Stomatal development in Arabidopsis

Abstract

Stomata consist of two guard cells around a pore and act as turgor-operated valves for gas exchange. Arabidopsis stomata develop from one or more asymmetric divisions followed by the symmetric division of the guard mother cell. Stomatal number is partly a function of the availability of smaller epidermal cells that are competent to divide asymmetrically. Stomata are spaced apart from each other by at least one neighbor cell. Pattern generation may involve cell-cell signaling that transmits spatial cues used to orient specific classes of asymmetric divisions. TOO MANY MOUTHS may function in receiving or transducing these cues to orient asymmetric divisions. TMM also is a negative or positive regulator of entry into the stomatal pathway, with the direction of the response dependent on organ and location. STOMATAL DENSITY AND DISTRIBUTION1 is a negative regulator of stomatal formation throughout the shoot and encodes a processing protease that may function in intercellular communication. FOUR LIPS apparently controls the number symmetric divisions at the guard mother cell stage. In some organs, such as the hypocotyl, the placement of stomata may be coordinated with internal features and involves genes that also regulate root hair and trichome formation. Other mutations affect guard cell morphogenesis, cytokinesis, and stomatal number in response to carbon dioxide concentration. The molecular analysis of stomatal development promises advances in understanding intercellular signaling, the control of the plane and polarity of asymmetric division, the specification of cell fate, and the regulation of cell differentiation and shape.

Figure 1.

Morphology and Distribution of Arabidopsis Stomata.(Left) Two kidney-shaped guard cells surround a pore. Transmission electron micrograph from Zhao and Sack (1999).(Right) Cryo-scanning electron micrograph of maturing epidermis from a cotyledon. The larger, non-stomatal cells are pavement cells that are shaped like pieces of a jigsaw puzzle. Bars = 2 µm (left) and 30 µm (right).

Figure 2.

The Stomatal Pore Connects the Atmosphere and Air Spaces within the Leaf.(Top) Cross section through pore and substomatal cavity. Transmission electron micrograph from Zhao and Sack (1999).(Bottom) Cryo-scanning electron micrograph showing abaxial epidermis with one stoma at lower right, spongy mesophyll cells at center, and palisade mesophyll cells at upper left. Bars = 10 µm (top) and 40 µm (bottom).

Figure 3.

Spacing Pattern and Anisocytic Stomatal Complexes in Arabidopsis.Stomata are surrounded by non-stomatal epidermal cells. Thus guard cells are excluded from the first ring of cells (non-shaded cells around stoma “a”). The second ring can include guard cells (e.g. shaded guard cells in stomata “b” and “c”). Many stomatal complexes are anisocytic (a, e, g). Others are not because they have more than three neighbor cells (b, c, d), or the neighbor cells are comparable in size (f). Tracing (by Matt Geisler) from the abaxial epidermis of a mature cotyledon. Bar = 40 µm.

Figure 4.

Specialized Stomata in Wild Type Arabidopsis.Cryo-scanning electron micrographs showing stomata at the tips of nectaries (top) and in the abaxial epidermis of the anther (bottom). Arrowheads indicate stomata, some of which are in contact with each other, even in the wild type. Anther micrograph from Kim Findlay. Bars = 30 µm (top) and 100 µm (bottom).

Figure 5.

Diagram Showing Key Cell Types and Divisions in Arabidopsis Stomatal Development.The first asymmetric division (in a MMC) produces a meristemoid. Meristemoids can divide before converting into a guard mother cell (GMC). GMCs divide symmetrically producing two guard cells. Note that the process can reiterate; neighbor cells can re-enter the pathway by becoming MMCs and dividing asymmetrically to produce satellite meristemoids. The plane of division is oriented so that the new meristemoid is placed away from the pre-existing stoma or precursor cell.Yellow, meristemoid mother cells (MMCs); red, meristemoids.

Figure 6.

Living Abaxial Epidermis from Developing Leaf Showing Asynchrony in Stomatal Development and Different Cell Types.Confocal scanning laser micrograph of green fluorescent protein line that labels cell membrane (“Q8” from Cutler et al., 2000). Because the cell walls are thin, the two cell membranes appear as one line at this magnification. GMCs (white asterisks) tend to have convex walls, whereas meristemoids (red asterisks and arrowheads) are more triangular. The stomatal pore is not visible because of the sharp curvature of the pore wall or the plane of the optical section. Bar = 20 μm.

Figure 7.

Example of Dental Resin Data.Bright-field light micrographs of nail polish replicas of dental impressions showing the same field of cells over a 24-hour period (6–7 days after germination). Abaxial epidermis of cotyledon. The MMC (left) divided asymmetrically producing a satellite meristemoid (right). Another neighbor cell divided symmetrically. Figure from Matt Geisler. Bar = 25 µm.

Figure 8.

Key Events in Stomatal Development Shown in Dental Impression Series.The abaxial epidermis of a single cotyledon is shown through time. Cells “c” (day 7) and “f” (day 8) are MMCs because they divided asymmetrically to produce meristemoids. The initial asymmetric division of MMC “f” took place next to a pre-existing stoma and produced a satellite meristemoid. Both MMCs arose from smaller, less sinuous cells. One smaller cell (“e”, day 7) divided symmetrically by day 8. Meristemoid “a” (day 6) divided twice asymmetrically in an inward spiral. Two apparent meristemoids are adjacent (day 6, lower right); the upper one formed a stoma but the lower meristemoid did not progress in development (“b” and “d”). From Geisler et al. (2000).Yellow, meristemoid mother cells (MMCs); red, meristemoids; blue, guard mother cells; green, stomata.

Figure 9.

Asymmetric and Symmetric Divisions.(A) A newly formed meristemoid is not polarized cytologically and is much smaller than its sister cell.(B) The meristemoid shown was produced by two previous asymmetric divisions (1 & 2). It is now polarized prior to a likely third asymmetric division (arrows). The series of divisions occurs in an inward spiral.(C) A higher magnification of a polarized meristemoid with an asymmetrically placed nucleus. This nucleus is close to the future site of division as marked by a preprophase band of microtubules (shown in Zhao and Sack, 1999).(D) The division site in GMCs is marked by wall thickenings before symmetric division.(E) The GMC division site wall thickenings are still visible in a developing stoma (arrowheads).Adapted from Zhao and Sack (1999). Bars = 3 µm (A), 5 µm (B), 1 µm (C–E).

Figure 10.

Fates of Sister Cell to Meristemoid.The same asymmetric division that produces a meristemoid (red) also produces a larger sister cell (*). This cell is plastic in fate. It can divide asymmetrically to produce a satellite meristemoid. It can divide symmetrically to produce two new neighbor cells that in turn have the same plasticity in cell fate. It may stay relatively small and not divide, or it may enlarge and differentiate into a pavement cell (latter not shown).

Figure 11.

Hypotheses for the Generation of the Stomatal One-Celled Spacing Pattern.Hypotheses tested by analysis of dental resin impression series (Geisler et al., 2000).

Figure 12.

Satellite Meristemoid Formation.(Top) Dental resin series showing asymmetric division of an MMC (yellow).(Bottom) Cryo-scanning electron micrograph. Asterisks show a satellite meristemoid (red), or GMCs produced by satellite meristemoids (blue). Successive stages of pore development are shown at the upper left and lower center. Note that five of the six existing and future stomata shown are patterned by satellite meristemoid placement. Cells located next to two stomata and/or precursors (such as the cell to the right of the red asterisk) usually do not divide. Adapted from Geisler et al. (2000). Bar (bottom) = 10 µm.

Figure 13.

Position Independent and Dependent Events in the Stomatal Pathway.(A) and (B) The placement of the first precursor cell, the MMC, appears to be random so that MMCs sometimes form in contact (B).(C) The plane of asymmetric division in separated MMCs also seems to be random.(D) and (E) Divisions of MMCs in contact are randomly oriented and can produce meristemoids in contact.(F) The orientation of asymmetric divisions next to a single stoma or precursor is position-sensitive and patterns most stomata.(G) Asymmetric divisions of adjacent meristemoids can be oriented and thus space stomata.(H) Cells next to two stomata or precursors (asterisks) usually do not divide asymmetrically.(I) The arrest of an adjacent meristemoid (asterisk) spaces some stomata.Adapted from Geisler et al. (2000).Yellow, meristemoid mother cells (MMCs); red, meristemoids; blue, guard mother cell (GMCs); tan, cells whose fate is affected by their position.

Figure 14.

Many Satellite Meristemoids Are Produced During Leaf Development.Cryo-scanning electron micrograph of the abaxial epidermis of a cotyledon. The red arrows indicate satellite meristemoids. Some of the satellites have divided asymmetrically. The yellow asterisks mark small neighbor cells; such cells usually remain division competent (compare with Figure 10). Adapted from Geisler et al. (2000). Bar = 30 µm.

Figure 15.

Stomatal Precursor Cell Placement with Respect to the Mesophyll.Tracing from a pea stipule showing epidermal cells (broken lines), stomata and their precursors (parallel hatching), and the subjacent mesophyll (solid lines). Figure from Sachs (1978) who did not find a consistent spatial relationship between stomatal precursor cell placement and the anticlinal walls of the mesophyll. Bar = 30 µm.

Figure 16.

Stomatal Clusters in too many mouthsCryo-scanning electron micrograph of tmm abaxial cotyledon epidermis. Bar = 15 µm.

Figure 17.

Developmental Basis of Stomatal Cluster Formation in tmm(Top) Dental resin series from tmm (bottom row) illustrating that stomatal clusters result from several defects including (1) the randomization of the orientation of asymmetric division, (2) cells (*) next to two stomata and/or precursors normally do not divide but do in tmm, and (3) sometimes both products of division develop into GMCs (lower right). Top row shows cartoon of what would have happened in wild type.(Bottom) Differential interference contrast micrograph of tmm epidermis showing new meristemoids (red asterisks) that are in contact with developing stomata. Bar = 10 µm.

Figure 18.

Opposite Stomatal Phenotypes in tmmtmm has clustered stomata in cotyledons and leaves but no stomata in stems. Tracings from cleared tissue.

Figure 19.

tmm Alters Stomatal Production in an Organ-Specific MannerDiagram showing the distribution of stomata in the reproductive shoot of wild type (top) and tmm (bottom). The stomatal density is more or less uniform throughout the stem and floral organs in the wild type. In tmm, stomata are absent from the stem, the base of the flower stalk, the ends of the silique and the adaxial sepal. The abaxial tmm sepal has more stomata than the wild type. A stomatal unit is defined as a single stoma or a cluster of stomata in contact. The density of stomatal units is shown in color coding at the bottom left. Adapted from Geisler et al. (1998).

Figure 20.

Gradient in Stomatal Phenotypes in tmm Flower StalksCleared and stained flower stalks of wild type (left) and tmm (right). Dark blue ovals are stomata. Note the even distribution of stomata in the wild type. In contrast, tmm displays a gradient in phenotype from excessive stomata, many in stomatal clusters at the floral end (top) to the elimination of stomata at the base (bottom). Reprinted from Geisler et al. (1998). Bar = 200 µm

Figure 21.

Model for Where Gene Products Act During Stomatal DevelopmentSummary of major events disrupted by different mutations. Yellow represents the selection of an MMC fate. Meristemoids are red. Negative regulation is indicated by “T”-shaped lines, positive regulation is indicated when just the gene abbreviation is shown.

Figure 22.

sdd1 has a Higher Stomatal Density but Fewer Clusters than tmm(Top) Micrographs from leaves of wild type (left), tmm (middle), and sdd1-2 (right). sdd1-2 has a higher stomatal density but fewer stomata in contact than tmm. Bar at left = 50 µm for all three micrographs.(Bottom) Developmental basis of sdd1-1 phenotype. Adapted from dental resin series from Berger and Altmann (2000). Wild type (C24 ecotype) at top, sdd1-1 at bottom. sdd1 produces extra generations of meristemoids. One stomatal cluster develops from an ectopic satellite meristemoid (arrow, day 6). However, most extra stomata are correctly spaced.Yellow, meristemoid mother cells (MMCs); red, meristemoids; green, stomata.

Figure 23.

Paired Stomata in four lips(Top) flp-1 displays both unclustered and clustered stomata. Stomata visualized using KAT::GUS staining (promoter from Rebecca Hirsch and Michael Sussman, methods as in Larkin et al. 1997).(Bottom) Cryo-scanning electron micrograph showing paired stomata at right. Bars = 200 µm (top) and 10 µm (bottom).

Figure 24.

Phenotypes of mus and cyd1(Top) Differential interference contrast optics micrographs of wild type (left) and mustaches (right) stomata. In mus, the pore wall and the shape of the guard cells are skewed with respect to each other. Wild type guard cells are bilaterally symmetric.(Bottom) Transmission electron micrograph of cyd1 stoma with incomplete cytokinesis as shown by wall stubs at both end. The left wall stub has a small stomatal pore that is lacking in the stub on the right side (arrowhead). From Yang et al. (1999). Both bars = 5 µm.