Journal of Experimental Botany, Vol. 50, No. 337, pp. 1381–1391, August 1999
Spatial and temporal variation in gas exchange over thelower surface of Phaseolus vulgaris L. primary leaves
Tracy Lawson1 and Jonathan Weyers2
Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK
Received 30 December 1998; Accepted 14 April 1999
Abstract measurements for the appropriate mean irradiance. Itwas not possible to deduce from the relationships
This paper describes spatio-temporal variation in gasbetween pairs of variables which factors were most
exchange over the lower surface of primary leavesimportant in determining A and g
lat any given time or
of glasshouse-grown Phaseolus vulgaris L. plants.space, but g
ldid not appear to be the only factor
Simultaneous measurements of assimilation and waterlimiting A. It is hypothesized that the observed
vapour conductance were made with a small areavariation in gas exchange, the lack of close corre-
cuvette attached to an infra-red gas analyser. Thespondence between g
land A and the reduction in
plants were kept in glass chambers so that the externalphotosynthesis compared with the apparent potential
gaseous environment could be controlled. Observa-value are all phenomena that arise from differences in
tions are reported from four half-hour periods duringthe induction times for these variables following
a day in which the ambient PPFD, while variable, waschanges in conditions, interacting with other factors
close to saturating for photosynthesis. ‘Snapshot’associated with position on the leaf.
measurements of gas exchange were made at 20 posi-tions on the leaf surface using a stratified random out- Key words: Assimilation, conductance, heterogeneity,to-in strategy, which avoided disturbance of yet-to-be- infrared gas analysis, photosynthesis, stomata.measured sites. Data were mapped using the ‘Unimap’cartographic program. For any given measurement
Introductionperiod, gas exchange varied greatly over the leaf sur-face: typically, net assimilation (A) varied by over 4-fold Spatial variation in stomatal characteristics exists fromand leaf conductance (g
l) by over 3-fold. Estimated the scale of plant community down to the individual leaf
intracellular pressures of CO2
and leaf temperatures and cell (see reviews by Pospısilova and Santrucek, 1994;showed less relative variation both in space and time. Weyers and Lawson, 1997; Mott and Buckley, 1998). AtComparing measurement periods, the spatial patterns the whole leaf level, the existence of heterogeneity inof variation in A and g
lwere dissimilar. Moreover, at stomatal conductance (gs) has been evident for many
different sites on the leaf, the trends in a given variable years (e.g. Darwin, 1898). It is now recognized to occurcould be in opposite directions, while external condi- in ‘macro’ forms, involving trends across leaf surfacestions were relatively constant. Although the correlation and patches relating to areolae, as well as ‘micro’ variationbetween A and g
lwas significant overall, there was a among individual pores (Weyers and Lawson, 1997; Mott
large degree of scatter in the data and zones of high and Buckley, 1998).g
loften corresponded to areas of low A. Depending on In contrast, evidence for variation in photosynthetic
the basis of calculation, A was as much as 63% lower CO2 assimilation across the leaf blade has only accumu-lated recently (Solarova and Pospısilova, 1983). Anthan a value predicted on the basis of steady-state
1 Present address: Department of Biology, University of Essex, Colchester CO4 3SQ, UK.2 To whom correspondence should be addressed. E-mail: [email protected]: A, net assimilation rate; C
i, intercellular (sub-stomatal) CO
2concentration; F
v, variable fluorescence; F
m, maximum fluorescence; g
l, leaf
conductance (lower surface); Gl, leaf conductance (both surfaces); g
s, stomatal conductance; I, incident irradiance; PAR, photosynthetically active
radiation (400–1000 nm); PPFD, photosynthetically active photon flux density; qN, non-photochemical quenching; T
impetus for research was the discovery of patchy photo- imaging was a significant advance (Daley et al., 1989),permitting spatially detailed, non-destructive studies ofsynthesis associated with certain treatments causing rapid
stomatal closure (Downton et al., 1988; Terashima et al., the kinetics of photosynthetic activity. It is normallyassumed that local variation in non-photochemical1988). These results have been confirmed using non-
invasive, non-destructive methods (Daley et al., 1989; quenching (qN ) or in quantum yield of linear electrontransport (we) arises from local differences in internal CO2Mott et al., 1993) that have also been used to observe
temporal variation in the patterns (Cardon et al., 1994; concentrations, which in turn result from changes in gs.A body of evidence suggests that patterns of qN or weMott, 1995; Siebke and Weis, 1995).
Stomatal and photosynthetic heterogeneity at the leaf can be used, under certain circumstances, to predict gs,providing an opportunity for simultaneous non-scale and below are now acknowledged as important
phenomena. From an eco-physiological viewpoint, the destructive measurements of the two variables (Daleyet al., 1989; Mott et al., 1993; Cardon et al., 1994; Gentydynamic heterogeneous environment at this scale, for
example sun and shade flecks (Pearcy, 1990), create local and Meyer, 1994; Mott, 1995; Eckstein et al., 1996).However, there is evidence that A and gs might not be(i.e. sub-leaf ) and temporary differences in plant
responses to conditions, with consequences for productiv- as closely linked as is sometimes assumed (Farquhar andSharkey, 1982; Jones, 1998). By measuring Ci directly inity, water use efficiency and shade adaptation (Zipperlen
and Press, 1997). Even when external conditions are water-stressed plants, it has been inferred that photo-synthesis was reduced by lowered rates of photosyntheticostensibly uniform, zones of low photosynthetic activity
and/or high water loss have been observed, implying biochemistry, rather than by a stomatal limitation (Lauerand Boyer, 1992). Similarly, by reducing the theoreticalinefficiencies that might be important in an agricultural
context (Mansfield et al., 1990). From a practical point stomatal limitation on A by increasing the atmosphericCO2 concentration, it was deduced that patchiness in gsof view, attention has focused on difficulties posed by
heterogeneity when making valid estimates of leaf water was not the sole cause of patchy A, especially at highwater deficits (Wise et al., 1992). Furthermore, it hasvapour conductance (gl ), assimilation (A) and intracellu-
lar CO2 concentration (Ci ), (reviewed by Pospısilova and also been demonstrated that the differences in inductiontimes for responses of A and gl to PPFD resulted in anSantrucek, 1994; Weyers et al., 1997). Finally, the extens-
ive progress in understanding the molecular biology of uncoupling of the two variables in the short-term(Barradas and Jones, 1996).photosynthesis and guard cell function (Willmer and
Fricker, 1995) contrasts with the current lack of detailed In the light of the above, it is desirable that alternativetechniques be applied to investigate heterogeneity in gasunderstanding of the nature of heterogeneity and the
reasons why it exists (Weyers and Lawson, 1997). exchange, preferably providing direct, simultaneous meas-urements of A and gl. The availability of cuvettes of smallThere are many technical difficulties in making observa-
tions of gl and A over leaf surfaces (Weyers and Lawson, area attached to IRGA-based gas exchange instrument(Parkinson, et al., 1980; Parsons et al., 1997) in theory1997). Direct measurements of stomatal dimensions (van
Gardingen et al., 1989; Lawson et al., 1998a) are unfeas- makes this possible. The aim of this study is to use suchequipment to characterize the nature of spatial andible at this scale and gas exchange equipment has generally
employed cuvettes covering the whole leaf or a large part dynamic variation in gas exchange over primary leavesof Phaseolus vulgaris L.of a leaf thereby averaging responses (Long and Hallgren,
1993). Thermal imaging has been used as an indirectmeasure of gl over whole leaves (Hashimoto et al., 1984;
Materials and methodsJones, 1999), but is subject to difficulties in quantifica-tion, especially when taking account of the effects of Plant materiallocal differences in boundary layer conductance (van Seeds of Phaseolus vulgaris L. cv. Hardy (Nickerson Zwaan,Gardingen and Grace, 1991; Jones, 1999). The essentially Barendrecht, Netherlands) were sown directly into 100 mm pots
containing Levington’s Universal Extra compost (Levingtondestructive technique of vacuum infiltration providesHorticulture Ltd., Ipswich, UK) in a heated rooftop glasshouse.a direct indication of patchy conductance patternsObservations were made on fully expanded primary leaves from(Beyschlag and Pfanz, 1990; Beyschlag et al., 1992).plants that were 3–4-weeks-old. The temperature was main-
However, it depicts stomatal behaviour in a bimodal tained above 15 °C at night and rarely exceeded 35 °C duringfashion, which can misrepresent the underlying state of the day. The plants were well watered throughout using a
capillary matting system. Supplementary light (#100 mmolindividual stomata (Lawson et al., 1998b).photons PAR m−2 s−1) was provided from 08.00 h to 24.00 hSmall-scale measurements of photosynthetic activityby 400 W Na vapour lamps 0.75 m above the plants. Plantshave been accomplished by localizing the uptake of 14CO2 were transferred to a 0.75×0.45×0.61 m (0.21 m3) glass
or synthesis of starch (Downton et al., 1988; Terashima chamber under the same conditions 1 d before readings. Airet al., 1988), but these methods involve destructive har- from outside the glasshouse was supplied to these chambers at
a rate of 1.56 m3 h−1, this being pre-warmed by passage overvesting. The development of chlorophyll fluorescence
the hot water piping system within the glasshouse, and area in turn, a Plant Efficiency Analyser (PEA; Hansatech,King’s Lynn, UK) was used to make measurements of the ratiohumidified by passage over moist capillary matting. Typically,
the CO2 partial pressure was 35±1.2 kPa and the relative of variable fluorescence to maximum fluorescence (Fv/Fm).Samples of leaf material were then removed from the samehumidity 45±4%. All gas exchange readings were taken within
the chamber using access holes. The slight pressurization within areas using a cork borer. These were used to determinechlorophyll content. Pigments were extracted for 2–3 h fromthe chamber ensured that there was no possibility of the outside
atmospheric conditions, including the operator’s breath, homogenized discs in a solvent mixture of acetone: water:saturated MgCO3 solution (8051951, by vol.). After centrifu-influencing results.gation, absorption measurements were made on a UV 160Arecording spectrophotometer (Shimadzu Corp., Tokyo, Japan)
Gas exchange measurements and the chlorophyll (a+b) content calculated (Leegood, 1993).To investigate the effects of leaf temperature (Tl ) on A, the‘Snapshot’ readings of gas exchange and environmental variables
were made using a CIRAS portable infrared gas analyser (PP plant and CIRAS instrument were placed in a growth cabinetwhose temperature controls were manipulated while Tl wasSystems, Hitchin, UK). A rectangular (‘porometer’) cuvette of
area 125 mm2 (5×25 mm) was attached to leaves sequentially measured using a thermistor positioned within the broad leafCIRAS cuvette. I was kept at 2028 mmol photons PAR m−2 s−1at 20 set positions defined by a stratified random out-to-in
sampling protocol. This protocol ensured readings were spaced throughout, Ca was set at 36 Pa and the relative humidity wasmaintained between 57% and 63%. Initially, A was allowed toover the entire surface and avoided shading of yet-to-be sampled
sites by the cuvette apparatus. The porometer cuvette sealed reach a steady-state value at Tl=19 °C, then the external(growth cabinet) temperature was increased step-wise such thatthe upper surface of the leaf during measurements, but allowed
ambient light to reach the leaf surface. Cuvette reference (input) Tl reached 38 °C over a 43 min period.CO2 partial pressure (Ca) and water vapour deficit were setclose to those prevailing in the chamber (see above). Once the Mapping and statistical analysis of dataclamp had been placed on the leaf, the system was allowed to Two-dimensional contour maps illustrating gas exchange overstabilize (always by 1.5 min; Parsons et al., 1997), at which the leaf surface were generated using ‘Unimap 2000’ (Uniras,time a computer record was made of rates of gas exchange (A Slough, UK) and all maps represent a view from above theand gl ), leaf temperature (Tl) and incident PPFD (I). Each leaf. The input data consisted of sets of x, y, and z values. Themeasurement period thus lasted 30 min. Tl was normally x and y coordinates represent the spatial location of the mid-estimated using the ‘energy balance’ algorithm of the CIRAS point of the sampling area and the z value the datum for theinstrument, but for certain experiments, a thermocouple attach- variable to be visualized. Interpolation between the points wasment was used. The measurement systems employed in the carried out using the ‘bilinear’ routine, prior investigationsCIRAS meant that non-plant (i.e. electronic, gas-mixing etc.) having shown this to be the most appropriate method (Lawson,‘noise’ in typical readings was of the order of 3% or less. 1997). The x and y data for the leaf edge were treated as a
So-called ‘steady-state’ photosynthetic characteristics were ‘fault’ line which prevented data interpolation beyond thisdetermined, for mid-lamina positions, using a circular ‘broad region.leaf ’ cuvette of area 250 mm2. This cuvette made a combined Statistical analysis was carried out using Minitab@ (Minitabmeasurement of gas exchange on both surfaces of the leaf. A/Gl Inc., State College, Pennsylvania, USA). Curves were fitted toand A/I relationships were determined as previously described steady-state data using a non-linear regression function of(Parsons et al., 1997). In both cases, Ca=36 Pa CO2, VPD= Photosyn Assistant (Dundee Scientific, Dundee, UK) and the1.6 kPa and Tl=24 °C. The A/Gl relationship was obtained as fitted equations were used to obtain ‘predicted A’ values.stomata slowly opened over a 70 min period at I=2028 (mol The results illustrated in this report are representative ofphotons m−2 s−1, having previously been closed by placing the three complete studies carried out in a similar format, and ofplant in the dark for up to 2 h. The A/I relationship was several other experiments in which like observations were madeobtained on a leaf for which Gl remained >400 mmol (Lawson, 1997).H2O m−2 s−1 throughout and for which A had stabilized at2028 mmol photons m−2 s−1 for 30 min or longer. Neutraldensity filters were used to give I values of 0, 111, 171, 185, Results263, 291, 460, 558, 690, 943, and 2028 mmol photons m−2 s−1and readings were only taken once A had stabilized (<1 min Figure 1 details changes in I and Tl that occurred duringnormally). A randomized treatment regime was adopted, save the four 30 min measurement periods when CIRAS read-for the penultimate reading, which was always obtained at
ings were taken. The sky was relatively clear throughout2028 mmol photons m−2 s−1 and the last reading, alwaysthis particular day, but temporary cloud cover causedobtained in darkness.
To compare variation in photosynthetic capacity with vari- some transient reductions in I (Fig. 1). Within the cham-ation in snapshot readings over the leaf lamina, a plant was bers, I peaked at about 905 mmol photons PAR m−2 s−1first pre-darkened for 24 h. It was then placed in a glass and Tl ranged between 27.3 and 32.6 °C. The ‘predictedchamber (as described above) in the light for 2 h. Using the
A’ values were steady throughout the first two meas-CIRAS with the porometer cuvette, 14 snapshot readings ofurement periods (mean values 12.3 and 12.6 mmolgas exchange were taken over each leaf (as described above)
after which time the plant was returned to the dark in the CO2 m−2 s−1, respectively), but were more erratic duringlaboratory for a minimum of 0.5 h. Previously, an opaque measurement periods 3 and 4, when I was more variable.paper replica of the leaf had been prepared, having 28–30 Figure 2 presents 2-D contour map representations ofcircular 7 mm diameter areas punched out evenly over its
the spatial variation in A and gl over the primary leafsurface. Initially, strips of opaque paper covered these areas.surface for measurement periods 1 and 2, when I wasWhilst maintaining the plant in darkness, this replica leaf was
attached to the adaxial surface of the leaf. Uncovering each near-saturating at all times. A and gl are shown to vary
Fig. 1. Variation in environmental conditions during the course of observations. The readings were taken at the same times as the snapshot readingsof gas exchange. The solid line is irradiance and the dashed line the leaf temperature estimated by the energy balance program of the CIRAS. Eachsegment of the graph represents a 30 min time interval over which data sets were taken, but except for the first two periods, these were notcontinuous. Also shown (dotted line) is the ‘predicted A’ based on the fitted ‘steady-state’ A/I curve shown in Fig. 6.
considerably over the leaf surface: of the order of 4-fold variables are shown in Fig. 3 for five selected measurementpositions. Throughout the day, these positions had differ-in the case of A and 3-fold in the case of gl. In most
areas A was lower than that predicted from a steady- ent A values. While trends in gl seemed to mirror changesin A at positions 3 and 5, this was not the case at positionsstate A/I curve. For instance, during observation period
2, where I varied between 509 and 883 mmol photons 1, 2 and 4 (Fig. 3a, b). Thus, the trends in value of Aand gl depended on location and were frequently inPAR m−2 s−1, A values predicted from the steady-state
A/I curve (Fig. 5a) should have been in the range different directions. For the whole data set, it wasobserved that A and gl values at the same sampling site12.0–12.9 mmol CO2 m−2 s−1, but the range of observed
values was actually between 0.2–7.7 mmol CO2 m−2 s−1 moved in different directions between pairs of samplingtimes 20 times out of 60.(as illustrated in Fig. 2c).
Assuming each snapshot reading to be representative The among-site differences in estimated Ci and Tl wereof lesser relative magnitude and any trends appeared toof an equal proportion of the leaf area and thereby
approximating the mean observed A value, and taking be consistent at all the measurement zones (Fig. 3c, d).Differences in relative dispersions of the variables werethe average of the ‘predicted A’ values at the prevailing I
as an indication of the leaf ’s photosynthetic potential, confirmed by plotting coefficients of variation (CoVs) forthe whole data set through time (Fig. 4). Thus, CoVs forthen the leaf realized 37.0 and 41.4% of the potential A in
measurement periods 1 and 2, respectively. Alternatively, A and gl generally exceeded 27% and for A were as highas 66% at 14.45 h (315 min), whereas those for estimatedtaking the mean A value as a proportion of the maximum
A observed at any location on the leaf during that period, Ci and Tl were less than 7% throughout.Figure 5 illustrates the relationships between variablesthen the leaf as a whole realized 53.6% and 66.1% of its
photosynthetic potential in measurement periods 1 and from ‘snapshot’ readings over all measurement times andpositions, alongside data sets obtained under ‘steady-2, respectively.
A comparison of the contour maps for either A or gl state’ conditions with similar plant material. There was asignificant positive correlation between snapshot readingsbetween observation times (i.e. Fig. 2a with 2c; Fig. 2b
with 2d) reveals that the spatial patterns were not consist- of A and I (Fig. 5a) and between A and gl (Fig. 5b).However, from Fig 5a, it is evident that the snapshotent (also true of the readings taken at 11.45 h and 14.45 h,
for which maps are not shown). A comparison of the values for A always fell below those predicted from thesteady-state A/I curve. Even when the prevailing I shouldcontour maps at either measurement period (i.e. Fig. 2a
with 2b, Fig. 2c with 2d) suggests that there is a broad have been virtually saturating (i.e. above 600 mmolphotons PAR m−2 s−1, denoted by open symbols), Asimilarity between patterns shown for the two variables,
but the correspondence is by no means complete in either values were between 17% and 72% of those expected.An obvious candidate for limiting A in these cases isspatial or quantitative senses.
To illustrate these points, changes in the measured gl, since the ‘steady-state’ curve was specifically obtained
Fig. 2. Contour maps showing spatial variation in gas exchange at different times. a, b: values measured at 09.15 h±15 min; c, d: values measuredat 09.45 h±15 min; a, c: net photosynthesis (mmol CO2 m−2 s−1); b, d: leaf conductance to water vapour (mmol H2O m−2 s−1). The maps arebased on 20 readings taken over a 30 min period.
Fig. 4. Relative variability of gas exchange and related parameters.Coefficients of variation were calculated for all 20 readings taken overthe leaf lamina. Time zero is 09.00 h and data are plotted at the mid-time of the relevant measurement period. (- -$- -), Assimilation (mmolCO2 m−2 s−1); (···$···), leaf conductance to water vapour (mmolH2O m−2 s−1); (—+—), estimated Ci (Pa); (– –6– –), estimated leaftemperature (°C ).
when Gl>400 mmol m−2 s−1 and the stomatal limitationwould be relatively low. However, the plot of A versusleaf conductance (Fig. 5b) reveals that all snapshot glvalues fall well below the A/Gl curve obtained undersaturating PPFD. Because of differences in the design ofthe cuvettes used, the latter curve contains an elementrelating to the parallel conductance of the upper leafsurface, while the snapshot readings do not. However,both cuvettes used in the study gave similar values for Aand conductance when the same position was measuredin rapid sequence (data not shown). The similarity in gland Gl values probably arises from the facts that (a) theratio of stomata between the lower and upper surfaces inthese leaves was about 1251 and (b) the upper surfacegenerally had a proportionately lower gl than would beexpected from this ratio (Lawson, 1997).
It was concluded that gl cannot have been the solelimiting factor at the time of measurement in all cases.Two sets of four readings have been highlighted to providea quantitative demonstration of this. The readings show-ing the four lowest A values measured under effectivelysaturating I (open triangles, Fig. 5a, b; A=1.7–2.9 mmolCO2 m−2 s−1), while also having low gl values(88–189 mmol H2O m−2 s−1), nonetheless gave A read-ings that were just 20.2–33.8% of those expected fromthe fitted A/Gl curve in Fig. 5b. The four readings havingthe highest gl values (open squares, Fig. 5a, b; gl=Fig. 3. Temporal variation in gas exchange and related parameters for366–521 mmol H2O m−2 s−1) had A values that werefive selected positions on the leaf over the whole observation period.
Time zero is 09.00 h and data are plotted at the time of recording. (a) 55.5–64.8% of those expected from the fitted A/I curvenet assimilation (solid lines); (b) leaf conductance (dashed lines); (c)
in Fig. 5a. Also, because these readings were exceeded inestimated Ci (dotted lines); and (d) leaf temperature (dot-dash lines).A by several readings with substantially lower gl (Fig. 5b),The inset in panel (d) shows the sampling sites for each position 1–5
and the scale for this drawing can be gauged from the rectangles this indicates that gl was not the only factor limiting A.denoting cuvette (sampling) position, which represents the 5×25 mm
Further confirmation that the limitation on A was notcuvette opening. In each graph, symbols indicate data for position 1(&); data for position 2 ($); data for position 3 (+); data for position4 (,); and data for position 5 (2).
(a+b) concentration was 806 mmol m−2 (range 526–967mmol m−2) while the mean Fv/Fm ratio was 0.80 (range0.77–0.81). In neither case was the variability over theleaf able to explain the gas exchange results—the relativevariability in chlorophyll content and fluorescence kineticswas far lower than for gas exchange, the respectivecoefficients of variation for A (mmol CO2 m−2 s−1),chlorophyll (a+b) concentration (mmol m−2) and Fv/Fmbeing 68.4%, 15.6% and 1.37%, respectively. Furthermore,when mapped, the patterns of A, pigmentation and fluo-rescence were all different (Lawson, 1997).
The effects of Tl on A were investigated under saturatingI (Fig. 6). Between 19 °C and 30 °C, A increased with Tl,reaching a maximum of 10 mmol CO2 m−2 s−1 while glremained relatively constant. However, above 30 °C,A decreased sharply with Tl, falling below 4 mmolCO2 m−2 s−1 at 38 °C. Correlations between A and Tlreadings for measurement periods 1–3 were not significant(r=−0.326; −0.060; 0.048 respectively, n=20, P>0.05in all cases). For data collected during measurementperiod 4, there was a highly significant positive correlation(r=0.892, n=20, P<0.001). This result could have arisenbecause decreases in I occurred simultaneously with thereduction in leaf temperature during this measurementperiod (Fig. 1).
Discussion
This is the first study to report simultaneous directmeasurements of assimilation and conductance at differ-Fig. 5. Correlations between ‘snapshot’ observations of gas exchange
and environmental conditions. Accumulated data from four observation ent positions over the surface of an entire leaf. Theperiods at the times and conditions given in Fig. 1. In (a) the solid line investigation was made possible by the availability of arepresents the ‘steady-state’ A/I curve and in (b) the solid line represents
small area porometer cuvette attached to an IRGA,the A/Gl curve, obtained as described in Materials and methods formid-lamina positions. For both plots, all symbols (circles, squares and allowing numerous independent readings of gas exchangetriangles) represent data from individual ‘snapshot’ readings. to be taken within a short period (Parkinson et al., 1980;Considering all the snapshot data together, in (a) the correlation
Parsons et al., 1997). In ‘unstressed’ Phaseolus vulgarisbetween A and I values is positive and significant (r=0.343, n=80,P<0.01); and in (b) the correlation between A and gl values is also primary leaves, a high degree of spatial variation waspositive and significant (r=0.637, n=80, P<0.01). In (a) the data have observed across the leaf surface, even under conditionsbeen subdivided arbitrarily at a PPFD of 600 mmol photons
where the external gaseous environment and irradiancePAR m−2 s−1. Readings taken when the irradiance was above thisfigure (open symbols, n=45) were assumed to be effectively light- were essentially constant, and the irradiance was close tosaturated, those below (closed symbols, n=35), not light saturated. saturating. The patterns were not consistent through time,These codings have been retained in the plot shown in (b). Among the
altering considerably between measurement periods.‘light saturated’ data, two further groups of readings have beenidentified. Firstly, the set of four readings with the lowest A values Further evidence that the results represent true spatial(<2.9 mmol CO2 m−2 s−1), denoted by open triangles, and secondly the variation rather than an artefact arising from the timingset of four readings with the highest gl values (gl 360>mmol
of measurements is provided in Fig. 3, which shows thatH2O m−2 s−1), denoted by open squares. The position of theseparticular readings in the different plots is discussed in the text. the long-term trends in gas exchange depended on leaf
position.These results are not in themselves surprising, becausecaused by low gl comes from analysis of estimated Ci
values, which were in nearly all cases higher in snapshot other researchers have shown spatial and temporal vari-ation in either A or gl, or closely related variables, at thisreadings compared to those obtained in the A/Gl curve
(data not shown). scale (see reviews by Pospısilova and Santrucek, 1994;Weyers and Lawson, 1997). However, the lack of preciseVariation in photosynthetic capacity over the leaf
surface was investigated via sub-leaf measurements coincidence of the patterns of variation of A and gl,as visualized using cartographic software, was anof pigment concentration and fluorescence kinetics.
For a representative leaf, the mean local chlorophyll intriguing finding.
For much of the time when observations were made in Since it is known that A is generally sensitive to hightemperature (Larcher, 1995), it is possible that parts ofthis study, I was close to values giving near-maximal A
in ‘steady-state’ experiments, but observed A readings did the leaf were briefly at temperatures super-optimal for A.Measured Tl values sometimes exceeded 30 °C later in thenot fulfil this potential. The apparent reduction in the
overall productivity of the leaf was estimated to be of day (Fig. 1), while Fig. 6 indicates that the optimumtemperature for photosynthesis at saturating PPFD wasthe order of 34–63%, depending on the basis of the
calculation. about 28 °C. However, high temperature is unlikely tohave been a factor in measurement period 1 and thereforeEven though data presented as 2-D contour maps
(Fig. 2) should be interpreted with care (see below), the cannot be held to be a general cause. Importantly, statist-ical analysis of the snapshot data revealed no significantmajor conclusions outlined above are substantiated by
detailed analysis of the individual gas exchange readings. negative correlation between A and Tl in the first threemeasurement periods.Firstly, tracking A and gl through time for given sampling
points revealed differing trends: the same variable meas- Detailed examination of data obtained under essentiallynon-limiting I and gl, seemed to rule out the hypothesisured at different locations was often found to change
with dissimilar kinetics, even to the extent of moving in that lower-than-expected values of A arose from theadditive effect of these two variables, although it remainsthe opposite direction (Figs 2, 3). Secondly, while the
correlation between A and gl for snapshot readings was possible that other factors, including some not consideredhere, might have contributed to reduced A.significant, the degree of scatter was high, and most
readings fell below values expected from a study of the The main environmental variable altering within theexperimental chambers was I. While ‘predicted A’ basedrelationship between the two variables under well-
controlled conditions (Fig. 5). on steady-state observations is unlikely to have beengreatly affected by the observed variation in I (see above)No clear reasons for the spatial and temporal differ-
ences in gas exchange could be deduced. On the face of it is feasible that the speed and extent of minor changesin I were enough to set up interactions between A and glmeasurements of I (Fig. 1), combined with the ‘steady-
state’ A/I relationship (Fig. 5a), A is predicted to be close that may have resulted in certain of the trends observed.It has been noted for many species, including P. vulgaristo saturation by I at nearly all times (Fig. 1), so any
changes in I would not be enough to explain the observed (Barradas and Jones, 1996; Lawson, 1997) that whenchanges to I occur, the induction times for responses oftrends in A via a direct relationship. Furthermore, since
I would be near-constant over the relatively flat leaf A and gl are different. The response to A generally occurswithin 1 min, while that for gl only occurs after a lag ofsurface at any given time, an A/I relationship could not
explain the observed spatial variation either. several minutes, and may take tens of minutes to becomplete. It has also been noted that for gl, the timeThe fact that I was not limiting is confirmed by the
fact that the snapshot readings in Fig. 5a fall below those taken for responses to increases and decreases in I maydiffer, with the response to darkening generally beingobtained under steady-state conditions. Since the latter
curve was obtained with open stomata (Gl>400 mmol faster (Meidner and Mansfield, 1968; Willmer andFricker, 1995).H2O m−2 s−1), it is possible that gl limited A in snapshot
readings. However, comparison of the snapshot readings Under this hypothesis, a temporarily lowered I couldwith an A/Gl curve obtained at effectively saturating Iindicates that this too was unlikely to be a major limitingfactor, since A values were consistently lower thanexpected from the observed gl. Also, had gl limited Aduring any snapshot reading, this would presumably havebeen revealed by lower estimated Ci values than thosefound in the steady-state for the same A, and these werenot observed.
Further studies were carried out to investigate otherpotential reasons for the lower than expected A values.While present, systematic spatial variation in photosyn-thetic capacity, as determined by pigment measurementsand fluorescence kinetics, could not alone account for thevariation in A, (a) because of differences in the relativevariability of the data; (b) because of the non-coincidenceof the spatial patterns observed; and (c) because these
Fig. 6. Effect of leaf temperature on leaf conductance and rates of CO2patterns would be highly unlikely to change in the time assimilation. Solid circles ($) are A values; solid squares (&) are Glvalues. The fitted curve is the running mean of three neighbouring values.period involved.
initiate a stomatal movement, through either a direct geneity in A and gs for this and other species undereffect on stomata, or an indirect feedback effect acting relatively unstressed and stable conditions (Gunasekeravia increases in Ci following a reduction in A. The and Berkowitz, 1991; Lawson et al., 1998), it may notconsequent reduced stomatal conductance could then always be valid during treatments in which gl is likely tobecome a limiting factor to A when the light level returns be decreasing rapidly, where many researchers haveto the original level, because of the difference in induction reported ‘patchy’ distribution of A (for reviews seetimes. A temporal interaction between A and gl might Pospısilova and Santrucek, 1994; Mott and Buckley,then occur, resulting in overshoots and oscillatory behavi- 1998).our (Farquhar and Sharkey, 1982), with A and gl being Thirdly, when observations of gl and A are made without of phase, and lower than optimal or steady-state a total sampling time of the order of 20–30 min, there isvalues. Hence, in a complex series of sun- or shade-flecks, a possibility that some of the spatial variation observedit would be possible for both A and gl to be lower than might have resulted from uncontrolled temporal variationpredicted under steady-state conditions, as observed here. in environmental parameters. For this reason, care wasData that can be interpreted to support this notion have taken to avoid effects of the investigator’s breath bybeen presented (Siebke and Weis, 1995). enclosing the leaves in a glass chamber where the gaseous
However, this hypothesis alone could not easily account environment was derived from that in the bulk atmo-for spatial variation in these variables at any given time. sphere outside the glasshouse. Care was also taken toTo explain this, it is necessary to postulate that, in ensure that effects due to shading by the cuvette wereaddition, there are interactions of ‘response(s)’ with ‘posi- avoided, by using an out-to-in stratified random samplingtion on leaf ’, as noted previously (Siebke and Weis, strategy. Some changes in I occurred, but, as argued1995). Such interactions could easily result from local above, these would not alone explain either the temporaldifferences in, for example, leaf anatomy, including factors variation in A (because I was near-saturating most ofrelated to photosynthetic capacity (Miranda et al., 1981) time) nor, crucially, the spatial variation (because theand heterobaric areolae (Sharkey, 1990); leaf temperature whole leaf received essentially the same I ).(Raschke, 1965; Wigley and Clark, 1974; Hashimoto The results reported here indicate that there may beet al., 1984); boundary layer thickness (van Gardingen dangers in extrapolation from single readings made withand Grace, 1991; Schuepp, 1993) or leaf water relations small-cuvette gas exchange equipment. Truly representat-(Slavik, 1963). This is true for this study, where I was ive data will only be obtained from replicated observationsnear constant over the leaf surface at any given time. In involving appropriate sampling patterns that take accountnature, the likely scale of natural heterogeneity in I (i.e.
of the potential for both spatial and temporal variation.sun and shade flecks) might well be equivalent or lower
A second corollary is that photosynthetic charactersto leaf size and would presumably act as another sourcederived from steady-state experiments, while indicatingof variation.potential, may overestimate actual rates of assimilationA number of issues must be discussed in connectionin a changing environment, even when the changes arewith the methods used in this study.apparently subtle.Firstly, contour mapping requires certain assumptions
In conclusion, this report presents a number of import-and its use to display differences in gas exchange overant observations. It reinforces previous investigationsleaf surfaces should be approached with care. The mapsindicating a high degree of spatial and temporal hetero-presented here are based on a limited number of readingsgeneity in A and gl, even in relatively unstressed leavesand involve interpolation based on an arbitrary algorithm.maintained under well-controlled conditions; it showsAccordingly, extensive validation tests were carried outthat correlations between A and gl, while statisticallyprior to this study. These indicated that excellent repro-significant, are not as close as is sometimes assumed induction of patterns based on mathematical models couldthe literature; it demonstrates that the temporal non-be obtained by a similar sampling strategy to the onecoincidence between A and gl observed by other authorsused here (Lawson, 1997). However, it should be noted(Barradas and Jones, 1996) extends to spatial non-that although the cuvette area was small in relation tocoincidence; finally, it reveals that A values over largethe total leaf area (of the order of 2% or less), treatingparts of a leaf can be substantially lower than thosethis as a point source may have led to minor distortionobtained under steady-state conditions.of the maps.
An experimental approach will be required to determineSecondly, cuvette-based measurements mask the likelythe reasons for the above findings. A working hypothesis‘micro’ variation among stomata (for gs) or groups ofhas been presented here that could prime these investi-mesophyll cells (for A) and should thus be regarded asgations. Such research will be valuable, not only becauseindicating trends in local mean values, while contourit involves an interesting physiological problem at themapping implies a smooth transition between values over
the leaf surface. While this may fit with observed hetero- whole leaf scale, but also because it relates to an apparent
Lawson T, James W, Weyers JDB. 1998a. A surrogate measureloss of productivity that has eco-physiological andof stomatal aperture. Journal of Experimental Botany 49,agricultural relevance.1397–1403.
Lawson T, Weyers JDB, A’Brook R. 1998b. The nature ofheterogeneity in the stomatal behaviour of Phaseolus vulgaris
Acknowledgements L. primary leaves. Journal of Experimental Botany 49,1387–1395.
TL received a UK BBSRC quota studentship. We thank Leegood RC. 1993. Carbon metabolism. In: Hall DO, ScurlockProfessor RA Herbert for supporting our work and Professor JMO, Bolhar-Nordenkampf HR, Leegood RC, Long SP,HG Jones for constructive criticism of the manuscript. Richard eds. Photosynthesis and production in a changing environment:Parsons, Bill Berry, Wendy James, and Jean Crombie are a field and laboratory manual. London: Chapman andthanked for technical assistance. Hall, 129–165.
Long SP, Hallgren J-E. 1993. Measurement of CO2 assimilationby plants in the field and the laboratory. In: Hall DO,
References Scurlock JMO, Bolhar-Nordenkampf HR, Leegood RC,Long SP, eds. Photosynthesis and production in a changing
Barradas VL, Jones HG. 1996. Responses of CO2 assimilation environment: a field and laboratory manual. London: Chapmanto changes in irradiance: laboratory and field data and and Hall, 247–267.a model for beans (Phaseolus vulgaris L.). Journal of Mansfield TA, Hetherington AM, Atkinson CJ. 1990. SomeExperimental Botany 47, 639–645. current aspects of stomatal physiology. Annual Review of
Beyschlag W, Pfanz H. 1990. A fast method to detect the Plant Physiology and Plant Molecular Biology 41, 55–75.occurrence of non-homogenous distribution of stomatal Meidner H, and Mansfield TA. 1968. Physiology of stomata.aperture in heterobaric plant leaves. Experiments with Arbutus Blackie, Glasgow.unedo during the diurnal course. Oecologia 82, 52–55. Miranda V, Baker NR, Long SP. 1981. Anatomical variation
Beyschlag W, Pfanz H, Ryel RJ. 1992. Stomatal patchiness in along the length of the Zea mays leaf in relation toMediterranean evergreen sclerophylls. Planta 187, 546–553. photosynthesis. New Phytologist 88, 595–605.
Cardon ZG, Mott KA, Berry JA. 1994. Dynamics of patchy Mott KA. 1995. Effects of patchy stomatal closure on gasstomatal movements, and their contribution to steady-state exchange measurements following abscisic acid treatment.and oscillating stomatal conductance calculated with gas- Plant, Cell and Environment 18, 1291–1300.exchange techniques. Plant, Cell and Environment 17, Mott KA, Buckley TN. 1998. Stomatal heterogeneity. Journal995–1008. of Experimental Botany 49, 407–417.
Daley PF, Raschke K, Ball JT, Berry JA. 1989. Topography of Mott KA, Cardon ZG, Berry JA. 1993. Asymmetric patchyphotosynthetic activity of leaves obtained from video images stomatal closure for the two surfaces of Xanthium strumariumof chlorophyll fluorescence. Plant Physiology 90, 1233–1238. L. leaves at low humidity. Plant, Cell and EnvironmentDarwin F. 1898. Observations on stomata. Philosophical 16, 25–34.Transactions of the Royal Society, London 190, 531–621. Parkinson KJ, Day W, Leach JE. 1980. A portable system forDownton WJS, Loveys BR, Grant WJR. 1988. Stomatal closure
measuring photosynthesis and transpiration of graminaceousfully accounts for the inhibition of photosynthesis by abscisicleaves. Journal of Experimental Botany 31, 1441–1453.acid. New Phytologist 108, 263–266.
Parsons R, Weyers JDB, Lawson T, Godber IM. 1997. RapidEckstein J, Beyschlag W, Mott KA, Ryel RJ. 1996. Changes inand straightforward estimates of photosynthetic character-photon flux can induce stomatal patchiness. Plant, Cell andistics using a portable gas exchange system. PhotosyntheticaEnvironment 12, 559–568.34, 265–279.Farquhar GD, Sharkey TD. 1982. Stomatal conductance and
Pearcy RW. 1990. Sunflecks and photosynthesis in plantphotosynthesis. Annual Review of Plant Physiology 33,canopies. Annual Review of Plant Physiology and Plant317–345.Molecular Biology 41, 421–453.Genty B, Meyer S. 1994. Quantitative mapping of leaf
Pospısilova J, Santrucek J. 1994. Stomatal patchiness. Biologiaphotosynthesis using chlorophyll fluorescence imaging.Plantarum 36, 481–510.Australian Journal of Plant Physiology 22, 277–284.
Raschke K. 1965. Das Siefenblasenporometer (zur messung derGunasekera D, Berkowitz GA. 1991. Heterogeneous stomatalstomaweite an amphistomatischen blattern). Planta 66,closure in response to leaf water deficits is not a universal113–120.phenomenon. Plant Physiology 98, 660–665.
Sharkey TD. 1990. Photosynthesis in intact leaves of C3 plants:Hashimoto Y, Ino T, Kramer P, Naylor AW, Strain BR. 1984.physics, physiology and rate limitations. The Botanical ReviewDynamic analysis of water stress of sunflower leaves by51, 53–105.means of a thermal imaging system. Plant Physiology
Schuepp PH. 1993. Leaf boundary layers. New Phytologist76, 266–269.125, 477–506.Jones HG. 1998. Stomatal control of photosynthesis and
Siebke K, Weis E. 1995. Assimilation images of leaves oftranspiration. Journal of Experimental Botany 49, 387–398.Glechoma hederacea: analysis of non-synchronous stomataJones HG. 1999. Use of thermography for quantitative studiesrelated oscillations. Planta 196, 155–165.of spatial and temporal variation of stomatal conductance
Slavik B. 1963. The distribution pattern of transpiration rate,over leaf surfaces. Plant, Cell and Environment 22, (in press).water saturation deficit, stomata number and size, photosyn-Larcher W. 1995. Physiological plant ecology, 3rd edn. Berlin:thetic and respiration rate in the area of the tobacco leafSpringer.blade. Biologia Plantarum 5, 143–153.Lauer MJ, Boyer JS. 1992. Internal CO2 measured directly in
Solarova J, Pospısilova J. 1983. Photosynthetic characteristicsleaves. Plant Physiology 98, 1627–1635.during ontogenesis of leaves. 8. Stomatal diffusive conduct-Lawson T. 1997. Heterogeneity in stomatal characteristics. PhD
thesis, University of Dundee. ance and stomata reactivity. Photosynthetica 17, 101–151.
Terashima I, Wong S-C, Osmond CB, Farquhar GD. 1988. Society for Experimental Biology, Seminar Series Vol. 63.Cambridge University Press, 129–149.Characterisation of non-uniform photosynthesis induced by
abscisic acid in leaves having different mesophyll anatomies. Wigley G, Clark JA. 1974. Heat transport coefficients ofconstant energy flux models of broad leaves. Boundary LayerPlant and Cell Physiology 29, 385–394.
van Gardingen PR, Grace J. 1991. Plants and wind. Advances Meteorology 7, 139–150.Willmer CM, Fricker M. 1995. Stomata, 2nd edn. London:in Botanical Research 19, 189–253.
van Gardingen, PR, Jeffree CE, Grace J. 1989. Variation in Chapman and Hall.Wise RR, Ortiz-Lopez DH, Ort DR. 1992. Spatial distributionstomatal aperture in leaves of Avena fatua L. observed by
low-temperature scanning electron microscopy. Plant, Cell of photosynthesis during drought in field-grown and acclim-ated and non-acclimated growth chamber-grown cotton.and Environment 12, 887–888.
Weyers JDB, Lawson T. 1997. Heterogeneity in stomatal Plant Physiology 100, 26–32.Zipperlen SW, Press MC. 1997. Photosynthetic induction andcharacteristics. Advances in Botanical Research 26, 317–352.
Weyers JDB, Lawson T, Peng Z-Y. 1997. Variation in stomatal stomatal oscillations in relation to the light environment totwo dipterocarp rain forest trees. Journal of Ecology 85,characteristics at the whole leaf level. In: van Gardingen P,
Foody G, Curran P, eds. Scaling up from cell to landscape. 491–503.
