCH-YFP is expressed in many small cells, but fluorescence diminishes as cells become morphologically distinct meristemoids (2) (Fig. 2K). Relative to equivalently staged SPCHpro:: SPCH-YFP plants, SPCH variants with strong overproliferation phenotypes displayed increased numbers of YFP-positive cells early (Fig. 2L) and a trend toward increased protein persistence into meristemoid, GMC, and guard cell stages later (Fig. 2L and fig. S8). When expressed in a CA-YODA background (in which SPCH was predicted to be phosphorylated), full-length SPCH-GFP was not visible, nor could it promote stomatal development (figs. S2 and S9C). However, GFP-SPCHΔ49, which is missing phosphorylatable residues, was detectable and was able to drive asymmetric divisions (fig. S9D).

SPCH is closely related to two other bHLH transcription factors that control stomatal development. We have shown, however, that a novel domain of SPCH renders it uniquely subject to phospho-regulation by a group of kinases that have been demonstrated to transduce signals downstream of both cell-cell and plant-environment interactions (fig. S10). In general, the domain mediates repression of SPCH and does so in a quantitative manner; the more potential MAPK sites eliminated, the stronger the effect of the SPCH variant on stomatal development. However, one specific residue phosphorylated by MPK6, Ser193, is required positively for activity, which suggests that the MPKTD is the integration site for complex regulatory inputs. The MPKTD is of unknown origin; it is not present in Arabidopsis proteins other than SPCH but is found in SPCH homologs from a variety of plant species (fig. S11) (25), hence MAPK regulation of a stomatal bHLH is likely to be a widespread regulatory strategy.

SPCH solves a problem intrinsic to MAPK signaling—how is a set of generally used MAPKs recruited to a specific biological event?—by providing the important effector in a spatially and temporally restricted domain. From the perspective of stomatal control, SPCH guards the entry into the stomatal lineage, including the production of self-renewing cells that contribute to later flexibility in epidermal development. This important decision point is likely the target of developmental, physiological, and environmental regulation (26, 27). Coupling the MPK3/6 signaling module to the activity of SPCH provides a unified, yet tunable, output for the complex set of inputs from these sources. Understanding the elements of the MAPK/SPCH regulatory system that coordinate stomatal production with the prevailing climate may allow the production of food or bioenergy crops with the ability to respond and adapt to changes in that climate.

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