Stomatal Patterning and Differentiation by Synergistic Interactions of Receptor Kinases

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Science  08 Jul 2005:
Vol. 309, Issue 5732, pp. 290-293
DOI: 10.1126/science.1109710


Coordinated spacing and patterning of stomata allow efficient gas exchange between plants and the atmosphere. Here we report that three ERECTA (ER)–family leucine-rich repeat–receptor-like kinases (LRR-RLKs) together control stomatal patterning, with specific family members regulating the specification of stomatal stem cell fate and the differentiation of guard cells. Loss-of-function mutations in all three ER-family genes cause stomatal clustering. Genetic interactions with a known stomatal patterning mutant too many mouths (tmm) revealed stoichiometric epistasis and combination-specific neomorphism. Our findings suggest that the negative regulation of ER-family RLKs by TMM, which is an LRR receptor–like protein, is critical for proper stomatal differentiation.

The growth and development of multicellular organisms require both proliferative and asymmetric cell division, the latter of which generates two daughter cells with distinct cell fates. During epidermal development in higher plants, protodermal cells undergo proliferative division to form pavement cells, which protect tissue layers underneath and prevent water loss (1). In contrast, differentiation of stomatal complexes initiates with asymmetric divisions in a subset of protodermal cells called meristemoid mother cells, which give rise to small triangular meristemoids (2). All derivatives of the meristemoids are defined as stomatal-lineage cells. In Arabidopsis, meristemoids typically undergo one to three rounds of asymmetric division. After each division, the smaller daughter cell maintains the stem cell activity of a meristemoid. The meristemoid eventually differentiates into a round guard mother cell, which divides symmetrically to generate a pair of guard cells (2). The larger daughter cells generated by asymmetric division of the meristemoids will be defined here as stomatal-lineage ground cells (SLGCs). Although Arabidopsis SLGCs would eventually adopt some pavement cell–like characteristics (2), they can be clearly distinguished by their small size and shape as well as by their expression of stomatal-lineage markers (Figs. 1, 2, 3). Some SLGCs may further divide asymmetrically to generate satellite stomatal complexes. The orientation of asymmetric division is regulated by preexisting stomata, so that guard cells are very rarely found adjacent to each other (one-cell spacing rule) (Fig. 1, A and C). The coordinated spacing of stomata indicates that cell-cell signaling is likely very important in stomatal patterning (25).

Fig. 1.

ER-family genes act together in regulating stomatal density and clustering. (A and B) Cleared differential interference contrast images of abaxial epidermis of a mature rosette leaf of wild type (wt) (A) and er erl1 erl2 triple mutant (B) leaves. (C and D) Scanning electron microscopy images of silique epidermis of wild-type (C) and er erl1 erl2 triple mutant (D) leaves. Over-proliferation of stomata and formation of high-density stomatal clusters are evident in the triple mutant. Arrowheads indicate SLGCs. Scale bars in (A) and (B), 50 μm; in (C) and (D), 20 μm.

Fig. 2.

The epidermal phenotype of er-family mutants. Line drawings of mature epidermis of wild-type (wt) (A), erl1 erl2 (B), er (C), er erl1 (D), er erl2 (E), and er erl1 erl2 (F) pedicels [stage 17 (22)] are shown. Guard cells and SLGCs are false colored in green and pink, respectively. Scale bar, 50 μm.

Fig. 3.

ER, ERL1, ERL2, and TMM promoter activity. (A to D) Longitudinal sections of the shoot apex of 2-week-old, wild-type seedlings expressing ER::GUS (A), ERL1::GUS (B), ERL2::GUS (C), and TMM::GUS (D). The promoters of ER-family genes are active in the meristem (marked with asterisks) and entire leaf primordia (marked with black circles), whereas additional stomatal-lineage–specific signal can be seen in the leaf epidermis expressing ERL1::GUS and ERL2::GUS (arrowheads). TMM::GUS marks the protodermal-layer and stomatal-lineage cells. The images of the lower panels were taken with phase-contrast microscopy to show the tissues. Scale bar, 100 μm. (E to H) Adaxial surface of a developing rosette leaf expressing ER::GUS (E), ERL1::GUS (F), ERL2::GUS (G), and TMM::GUS (H). ERL1, ERL2, and TMM promoters are highly active in meristemoids (asterisks), guard mother cells (black circles), and immature guard cells (plus signs); somewhat active in SLGCs and mature guard cells (arrowheads); and not active in pavement cells. Scale bar, 50 μm. (I and J) SLGCs in er pedicel epidermis express moderate levels of stomatal-lineage markers ERL1::GUS (I) and TMM::GUS (J). Scale bar, 25 μm.

A single loss-of-function mutation in Arabidopsis STOMATAL DENSITY AND DISTRIBUTION1 (SDD1), TOO MANY MOUTHS (TMM), or YODA (YDA) genes confers increased stomatal density and clusters (68). SDD1, TMM, and YDA encode a putative subtilicin-like extracytoplasmic protease, a transmembrane leucine-rich repeat (LRR) receptor-like protein, and a mitogen-activated protein kinase kinase (MAPKK) kinase, respectively (79). Based on genetic analyses, a model has been proposed in which SDD1 modifies a ligand for TMM, and the activated receptor signals to downstream MAPK cascades via YDA to repress stomata formation (8, 10, 11). Because TMM lacks any recognizable intracellular domain, signal transduction is thought to occur via interaction with its coreceptor kinase, which has not yet been identified (9).

Arabidopsis ER and its functional paralogues ERL1 and ERL2 show synergistic interaction in promoting above-ground organ growth (12, 13). These three genes encode members of the large family of plant LRR receptor-like kinases (RLKs) (13, 14). In the cortex, the three ER-family proteins act together to coordinate proliferative cell division (13). We investigated the roles of ER-family proteins in epidermal cell patterning to gain insight into their function in tissues that give rise to distinct cell types by asymmetric division. Complete loss of function of all three ER-family genes led to the generation of high-density stomatal clusters (Fig. 1, B and D, and fig. S1) and a 50 to 200% increase in the stomatal index (table S1). Together, the ER-family LRR-RLKs act as negative regulators of stomatal development.

To dissect specific roles for each ER-family protein during stomatal complex formation, we examined epidermal patterning in three single er-family mutants, three combinations of double mutants, and a triple mutant (Fig. 2). The phenotypes of er-family mutations were uniform in all epidermal tissues that normally produce stomatal complexes, including cotyledons, leaves, stems, pedicels, and siliques (Fig. 2; figs. S1, S2, S4; and S5, and table S1).

The growth phenotypes of erl1 and erl2 single-mutant plants, as well as those of erl1 erl2 double-mutant plants, did not differ from those of the wild type (12, 13). These mutants conferred a reduction in the number of SLGCs. Consequently, many stomata were unaccompanied by SLGCs (Fig. 2B and fig. S1). ERL1 and ERL2 may prevent precocious differentiation of meristemoids into guard mother cells or promote continued asymmetric division of the meristemoid to produce a stomatal complex that includes adequate SLGCs.

In contrast, the er single mutation conferred an increased number of SLGCs that failed to differentiate into stomata (Fig. 2C and figs. S1 and S2). This failure led to characteristic patches of two or three small cells surrounded by larger pavement cells (Fig. 2C and fig. S1). If each patch of SLGCs is derived from a series of asymmetric divisions of a mersitemoid, then the er epidermis must produce substantially more meristemoids. We analyzed the cotyledon epidermis at different developmental time points and observed that er undergoes excessive asymmetric division and meristemoid formation (fig. S2). An additional erl1 or erl2 mutation had different effects on er (Fig. 2, D and E). With an additional erl1 mutation, guard mother cell differentiation resumed in all of the more numerous stomatal complexes seen in er (Fig. 2D), so that the stomatal index was elevated in er erl1 double mutants (table S1). In contrast, an additional erl2 mutation enhanced the number of SLGCs seen in er (Fig. 2E). The presence of any functional ER-family gene was sufficient to enforce the one-cell spacing rule, because stochastic stomatal cluster formation occurred only in the er erl1 erl2 triple mutant (Figs. 1, B and D; and 2F; and fig S1).

Promoter activity of ER, ERL1, and ERL2 in the epidermis of wild-type developing leaves was analyzed to determine whether the timing and location of expression corresponded to the proposed site of function. Initially, all three promoters were uniformly active in the protoderm of wild-type leaf primordia (Fig. 3, A to C). The expression of ER::GUS (β-glucuronidase) diminished to below detectable levels before epidermal cells differentiated (Fig. 3, A and E). Meanwhile, ERL1::GUS and ERL2::GUS showed strong expression in stomatal-lineage cells in developing leaves: high activity in meristemoids, guard mother cells, and immature guard cells; reduced activity in mature guard cells and SLGCs; and no activity in pavement cells (Fig. 3, B, C, F, and G). These expression patterns were very similar to TMM::GUS expression (Fig. 3, D and H), so they were used as molecular stomatal-lineage markers to characterize the patches of SLGCs (those small and irregular cells that appear to derive from asymmetric division) seen in the er epidermis (Fig. 2C). Indeed, these cells expressed moderate levels of ERL1::GUS and TMM::GUS, supporting their molecular identity as stomatal-lineage cells (Fig. 3, I and J).

ER-family RLKs regulate two critical steps during stomatal development (fig. S3). First, ER, ERL1, and ERL2 together govern the initial decision of protodermal cells to enter proliferative division to produce pavement cells or asymmetric division to generate stomatal complexes. Second, ERL1 and, to a lesser extent, ERL2 are important for maintaining stomatal stem cell activity and preventing terminal differentiation of the meristemoid into the guard mother cell.

The identification of three LRR-RLKs involved in stomatal patterning facilitates further understanding of cell-cell signaling in this process. Among known players in stomatal patterning, TMM LRR-receptor–like protein is thought to associate with a coreceptor kinase. TMM has contrasting effects in different organs: tmm leaves and siliques develop stomatal clusters, whereas tmm stems produce no stomata at all (3, 15). We genetically tested the interaction of the ER family and TMM and discovered the ER-family–dependent basis for TMM function in different organs. Like tmm, tmm er and tmm erl2 formed no stomatal-lineage cells in the stems, indicating that TMM is epistatic to ER and ERL2 (Fig. 4, A and B, and fig. S4). TMM is partially epistatic to ERL1, because tmm erl1 led to the formation of stomata, albeit at a very low density (fig. S4). tmm er erl1 conferred recovery of stomatal differentiation at normal density (Fig. 4C and fig. S4). Finally, the tmm er erl1 erl2 quadruple mutant formed stomatal clusters similar to er erl1 erl2, indicating that together, the three ER-family genes are epistatic to TMM (Fig. 4D and fig. S4). The epistasis of TMM to ER and ERL2 in the stem is dependent on the presence of functional ERL1. Therefore, TMM may primarily act to inhibit the activity of ERL1 in repressing asymmetric division in the stomatal pathway. The stoichiometric dynamics of epistasis suggest that these four putative receptors act in close proximity.

Fig. 4.

Genetic interactions of ER-family RLKs and TMM. (A to D) Stoichiometric nature of epistasis between ER-family genes and TMM in stem epidermis. tmm (A) and tmm er (B) do not differentiate stomata. In contrast, tmm er erl1 (C) confers a recovery of stomatal differentiation (asterisks). A tmm er erl1 erl2 quadruple mutant (D) produces high-density stomatal clusters (dashed brackets). (E to H) A combination-specific neomorphism revealed by interactions of TMM with ER-family genes in silique epidermis. tmm (E) produces guard cells (asterisks) with a mild clustering, whereas er (F) confers occasional failure of guard mother cell differentiation (bracket). In tmm er double mutants (G), all stomatal-lineage cells adopted SLGC cell fate (brackets). Again, guard cells (asterisks) differentiate in tmm er erl1 (H). Scale bar, 50 μm.

In cauline leaves and siliques, where tmm and er erl1 erl2 independently form clustered stomata, specific combinations of tmm and er-family mutations created a novel phenotype. Whereas tmm produced some clustered stomata (Fig. 4E) and er produced stomatal complexes with and without guard cells (Fig. 4F), both tmm er and tmm er erl2 differentiated SLGCs but no stomata (Fig. 4G and fig. S5). However, an additional erl1 mutation caused stomatal differentiation to resume as tmm er erl1 formed stomata (Fig. 4H). tmm er erl1 erl2 silique epidermis resembled er erl1 erl2 epidermis (fig. S5). The observed neomorphism in tmm er and tmm er erl2 indicates that TMM is conditionally required for stomata formation in the absence of ER. Again, TMM may inhibit the activity of ERL1, this time in repressing guard mother cell differentiation.

The complex interaction of TMM and ER-family genes suggests that their products are not simply upstream or downstream of each other but work in combination to determine stomatal-lineage cell fate. TMM negatively regulates specific ER-family members at both critical steps in stomatal differentiation, first at the initial decision of a protodermal cell to divide asymmetrically to enter the stomatal lineage and then at the later decision of a meristemoid to terminally differentiate into a guard mother cell (fig. S3). The mechanism for regulation of the ER family by TMM is unknown. One possibility is that TMM forms a receptor heterodimer with ER-family RLKs, most likely with ERL1, and prevents signaling. The lack of a signal transducer domain in TMM supports this inhibitory function. The association of an LRR-RLK with an LRR receptor–like protein has been proposed for shoot apical meristem development, in which case CLAVATA1 (CLV1) and CLV2 may form a functional heterodimer to signal (16, 17). Alternatively, TMM may titrate the same ligands as the ER family, hence repressing the ER-family signaling pathway. In the absence of TMM, ER, and ERL2, remaining ERL1 could become overly active as an inhibitor of guard mother cell differentiation because of excessive ligand availability.

We have shown that three ER-family RLKs act in overlapping but unique manners during stomatal stem cell fate specification and differentiation. Landsberg er was isolated nearly half a century ago and has since been used as a wild type (18). However, the effects of er on epidermal patterning have not been documented until now. Using distinct combinations of er-family mutants, we have elucidated a specific function for TMM during stomatal development in different organs. The diverse and overlapping functions of ER-family RLKs highlight the versatile nature of this receptor subfamily and emphasize the universal theme of receptor signaling in plants and animals. In animals, key signaling components, such as bone morphogenetic protein and Wnt, act as both mitogens and morphogens in various developmental processes, and their signaling pathways are used reiteratively during development (1921). Similarly, ER-family RLKs regulate cell proliferation and differentiation, during both plant organ growth and epidermal patterning. Now that a possible receptor partner for stomatal differentiation has been identified, we can begin to elucidate how specific functions of ER-family RLKs are manifested at the molecular and cellular level. The use of the same signaling components in different combinations for unique developmental processes has likely been a key factor in the diversification of plants.

Supporting Online Material

Materials and Methods

Figs. S1 to S5

Table S1

References and Notes

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