Stomatal patterning in angiosperms


Stomatal patterning in angiosperms1

Judith L. Croxdale2,0

0 Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 USA

Received for publication November 5, 1999. Accepted for publication April 20, 2000.

My thesis is that understanding stomatal patterning requires a holistic perspective. Since stomata are structures critical to the survival of terrestrial plants, they need to be viewed in relation to their function and their interface with other structural components. With this outlook, I begin by discussing pattern types, means of measuring them, advantages of each type of measurement, and then present patterning from evolutionary, physiological, ecological, and organ views. I suggest areas where I believe profitable studies might enable us to better understand stomatal patterning. The final sections of the paper review stomatal patterning on angiosperm leaves and present a theory of patterning. With the abundance of molecular information, and coming genomic sequences and new tools, an opportunity exists to dissect the process of how cells are selected to become different from their neighbors and assume a fate critical to plant survival. Understanding this biological process at the molecular level requires comprehending the broad base on which stomatal patterning rests.

Key Words: angiosperms • distribution • evolution • organ form • physiology • stomata • theory

Source: American Journal of Botany. 2000;87:1069-1080.



Designs and patterns in nature are not only aesthetically pleasing, but they also arouse curiosity. Why do such patterns exist? Are they essential for function? How are patterns established? Patterns are found at all biological levels, from ecosystems to molecules, and tools used to assess pattern at one level may be useful in understanding pattern at other levels. For example, ecologists have long used nearest neighbor analysis to understand plant communities; I and others have used such analysis to understand stomatal pattern, and molecular biologists now apply this method to understand genomic evolution. What is important is the realization by researchers at other levels that such analysis may be useful in understanding their problem.

This paper examines stomatal distribution as a pattern for study and is not concerned with differentiation of stomata and stomatal complexes, except for those cells that enter the stomatal pathway, but fail to fully differentiate. Stomata are well suited for investigation because the patterns are two dimensional, which simplifies analysis, and because stomata occur on organ surfaces, which makes them readily accessible. In spite of these advantages, information on stomatal patterning is sparse. Likewise, information on trichome patterning is also limited, in spite of growing molecular information on trichome differentiation. While trichome and stomatal patterning might be considered together in a paper on epidermal patterning, I choose to highlight stomatal patterning and present information in depth on these critically important cells, stomata.

In this paper, I provide a broad context for understanding pattern because I believe it is an essential backdrop necessary to understand theories and possible mechanisms of patterning. Stomatal patterning can be considered from many different reference points and based on these varied perspectives, I pose a number of questions to which we do not have the answers. I ask these questions to stimulate the community of researchers and suggest an avenue of study to tackle important problems in stomatal patterning. I begin first with a general discussion of pattern and common means by which stomatal pattern is assessed (see below, Pattern and Pattern Assessment). Stomatal patterning cannot be considered in isolation, but needs to be understood in terms of evolution, physiology, and organ form (Stomatal Pattern and Evolution, Stomatal Pattern and Physiology, Stomatal Pattern and Organ Form). Next, monocot and dicot leaf growth are reviewed and followed by existing theories explaining stomatal patterning in these two groups (Stomatal Patterning in Angiosperms). Lastly, I propose a unifying theory of stomatal patterning in Angiosperms, with a heading of the same name, followed by a brief summary.

Pattern and pattern assessment

Some patterns are easy to recognize—a checkerboard, the hexagonal units of a beehive, stripes of alternating colors on a flag, but biological patterns are often more subtle and less easy to discern. As Gould (1991) has pointed out, however, our visual perception of patterns tends not to be accurate. Discriminating the type of pattern is difficult. As we search to understand pattern, we may believe we see figures or objects in random patterns that are not there, like the mythological characters, objects, or animals in constellations. Random arrangements often look like clustered patterns (Fig. 1A) and ordered patterns may look random (Fig. 1B). Our minds, or those of most, are incapable of intuitive probability calculations while viewing a pattern. We perceive patterns where none exist, as viewers of modern art have done for years. We look for and find the familiar in the unfamiliar. Moreover, our terms for pattern have mathematical as well as common meanings, and unless we use language precisely, confusion may result. For example, we might describe the leaves of a palm tree as clumped at the top of the stem. In reality the leaves are present in a precise and regular order, albeit at the top of the stem.

Two-dimensional patterns, such as random, clustered, and ordered, are easy to grasp and to study. In part, this ease comes about because patterns can be reduced to dots on a sheet of paper, and, in part, because examination is simpler than for three-dimensional patterns. Patterns, even simple two-dimensional ones, are difficult to assess when they form over time. At an early stage, no pattern may be apparent, for example, three data points on a sheet of paper. Or, the pattern may change so its final configuration differs from its earlier form. Consider, for example, seat occupancy on an early morning bus. Passengers at the beginning of the bus route may select seats on a more or less random basis. If two neighbors get on at the same stop and take adjacent seats while they continue a conversation, the pattern takes on a clustered aspect. When the bus is at the end of its route and passengers fill all the seats, the pattern is highly ordered. Stomatal pattern on dicotyledonous leaves is the biological equivalent of bus occupancy. The kinetic development of the pattern confounds our ability to understand the patterning mechanism. Our knowledge of pattern is not yet sophisticated enough that we can detect the placement of the bus seats and thereby predict the final pattern.

Random vs. nonrandom
Distribution of developing stomata is reportedly random, although an exclusionary distance is present around each stoma (Sachs, 1974 ; Rasmussen, 1986 ). This may be an instance where random is being used in the everyday sense of the word, meaning that an ordered pattern is not visually obvious, rather than in the mathematical sense. Asserting that a pattern is mathematically random must be based on a standard statistical method.

A statistical method developed to assess plant distribution in ecological studies (Clark and Evans, 1954 ) is often used to determine whether stomatal distribution is ordered, random, or clustered. The mean neighbor distance between stomata in a population is compared to that in a randomly distributed population; the ratio of these two distances is called the R value. The two populations must have the same density, samples per unit area, but not necessarily the same size. When the sample distribution is random, the R value is 1, when distribution is clustered, the R value is near 0, and when distribution is ordered, the R value is significantly greater than 1. For example, if stomata were arranged in precise hexagonal arrays, the R value would be 2.1491. R values reported for stomatal distribution range from ~1.4 to 1.6. The R value indicates the type of pattern, but yields no information on dimensional or space-filling aspects of the pattern.

Arrested stomata and stomatal distribution
Published data indicate that stomata are ordered initially and become more ordered as leaves mature (Sachs, 1988 ; Kagan and Sachs, 1991 ; Croxdale et al., 1992 ; Boetsch, Chin, and Croxdale, 1995 ; see Tessellation section below). The increase in order results from stomata that fail to complete their development, so-called aborted, arrested, or immature stomata. Their presence on leaves is very common, if not universal. In Tradescantia, arrested stomatal initials can be identified shortly after the initials form. When developing stomatal initials have the first pair of subsidiary cells, arrested initials in the same cell file lack them (Boetsch, Chin, and Croxdale, 1995 ). The strict basipetal development of stomatal complexes in a cell file permits the early identification of arrest. In other genera, arrested stomata have been identified primarily by their structural configuration on mature leaves. Depending on when stomata arrest and their subsequent development, they might not be recognizable on mature organs. Based on illustrations of monocot and dicot stomata, 10–50% of the total number of initiated stomata arrest. In Ruscus and Sansevieria, 30–40% of stomata arrest (Kagan and Sachs, 1991 ; Sachs, Novoplansky, and Kagan, 1993). Such a high rate of failure is felt to be noncompetitive in evolutionarily successful plants and unlikely to result merely from developmental failure. Instead, it has been argued that patterning is epigenetic and Darwinian.

The change in R values reveals the relationship between stomata that arrest and those that complete their development. Since R values increase with leaf maturation, stomatal arrest is based on position. If arrest were random, R values measured at maturity would not differ from R values measured on immature leaves. Although R values change during leaf development, whether the change in pattern is statistically different rarely is tested. Standard procedures are available for testing whether values are significantly different from one another, including one by the authors of R values (Clark and Evans, 1954 ), which should be used routinely.

Does this adjustment in the stomatal array yield clues to stomatal patterning? In Tradescantia stomata arrest only at the earliest stage of stomatal development (Chin et al., 1995 ; Boetsch, Chin, and Croxdale, 1995 ), but in Pisum stipules stomatal arrest occurs at several stages of development (Kagan, Novoplansky, and Sachs, 1992 ). Variation in the time of arrest indicates there are redundant pathways of arrest. In Tradescantia (Boetsch, Chin, and Croxdale, 1995 ) the position of an arrested stoma is linked to the distance of only the nearest of the five closest stomata. Although proximity does not prove a causal link in arrest, the location of possible participants in the process is revealed. Similar positional analysis has not been done in other taxa, but a group of stomata rather than a single stoma might be necessary to suspend development of stomata in other genera. The presence of arrested stomata indicates that guard mother cells (GMC) or stomatal initials are committed, but not determined in the stomatal pathway. Nevertheless, to understand how cells are patterned, as a developmental biologist, I am interested not simply in a mathematical description of pattern. I am interested in a theory of patterning that accounts for the origin of all stomata, including those that arrest.

Scale, boundaries, and pattern
Assessment of pattern is best done on an entire organ or on large areas of an organ. Simply looking at small fields of pattern (cf. Fig. 1C, D, which are subsections of Fig. 1A, B) shows that pattern can appear ordered when it is random or it can appear random when it is ordered. These extreme examples show how erroneous conclusions might be reached using data from small-sized samples. The effects of scale on stomatal distribution have not been studied, but clearly are important in our understanding of patterns.

When the physical size of the sample is small, scale and the possibility of edge effects on pattern also must be considered. While the leaf edge is an obvious physical boundary, the areas above the midrib and the vascular bundles generally lack stomata and serve as a boundary. Pisum stipules (Kagan, Novoplansky, and Sachs, 1992 ) and Avena coleoptiles are exceptions to this general rule for stomata lie above their vascular bundles (Dollahon et al., 1988 ); exceptions may exist in other species. Since stomata are not the only differentiated cell type in the epidermis, lithocysts, trichomes, or papillae that differentiate earlier than stomata will serve as absolute barriers (Smith and Watt, 1986 ; Rasmussen, 1986 ; Larkin et al., 1996 ). Because of the labor involved in evaluating pattern on entire organs, we do not yet know whether pattern is adjusted near boundaries and is different from pattern in boundary-free areas.

A common and useful measure of stomatal distribution is frequency, although it provides no information on pattern type. Frequency measured on an area basis (stomatal number per unit area) simplifies and speeds the collection of data but restricts its usage to organs of the same developmental age and the same taxon. Even within the same taxon, one cannot make valid comparisons between stomatal frequency of young and mature leaves because of cell size changes that take place during leaf expansion. Measuring frequency on an index basis (100 cells) permits leaves of different age, but not different genera to be compared. Additionally, experimental treatments that influence stomatal frequency might also influence cell size so frequency comparisons between treatments also must examine cell size to ensure uncensored data.

The mode of organ growth may also complicate analysis of pattern. Generally speaking, entire (simple) leaves are reasonably straightforward to study, although there are exceptions such as Tropaeolum, which is a simple leaf at maturity but bears leaflets early in development (Fuchs, 1975 ). Compound leaves generate leaflets in ways that complicate analysis. For example, leaflets may be generated basipetally or acropetally or from the middle toward the two extremes of the leaf blade region. Thus, the leaflets of an immature leaf are not developmentally equivalent to one another; each leaflet will develop according to a timetable established at its initiation. This information as well as the growth mode of the leaflet itself must be known before embarking on a study of stomatal pattern. Leaves of dicotyledons have scattered clonal growth with new cells being intercalated between existing cells. Such growth may be rhythmic, and comparisons of stomatal frequency during leaf expansion may not be valid. Fern, gymnosperm, and monocotyledon leaves exhibit polarized growth, simplifying analysis of their patterns.

Frequency determinations on a cell or index basis permit comparison of leaves or leaf areas at different developmental stages (Radoglou and Jarvis, 1990 ; Croxdale et al., 1992 ). Again, this measure provides no information on two-dimensional order (pattern), but a sense of spatial coverage can be gleaned using the index basis. These measures also can be misleading, however, unless the investigator is familiar with the typical configuration of the stomatal complexes in the species. For example, Tradescantia plants grown in elevated CO2 have an increased stomatal frequency on an index basis, 27.2 vs. 23.9 in ambient CO2 (Boetsch et al., 1996 ). However, the number of subsidiary cells associated with nearly half the stomatal complexes (44%) increases from four to six or seven cells. The additional cells are recruited from the epidermal cell population. The shift in cells from the epidermal population to the stomatal complex population increases the stomatal index, although stomatal frequency on an area basis remains the same, 23.1 in elevated CO2 vs. 23.9 in ambient CO2. This example demonstrates that no single measure of pattern is adequate in all circumstances.

Regardless of whether frequency is measured on an area or an index basis, reports must state whether the stomata have subsidiary cells and whether they are counted as part of the stomatal complex or as epidermal cells. Although distinguishing subsidiary cells from neighboring cells has been a controversial issue (Baranova, 1992 ), authors need to state how they assessed cells surrounding each stoma. Information on the size and minimum number of stomata in the sample field is useful in all reports of stomatal frequency. Additionally, the number of plants and leaves sampled needs to be statistically relevant. Individual plants and individual leaves will show variance in stomatal frequency, and the sample size must be adequate to provide meaningful data. Because of plasticity in stomatal pattern, studies should report whether the plants were grown at the same time or at different times to account for possible sources of variation. Investigators always should be aware of possible genetic changes that may be carried forward in vegetatively propagated clonal material or be found in sexually produced seed. New individuals resulting from either means of propagation should be surveyed to determine whether stomatal frequency is within the confidence limits established in earlier experiments. The reporting of stomatal frequency has limited utility for understanding stomatal patterning. Investigators need to examine stomatal pattern, not as a mathematical exercise, but in ways that lead to testable hypotheses about the mechanism of stomatal patterning.

Tessellation is a powerful visual and quantitative means of assessing stomatal pattern. Tessellation consists of dividing a region into tiles using markers (stomata for example) as tile centers and drawing lines halfway between tile centers to form the tile edges. We have photographed entire blade panels, marked relevant features, and assessed relationships using tessellation programs. Figure 2 is an example of a tessellated leaf blade panel with stomata, arrested stomata, veins, and leaf margins marked. The latter two are marked because they are boundaries. If stomata and arrested cells are the markers of the tessellation, a portion of the tiled leaf area looks like that in Fig. 2A. Tiling changes visually (becomes more ordered) when tessellation uses only stomatal positions (cf. Fig. 2A to B), and order can be readily assessed statistically. The distribution curves of tile areas for the two populations differ markedly (Fig. 2C). The stomata and arrested cell population contains a large number of small areas while in the stomata population tile areas are shifted to larger values. The curve for the stomatal population is more symmetrical and has a lower maximum than that for the mixed population, which peaks at small tile areas but slowly trails off at larger areas. Tile areas and their distributions can be analyzed by common statistics—averages, standard deviations, variance, normal probability distributions, skewness, and kurtosis. Tessellation provides spatial and dimensional aspects of pattern that R values and frequencies do not. Assembling the raw data is tedious, but the visual immediacy of tessellations is memorable and the power of statistics provides a robust platform for pattern analysis.

Stomatal patterning

Stomatal pattern and evolution
Given the critical role of stomata in plants' successful exploitation of the terrestrial environment, current-day patterns likely are derived from those of fossil land plants, even though the arrays in fossils may represent imperfect solutions to the problem of gas exchange in today's atmosphere. A comprehensive study of fossil stomatal arrays is not available, but would be valuable to compare patterns of plants grown in different atmospheric conditions, in spite of the shortcomings and bias in the fossil record. The range of fossil patterns may yield clues to the regulation of pattern in extant plants. In fact, the patterns could be compared with recent evidence on the interplay between atmospheric gas concentrations and stomatal frequency (Woodward, 1987 ; Beerling and Chaloner, 1993 ; McElwain and Chaloner, 1995 ; Edwards, Kerp, and Hass, 1998 ).

Cooksonia, known from late Silurian deposits (Edwards, Davies, and Axe, 1992 ), is the most ancient vascular land plant with stomata. Stomata appear on the reproductive axis, but because of axis distortion in the compression, their distribution is not known. In other genera from the same deposit, stomata are present in oblique bands at sporangial bases or scattered across sporangial surfaces (Edwards, Kerp, and Hass, 1998 ). Stomata occur in other ancient plants such as Rhynia, Zoosterophyllum, Sawdonia, and Agalophyton, but no information is available regarding order (Willmer and Fricker, 1996 ).

Comprehensive reviews of stomatal evolution (Ziegler, 1987 ) and structure in extant angiosperms (Baranova, 1992 ) are available, for their structure does differ over time, but such changes are peripheral to the central issue of this paper on stomatal pattern. Structural aspects of stomatal development in extant plants are very plastic, up to five different modes of development are reported to occur on a single organ (Baranova, 1992 ). This plasticity will complicate understanding the mechanism of patterning and the evolution of pattern.

Leaves, as opposed to the naked axes and sporangia discussed above, from fossil lycopods (Baragwanathia abitibiensis, Drepanophycus spinaeformis) reportedly have randomly placed stomata (Taylor and Taylor, 1993 ). Photographs of the lyocopod Lepidophylloides show stomata distributed near shallow grooves that flank the single vascular bundle (Taylor and Taylor, 1993 ), similar to some present-day grasses like barley. In the Sphenophyllales stomata reportedly are random (Sphenophyllum multirame) or randomly placed but with their long axes parallel to the long axis of the leaf (Asterophyllites characformis) (Taylor and Taylor, 1993 ). On the other hand, leaves of fossil conifers (Pseudofrenelopsis, Swillingtonia, Podocarpus,and Sciadopityoides microphylla) and a fossil palm (Sabal dortchii) have stomata in linear series (Taylor and Taylor, 1993 ), much like those of Tradescantia. Similarly, surveys of 21 species of fossil Agathis (Stockey and Atkinson, 1993 ) and four species of fossil Dacrydium (Stockey and Ko, 1990 ) report that stomata occur in discontinuous rows, a common feature of monocot leaves. Although there are few reports on fossil stomatal patterns, the published pattern descriptions are not markedly different from those of extant plants.

Stomatal pattern(s) may have evolved as the plant body elaborated into varied forms over geological time. Therefore, patterns on fossil axes or stems (cylinders) may differ from patterns on fossil leaves (sheets). These pattern differences may reflect changes in organ form as well as changes in the ancient atmospheres. Mechanisms of patterning also may have diverged in response to these changes, and given the time frame involved, distinguishing the mechanism or mechanisms may be complicated.

Stomatal pattern and physiology
Although the adaptive responses of stomata have long been of interest to physiologists, stomatal patterns and their relationship to photosynthesis and transpiration have not received adequate attention (Jarvis and Mansfield, 1981 ; Mansfield, Davies, and Leigh, 1993 ). Since photosynthesis operates in one of three fixation pathways (C3, C4, and CAM), stomatal pattern may vary because each pathway has particular advantages or properties that might affect gas exchange. The physiological differences (photorespiration in C3 vs. C4 plants) and the temporal (CAM) and spatial operation (C4) of these pathways may influence or be associated with particular stomatal arrays. Broad surveys of stomatal pattern and fixation pathway are not available. Some plants, maize for example, are C3 as juveniles, but become C4 plants as adults. CAM plants also revert to C3 photosynthesis when well watered. Such plants provide a special opportunity for understanding the relationship between physiology and stomatal pattern.

In addition to differences in photosynthetic pathway, surface features, light penetration (Martin et al., 1989 ; Cui, Vogelmann, and Smith, 1991 ), and the internal architecture of leaves (Terashima and Inoue, 1984, 1985a, b ; Vogelmann, Bornman, and Josserand, 1989 ; Terashima, 1992 ) may impact on stomatal distribution as well as function. These parameters may explain the heterogeneity of stomatal responses in leaves (Omasa and Onoe, 1984 ; Daley et al., 1989 ; Raschke et al., 1990 ; Beyschlag, Pfanz, and Ryel, 1992 ; Malone et al., 1993 ; Weyers and Lawson, 1997 ; Mott and Buckley, 1998 ).

Numerous studies have surveyed the relationship among environmental gasses (CO2, O3, SO3, O2) and stomatal numbers and function (Mansfield, 1976 ; Omasa et al., 1985 ; Fowden, Mansfield, and Stoddart, 1993 ; Ramonell, Crispi, and Musgrave, 1997 ), but not their effect on stomatal pattern. Stomatal frequency (Tichá, 1982 ) and chlorophyll fluorescence (Schriber, Fink, and Vidaver, 1977 ) are known to differ on upper and lower leaf surfaces and across the leaf surface (Croxdale and Omasa, 1990 ). Is this related to environmental conditions, photosynthetic pathway, or internal leaf geometry? As of yet, there are no answers to these fundamental questions for stomatal pattern on leaves or other organs.

How plastic is stomatal pattern on a given species growing in a given habitat? Do plants within an environment share stomatal characteristics? One study of stomata on mangrove species (Stace, 1966 ) concluded that there was no evolutionary convergence of stomatal types on an interfamilial basis. Instead, stomatal characteristics in a species were more similar to species with the same family than species in the same environment. If a community's species were examined throughout the growing season, would each or only some species exhibit changes in stomatal numbers or pattern? Would these changes correlate to season? Can terrestrial species, especially those located near aquatic communities, adapt to inundation? If so, are the changes similar to those exhibited by amphibious species that grow in both environments? Adaptation of stomatal pattern to habitat and changes in stomatal pattern on a seasonal basis are areas ripe for study.

Stomatal pattern and organ form
Stomatal pattern on leaves and stems is likely related to internal and external organ architecture. Given the functional connection between stomata and photosynthesis, the distribution of photosynthetic cells and the topography of the air space system (Turrell, 1936 ; Sifton, 1945 ) may be linked to stomatal pattern. Studies relating internal geometry to different stomatal patterns would be of great value in understanding the relationship—if there is one—among structure, function, and patterning.

Organ geometry and growth (Payne, 1979 ) may be important as well. Cylindrical stems may have arrays of stomata that differ from flattened stems, but mirror those of cylindrical leaves. The stomatal patterns found on flattened stems may be similar to those of bifacial leaves while the unifacial tip of monocot leaves may exhibit a pattern analogous to cylindrical stems. Patterns on species with decurrent leaf bases in which the stem and leaf base extend together would be of special interest. A survey comparing stomatal patterns on different organ forms would provide a measure of pattern plasticity in relation to organ geometry.

Patterning mechanisms
Although the molecular mechanisms of patterning in plants and animals are probably similar, the closed growth of animals vs. the open growth of plants has repercussions on understanding and tackling patterning problems. In animals, critical patterning decisions occur during embryogenesis, whereas in plants the timing of these decisions spreads over the lifetime of the organism and the location of these decisions varies by organ. In animals, patterning commonly occurs by three different physical modes: a soluble signal that diffuses to a competent cell, cell matrix contact to induce specification of an adjacent cell, and direct cell-cell contact. Similar modes may exist in plants, but the presence of cell walls, the absence of cell movement, and the status of symplasmic domains may complicate understanding them. In considering molecular mechanisms of patterning, we need to realize the infancy of the field. However, in Drosophila embryos, the presence of Notch and delta, both transmembrane proteins, are required to pattern the epidermis into hypodermal cells (skin) and neuroblasts. When either are missing, all surface cells become neuroblasts. This information is interesting for several reasons—all surface cells can assume the same fate, but Notch and delta are required for some of them to become different. Trichome patterning may work in a similar fashion with two or more genes. Most interesting is the observation that one of the genes (GLABROUS) is expressed throughout the epidermis and later is restricted to trichome precursors (Marks, 1997 ). What turns this gene off in most epidermal cells may be the product of another gene in the pathway. At this writing, equivalent molecular information is not yet available for stomatal patterning.

Stomatal patterning in angiosperms
Angiosperm leaves exhibit two fundamentally different modes of growth and development. Monocot leaves have polarized growth from a single point source of cells at or near the leaf base, creating a leaf blade with the oldest cells at the tip. The epidermis consists of regular longitudinal files of cells, whose cells differentiate basipetally, providing a continuum of stages along the blade length. Stages in the origin of stomatal precursors and their development into mature stomata are readily followed. Dicot leaves on the other hand grow from multiple point sources in a patchwork quilt fashion, with clones of new cells forming throughout growth and development. At maturity, the epidermis consists of irregularly shaped cells interspersed with stomata. Evidence for this type of growth is based on marked mesophyll cells (Poethig and Sussex, 1985 ), and it seems likely that epidermal cell growth is also clonal in order to keep pace with divisions in the underlying cell layers. Superimposed on the pattern of new cell origin in dicot leaves is the basipetal pattern of cell expansion (Wolf, Silk, and Plant, 1986 ). This addition of new cells to the leaf blade and their subsequent expansion complicates understanding stomatal patterning.

Bünning (1956) observed that new stomata developed between mature complexes in dicotyledonous leaves and hypothesized that mature stomata released an inhibitor to prevent new stomata from arising nearby (Fig. 3A). Only when new growth exceeded the inhibitory influence would new stomata arise. Beyond Bünning's original observations, no measurements have established the size of the inhibitory field in any species and no inhibitor has been isolated.

Support for Bünning's inhibition theory comes from studies using a wide variety of species (Pelargonium zonale and Sedum stahlii—Korn, 1972 ; Anagallis arvensis—Marx and Sachs, 1977 ; Aeonium—Sachs and Benouaiche, 1978 ; Vinca major—Sachs, 1979 ; Pilea cadierei—Smith and Watt, 1986 ; Pisum sativum—Kagan, Novoplanksy, and Sachs, 1992 ). However, the presence of aborted stomata in these species indicates that Bünning's theory is imperfect. Inhibition failed to prevent the origin of stomata that ultimately arrest (Anagallis arvensis—Marx and Sachs, 1977 ; Aeonium—Sachs and Benouaiche, 1978 ; Vinca major—Sachs, 1979 ), and their existence is cause to re-examine the foundation of Bünning's theory.

More recently, Sachs (1994) , Larkin et al. (1996) , and M. Geisler and F. Sack (Ohio State University, personal communication) have indicated that stomatal distribution results from an interactive process that occurs during leaf growth and might be attributed to cellular interactions. While it seems likely that some form of cellular communication is involved in stomatal patterning, the evidence to date is limited to cell division patterns that serve to separate stomata from one another. Analysis of tomato chimeras and intergeneric chimeras from members of the nightshade family (Solanaceae) shows that the genotype of the epidermal layer regulates the frequency of specialized cells, trichomes and stomata (Szymkowiak and Sussex, 1996 ; E. Szymkowiak, University of Iowa, personal communication). However, in maize the Knotted genotype in the mesophyll induces divisions in the epidermis that are characteristic of Knotted rather than wild type (Hake and Freeling, 1986 ). These disparate results indicate that cell fate is not dependent solely on cell lineage, but results from the integration of signals within and between cell layers, as is known to occur during floral organ determination (Bouhidel and Irish, 1996 ).

Indeed, many events in dicot leaf development (early trichome initiation, scattered islands of growth, clonal development of photosynthesis, and delayed photosynthetic competence in late arising clones) mean that final stomatal pattern is derived by new stomata being inserted into an existing network. Given this pattern of leaf growth, new stomata may arise according to the development of individual clones and not by exceeding the sphere of influence from existing stomata. Although existing stomata may appear to inhibit new stomata, new stomata may be arising in association with the formation of new epidermal cells (within the clone of new cells), independent of influence from existing cells. Stomatal patterning could be clonal autonomous. Anomalous clusters of Arabidopsis stomata, like those in the mutants tmm and flp (Yang and Sack, 1995 ) or those that occur under certain growth conditions (Serna and Fenoll, 1997 ), are reportedly clonal. However, study of another specialized epidermal cell type, trichomes, concluded that cell lineage or clonal growth was not responsible for trichome distribution (Larkin et al., 1996 ). Considering that dicot leaves exhibit clonal growth scattered across the leaf, the possibility of flawed logic in Bünning's inhibitor theory is obvious.

If we assume that existing stomata regulate the placement of new stomata by cellular interactions, then what is the route of communication? Plasmodesmata between intervening cells might serve as a structural link for easy passage of signals. If a structural bridge between cells is not present, then signals have to traverse cell membranes and cell walls to affect development. In oat, developing stomata are in symplastic continuity, but at maturity are symplastically isolated from their epidermal neighbors (Palevitz and Hepler, 1985 ). The fundamental relationship among leaf growth, symplastic domains, and stomatal commitment in dicotyledonous species must be studied to understand the level at which the regulation of stomatal placement might take place.

Clonal and polar growth in dicot species
The earliest study reflecting the impact of clonal growth and stomatal patterning may be that of Leick (1955) , who reported that in selected dicots (Berberis aquifolium, Sedum confusum, Silene nutans, and Choisya ternata) that stomatal number increases in waves, while in other dicot species (Globularia willkommii, Emex spinosa, Polygonum convolvulus, Penstemon barbatus, Viola, and Jovis), a large number of stomata are present when the leaf is young and the stomatal index or frequency declines as the leaves expand. The latter leaves and at least some members of the Caryophyllaceae (Pappas et al., 1988 ; Fahn, 1990 ) grow in a polarized manner similar to those of monocots. In Arabidopsis leaves stomatal numbers also increase in non-steady-state kinetics, similar to waves, at predictable times (hours after imbibition) or at given numbers of leaf cells (M. Geisler and F. Sack, Ohio State University, personal communication). The non-steady-state kinetics of stomatal initiation likely reflects initiation of new stomata during simultaneous growth of clones.

Polarized growth makes monocot leaves well suited for stomatal patterning studies because the developmental history of the leaf can be traced down its length. New blade cells and stomatal precursors originate in polarized fashion at the leaf base and are present in a continuum of stages from the leaf tip (mature) to the leaf base (immature). Unlike the morning bus example cited previously, stomatal origin in monocotyledons is similar to passenger loading in airplanes, from the rear to the front of the aircraft. Stomatal pattern propagates as a wave down the leaf as the blade grows.

Young monocot leaves have no stomata, although all three axes of symmetry are in place. The proximal-distal axis is established when the blade can be distinguished from the sheath, readily apparent in species with ligules (Sylvester, Cande, and Freeling, 1990 ). The central-lateral axis is apparent internally by the midvein and lateral bundles, and in some species by crystal-containing cell files adjacent to the bundles (Croxdale et al., 1992 ; Croxdale, 1998 ). Externally, the epidermis is organized into longitudinal tracts, each tract bearing similar cells and sometimes papillae (Rasmussen, 1986 ). The dorsal-ventral axis may be designated by a ligule or differential growth of the vascular tissue or mesophyll cells. Divisions responsible for generating stomata occur late in leaf development (Croxdale et al., 1992 ) and, as shown by the corn mutant tangled 1, are under separate regulatory control from generic epidermal divisions (Smith, Hake, and Sylvester, 1996 ).

Stomata in the monocot barley appear in visibly ordered arrays adjacent to cell files near vascular bundles. The predictable placement of stomata led Bünning to suggest that cell divisions in already ordered cells established stomatal distribution. Bünning's theory, which also is called the cell lineage theory, does not address how cells are designated into the stomatal pathway, only with the predictable sequence of divisions that generate stomata from existing cells (Fig. 3B). He is suggesting that stomata are prepatterned, an interpretation disputed by Sachs (1994) , who contends that stomata are not prepatterned in either monocotyledons or dicotyledons because stomatal arrest occurs in each group.

Although results from new studies (Sachs, 1974 ; Rasmussen, 1986 ; Croxdale et al., 1992 ) support the contention that stomata arise by divisions in blocks of ordered cells, Bünning's theory fails to explain several aspects of stomatal distribution. First, not all cells in a block undergo division to yield stomata. Selected cell files have no stomata, while neighboring files are enriched with stomata. Second, sometimes stomata are grouped in linear series, separated from one another by single epidermal cells, and other times stomata are separated by multiple epidermal cells. Lastly, the theory does not explain arrested, immature, or aborted stomata, which are common on these leaves.

Sachs uses the presence of arrested stomata as evidence that stomatal patterning takes place during development, in leaves of Sansevieria and phylloclades of Ruscus hypoglossum for example (Kagan and Sachs, 1991 ; Sachs, Novoplansky, and Kagan, 1993 ). The correctness of his interpretation depends on one's definition of stomatal patterning. If one views stomatal patterning as all events that contribute to the final pattern of stomata, his interpretation is accurate. If one views stomatal patterning as being concerned only with the specification of cells into this specialized pathway, then it might be said that arrest of stomata is a differentiation failure, not an aspect of patterning. Since stomatal arrest yields a more ordered pattern than would have been present had all stomata matured, patterning has been termed Darwinian (survival of the fittest) (Edelman, 1987 ; Held, 1992 ) or epigenetic (Sachs, 1988 ; Kagan and Sachs, 1991 ). However, stomatal development is not complete when they arrest, so fitness is not related to functional ability. Therefore, selection does not occur based on functional fitness, but on position, which is reflected in increasing stomatal order.

Developmental arrest might result from inhibition or from the scarcity of factors essential for continued development. Such factors might be available in differentiating leaf regions and account for epigenetic selection. The stoma that begins to develop first may outcompete those that arise slightly later. The latter is a simpler and, in this author's view, more parsimonious solution to regulating stomatal development because only molecules necessary for stomatal differentiation are required (Boetsch, Chin, and Croxdale, 1995 ). If inhibition regulated stomatal development, both an inhibitor as well as a receptor for the inhibitor would be necessary. The synthesis of these components, and their appropriate temporal expression, would also need to be regulated. Inhibition may be a convenient way of describing stomata that cease to develop, but use of this term tends to restrict one's thinking so that other equally or more likely possibilities are not considered. Stomatal initials that arrest in Tradescantia do not remain undifferentiated; they enter the epidermal cell pathway as evidenced by their expansion and accumulation of secondary metabolites (Boetsch, Chin, and Croxdale, 1995 ).

Korn (1993) uses modeling to understand the growth dynamics of different epidermal cell types, the timing of stomatal origin, and the rate of stomatal induction necessary to generate the spatial pattern present on mature leaves. He established models to account for mature stomatal distribution in Chlorophytum cosmosum, Iris pumila, and Allium cepa. These studies predict spatial aspects of stomatal arrangement, but not how particular cells are targeted to the stomatal pathway. Patterning by inhibition is supported in the dicot Pelargonium by either a mathematical or geometric model (Korn, 1993 ), although the kinetics of pattern was not considered (Sachs, 1994) . Korn's growth models contribute valuable information to our understanding of the epidermal layer. However, my goal is to understand the mechanism of patterning, so my focus must be trained on cells and how they become different from their neighbors.

Charlton (1990) suggested the cell cycle regulated stomatal patterning based on two pieces of evidence: the position of stomatal initials at a common boundary in the leaf base and the tendency of stomata and of epidermal cells to occur in linear groups (Fig. 3C). He postulated that a zone existed at the base of the leaf where cells are targeted for the stomatal pathway and that the fate of a cell entering this area is dependent on the cell's position in the division cycle. Those cells closest to mitosis are more likely to divide forming a stomatal initial than cells distant from mitosis. Since sister cells maintain a high degree of cell cycle synchrony for several cycles (Webster, 1979 ), sister cells and their derivatives would be close to mitosis upon entering this zone and would be patterned to the same fate (Charlton, 1990 ). Thus, a long string of cells patterned to a common fate will be of common ancestry.

In Tradescantia, support for the involvement of the cell cycle was found in precursors of stomatal strings, groups of stomata that occur in linear series (Chin et al., 1995 ). Strings of stomatal precursors can be identified by their position near the leaf base and proximal to the stomatal initial region and by their replicated DNA (Chin et al., 1995 ). Cells in actively dividing regions of Tradescantia rest in G1 (Webster, 1979 ; Kudirka and Van't Hof, 1980 ), while cells with replicated DNA go on to divide. Using DAPI, we identified stomatal strings based on intensity of nuclear staining and location, in support of Charlton's original suggestion that cell cycle position is a critical aspect of stomatal patterning and could result in a string of cells being targeted to the same pathway. Their common position in the cell cycle indicates cells are at a similar point in the division cycle when they are specified to the stomatal pathway and stomatal groups develop in synchrony. Tradescantia leaves, as do maize leaves, bear epidermal cells in linear series (Croxdale et al., 1992 ). The distribution of string lengths is the same in the mature leaf as at the leaf base indicating our correct identification of stomatal precursor strings and the absence of subsequent divisions altering original string length (Chin et al., 1995 ).

A unifying theory of stomatal patterning in angiosperms
Charlton's theory of monocot stomatal patterning explains that cells are selected to become stomata based on their position in the cell division cycle as they are displaced through the patterning region. Now there is evidence of the cycle's importance in Tradescantia stomatal development. The involvement of the cell cycle provides a means of explaining the species-specific variation in stomatal patterning because each array would reflect that species' cell cycle dynamics and the longevity of the patterning signal. The scattered intercalary growth of dicot leaves complicates their use in stomatal patterning studies, but a recent observation is consistent with the possibility that cell cycle position may also be critical in this group (Fig. 4). Arabidopsis cotyledons generate stomata in non-steady-state kinetics (M. Geisler and F. Sack, Ohio State University, personal communication), and it has long been known that dicot meristemoids divide a variable number of times before producing a guard mother cell (GMC). The position in the cell cycle when the patterning signal is present may dictate whether a GMC is produced. This would account for the waves of GMCs throughout the leaf and link their patterning to the monocotyledon type where cell cycle position dictates whether a cell responds when a stomatal-inducing signal is present.

Recent reports highlight unusual features of the cell cycle in stomatal patterning of dicotyledonous leaves. In Arabidopsis, trichomes and epidermal cells enter the endomitotic cycle and acquire DNA levels up to 64C while stomata remain diploid and remain in the mitotic cycle (Melarango, Mehorotra, and Coleman, 1993 ). Even at maturity Arabidopsis guard cells express genes indicating competency for cell division (Serna and Fenoll, 1997 ), an unusual feature of a terminally differentiated cell. Remaining within the cell division cycle may explain why sugar beets were easily transformed from guard cell protoplasts (Hall, Riksen-Bruinsma, and Weyens, 1996 ) and why isolated stomata are able to assume several different fates and express different gene products when cultured (Taylor et al., 1998 ). While one might argue that the relationship between stomata and the cell cycle is merely circumstantial, the involvement of the cycle in stomatal patterning is a testable hypothesis.

If the cell division cycle is a component of dicotyledon stomatal patterning, then the expression of cycle genes will correlate with GMC formation. Since GMC formation takes place episodically in the Arabidopsis leaf, this expression will occur episodically throughout the leaf epidermis. GMCs also should undergo equal divisions to form guard cells across the leaf in near synchrony. The nature of the molecule that signals or induces particular cells to enter the stomatal pathway is not yet known.

Our understanding of the plant cell division cycle, its regulation, and interaction of gene products is expanding, following advances made primarily in yeast. Although there appear to be differences in cycle regulation by kingdom (Fobert et al., 1996 ; Zhang, Letham, and John, 1996 ), many features of cycle regulation are common among plants, algae, yeast, and animals (John, Sek, and Lee, 1989 ; John et al., 1990 ). Indicators and localization of specific cell-division-cycle products have been detected in plant protoplasts (Ferreira et al., 1994 ), tissue cultures (Savoure et al., 1994 ), and intercalary (Sauter and Kende, 1992 ; Lorbiecke and Sauter, 1997 ) and apical meristems (Fobert et al., 1994 ). Transcriptional regulation of numerous gene products that function in independent regulatory pathways has been associated with the cell cycle (Francis and Halfor, 1995 ; Magyar et al., 1997 ) and cycle products appear in systems developing naturally (Ferreira et al., 1994 ) or when stimulated by specific factors (Savoure et al., 1994 ) or plant hormones (Sauter and Kende, 1992 ; Zhang, Letham, and John, 1996 ; Lorbiecke and Sauter, 1997 ).

Since the basic patterns of cell division and stomatal formation are known in angiosperm leaves, localization of cycle-specific gene products would reveal the relationships between cell division and cell fate. Because stomata are highly specialized cell types that are critical to plant survival and show a unique association to the cell division cycle, they make good candidates for study.


Stomatal patterning can be viewed from different perspectives and used as a model system for investigations in developmental biology, morphology, evolution, ecology, and physiology. The function of these specialized pores—and probably their distribution—has played a critical role in plant evolution and the successful exploitation of the terrestrial environment. Stomatal patterns are ordered indicating position complements the internal geometry of the plant organ and its photosynthetic function. Although quantitative aspects of stomatal pattern (degree of order and absolute number of stomata) in extant plants may permit adaptation to changing atmospheric conditions, the mechanism and plasticity of patterning are central in understanding the evolution of land plants.

The critical questions in stomatal patterning today revolve around two issues: the cell cycle and cellular communication. As the evidence presented here indicates, the patterning of stomata in angiosperms is likely coupled to the cell cycle. This connection can be tested directly by altering the cycle dynamics and noting the final stomatal pattern. The second critical question is centered on cellular connections because the molecule that specifies stomatal fate may come from a cell other than the one that will be specified. Knowing which cells are symplastically connected during the time of specification tells us which routes are available for specification. Cells that are symplastically isolated from their neighbors may still receive signals from them, but the mechanics of the process would differ. Knowing the routes of travel provides essential information regarding the possible pathways of signal movement.

The ultimate goal of all developmental biology studies is to understand how undifferentiated cells acquire their fate. Patterning decisions are made throughout plant growth, from the zygote onward. What is learned about distribution of stomata may be applicable to other situations, such as trichomes, root hairs, and crystal distribution, for patterning mechanisms are likely conserved. What is successful in one circumstance is likely successful in another; evolution works with the tools at hand.


1 The author thanks Tsvi Sachs and Fred Sack for stimulating discussions regarding patterning; Kandis Elliot and Claudia Lipke for assistance with illustrations; and M. Christianson for providing constructive comments on an early version of the manuscript. 

2 Author for correspondence ([email protected] ).

Literature Cited

Baranova, M. 1992 Principles of comparative stomatographic studies of flowering plants. Botanical Review 58: 49–99.

Beerling, D. J., and W. G. Chaloner. 1993 Stomatal density responses of Egyptian Olea europaea L. leaves to CO2 change since 1327 BC. Annals of Botany 71: 431–435.

Beyschlag, W., H. Pfanz, and R. J. Ryel. 1992 Stomatal patchiness in Mediterranean evergreen sclerophylls: phenomenology and consequences for the interpretation of the midday depression in photosynthesis and transpiration. Planta 187: 546–553.

Boetsch, J., J. Chin, and J. Croxdale. 1995 Arrest of stomatal initials in Tradescantia is linked to the proximity of neighboring stomata and results in arrested initials acquiring properties of epidermal cells. Developmental Biology 167: 28–38.

———, ———, M. Ling, and J. Croxdale. 1996 Elevated carbon dioxide affects the patterning of subsidiary cells in Tradescantia stomatal complexes. Journal of Experimental Botany 47: 925–932.

Bouhidel, K., and V. F. Irish. 1996 Cellular interactions mediated by the homeotic PISTILLATA gene determine cell fate in the Arabidopsis flower. Developmental Biology 174: 22–31.

Bünning, E. 1956 General processes of differentiation. In F. Milthorpe [ed.], The growth of leaves, 18–30. Butterworths, London, UK.

Charlton, W. 1990 Differentiation in leaf epidermis of Chlorophytum comosum Baker. Annals of Botany 66: 567–578.

Chin, J., W. Yong, J. Smith, and J. Croxdale. 1995 Linear aggregations of stomata and epidermal cells in Tradescantia leaves: evidence for their group patterning as a function of the cell cycle. Developmental Biology 167: 39–46.

Clark, P. J., and F. C. Evans. 1954 Distance to nearest neighbor as a measure of spatial relationships in populations. Ecology 35: 445–453.

Croxdale, J. 1998 Stomatal patterning in monocotyledons: Tradescantia as a model system. Journal of Experimental Botany 49: 279–292.

———, and K. Omasa. 1990 Chlorophyll a fluorescence and carbon assimilation in developing leaves of light-grown cucumber. Plant Physiology 93: 1078–1082.

———, J. B. Johnson, J. Smith, and B. Yandell. 1992 Stomatal patterning in Tradescantia: an evaluation of the cell lineage theory. Developmental Biology 167: 39–46.

Cui, M., T. C. Vogelmann, and W. K. Smith. 1991 Chlorophyll and light gradients in sun and shade leaves of Spinacia oleracea. Plant Cell and Environment 14: 493–500.

Daley, P. F., K. Raschke, J. T. Ball, and J. A. Berry. 1989 Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence. Plant Physiology 90: 1233–1238.

Dollahon, N. R., A. B. Maksymowych, M. Galant, and J. A. J. Orkwiszewski. 1988 Scanning electron microscope studies of Avena coleoptiles during primary leaf emergence. Acta Societatis Botanicorum Poloniae 57: 431–445.

Edelman, G. M. 1987 Neural Darwinism: the theory of neuronal group selection. Basic Books, New York, New York, USA.

Edwards, D., K. L. Davies, and L. Axe. 1992 A vascular conducting strand in the early land plant Cooksonia. Nature 357: 683–685.

———, H. Kerp, and H. Hass. 1998 Stomata in early land plants: an anatomical and ecophysiological approach. Journal of Experimental Botany 49: 255–278

Fahn, A. 1990 Plant anatomy, 4th ed. Pergamon, Oxford, UK.

Ferreira, P. C. G., A. S. Hemerly, J. D. Engler, M. Van Montagu, G. Engler, and D. Inze. 1994 Developmental expression of the Arabidopsis cyclin gene cyc2At. Plant Cell 6: 1763–1774.

Fobert, P. R., E. S. Coen, G. J. P. Murphy, and J. H. Doonan. 1994 Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants. European Molecular Biology Organization 13: 616–624.

———, V. Gaudin, P. Lunness, E. S. Coen, and J. H. Doonan. 1996 Distinct classes of cdc2-related genes are differentially expressed during the cell division cycle in plants. Plant Cell 8: 1465–1476.

Fowden, L., T. Mansfield, and J. Stoddart. 1993 Plant adaptation to environmental stress. Chapman and Hall, London, UK.

Francis, D., and N. G. Halfor. 1995 The plant cell cycle. Physiologia Plantarum 93: 365–374.

Fuchs, C. 1975 Ontogenèse foliaire et acquisition de la forme chez le Tropaeolum peregrinum L. Annales des Sciences Naturelles A. Botanique Series 12, 16: 321–389.

Gould, S. J. 1991 Bully for brontosaurus. Norton Press, New York, New York, USA.

Hake, S., and M. Freeling. 1986 Analysis of genetic mosaics shows that extra epidermal cell divisions in Knotted mutant maize plants are induced by adjacent mesophyll cells. Nature 320: 621–623.

Hall, R. D., T. Riksen-Bruinsma, and G. J. Weyens. 1996 A high-efficiency technique for the generation of transgenic sugar beets from stomatal guard cells. Nature Biotechnology 14: 1133–1138.

Held, L. I. 1992 Models for embryonic periodicity. Karger, Basel, Switzerland.

Jarvis, P. J., and T. A. Mansfield. 1981 Stomatal physiology. Cambridge University Press, New York, New York, USA.

John, P. C. L., F. J. Sek, J. P. Carmichael, and D. W. McCurdy. 1990 P34-cdc2 homologue level, cell division, phytohormone responsiveness and cell differentiation in wheat leaves. Journal of Cell Science 97: 627–630.

———, ———, and M. G. Lee. 1989 A homolog of the cell cycle control protein p34cdc2 participate in the division cycle of Chlamydomonas, and a similar protein is detectable in higher plants and remote taxa. Plant Cell 1: 1185–1194.

Kagan, M. L., N. Novoplansky, and T. Sachs. 1992 Variable cell lineages form the functional pea epidermis. Annals of Botany 69: 303–312

———, and T. Sachs. 1991 Development of immature stomata: evidence for epigenetic selection of a spacing pattern. Developmental Biology 146: 100–105

Korn, R. W. 1972 Arrangement of stomata on the leaves of Pelargonium zonale and Sedum stahlii. Annals of Botany 36: 325–333

———. 1993 Evidence in dicots for stomatal patterning by inhibition. International Journal of Plant Sciences 154: 367–377

Kudirka, D. T., and J. Van't Hof. 1980 G2 arrest and differentiation in the petal of Tradescantia clone 4430. Experimental Cell Research 130: 443–480

Larkin, J. C., N. Young, M. Prigge, and M. D. Marks. 1996 The control of trichome spacing and number in Arabidopsis. Development 122: 997–1005

Leick, E. 1955 Periodische Neuanlage von Blattstomata. Flora 142: 45–64.

Lorbiecke, R., and M. Sauter. 1997 A ribosomal 5S-rRNA-binding protein gene from rice (Oryza sativa L.) is regulated in a cell cycle phase-specific manner and in response to gibberellin. Journal of Plant Physiology 151: 334–338

Magyar, Z., T. Meszaros, P. Miskolcz, M. Deak, A. Feher, S. Brown, E. Kondorosi, A. Athanasiadis, S. Pongor, M. Bilgin, L. Bako, C. Koncz, and D. Dudits. 1997 Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells. Plant Cell 9: 223–235

Malone, S. R., H. S. Mayeux, H. B. Johnson, and H. W. Polley. 1993 Stomatal density and aperture length in four plant species grown across a subambient CO2 gradient. American Journal of Botany 80: 1413–1418

Mansfield, T. A. 1976 Effects of air pollutants on plants. Cambridge University Press, Cambridge, UK.

———, W. J. Davies, and R. A. Leigh. 1993 The transpiration stream: a theme. Royal Society, London, UK.

Marks, M. G. 1997 Molecular genetic analysis of trichome development in Arabidopsis. Annual Review of Plant Physiology and Plant Molecular Biology 48: 137–163.

Martin, G., S. A. Josserand, J. F. Bornman, and T. C. Vogelmann. 1989 Epidermal focussing and the light microenvironment within leaves of Medicago sativa. Physiologia Plantarum 76: 485–492.

Marx, A., and T. Sachs. 1977 The determination of stomata pattern and frequency in Anagallis. Botanical Gazette 138: 385–392.

McElwain, J. C., and W. G. Chaloner. 1995 Stomatal density and index of fossil plants track atmospheric carbon dioxide in the Paleozoic. Annals of Botany 76: 389–395

Melarango, J. E., B. Mehorotra, and A. W. Coleman. 1993 The relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis. Plant Cell 5: 1661–1668.

Mott, K. A., and T. N. Buckley. 1998 Stomatal heterogeneity. Journal of Experimental Botany 49: 407–418

Omasa, K., Y. Hashimoto, P. J. Kramer, B. R. Strain, I. Aiga, and J. Kondo. 1985 Direct observation of reversible and irreversible stomatal responses of attached sunflower leaves to SO2. Plant Physiology 79: 153–158

———, and M. Onoe. 1984 Measurement of stomatal aperture by digital image processing. Plant and Cell Physiology 25: 1379–1388

Palevitz, B. A., and P. K. Hepler. 1985 Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer Yellow. Planta 164: 473–479

Pappas, T., P. McManus, P. Vanderveer, and J. Croxdale. 1988 Characterization of stomatal development in Dianthus chinensis. Canadian Journal of Botany 66: 142–149.

Payne, W. W. 1979 Stomatal patterns in embryophytes: their evolution, ontogeny and interpretation. Taxon 28: 117–132

Poethig, R. S., and I. M. Sussex. 1985 Cellular parameters of leaf development in tobacco: a clonal analysis. Planta 165: 170–184

Radoglou, K. M., and P. G. Jarvis. 1990 Effects of CO2 enrichment on four poplar clones. II. Leaf surface properties. Annals of Botany 65: 627–632

Ramonell, K., M. Crispi, and M. E. Musgrave. 1997 Changes in stomatal density in Arabidopsis thaliana L. Heynh. Grown under low oxygen atmospheres. Eighth International Conference on Arabidopsis Research Meeting Schedule and Abstracts, 6–22.

Raschke, K., J. Patzke, P. Daley, and J. Berry. 1990 Spatial and temporal heterogeneities of photosynthesis detected through analysis of chlorophyll-fluorescence images of leaves. Current Research in Photosynthesis 4: 573–578.

Rasmussen, H. 1986 Pattern formation and cell interactions in epidermal development of Anemarrhena asphodeloides (Liliaceae). Nordic Journal of Botany 6: 467–477

Sachs, T. 1974 The developmental origin of stomata pattern in Crinum. Botanical Gazette 135: 314–318.

———. 1979 Cellular interactions in the development of stomatal patterns in Vinca major L. Annals of Botany 43: 693–700

———. 1988 Epigenetic selection, an alternative mechanism of pattern formation. Journal of Theoretical Biology 134: 547–559

———. 1994 Both cell lineages and cell interactions contribute to stomatal patterning. International Journal of Plant Sciences 155: 245–247

———, and P. Benouaiche. 1978 A control of stomata maturation in Aeonium. Israel Journal of Botany 27: 47–53

———, ———, and M. L. Kagan. 1993 Variable development and cellular patterning in the epidermis of Ruscus hypoglossum. Annals of Botany 71: 237–243.

Sauter, M., and H. Kende. 1992 Gibberellin-induced growth and regulation of the cell division cycle in deepwater rice. Planta 188: 362–368

Savoure, A., Z. Magyar, M. Pierre, S. Brown, M. Schultz, D. Dudits, A. Kondorosi, and E. Kondorosi. 1994 Activation of the cell cycle machinery and the isoflavonoid biosynthesis pathway by active Rhizobium meliloti nod signal molecules in Medicago microcallus suspensions. European Molecular Biology Organization 13: 1093–1102.

Schriber, U., R. Fink, and W. Vidaver. 1977 Fluorescence induction in whole leaves: differentiation between the two leaf sides and adaptation to different light regimes. Planta 133: 121–129

Serna, L., and C. Fenoll. 1997 Tracing the ontogeny of stomatal clusters in Arabidopsis with molecular markers. Plant Journal 12: 747–755

Sifton, H. B. 1945 Air-space tissue in plants. Botanical Review 11: 108–143

Smith, D. L., and W. M. Watt. 1986 Lithocysts, trichomes, hydathodes and stomata in leaves of Pilea cadierei Gagnep. and Guill. (Urticaceae). Annals of Botany 58: 155–166

Smith, L. G., S. Hake, and A. W. Sylvester. 1996 The tangled-1 mutation alters cell division orientations throughout maize leaf development without altering leaf shape. Development 122: 481–489

Stace, C. A. 1966 The use of epidermal characteristics in phylogenetic considerations. New Phytologist 65: 304–318

Stockey, R. A., and I. J. Atkinson. 1993 Cuticle micromorphology of Agathis Salisbury. International Journal of Plant Sciences 154: 187–224

———, and H. Ko. 1990 Cuticle micromorphology of Dacrydium (Podocarpaceae) from New Caledonia (South Pacific Ocean). Botanical Gazette 151: 138–149

Sylvester, A. W., W. Z. Cande, and M. Freeling. 1990 Division and differentiation during normal and liguless-1 maize leaf development. Development 110: 985–1000.

Szymkowiak, E. J., and I. M. Sussex. 1996 What chimeras can tell us about plant development. Annual Review of Plant Physiology and Plant Molecular Biology 47: 351–376.

Taylor, J. E., B. Abram, G. Boorse, and G. Tallman. 1998 Approaches to evaluating the extent to which guard cell protoplasts of Nicotiana glauca (tree tobacco) retain their characteristics when cultured under conditions that affect their survival, growth, and differentiation. Journal of Experimental Botany 49: 377–386

Taylor, T. N., and E. L. Taylor. 1993 The biology and evolution of fossil plants. Prentice Hall, Englewood Cliffs, New Jersey, USA.

Terashima, I. 1992 Anatomy of non-uniform leaf photosynthesis. Photosynthesis Research 31: 195–212.

———, and Y. Inoue. 1984 Comparative photosynthetic properties of palisade tissue chloroplasts and spongy tissue chloroplasts of Camellia japonica L.: functional adjustment of the photosynthetic apparatus to light environment within a leaf. Plant and Cell Physiology 25: 555–563

———, and ———. 1985a Palisade tissue chloroplasts and spongy tissue chloroplasts in spinach: Biochemical and ultrastructural differences. Plant and Cell Physiology 26: 63–75

———, and ———. 1985b Vertical gradient in photosynthetic properties of spinach chloroplasts dependent on intra-leaf light environment. Plant and Cell Physiology 26: 781–785

Tichá, I. 1982 Photosynthetic characteristics during ontogenesis of leaves. 7. Stomata density and sizes. Photosynthetica 16: 375–471

Turrell, F. M. 1936 The area of the internal exposed surface of dicotyledon leaves. American Journal of Botany 23: 255–264

Vogelmann, T. C., J. F. Bornman, and S. Josserand. 1989 Photosynthetic light gradients and spectral regime within leaves of Medicago sativa. Philosophical Transactions of the Royal Society of London B 323(1216): 411–422.

Webster, P. 1979 Variation in sister-cell cycle durations and loss of synchrony in cell lineages in root apical meristems. Plant Science Letters 14: 13–22

Weyers, J., and T. Lawson. 1997 Heterogeneity in stomatal characters. Advances in Botanical Research 26: 318–352.

Willmer, C. M., and M. D. Fricker. 1996 Stomata, 2nd ed. Chapman and Hall, London, UK.

Wolf, S. D., W. K. Silk, and R. E. Plant. 1986 Quantitative patterns of leaf expansion: Comparison of normal and malformed leaf growth in Vitis vinifera cultivar Ruby Red. American Journal of Botany 73: 832–846

Woodward, F. I. 1987 Stomatal numbers are sensitive to increases in CO2 from pre-industrial levels. Nature 327: 617–618

Yang, M., and F. D. Sack. 1995 The too many mouths and four lips mutations affect stomatal production in Arabidopsis. Plant Cell 7: 2227–2239

Zhang, K., D. S. Letham, and P. C. L. John. 1996 Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34-cdc2-like H1 histone kinase. Planta 200: 2–12

Ziegler, H. 1987 The evolution of stomata. In E. Zeiger, G. Farquhar, and I. Cowan [eds.], Stomatal function. Stanford University Press, Stanford, California, USA.



Fig. 1. Two-dimensional spatial patterns. (A) Random distribution; (B) Ordered distribution; (C) a magnified view from Fig. 1A ; (D), a magnified view from (B)

figure 1


Fig. 2. Leaf tessellations from a portion of a blade panel with the margin at the lower left and the area near the midrib on the right. The position of an underlying vascular bundle is indicated as a solid line within the tessellation. Markers (cell types in this case) establish tile centers and tile edges are lines drawn midway between adjacent tile centers. (A) Tessellation based on stomata and arrested stomata (shaded) as markers. (B) Tessellation based only on stomata. (C) Relative tile distributions from the above tesselations. The relative values of tile areas are plotted as a function of count (number)

figure 2


Fig. 3. Existing theories of stomatal patterning. (A) The inhibition theory, which was proposed by Bünning to explain patterning in dicot leaves. He postulated that mature stomata released an inhibitor that created a field in which no new stomata could arise. When growth occurred between existing stomata, the effectiveness of the inhibitory field was exceeded and new stomata could originate. The darkened cells are meristematic cells capable of becoming stomata after enough growth has occurred to separate existing stomata from one another. (B) In the cell lineage theory, which Bünning put forward to explain stomatal patterning in monocot leaves, he stated that the origin of stomata was based on previously ordered divisions and their distribution was previously designated. (C) The cell cycle theory by Charlton was advanced to explain linear groups and individual stomata in monocots. The cell cycle positions are shown in diagramatic form in the upper left. Either a single cell or a series of sister/cousin cells might all be at a similar position in the cell cycle and become specified to the same fate. Beginning at the figure's lower left a single cell and its derivatives, which tend to undergo synchronous cell divisions, create a string of cells which could all become stomata over three cell cycles

figure 3


Fig. 4. A unified theory of stomatal patterning in angiosperms is predicated on the cell cycle theory of Charlton. In Arabidopsis, the dicot example, leaves have scattered, clonal growth, and only proliferating cells in the appropriate position of the cell division cycle (red) are capable of producing a stoma; other groups of proliferating cells (blue) are not capable of being specified to the stomatal fate. As the leaf grows, new groups of proliferating cells arise and some of the existing proliferating cells are now at the appropriate position in the cycle to become stomata. In Tradescantia, the monocot example, leaves have polarized growth and the proliferating cells are massed at the leaf base (blue). Some of these cells are at the appropriate position in the cell division cycle and become stomatal precursors (red). As the proliferating cells produce additional derivatives, the stomatal precursors divide unequally to produce a small stomatal initial (red) and a large epidermal cell. With further growth, the stomatal initials divide and differentiate as guard cells. Mature stomata first appear at the leaf tip and advance basipetally

figure 4