[advances in botanical research] advances in botanical research volume 26 volume 26 || heterogeneity...

Heterogeneity in Stomatal Characteristics

JONATHAN D . B . WEYERS and TRACY LAWSON

Department of Biological Sciences. University of Dundee. Dundee DDl 4HN. UK

I . Introduction ............................................................................. A . Rationale and Scope ........................................................... B . Relevant Stomatal Characteristics .......................................... C Historical Context . ..............................................................

11 . Terminology ............................................................................. A . Definitions of “Micro” and “Macro“ Heterogeneity ................. B. Discriminating between “Macro” Forms of Heterogeneity .........

111 . Methods .................................................................................. A . Anatomical Characteristics B . Stomatal Behaviour ............................................................ C . Data Analysis and Presentation ............................................

...................................................

IV . Evidence .............. .............................................................. A . Spatial Heteroge y in Anatomical Characteristics ................. B . C .

.......................... Spatial Heterogeneity in Stomatal Behaviour Temporal Heterogeneity in Stomatal Behaviour .......................

V . Conclusions ............................. ............................................ A . Causes of Stomatal Heterogeneity ..... ............................... B . Relevance of Heterogeneity in Stomatal Characteristics ............

D . Summary ...................................................................

Acknowledgements ........................................... ............ Appendix ................................................................................

........................... C . Topics Requiring Further Investigation

References ........................................... ...............................

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Advances in Botanical Research Val . 26 incorporating Advances in Plant Pathology ISBN 0-12-C0592&6

Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved

318 J. D. B. WEYERS and T. LAWSON

I . INTRODUCTION

A. RATIONALE AND SCOPE

The fact that stomata are variable in many attributes is evident to any scientist who has researched their structure or physiology. Heterogeneity in stomatal characteristics (or related variables, such as stomatal conductance, g s ) is found at many levels, from the size, frequency and behaviour of individual guard cells to the gas exchange of whole plants or stands of plants (TichB, 1982; SolBrovh and PospiSilova, 1983; Spence 1987; Weyers and Meidner, 1990; PospiSilovi and SantrGtek, 1994; Willmer and Fricker, 1996). Adding to this complexity, variability is found in both spatial and temporal senses at all these scales (PospiSilova and Santr%ek, 1994; Weyers ef al. , 1997).

While this topic might initially seem narrow and even obscure, an understanding of the nature of stomatal heterogeneity and its origins is important. The phenomena involved might provide plants with some functional advantages and disadvantages as yet not fully determined. Knowledge of how heterogeneity influences plant productivity and water use efficiency may allow us to manipulate relevant characteristics in crop plants. In addition, heterogeneity in stomatal characteristics, where developmentally controlled, provides insights into mechanisms of cell differentiation and their control. Finally, stomatal heterogeneity impacts on experimental protocols and the analysis of data in a wide range of investigations and knowledge of it is therefore of great practical use.

In the light of the above, the aims of this review are four-fold: firstly, to provide working definitions for the main types of variability found at and below the whole-leaf level, and to discuss problems associated with distin- guishing these forms; secondly, to review the methods used to observe heterogeneity in stomatal characteristics, and to discuss their advantages and shortcomings; thirdly, to summarize current knowledge about the nature of stomatal heterogeneity; and fourthly, to outline where research in this area could profitably be directed in future. Where appropriate, illustrative examples are drawn from the authors’ own research chiefly involving the species Commelina communis L. and Phaseolus vulgaris L.

Relevant stomatal characteristics will be held to include anatomical traits that have a potential effect on gas exchange (see below). Variation in transpiration itself will be considered, as will photosynthesis, insofar as measurements involve an implied or measured stomatal influence. Heterogeneity will be considered in both spatial and temporal senses and at levels from the individual pore, through whole leaves to whole plants. Variation at the canopy, species or community level will not be covered in detail - this subject has recently been reviewed by Jarvis and McNaughton (1986), McNaughton and Jarvis (1991) and Kruijt et al. (1997).

HETEROGENEITY IN STOMATAL CHARACTERISTICS 319

B. RELEVANT STOMATAL CHARACTERISTICS

Building on the early work of van den Honert, modern plant physiologists have adopted the electrical circuit analogy as a logical and quantitatively substantiated model for describing influences on liquid and gas fluxes to, within and from plants (see e.g. Nobel, 1991; Jones, 1992). In particular, the conductance/resistance concept has been found to be useful to describe properties of each pathway component relating to mass transfer of water. The extent to which the anatomy of an individual stomatal pore defines its potential conductance g, in units of mol m-2 s-’ can be approximated from:

where Pa is the atmospheric pressure (Pa = J IT-^), D , is the water diffusivity in air (m2s-’), A , is the mean pore area (m2), R is the gas constant (J mol-* K-I), T is the mean of leaf and air temperature (K), d is the pore depth (m) and c is the “end correction” (m). Extending this approach to estimate the conductance of a small patch of stomata on a single leaf surface (gs), we have:

Pa D,A,SF ”= RT(d+2c)

where SF is the stomatal frequency (mP2). The above equations represent simplified models of gas exchange through

narrow pores. There is some disagreement in the literature about the method used to calculate the end correction (Nobel, 1991): while errors arising from this may be relatively trivial at narrow pore widths, they may become appreciable at wider apertures (see Weyers and Meidner, 1990). Account should be also taken of changes in Dw due to wall collisions in narrow channels; the necessary correction factor is approximately 0.9 at wide pore widths, reducing to 0.67 when pores become narrow (see Cowan and Milthorpe, 1968). In studies where high accuracy is required, the complex geometry of the ventral pore wall can be taken into account (e.g. Bange, 1953), as can interactions among diffusing species and the influence of mass flow (Leuning, 1983).

At the patch scale we can conclude that g, varies directly with stomatal area and stomatal frequency (the product of which gives the cross-sectional area available for gas fluxes), and that it varies inversely with pore depth. Figure I uses equation 2 to model for C. cornrnunis the sensitivity of g, to changes in component variables within empirically derived ranges. This presentation shows clearly that the main adjustment tog,is likely to be made physiologically, by way of pore width or aperture ( A ) . Changes in cell anatomy and spacing also affect g,, though to a lesser extent. Of these factors, stomatal frequency exerts


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Fig. 1. Predicted sensitivity of stomatal conductance to changes in pore dimensions and frequency within empirically derived ranges. The analysis employed the following typical ranges of values derived from observations of Commelina communis: stomatal aperture, 0-15 pm; stomatal frequency, 35-65 mm-2; pore length, 33.3- 40 pm; pore depth, 15-25 pm. Values within each range were used in equation 2 to predict stomatal conductance, assuming the pore to be elliptical and its area to be (P AL)/4. The double end correction was taken as (A/P)’.’ (Nobel, 1991). The vertical line represents the conductance obtained using the median values for each variable; this was 346 mmol m-* s-’. Effects of adjusting each anatomical characteristic within its estimated range were calculated, keeping the other values constant, and expressed as a percentage of the “median value”.

the most effect over its normal range. The effect of pore depth is next largest and pore length (L) has the least effect within its normal range. In terms of the current discussion, the important anatomical characteristics governing the stomatal contribution to gas flux are thus considered to be: stomatal aperture, pore depth and length, and stomatal frequency.

It should be noted that the gas flux density through a patch will depend not only on g,, but also on the relative sizes of the intercellular air space, cuticular and boundary layer conductances, as well as the chemical potential gradient for the gas under consideration. Moreover, as gas exchange is modelled at increasingly larger scales, the influence of individual pores (and hence of their anatomy and physiology) diminishes, though it can remain important in certain cases such as high wind speeds and isolated specimens (Jarvis and McNaughton, 1986; McNaughton and Jarvis, 1991).


C. HISTORICAL CONTEXT

Heterogeneity in stomatal activity was first observed nearly a century ago. An important observation was made by Francis Darwin (1898), who observed while testing his horn hygrometer device (a simple diffusion porometer): “in some leaves, particularly those of monocotyledons, the hygroscope gives very different readings in different parts of the leaf ’. Later, Knight (1916), who pioneered the use of viscous flow porometry , concluded that differences in conductivity over the surface of Ficus leaves were minimal. It was only in the 1960s, when a series of more sophisticated measurement techniques became available, that reports appeared showing spatial variation at the whole-leaf scale in measurements of g s , leaf temperature and stomatal frequency (e.g. Slavik, 1963; Cook et al., 1964; Raschke, 1965). However, these studies involved relatively few samples, and they were not subjected to sophisticated analysis. Hence, in a major review, Solarova and PospiSilova (1983) concluded that “little attention has been paid to heterogeneity of the leaf blade with respect to gas exchange”.

The findings of Downton et al. (1988) and Terashima et af. (1988) prompted renewed interest in this topic. These research groups showed that the assumption of uniform stomatal aperture had given rise to the erroneous conclusion that abscisic acid (ABA) affected photosynthesis directly rather than acting solely via stornatal closure. They concluded that “patchy” conductance was the cause of this error, as had been predicted by Laisk (1983). Their observations have been substantiated using a variety of methods and treatments (see discussion below), yet, while many authors have convincingly demonstrated patchy stomatal behaviour among regions on a leaf, others have reported variations of similar range but have interpreted them as involving smooth rather than abrupt transitions between zones (Hashimoto et al . , 1984; Smith et al., 1989). Meanwhile, the literature indicates some confusion over what exactly constitutes patchy behaviour and there is a need to clarify terminology and methods of analysis.

Researchers interested in the anatomy and distribution of stomata appear to have appreciated from the outset that a high degree of variability is present in relevant characteristics. In his seminal paper of 1927, Salisbury highlighted the fact that different species, plants, leaves and parts of leaves could all have different stomatal frequencies and, moreover, different proportions of guard cells to other cells in the epidermal layer. Salisbury coined the term stomatal index ( S I ) to quantify the latter ratio:

S F x 100 SF + ECF

SI = ( 3 )

where ECF is the frequency of cells other than guard cells. Ticha (1982) provided a comprehensive review of a large number of studies

322 J . D. B. WEYERS and T. LAWSON

concerning differences in stomatal frequency and index within and between plants. Research in this area appears to have been motivated by the potential for manipulating these characteristics via plant breeding to influence crop productivity and water use efficiency (Jones, 1987) and their use as a model system for studying control of plant cell differentiation (Sachs, 1991). In recent years, interest has been further prompted by the observation of Woodward (1987) that atmospheric pC02 can influence stomatal frequency and index and that there is a correlation between the post-industrial increase in atmospheric pC02 and these characteristics.

In contrast to the above-mentioned research themes concerning heterogeneity in stornatal behaviour and stornatal frequency, relatively little interest has been shown in differences in guard cell size, particularly pore depth, despite the fact that it is a relatively important determinant of pore conductance (Fig. 1).

11. TERMINOLOGY

Before proceeding, it will be valuable to define certain terms which have been current in the literature, but which have not necessarily been used in a consistent manner. For the purposes of this review, the term “heterogeneity” will encompass differences in a given stornatal characteristic at a defined scale of study. The term “variability” is regarded as a synonym and the adjectives “heterogeneous”, “variable” and “non-uniform” are used interchangeably to describe variable characteristics. The additional terminology suggested below is exemplified in relation to stornatal aperture/conductance at the scale of the whole-leaf and below, but may apply to other characteristics as well.

A. DEFINITIONS OF “MICRO” AND “MACRO” HETEROGENEITY

I . This form of heterogeneity (Fig. 2), referring to the differences in a characteristic (e.g. aperture) of individual stomatal pores in a given area, is effectively random or pseudo-random scatter about a local mean. It can also be termed “noise”. The sample area used to obtain a local mean value should be defined with reference to the assumed “macro” pattern of variance (see below) and could, for example, refer to a field of view or an areola (area between minor veins).

Noise should be measured on an appropriate “micro” scale that would normally involve measurements related to individual pores. It can be expressed as a statistic of dispersion, possibly relative to the local mean value (e.g. a coefficient of variation). However, the shape of frequency distributions of samples of stornatal characteristics may not be symmetrical (as in

“Micro” Heterogeneity: Variability among Adjacent Stomata


0 2 4 6 8 10 12 14

Distance from leaf margin (mm)

Fig. 2. Example of “noise” in a stomatal characteristic sampled in transect form. Individual stomatal apertures were measured in a 1.15 mm transect from the margin (left) to the mid-rib (right) of a Commelina communk L. leaf. The dashed line is the running mean value for each 500 pm segment of transect. Pairs of vertical lines indicate the position of veins crossing the transect. Redrawn from data originally presented by Smith et al. (1989).

the case of aperture, see Laisk et a l . , 1980), a fact that should influence the choice of descriptive statistics used and the type of statistical test employed to compare fields or areas.

2. These forms of variation occur at a scale between the individual pore and the whole leaf and involve “non-random” differences in the measured characteristic within the area considered. It is possible to contrast two possible forms of macro-variation: patches and trends:

Patches (Fig. 3a,b) are commonly understood in this context to constitute macroscopic areas (i.e. patches of leaf) each having a distinctive pore width or conductance that differs from that of neighbouring areas. The natural anatomical area to consider at the whole-leaf level is the areola. Patchiness can thus be defined in a more statistical sense as “adjacent regions differing by exhibiting mean values that differ significantly in the context of local variability”. It is implied that the local mean value is relatively constant over

“Macro” Heterogeneity: Patches and Trends


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Fig. 3. Diagrams illustrating idealized transect and contour map views of different forms of heterogeneity in stornatal characters. For simplicity, only four contour bands are shown in each case. (a) Transect across a leaf showing patchiness; (b) predicted “mosaic-like” contour map for a patchy stomatal characteristic; (c) transect across a leaf showing trends; (d) predicted “parallel line” contour map for a trendy stomatal characteristic.

the patch area, especially where patches are small; consequently, there is an expectation of relatively sharp transitions between patches (Fig. 3a). In a 2-D view, patches would be represented as a mosaic-like pattern over the leaf surface (Fig. 3b).

Trends (Fig. 3c,d) can be defined as involving “areas differing from others where there is a continuous and smooth transition between zones”. In contrast to patchiness, few sharp transitions between zones of leaf are expected, and, by definition therefore, little or no correlation with leaf venation pattern (Fig. 3c). In a 2-D view, trends would be represented by more-or-less parallel contour lines (Fig. 3d).

The term “patchiness” has been used to refer to either noise (e.g.


Cheeseman, 1991) or to general forms of micro- and macro-variation (PospiSilovri and Solarova, 1994), but we suggest that it might be better reserved for the specific meaning defined above.

B. DISCRIMINATING BETWEEN “MACRO” FORMS OF HETEROGENEITY

The goal of formulating a standard technique for quantifying the degree of non-uniform or patchy stornatal closure (van Kraalingen, 1990) requires a decision about the precise type of macro-heterogeneity that is involved. Where this appears to depend on species and treatment (see PospiSilova and Santrfieek, 1994; and discussion below), we need to make a quantitative distinction between the two types that is statistically based rather than one that is merely qualitative and possibly subjective. This is not only crucial for the analysis and presentation of data, but also for interpreting the effects of treatments on patterns and determining their physiological origin. Once again, although this matter is of prime importance for the analysis of variation in stomatal aperture and conductance, the principles are the same for other characteristics.

While patches and trends can be viewed as alternative forms of macro- variance, they represent extreme hypothetical cases, and the distinction between them might be blurred. For example, supposing the true variation to be “trendy” in nature, there could be a sharp transition between zones that might be interpreted as a division between patches. On the other hand, if patches exist, and the characteristic is not absolutely constant within each patch, the boundaries between patches may not be clear, making the variation appear trendy. The possibility that the form of heterogeneity on an individual leaf may alter through time or as conditions change (see below) further complicates any decisions.

To assess these possibilities quantitatively, it is necessary to take into account noise in the basic elements contributing to conductance at this scale - the stomata - and this requires that measurements are available at the individual pore scale. To date, there have been few reports providing sufficient information (Pospiiilova and SantrGEek, 1994). Technical problems are involved in obtaining data of sufficient quality (see below), and these will need to be overcome before the precise nature of macro-heterogeneity in stomatal characteristics can be resolved for specific cases.

111. METHODS

The intention in this section is to present a critical review of the methods available for assessing variation in stornatal characteristics at the whole-leaf scale and below. Stomata1 aperture, guard cell dimensions and the distances between stomata are all in the p.m range and hence must be measured using


some form of microscopy. A compromise must be struck between the resolution of the measurement and the convenience (and expense) of the instrument including any preparative and maintenance procedures. The best resolution (about 50 nm in practice) can be obtained with a scanning electron microscope (e.g. van Gardingen et al., 1989), but this technique is not convenient for large areas of leaf. The resolution of the standard light microscope (about 0.5-1 pm in practice) is satisfactory to measure some stomatal characteristics, but may lead to significant measurement error where the dimensions involved are below 10 p.m or so.

A. ANATOMICAL CHARACTERISTICS

Anatomical characteristics considered here will be chiefly those concerned with guard cell size and spacing. Relevant dimensions can be measured on fresh, preserved or replicated material. Fresh and preserved leaf material may be difficult to view microscopically because of difficulties of light transmission and scatter. There is also a danger of spatial distortion due to dehydration of the material if measurements are made over a prolonged period. These problems can be reduced with certain replication methods (Weyers and Meidner, 1990). Difficulties of interpreting negative replicas, such as obtained by applying nail varnish, can be overcome by using a two-stage technique (Fig. 4; Samson, 1961; Weyers and Johansen, 1985).

Measurement technique, sampling procedures and methods of data analysis must all be considered with care. Pore and guard cell length are relatively straightforward in definition (Fig. 5a) and are easy to measure directly via the microscope eyepiece, a projected image, a video monitor image or a photograph. Stomata1 frequency may appear to be a simple measure of the aerial density of stomata over the leaf surface, but there are a number of problems associated with obtaining relevant data. The normal method is to count the number of stomata within a field of view and divide the count by the field’s area. As Kubinova (1994) pointed out, an unbiased decision needs to be made about stomata appearing on the edges and corners of the field. One possible protocol for rectangular fields (Fig. 5b) is to include all stomata intersecting with two predefined sides and the corner between them, but to exclude those intersecting with the two remaining sides and three remaining corners.

A further problem arises due to the fact that there are generally no stomata on the epidermis which lies over veins (Fig. 4). A decision is required as to whether these areas are included when sampling or whether samples are restricted to interveinal areas. From the point of view of using stomatal frequency data to predict or model whole-leaf gas exchange, a value that includes vein areas would appear to be most valid, but the frequency distributions of data would almost certainly be skewed due to counting of fields within or overlapping vein areas which had low or zero stomatal


Fig. 4. Field of view of a silicone rubber impression of Commelina comrnunis L. leaf epidermis. Xantopren impression material was mixed with hardener (Bayer Ltd, Leverkusen, Germany) and applied to the lower leaf surface with minimal force. When hard, this negative replica was detached and placed on clear nail varnish freshly applied to a microscope slide. Note that individual pore widths can be measured, and that they are not uniform, even over this small area, although individual pores are roughly symmetrical about their longitudinal axis. Also, although the stomata appear to be relatively evenly spaced, there is none present in the epidermis lying over veins (top of field). The scale bar is approximately 100 km in length.

frequency. I t might be desirable to remove this source of variation in studies focused on stomatal development.

The appropriate size, number and arrangement of samples will depend on the aim of the research, the assumptions that can be made, and the time available for measurement. To provide samples covering a whole leaf, the number of observations required is large: there are 500000 stomata on a typical medium-sized leaf having an area of 5000mm' and mean stomatal frequency 100mm-'. In this example, even sampling 1 % of the stomata would involve 5000 measurements. If it is unrealistic to measure the whole population of stomata, due regard must be given to sampling strategy. In either light or electron microscopy, computerized counting and measurement technology can be used to reduce the tedium of making large numbers of measurements, while automatic image analysis has also been used for measuring stomatal aperture (Omasa ef al. , 1983; Omasa and Onoe, 1984; van Gardingen ef af., 1989).

328 J . D. B . WEYERS and T. LAWSON

Because of the inherent variability among adjacent stomata, it is normal to take samples representative of small areas on the leaf. The relatively high stomatal frequency (20400 per mm2, depending on species) means that it is generally unlikely that any macro-form of variation will influence samples so long as the sampling area is relatively small. To reduce unconscious operator bias, it is preferable to have a sampling protocol that spreads readings evenly in some predetermined way over the sampling area. The number of readings required per area to show predetermined differences can be estimated using objective statistical criteria (see e.g. Poole et al . , 1996).

Where data are to be mapped, the number and pattern of samples required per leaf depends on the type of intrinsic macro-variation and the algorithms used by the mapping program and should be determined at the outset of any investigation. Contrary to intuition, the largest number possible may not be ideal, as a mapping algorithm that assumes zero error in the measured variable may introduce pattern which is an artefact of natural sample variation. It might be better to have fewer, more accurate, values.

B. STOMATAL BEHAVIOUR

If stomatal behaviour is to be measured, researchers should be aware of a number of potential artefacts arising from their presence while taking readings. In the first place, human breath contains high partial pressures of C o t and H20; secondly, the operator or instrument might affect the PPFD incident on the leaf under study; and thirdly, the operator or instrument might affect the wind currents (and therefore the boundary layer) close to the leaf. All these possible changes in conditions might result in stomatal movement. For some studies, it may therefore be beneficial to enclose the plant under controlled conditions where these artefacts are minimized. Otherwise, the operator should always stand downwind and avoid casting shade over the plant.

Methods of measuring stomatal behaviour can be classified on the basis of whether they involve “direct” measures of stomatal pores or “indirect” measures which sum the activity of small populations of pores (Weyers and Meidner, 1990; Weyers et al., 1997). Direct measurements provide the

Fig. 5 . (Opposite) Anatomical measurements on stomata. (a) Plan and TS diagrams of a pair of guard cells, showing the different stomatal dimensions that are relevant (after Weyers and Meidner, 1990); (b) Diagram illustrating rules for unbiased measurement of stomatal frequency: the rectangular area represents an area within a field of view; filled pores represent stomata that should be included in the field count, open pores those that should not. Any two sides and one corner could be chosen (a priori) for the inclusion rule (after Kubinova, 1994).

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information required to assess variability at the individual pore level but are tedious to obtain. Indirect methods are usually more convenient, but averaging the responses of many stomata may obscure important information and can lead to artefacts. The destructive nature of some techniques precludes their use in time series measurements. Even non-destructive methods may cause artefacts when measurements are repeated on the same area one or more times. The fundamental problem is due to the act of measurement affecting the process being measured.

1. Measurements of Stomata1 AperturelArea The reports by Saxe (1979), Omasa et al. (1983), Kappen et al. (1987), and Gorton et al. (1989a,b) illustrate that useful information can be obtained by repeated direct microscopic measurement of pore dimensions, but the area that can be studied is small and the environmental conditions are not naturally determined. To study larger areas and obtain an indication of apertures in vivo, there is a need to “freeze” the stomata at one moment in time. Van Gardingen et al. (1989) successfully measured stomata1 dimensions from frozen specimens, while Weyers and Johansen (1985) succeeded in measuring stomata1 apertures from positives made from silicone rubber impressions. The latter method was deemed more applicable to areas at the whole-leaf scale, but it is potentially subject to measurement bias and requires that the species studied has relatively large stomata (Weyers et al. , 1997). For both techniques, the plane of focus is crucial (see Fig. 5a). This, combined with refraction and lens-like light-focusing effects within the cells of the epidermis (Poulson and Vogelmann, 1990), accounts for the paucity of reports involving computerized image analysis techniques.

2. Spot measurements of g, can be obtained with various forms of porometer. In general, diffusion porometers are easier to use, faster, and less subject to artefact (see Weyers and Meidner, 1990). Newer models of infra-red gas analyser (IRGA) also permit such measurements when cuvettes of small area are attached. These machines have the advantage of providing much more information than a simple porometer, including rates of net photosynthesis and estimates of leaf temperature and internal pC02 (Ci).

Three important difficulties arise with respect to the porometer/IRGA cuvette when attempting to measure variation in measured parameters over a whole leaf:

Measurements of Stoma ta 1 Con ductan ce

(a) Size of sampled area. Cuvettes of area 100-300 mm2 are now available; these are small enough to provide a reasonable number of spatially separate readings on a leaf of area 5000mm2 and above. However, for leaves below this size, the number of independent readings that can be made may be less than ideal;

HETEROGENEITY IN STOMATAL CHARACTERISTICS 33 1

(b) Clamping artefacts. Care must be taken to avoid artefacts that might arise specifically due to clamping, such as shading other areas of leaf, reaction to mechanical disturbance or changes in the wind-flow pattern; and

(c) Timing dificulties. Both diffusion porometers and IRGAs are capable of providing “snapshot” readings in 1-2min (in the former case, depending on actual conductance). Clearly then, a sample of 30 or so readings will take at least 30 min to obtain, and in this time it is possible for stomatal movements to occur and the pattern of heterogeneity to have changed.

3. The Infiltration Technique Liquid infiltration has long been used to estimate g,. Measurements are normally based on the rate of infiltration of a defined hydrocarbon liquid through the stomatal pore, or the viscosity of the liquid that is able to penetrate in a defined time (see Weyers and Meidner, 1990, for review). In a modern “whole-leaf” version of the technique (Beyschlag and Pfanz, 1990; Beyschlag et al., 1992), water or another liquid is forced into a detached leaf by an applied pressure difference across the epidermis. When back-lit, infiltrated areas appear lighter in colour (Plate 3). The correlation between g, and extent of infiltration is good, and the method reveals what appears to be “patchy” stomatal opening. Problems with this technique include:

(a) the need to detach the leaf and expose it to water or other solvents, both of which may result in rapid stomatal movements (Meidner, 1965; Weyers and Meidner, 1990);

(b) the fact that infiltration is an all-or-nothing event, which means that the technique essentially classifies groups of stomata either as “open” or “closed” according to whether some or all of them are above or below some liquid conductance threshold. In reality. there is likely to be a range of apertures present (Fig. 2) and the method cannot show this;

(c) difficulties in applying a consistent pressure difference across the epidermis, especially in the field, which may lead to difficulties in comparing results; and

(d) the fact that not all species are amenable to the technique (Beyschlag and Pfanz, 1990).

4. Measurement of Leaf Temperature Leaf temperature is a well-established indirect measure of stomatal activity, the underlying theory being that active transpiration causes cooling due to the latent heat of evaporation of water (Nobel, 1991). Differences between areas of high and low transpiration are of the order of 1-5 “C. Measurements have been made using thermistors (Cook et a[., 1964; Wigley and Clark, 1974) and infra-red thermometry (Hashimoto rt al. , 1984).

To be sure that leaf temperature reflects the rate of transpiration, it is


important that the incident PPFD has been constant over the leaf surface for a period before measurements begin. Passage of sunflecks over the leaf surface in the recent past might lead to local temperature increases due to absorbed radiation. Even if the PPFD is constant, leaf temperature may reflect differences in the boundary layer conductance rather than that of the stomata (Wigley and Clark, 1974).

5 Indirect Measures via Rates of Photosynthesis l4CO2 uptake and starch-iodine tests constitute destructive methods of measuring photosynthetic activity; by assuming that assimilation varies with g, under the experimental conditions, they can be used to demonstrate stomatal heterogeneity (Downton et a l . , 1988; Terashima et a l . , 1988; Gunasekera and Berkowitz, 1992). Similar assumptions lie behind analysis of photosynthetic activity via studies of non-photochemical quenching of chlorophyll fluorescence. In this technique, low Ci resulting from stornatal closure reduces the rate of electron transport through photosystem 11. This is monitored via the resulting increase in non-photochemical fluorescence quenching (Daley et al . , 1989). Stomata1 movements can be followed through time (Cardon et a l . , 1994) and the technique is essentially non-destructive, although the long-term effects on stomata of low pOz atmospheres and short flashes of “saturating” PPFD have not been fully reported. Finally, the method requires expensive and complex equipment.

The-e methods do not provide direct quantitative information about g,. Although the assumptions they are based on appear to be sound, the possibility that photosynthetic reactions might be affected by variables other than those controlled by local Ci or local g , should be considered, especially when relatively severe treatments are given. Perhaps more importantly, they do not provide information at the scale of individual pores. This could be a benefit in some studies as the local Ci is likely to be determined by a group of stomata, and in any case researchers may be more interested in effects on photosynthesis than on stomatal responses themselves. The downside is that a quantitative description of the type of stornatal heterogeneity involved is not always possible, although the frequency distributions of pixel intensities can be analysed (e.g. Mott, 1995).

C. DATA ANALYSIS AND PRESENTATION

Measurements of stornatal dimension, including aperture, are readily amenable to statistical analysis. This should take into account skewness in data, which might occur with randomly sampled stornatal frequency data or stornatal apertures. Indirect methods may provide images that are less easy to quantify (e.g. infiltration, chlorophyll fluorescence quenching); however, if the results are digitized, there are a number of powerful image analysis programs available that might allow a quantitative treatment of results.


Where spot readings are taken, these can be analysed using geographical mapping programs. These require data in some ( x , y, z ) format, where x and y are Cartesian co-ordinates indicating the position of the sample on the leaf surface, and z is the measured variable. A set of x , y data can usually be entered to indicate the leaf margin. Data can be represented as two-dimensional contour maps using “iso-character” lines, or as three- dimensional diagrams. Care must be exercised in choosing the algorithm used to generate and smooth the contours, as this may take either “too literal” or “too liberal” account of individual data points, giving rise to artificial patterning. It is also generally necessary to modify automatic settings for contour frequency, colouring and perspective, to suit the needs of the study.

As an illustration of the influence that measurement and analysis techniques can have on the appearance of data, we provide two representations of stomatal heterogeneity in a single leaf of P. vulgaris (Plate 3). The contour map (Plate 3a) assumes smooth trends over the leaf surface; the image of the infiltrated leaf (Plate 3b) appears to indicate that there are patches of open and closed stomata correlating with vascular boundaries within the leaf. The two representations are broadly similar, but which is the more realistic? This question can only be answered with access to data about individual pores, which is technically very difficult for this species, because of its small stomata. Surrogate measurements of inter-dorsal wall distance (Fig. 5a), which show a good correlation with stomatal aperture and are easier to measure (T. Lawson and J. D. B. Weyers, unpublished), indicate that individual stomata of P. vulgaris were similarly variable to those of C. communis, as shown in Fig. 1. Further analysis, using such data, is required to assess which form of macro-heterogeneity and which method of determining it best represents the real-life situation.

IV. EVIDENCE

A. SPATIAL HETEROGENEITY IN ANATOMICAL CHARACTERISTICS

1. Spacing of Stomata The stomata are not randomly distributed over the leaf surface, but appear to be more evenly spaced than would be expected by chance (Bunning and Sagromsky, 1948). This can be illustrated graphically by plotting the frequency of occurrence of adjacent pores against distance away from a central pore (Sachs, 1991; Fig. 6). If stomata were truly randomly distributed, this plot would be a horizontal line intersecting the frequency axis. In fact, there appears to be a “stomata-free’’ zone where few guard cells have differentiated. In the case of C. communis, this zone is approximately 80 Fm in radius (see also Fig. 4). Examining closely the data presented in Fig. 6,


Distance from Central Stoma (Fm)

Fig. 6. Frequency (pores mm-2) of Commelina communis L. stomata as a function of distance from a central stoma, plotted in one dimension. Measurements were made on photographs; one pore was selected arbitrarily and the distances (and directions) of all other pores in the field determined. Forty-two fields were examined involving about 1100 pores. Frequencies were determined for distance class widths of 30 km. Data of G. Hill and J . D. B. Weyers, unpublished.

there are high and low likelihood distances for stomata to occur beyond this zone of inhibition. When the frequency of stomata is plotted in two dimensions (Plate 5 ) it can be seen further that there are preferred spatial positions for the guard cells. This appears to be a more orderly arrangement than that described for the related monocotyledonous genus Tradescantia (Croxdale et af., 1992), where stomata were either more closely or more distantly arranged than expected by chance and there appeared to be no relationship between stomata in neighbouring files of cells.

Moving up to the whole-leaf scale, stomata1 frequency is certainly not found to be constant over the leaf surface (Salisbury, 1927). In some species, stomata are more dense near to the margins and tip of the leaf (e.g. Plate 4a), but in others they are more dense at the base of the leaf (see Ticha, 1982 for review). Differences can be of the order of two- to three-fold. The use of large numbers of samples combined with analysis via geographical mapping programs (e.g. Smith et a l . , 1989; Poole et af., 1996) has allowed the extent of variation to be seen more clearly.


TABLE I Leaf characters at different insertion points on the main axis of well-watered plants

of Commelina communis

Stomatal Potential Leaf insertion frequency stomatal number from Leaf area (pores Stomatal Guard cell aperture base of plant (mm2) mm-?) index (YO) length (pn ) (YO leaf area)

10 9 8 7

1620 96.8 10.0 55.7 7.00 1760 90.4 10.2 57.0 6.83 1730 80.6 10.3 59.6 6.66 1660 70.7 10.2 62.4 6.41

Modified from Peng (1995). Potential stornatal aperture was calculated assuming pores were circular, with maximum aperture defined as two-thirds of guard cell length. Data are means from three leaves. Samples were taken from similar mid-lamina areas.

In the light of the above results, a degree of caution would appear to be required when specifying a mean stornatal frequency for any given leaf. Most authors account for this problem by specifying a particular area of leaf for sampling (e.g. a position on the leaf lamina half-way from tip to base and half-way from mid-rib to margin). However, the observation that in some species patterns in stomatal frequency may not be the same in leaves of the same insertion point (Poole et al . , 1996), suggests that even this practice may not be wholly reliable. The complexity of the patterns observed in that study indicates that a large number of stratified samples would be required to obtain a truly representative value.

Although reports usually beg certain of the above questions related to sampling, there has been much work on stornatal frequency differences among leaf insertion positions. Summarizing the results of many papers, Ticha (1982) stated that for fully expanded leaves of both herbaceous plants and broadleaf trees, there is a regular increase in the frequency of stomata from the base to apex, as can be seen in Table 1. This is true for either leaf surface (if stomata were present). The upper and lower surfaces of leaves generally have different stornatal densities, but the differences over the leaf surface are generally found to be analogous (Slavik, 1963), though not always (TichB, 1982).

Stomatal frequency is known to be affected by many environmental variables (Ticha, 1982). The main factors and their effects can be summarized as follows:

(a) Plant water status. Water stress generally acts to increase the stornatal frequency and steepen the insertion gradient. Stornatal size also declines under stress, so the general effect is for the potential stomatal aperture to be relatively stable;

(b) Zrradiance. High irradiance generally increases stomatal frequency and


steepens the insertion gradient. “Sun” leaves have higher stomatal frequencies than “shade” leaves. Light quality can also influence stornatal development; and

(c) pC02. Stomatal frequency decreases with increasing p C 0 2 , but not necessarily in all species (Korner, 1988; Malone et al., 1993; Ferris and Taylor, 1994).

Salisbury (1927) claimed that stomatal index was relatively constant under all environmental conditions. Because of this, differences in stornatal frequency between sun or shade leaves or between well-watered and stressed plants were attributed to differences in cell size leading to changes in overall leaf area. However, this is not the whole story, as stomatal index can be affected by stress (Quarrie and Jones, 1977), irradiance (Schoch et al., 1980; Poole et al., 1996) and p C 0 2 (Woodward, 1987). Poole et al. (1996) reported a 2.5-fold difference in stomatal frequency and a two-fold variation in stomatal index over “sun” leaves of Alnus glutinosa. The co-variation between stomatal frequency and stomatal index found by these authors at different positions on the leaf indicated that the spatial variation in stornatal frequency might be due to localized differences in stomatal differentiation rather than in leaf expansion.

2. Guard Cell Size Ticha (1982) collated many reports and concluded that stomatal size decreased from the base to the apex of plants, there generally being an inverse relationship between stornatal size and frequency (Table I). This relationship can also be seen at the individual leaf level (compare Plate 4a and b). There have been no reports concerning variation in pore depth, despite its apparent relative importance in determining pore and patch conductance (Fig. 1).

B. SPATIAL HETEROGENEITY IN STOMATAL BEHAVIOUR

1. The individual stomatal pore shows a high degree of symmetry on either side of its long axis (Fig. 4). Careful measurement and analysis of a sample of half-widths of pairs of C. cornmunis guard cells demonstrates that the pores are more symmetrical than would be expected on the basis of variation in overall pore width (Table 11). The variation in aperture among adjacent pores (as seen in Fig. 4) accounted for over 45% of the total variance. However, the analysis of variance also shows that there are significant differences among different fields within this sampling area (Table 11), indicating that even at this small scale there were trends or patches evident.

For C. communis, the relative variation of stornatal apertures among adjacent pores is greater than that of other stornatal traits such as size and

“Micro” Variation: Direct Measurements of Stomatal Aperture


TABLE I1 Analysis of variance arising from different sources in epidermal strips of

Commelina communis

Degrees of Sum of Mean Source of variation freedom squares squares F

Among fields of view 4 73.8 18.44 4.80 Among pores within 25 96.0 3.84 5.52

Within pores (i.e. 30 20.9 0.70 fields

half apertures) - -

Total 59 190.7

Proportion of total variance

( Y o )

34.9 45.1

20.0

-

100.0

Measurements of the half pore widths of a sample of Commelina comrnunis stomata were made directly from enlarged photographs. Five interveinal fields of view were observed at the centre and corners of a 3mm x 3mm area of a silicone rubber impression as shown in Fig. 4 and six stomata were randomly selected for observation within each field. Following analysis of variance on the data, it can be concluded (a) that there were significant differences among the different fields of view compared with variation among pores within fields (P < 0.01); and (b) that the variation in guard cells between pores in the same field of view is significant compared with the difference between the half-apertures of a pair (Pc0.01). Data obtained by Y. Lindsay and J. D. B. Weyers and analysed by R. A’Brook.

distance apart. Weyers and Meidner (1990) reported the respective coefficients of variation for these characteristics to be 23.0%, 7.2% and 15.4%. Spence (1987) concluded that the dispersion of stomatal aperture data (in epidermal strips) depended both on plant species and pretreatments. Laisk et al. (1980) noted that the frequency distributions of stomatal apertures in Vicia faba were symmetrical and bell-shaped, except when the mean aperture approached zero, when the distribution was skewed to the right (Fig. 7). Essentially similar results concerning the statistical distribution of micro- variation data were reported by van Gardingen et al. (1989) and PospiSilov6 and SantrGtiek (1994).

Laisk et al. (1980) suggested that differences in apertures among individual stomata might be conserved as the mean value altered and the frequency distribution was translated along the aperture axis (Fig. 7). This view was confirmed by Saxe (1979) and Kappen et al. (1987), who reported that while small samples of stomata (of C . comrnunis and V. faba, respectively) in a small field of view had greatly differing initial stomatal apertures, they all responded similarly to changes in humidity or p C 0 2 .

2. Only one report has involved direct measurements of stomatal aperture over an entire leaf - that of Smith et al. (1989). These authors showed that pore

“Macro” Variation over Individual Leaf Surfaces


Stomata1 aperture class mid value (pm)

Fig. 7. Selected frequency distributions of samples of Vicia faba stomata. Redrawn data of Laisk et al. (1980). The number beside each curve is the leaf number quoted in Table 1 of Laisk et a f . , in which full details of methods can be obtained. For leaves 2, 6, 8 and 9, the respective mean apertures and numbers of stomata per sample are: (1.6 pm, 249), (2.7 pm, 327), (5.4 pm, 294) and (10.8 pm, 180).

width was generally lower near to the leaf perimeter, though this rule was not absolute (Plate 4c). Evidence for macro-heterogeneity in conductance over single leaf surfaces has now accumulated from a variety of indirect methods (PospiSilova and Santrikek, 1994). These studies have involved a wide range of species and techniques. Although the qualitative nature of some of the assessments must be taken into account, the main conclusions that can be drawn are:

(a) The form of macro-heterogeneity observed depends on the conditions experienced by the plant. When patchiness is observed, it may only occur when relatively traumatic conditions prevail, such as treatment with high concentrations of ABA (Downton et al., 1988; Terashima et al., 1988), rapid water stress (Gunasekera and Berkowitz, 1992; Wise et a l , 1991; Matthews and Omasa, 1992), sudden exposure to low air humidity (Bunce, 1988; Mott and Parkhurst, 1991; During, 1992), or exposure of roots to salt water (Flanagan and Jeffries, 1989). Beyschlag and Pfanz (1990), working with Arbutus unedo, showed that both the number and


size of non-infiltrated leaf patches altered through the day. Perhaps significantly, the patchiness was most pronounced at times of rapid stornatal movement before and after midday closure.

(b) The form of macro-heterogeneity observed appears to depend on the leaf anatomy of the species studied. In this respect, the division of leaves into heterobaric and homobaric forms (Sharkey, 1990) seems to be important. The former type, where the areolae are pneumatically isolated due to the presence of bundle-sheath extensions, tend to show patchy patterns of variables related to g, (see discussion below). Hence, Terashima et a f . (1988), using starch accumulation as an assay of local conductance, observed distinct patches with the heterobaric species Helianthus annuus, but the patterns observed with the homobaric species Viciufaba involved smoother transitions between zones of high and low starch accumulation. Similarly, Gunasekera and Berkowitz ( 1992) found patchy accumulation of radioactivity from 14C02 in the heterobaric species P. vulgaris, but not with Spinacea oleraceu or Triticurn aestivum. Loreto and Sharkey (1990) found that patches in I4CO2 accumulation were observed in young leaves of Olea europea, but not in mature leaves.

3. Differences between Leuf Surfaces Variability between the stornatal responses of the upper and lower leaf surfaces has long been acknowledged (Turner, 1979). Since the frequency of stomata on either surface of amphistomatous leaves is rarely equal, differences in conductance between the sides of a leaf are expected on this basis alone. However, research shows that the stomata on each surface also have different sensitivities to stimuli and there are many reports of independent behaviour on the two surfaces (Turner, 1979). In the context of non-uniform stomatal movements, Farquhar et al. ( 1987) demonstrated that ABA treatment caused complete stomatal closure on the upper surface of H. annuus leaves, whereas the stomata on the lower surface were open in some patches and closed in others. Mott el al. (1993) extended these observations by showing that patches on the upper and lower surfaces of Xanrhium strumarium leaves are not always spatially correlated.

4. Within-plan[ and Within-canopy Vuriution in Stomata1 Conductance Solarova and PospiSilova (1983) reviewed many papers concerned with g, at different leaf insertion points within plants. One approach has been to determine maximum values of g, at different positions in the canopy; another to follow maximum values for the same leaf as it matures. In general, maximum g, rises rapidly with leaf age; it may then remain static, but will eventually decline as the leaf senesces. Thus, in a canopy, the highest potential conductances are found in the upper zone of mature leaves. Under various stresses, the effect on these gradients, if any, was to amplify them.


The instantaneous g, values within the canopy are, of course, susceptible to differences in environment, particularly shading and wind speed (Pearcy , 1990; van Gardingen and Grace, 1991). To arrive at a canopy or stratum conductance, the approach is generally to use a parallel sum of individual leaf conductances; alternatively, a value can be estimated via the Penman- Monteith equation (Jones, 1992). More complex procedures were discussed by McNaughton (1994). The degree of influence of leaf or canopy conductances on evaporation to the bulk atmosphere depends on the boundary layer conductance and efficiencies of heat and mass transfer between the canopy “surface” and the bulk atmosphere (Jarvis and McNaughton, 1986; McNaughton and Jarvis, 1991).

C. TEMPORAL HETEROGENEITY IN STOMATAL BEHAVIOUR

The studies by Saxe (1979) and Kappen et al. (1987) provide direct evidence that individual pores in small groups, although differing in absolute apertures, tend to follow very similar patterns of movement through time over periods of hours, a conclusion that was also reached by Laisk et al. (1980) studying larger populations of pores. In these investigations, the stomata were responding to changes in various stimuli; however, when conditions were maintained constant over a timescale of days, Gorton et al. (1989b) observed that endogenous rhythms of individual pores were not coordinated.

When observing temperature variation over Helianthus annuus leaves, Hashimoto et al. (1984) found that trends in leaf temperature remained very similar in pattern over a 2.5 h period under constant, defined conditions. Over a longer timescale in “natural” conditions, the observations of Smith et al. (1989), Beyschlag and Pfanz (1990) and Beyschlag et al. (1992) illustrate the well-known daily course of stomatal movements (Meidner and Mansfield, 1968), but these authors also observed a change in the patterns of conductance at different times. Smith et al. (1989) found that there was little consistency in iso-aperture maps through time, although stomata were always relatively closed at the leaf tip and base. Beyschlag and Pfanz (1990) found that “randomly” patchy leaf infiltration was most evident when the stomata were opening or closing; otherwise the pattern of infiltration was constant or the patchiness itself showed trends.

Measurements of non-photochemical chlorophyll fluorescence quenching have also provided information on the dynamics of stomatal heterogeneity. Cardon et al. (1994) demonstrated that the behaviour of individual patches is dynamic and sometimes independent. Two contrasting sets of results were presented in which patches either oscillated in concert, such that the overall gas exchange for the area oscillated in phase, or independent patchy behaviour was observed which “cancelled out” to give a relatively constant


rate of gas exchange. Mott (1995) found that treatment with ABA initiated patchy stomatal closure as found with destructive methods (e.g. Downton ef a f . , 1988; Terashima et al., 1988), but was able to show that the patchiness could be of a transient nature, depending on the concentration of ABA applied.

V. CONCLUSIONS

The overall picture that emerges from these studies is one of considerable heterogeneity in stomatal characteristics at scales from individual pore to whole leaves and above. In the case of g, , or parameters related to it, the patterns appear to be dynamic. The causes and consequences of this spatial and temporal heterogeneity will now be explored.

A. CAUSES OF STOMATAL HETEROGENEITY

1. Spacing of Stomata It is tempting to speculate that the space around stomata where it is less likely that another pore will be situated is due to some “influence” emanating from the differentiating guard cells which prevents other protodermal cells developing into stomata (Biinning and Sagromsky, 1948). However, at least part of the non-random spacing of stomata can also be attributed to the fact that pairs of guard cells do not form independently of other cells in the epidermis (Sachs, 1991). The abutting cells may share a common mother cell or arise from other protodermal cells through a specific series of divisions. This is true whether a stornatal complex is formed or not, and it effectively creates a minimum distance between pores (and a maximum stornatal frequency).

Sachs (1991) therefore concluded that both “cellular interaction” and “cell packing” (cell lineage) mechanisms accounted for the distribution of stomata. He suggested that further complexity in the mechanisms of stomatal development was required to account for: (a) the common orientation of stomatal pores in certain species, especially monocotyledons; (b) the existence of stomata in certain files of cells but not others, again especially in monocotyledons; and (c) the arrangement of stomata in small groups in certain genera, such as Begonia.

Sachs (1994) cautioned that the mature state of stomata provides little reliable evidence about how a stornatal pattern has formed; detailed studies of stornatal development processes are also required. In general, it appears that stomatal differentiation is determined at an early stage in leaf development during a “competence window”. While treatments that affect stornatal frequency might also operate by affecting the expansion of the leaf (either as a whole or locally), if they concurrently or co-spatially affect stornatal


index, then one might suspect an influence acting via the cell interaction mechanism.

2. The fact that stomatal size and frequency are generally inversely related, while stomatal index remains relatively constant (Salisbury, 1927; Ticha, 1982; see also Table I , Plate 4), indicates that differences in leaf cell expansion may be important in modifying stomatal frequency, with total cell numbers remaining relatively constant. On the other hand, intra-leaf differences in stornatal frequency in Alnus glutinosa were interpreted to result from local differences in cell differentiation rather than differential leaf expansion (Poole et al., 1996). The fact that there are differences between cultivars in these parameters, coupled with the fact that stornatal size and frequency alter according to the level of ploidy in some species (see Willmer and Fricker, 1996), indicates that genetic control is possible. However, there also are numerous accounts of all three parameters being susceptible to growth conditions (see above), showing that a degree of environmental control is likely too.

Stornatal Size, Frequency and Index

3. Heterogeneous Stornatal Behaviour We can conclude from the results discussed above that the main “unit of variability” is the stomatal pore rather than the individual guard cells (Table 11). The reason for this is unclear, but the greater-than-expected degree of pore symmetry could reveal some mechanism that tends to equilibrate the individual guard cells in a pair, possibly acting via connections across the common wall area (Willmer and Fricker, 1996). The reason for inter-pore variability also remains obscure. Spence (1987) identified variability in guard cell size as a factor leading to aperture variance in certain species, and this could act at both the patch and whole-leaf scales. However, for C. cornmunis at least, this is unlikely to be the only factor involved, because the coefficient of variation of stornatal aperture is at least three-fold greater than that of guard cell length (see above).

Guard cells are unusual in the plant body in having no plasmodesmatal connections to other cells when mature (Sack, 1987). One possible reason for this is a need for electrical independence from other cells so that they can transport solutes efficiently. This independence may result in heterogeneity. While authors have speculated in general terms on optimal stomatal behaviour (Farquhar and Sharkey, 1982), the consequences of this in terms of heterogeneity have not been fully explored. If each individual pore is capable of optimizing its own behaviour with respect to prevailing conditions, and if the conditions themselves are (a) heterogeneous and (b) dynamic, then this might lead to non-uniform stornatal behaviour. The movements of individual pores will constitute “hunting” towards some “ideal” state and involve the short-term oscillations typical of such responses.


If the rhythms adopted by individual pores are out of phase, this would explain the high degree of inter-pore variation that is found. Somewhat against this notion is the finding discussed above, that small numbers of neighbouring pores tend to respond together to stimuli, retaining differences among themselves that were initially present, although this behaviour might be predicted if the environment, though changing, is homogenous at this scale.

At a slightly higher scale, the stornatal movements observed in different areas of leaf will certainly be influenced by spatial and temporal heterogeneity in micro-meteorological conditions. Examples could include local differences in the boundary layer thickness (van Gardingen and Grace, 1991), leaf temperature (Jones, 1992), and PPFD (Pearcy, 1990). Such micro- meteorological variation would lead, ex hypothesis, to the stomata in different zones hunting towards a different optimal aperture, giving rise to different conductances in different areas. It should be noted that there would be complex feedback effects (Raschke, 1979) operating in such a system.

Another possible cause of within-leaf heterogeneity is physiological variation, both in the underlying mesophyll tissues and in the guard cells themselves. Thus, even if conditions in two areas of leaf were the same, differences in the sensitivity of the stomata to the various stimuli present could lead to differences in their conductance. Alternatively, differences in photosynthetic characteristics might also result in different local C, values. These sources of variation could arise due to, for example, differences in the maturity of stomata or mesophyll tissues in different zones, differences in underlying photosynthetic capacity/efficiency, or differences in stomatal size and frequency, all of which have been described at the whole-leaf level (e.g. Miranda et al., 1981). At a first approximation, it would appear that observed intra-leaf variations in conductance and assimilation are too great to be caused solely by such factors. although a quantitative estimate of their contribution would be valuable.

Stomata1 apertures are determined by a relationship between the turgors of guard and neighbouring cells (Raschke, 1979). Heterogeneous influences on guard and/or neighbouring cell turgors might thus result in heterogeneous apertures. The chief of these influences would be guard cell solute potential and guard cell water potential. Guard cell solute levels alter as part of their physiological responses to external and internal stimuli and their turgors are also affected by the vapour pressure deficit and leaf water potential. Slavik (1963) observed differences in water potential over the leaf surface that could influence guard cell turgor. The proximity and position of a particular stoma in relation to the vascular system could be important for this reason, and it may also influence behaviour due to physiologically active solutes delivered via the transpiration stream, such as ABA. K + and Ca2+ (Mansfield et al. , 1990), which might bring about changes in cell solute potential. Laisk et al. (1980) proposed that variability in aperture reflected differences in guard cell


solute potentials (as observed in characteristic sigmoidal plasmolysis curves); these authors accounted for changes in the shape of the aperture-frequency distribution by assuming guard cells below a certain turgor threshold would all have zero aperture, while those close to the upper extreme would encounter wall stiffening.

Effects of heterogeneity in the rate of photosynthesis on stomata (or vice versa) might act primarily via Ci. Local changes in Ci would be expected to be transmitted rapidly via gas-phase diffusion. Therefore, the degree of gas- phase connection within the mesophyll might influence the patterns found. The key anatomical feature is the presence of a bundle-sheath extension between areolae (Sharkey, 1990). It should be noted that this is a quantitative characteristic: in any given species, a characteristic proportion of the minor vein length will carry a bundle-sheath extension (Mauseth, 1988; Fahn, 1990).

While evidence is thin, trends in stomatal behaviour might be expected for the homobaric type of leaf in which the majority of minor veins do not have bundle-sheath extensions (Gunasekera and Berkowitz, 1992). In such leaves the gas phase within the leaf is free to mix and can therefore be expected to show smooth transitions resulting from local differences in, for example, stornatal C 0 2 supply or photosynthetic demand. If stomata respond individually to these trends, then their conductance will follow the trend. Patchy stomatal behaviour has been associated with the heterobaric form of leaf anatomy in which the bundle-sheath extensions of minor veins subdivide the leaf air space into more-or-less gas-tight compartments. It can be speculated that the gases in the intercellular air spaces of these compartments do not mix and thus can maintain independent partial pressures of C 0 2 or water vapour, which can in turn drive independent stornatal behaviour in that patch.

B. RELEVANCE OF HETEROGENEITY IN STOMATAL CHARACTERISTICS

The following important issues have emerged from the foregoing discussion regarding the relevance of heterogeneity in stomatal characteristics:

(a) In view of its prevalence at all scales of study, a full understanding of the anatomical and physiological origins of stornatal heterogeneity is necessary for a complete understanding of stornatal function. Further, an awareness of the benefits and disadvantages of stornatal heterogeneity to the plant may add to our understanding of the evolution of these phenomena and the physiological systems that sustain them.

(b) Certain types of heterogeneous stomatal behaviour are correlated with what appear to be short-term local inefficiencies in photosynthesis that could reduce crop productivity (Mansfield et al. , 1990). Moreover, variation in stornatal anatomy and physiology may have consequences


for the transpiration and water use efficiency of leaf patches, whole leaves, plants and canopies, with obvious ecological and agricultural importance.

(c) Heterogeneity is important in the specific case of estimating the internal COz concentration in photosynthesizing leaves. Many authors have pointed out the systematic error in estimates of Ci arising from this source (Laisk, 1983; Downton et al . , 1988; Terashima et al . , 1988) which gave the erroneous impression of non-stomata1 inhibition of photosynthesis. This bias appears to be more important during traumatic treatments causing non-uniform stornatal closure (e.g. ABA treatments) and may be transient in some cases (Mott, 1995).

(d) Stomata1 frequency has been considered a potentially useful characteristic in breeding for drought resistance in crop plants (Jones, 1987). It has also been suggested that this parameter could be used to predict past environments from anatomical data derived from fossil or subfossil leaves (e.g. McElwain and Chaloner, 1995). Consideration of heterogeneity in stomatal characteristics should be an important component in studies based on these proposals.

(e) The leaf epidermis constitutes a model system in developmental control and patterning (Palevitz, 1981; Sachs, 1991). Cells on the leaf epidermis are relatively easy to measure and this essentially two-dimensional tissue is easier to describe, analyse and model than more complex three- dimensional systems. Studies of differences in cell differentiation and distribution over the leaf surface may reveal local differences in development patterns (Sachs, 1991; Croxdale et al . , 1992; Poole ef al . , 1996).

(f) The precise nature of heterogeneity in stornatal characteristics has consequences for sampling strategy, experimental design and data analysis. It is therefore important for all research involving measurements of stornatal development or activity. Firstly, either experimental design or sampling strategy should take into account variability at relevant scales. As a result, numbers of measurements and their location must be carefully chosen. Secondly, methods of statistical analysis must be suitable for the distribution pattern of the data. Thirdly, methods of data presentation should be appropriate to the underlying pattern of heterogeneity. Without due consideration of the various phenomena involved, conclusions might be biased or erroneous.

C. TOPICS REQUIRING FURTHER INVESTIGATION

Further observations and measurements are required to establish fully the nature of stornatal heterogeneity and the reasons for it.

At the individual pore level, we need to understand better how the high


degree of pore symmetry is achieved, what factors control the distribution of stomata, and why there is so much inter-pore variability in small areas of leaves. At the whole-leaf scale, quantitative anatomical studies linked to observations of macro-heterogeneity in conductance are required to elucidate fully the anatomical and treatment factors involved in patchiness. We need to use a wider range of species to assess whether patchy stomatal behaviour is confined to heterobaric leaf forms and the conditions under which it will appear.

Further work is required to improve techniques used to investigate macro-heterogeneity. We need to know whether macro forms of heterogeneity visualized by different techniques really represent the underlying pattern of individual pore behaviour. Non-destructive techniques are required to resolve heterogeneity in space and time. The extent to which patterns of macro-heterogeneity are initiated and sustained by heterogeneity in the micro-meteorology of the leaf should be investigated. This may require development of new non-invasive sensing techniques.

Most of the studies considered in this review were laboratory-based. We require more information about the prevalence of stomatal heterogeneity in the field. It would also be desirable to widen the range of species that have been investigated, correlating the results with micro-meteorological measurements, ecological habitat and leaf form. The mechanisms whereby certain environmental factors (e.g. stress, pCOz) affect stomatal distribution and differentiation require to be fully elucidated. The consequences of this form of developmental plasticity for plant water use and productivity should also be modelled.

Small-scale stomatal heterogeneity should be considered in models of gas exchange at higher scales (Weyers et al . , 1997). On the one hand, the phenomena discussed in this review are likely to affect the accuracy of estimates used in models and should influence sampling strategies; on the other hand, as scale increases, their influence over the output of models is likely to diminish.

D. SUMMARY

We have outlined the major forms of heterogeneity to be found in characteristics associated with the development and function of stomata. A variety of methods has been used to study these phenomena, each having its advantages and disadvantages. For this reason alone, caution is required in the interpretation of data. Furthermore, the high variability of stomatal characteristics indicates that conclusions should be based on sound statistical analysis. It is possible to speculate on the causes of this heterogeneity, but much more research is required before it will be possible to make firm conclusions.


The growing appreciation of the complexity revealed by studies on stomatal heterogeneity is evident in the primary literature, reviews and texts. There is increasing interest in global ecosystems and a realization of the fact that scaling between “levels” in such systems is not a simple process (van Gardingen el al . , 1997). For physiological processes, the focus is also shifting from consideration of spatial variability as a constant phenomenon towards considering it as a dynamic process. However, the study of temporal heterogeneity is difficult due to the interaction of measurement techniques with the processes under study. At the whole-leaf scale there is a challenge to incorporate such complex influences as venation patterns, surface topography, hairs, leaf shape, wind direction and turbulence. At the canopy level and beyond there is the need to consider further details such as the placement of the individual plant within a community and the influence of the community’s activities on the individual.

As discussed above, there are numerous reasons why we should regard an understanding of stomatal heterogeneity as important. Perhaps the most important rationale for working on this topic arises because it is fundamen- tally related to our understanding of the stornatal mechanism itself. Although much progress has been made on the physics, biochemistry and molecular biology of guard cells, we cannot pretend that we know anything like the full story while we remain ignorant of the reasons why two adjacent pores, receiving essentially the same stimuli, have different apertures.

ACKNOWLEDGEMENTS

We would like to acknowledge gratefully many collaborators who have painstakingly obtained data and discussed ideas regarding variation in stornatal characteristics. Richard A’Brook, Graham Hill, Zhi-Yong Peng and Imogen Poole are singled out for particular mention as their work features heavily in this chapter. Neil Paterson provided valuable criticism of the manuscript. Bill Berry and Chris Hutchison are thanked for help with mapping and image analysis, Other co-workers include Stephen Barr, Martin Davidson, Karen Findlay, Audra Hunter, Wendy James, Yvonne Lindsay, Alison Roberts, Richard Parsons, Susan Smith and Andrew Yool. TL was supported by the UK BBSRC.

APPENDIX

SYMBOLS AND ABBREVIATIONS

A pore width/stomatal aperture (m) A , mean pore area (m’) ABA abscisic acid

348

C

Ci d D w ECF gs

g P L p a

PCO2 PPFD R SF SI T

J . D. B. WEYERS and T. LAWSON

“end correction” (m) intercellular air space pCO2 (Pa) pore depth (m) water diffusivity in air (m2s-l) frequency of cells other than guard cells (m-2) conductance of a group of stomata on a single leaf surface (mol m-2 s-l)

conductance of a single stoma (mol mP2 s-’) pore length (m) atmospheric pressure (Pa = J m-3) partial pressure of carbon dioxide (Pa) photosynthetically active photon flux density gas constant (J mol-’ K-’) stornatal frequency (m-2) stomatal index (YO) mean of leaf and air temperature (K)

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