The profile of photosynthetic rate with depth through a leaf depends on the profiles of light absorption and photosynthetic capacity. Using a combination of several techniques, a comprehensive description of spinach leaves has been obtained. Profiles of CO2 fixation were obtained by exposing leaves to 14CO2 for 10 s under blue or green light before freeze clamping and paradermal sectioning. Profiles of light absorption were measured on adjacent parts of the leaf by quantifying chlorophyll fluorescence images of the transversely cut face obtained when blue or green light was applied to the adaxial or abaxial surface. The profile of CO2 fixation was modelled using the measured profiles of light absorption and photosynthetic capacity. There was remarkably good agreement between the observed and modelled CO2 fixation profiles for the eight combinations of colour, orientation and irradiance tested. Gas exchange of an intact leaf was also measured concurrently with conventional chlorophyll fluorescence under blue or green light. These data were consistent with the multi-layer leaf model with the exception of blue light applied to the abaxial surface, where chlorophyll fluorescence appeared to come from layers deeper than expected. Photosynthetic capacity matched the profile of green but not white light absorption through the leaf.
Extensive measurements of the gas exchange of leaves have been made and, by making some simplifying assumptions, it has been possible to describe the photosynthetic characteristics of a leaf with a handful of key parameters that reflect the underlying biochemistry of the leaf (Farquhar, von Caemmerer & Berry 1980). The mathematical description of biochemistry at the level of a chloroplast was scaled up to that of a leaf by assuming a uniform intensity of light and a constant biochemical composition through a given leaf. This assumption is clearly no longer tenable given our increasingly detailed knowledge of leaves. First, Terashima & Saeki (1983) showed that monochromatic light was attenuated in direct proportion to the cumulative chlorophyll content of a leaf. Second, by painstakingly paradermally sectioning leaves, Terashima & Inoue (1984, 1985a, b) demonstrated that chloroplasts differed through a leaf both biochemically and ultrastructurally. However, scaling from single chloroplasts to a leaf is still possible, by changing the assumption of uniform light and constant biochemical composition to one in which biochemical capacity divided by light absorption is constant for all layers (Oja & Laisk 1976; Terashima & Saeki 1985; Farquhar 1989).
Obtaining the profile of light absorption through a leaf has until now been difficult. One approach, developed by Vogelmann and coworkers (Vogelmann & Björn 1984; Vogelmann et al. 1988; Vogelmann, Bornman & Josserand 1989; Cui, Vogelmann & Smith 1991) has been to insert fibre-optic microprobes through leaves at different angles. This allows gradients in space irradiance [quantum flux in all directions through a point (Vogelmann & Björn 1984), cf. irradiance which is quantum flux incident onto a plane surface] to be calculated. Unfortunately, the parameter that is needed is absorptance, the amount of light absorbed at a given depth and this cannot be simply derived from space irradiance. It has been possible to combine multiple optical measurements with estimates of the spatial pattern of chlorophyll through leaves to calculate the light regime within a leaf through a complex model (Richter & Fukshansky 1996a, b). This approach suffers from two problems. The main limitation is that optical information obtained via fibre-optic microprobes is spatially very explicit and data from many insertions need to be combined to reach a spatially averaged estimate. The second limitation is the requirement for a profile of chlorophyll. Obtaining a spatial profile for chlorophyll has not been straightforward until recently (Vogelmann & Evans 2002).
A second approach to obtain profiles of light absorption through leaves has been to use a ray-tracing model (Ustin, Jacquemoud & Govaerts 2001). This requires some general assumptions about the average geometry of the mesophyll and also depends on the profile assumed for chlorophyll distribution through the leaf as well as extinction coefficients. An advantage of this approach is the ability to investigate the effect of angle of incidence, which is not easily addressed by other methods.
A third approach to obtain profiles of light absorption through leaves, has been to image chlorophyll fluorescence from a transversely cut surface of a leaf receiving laser light to the adaxial (top) surface (Takahashi et al. 1994). Profiles of scattered light, which relate to mesophyll cell anatomy and space irradiance, differed markedly from those of fluorescence. As would be expected, more weakly absorbed wavelengths penetrated further into the leaf such that the peak for fluorescence occurred deeper in the leaf with 515 compared to 477 nm light (Koizumi et al. 1998). Vogelmann & Han (2000) used a similar but simpler approach to measure chlorophyll fluorescence profiles through spinach leaves under blue, green and red light. These profiles are relatively easy to obtain, provide a good spatial average and seem to provide a valid measure of light absorption through a leaf (Vogelmann & Evans 2002).
Profiles of carbon fixation through leaves have been measured in Vicia faba (Outlaw & Fisher 1975; Jeje & Zimmermann 1983) and Spinacia oleracea (Nishio, Sun & Vogelmann 1993; Sun, Nishio & Vogelmann 1998). The peak fixation shifted to greater depths when measured with less strongly absorbed wavelengths. Nishio et al. (1993) argued that ‘the carbon fixation gradient did not follow the leaf internal light gradient’ and that ‘the light gradient is disconnected from CO2 fixation’. However, re-analysis of their data showed that the carbon fixation profiles were consistent with the expectations based on light absorption obeying the Beer–Lambert law in combination with the distribution of Rubisco through the leaf (Evans 1995, 1999). Despite the close agreement between experimental data and model predictions, this interpretation is still debated, e.g. (Nishio 2000).
Consequently, we have set out to obtain a comprehensive set of new data. We measured chlorophyll fluorescence profiles to estimate the profile of light absorption and 14CO2 fixation profiles under a range of conditions to test against model predictions. The methodology and justification for equating fluorescence profiles with light absorption has been described in detail in the companion paper (Vogelmann & Evans 2002). Here, we present the 14CO2 fixation profiles and show them to be consistent with predictions made using the profiles of light absorption and photosynthetic capacity. A further test is made using gas exchange and conventional chlorophyll fluorescence collected from the surface receiving the actinic light. The multi-layer leaf model agreed with the gas exchange data and was largely consistent with conventional chlorophyll fluorescence data.
MATERIALS AND METHODS
Spinach plants (Spinacia oleracea L.) were grown in soil in controlled environments under a 12 h photoperiod of 600 µmol quanta m−2 s−1 and a day/night temperature of 20/15 °C. Plants were watered daily and supplied with a slow-release fertilizer (Osmocote; Scotts Australia, Castle Hill, NSW, Australia). A detailed description of the fluorescence imaging methodology is given in Vogelmann & Evans (2002).
The 14CO2 labelling was performed in a fume hood in a 3-cm-square clear acrylic housing with a 15-mm diameter chamber and glass windows. The chamber volume was 1.6 cm3. The two halves of the chamber were either clamped onto a detached leaf or leaf pieces were placed on a mesh support inside the chamber. Leaves were collected from plants held under 1000 µmol quanta m−2 s−1 so that labelling could begin within 30 s of closing the chamber. The chamber was clamped into position under a fibre-optic light guide that provided 150 or 600 µmol quanta m−2 s−1 of blue (450 ± 19 nm) or green (567 ± 24 nm) light. Labelling began by injecting 1 cm3 of room air containing 7.4 kBq 14CO2 into the lower half of the chamber. After 10 s the chamber was removed and a 6-mm-diameter leaf disc was punched and frozen with a clamp that had been pre-chilled in liquid nitrogen, a process taking up to 5 s. Two samples were labelled from each leaf. One-half of the leaf was oriented normally, receiving light on the adaxial (top) surface (see Fig. 1, arrow 2). The other was oriented upside down to deliver light to the abaxial (bottom) surface (Fig. 1, arrow 3). The surface towards the light was marked with ink just prior to freeze clamping so that it could be correctly oriented during subsequent paradermal cryosectioning.
Additional leaf material was cut (2 × 6 mm paired pieces) and mounted vertically in the chamber so that it received light perpendicular to the transverse face (Fig. 1, arrow 4, denoted edge labelling). These were exposed to 22.2 kBq 14CO2 for 10 s under 2000 µmol quanta m−2 s−1 before freeze clamping. One piece of each pair was sectioned from opposite sides.
Frozen leaf pieces were transferred to a cryomicrotome and 40-µm-thick sections were collected and placed into scintillation vials. Fifty microlitres of acetic acid were added to liberate any remaining CO2, then 100 µL of ethanol and 1 mL of scintillant (Ultima gold XR; Packard Bioscience BV, Groningen, The Netherlands) were added and the c.p.m. were measured for each vial in a liquid scintillation counter. Given the small amount of leaf material assayed, quenching was very low and was similar for all vials.
Concurrent gas exchange and chlorophyll fluorescence measurements were made with a special chamber (4.15 cm2) attached to a LiCor 6400 gas exchange system (LiCor Inc., Lincoln NE, USA). The rate of electron transport, J, was calculated according to von Caemmerer & Farquhar (1981):
J = (A + R)(4C + 8Γ*)/(C − Γ*)(1)
where A and R are the rates of CO2 assimilation and respiration, C is the calculated intercellular CO2 partial pressure and Γ* is the CO2 photocompensation point [36.9 µbar (von Caemmerer et al. 1994)]. Actinic light was supplied via a multi-armed fibre-optic bundle with four other arms being utilized for chlorophyll fluorescence measurements using a modified PAM 101 (Walz, Effeltrich, Germany). The modulated light-eliciting fluorescence was supplied by a blue light-emitting diode (LED) (470 nm) and monitored by two detectors. Either the normal detector (730 nm) or one which had a 680 nm interference filter were monitored. Saturating pulses (1 s, 10 000 µmol quanta m−2 s−1) were delivered to determine Fm′. Photochemical efficiency of photosystem II was calculated according to (Genty, Briantais & Baker 1989) as 1 − F/Fm′. Reflectance and transmittance of the leaf were subsequently measured with a Taylor integrating sphere to enable data to be presented on an absorbed light basis.
A multi-layer model was used to predict CO2 fixation profiles (Evans 1995, 1999). The irradiance response function for each layer is given by:
P = (φI + Pm − [(φI + Pm)2 − 4ΘφIPm]0.5)/2Θ(2)
where Θ is a curvature factor (0.85), φ is the maximum quantum yield [0.5 mol e–(mol quanta)−1], I is the product of actinic irradiance with the fraction of that irradiance absorbed by the layer (from fluorescence profiles) and Pm is the photosynthetic capacity of the layer (from edge labelling, the sum of Pm for the leaf was 179 µmol e– m−2 s−1). Because the 14CO2 labelling is not in absolute units, the sum of all the layers is set to one.
The model was also used in the reverse direction, taking 14CO2 fixation profiles and seeking profiles of light absorption and photosynthetic capacity that minimized the variance. First, the profiles obtained under 150 µmol quanta m−2 s−1 were used to calculate the profile of light absorption for each colour and leaf orientation. Then the four 14CO2 profiles obtained under 600 µmol quanta m−2 s−1 were used simultaneously along with the derived light absorption profiles to derive a profile of photosynthetic capacity.
In order to calculate the profile of CO2 fixation through a leaf, the profiles of photosynthetic capacity and light absorption are needed. We estimated photosynthetic capacity by labelling leaf pieces oriented vertically so that light fell normal to the transversely cut surface (Fig. 1, arrow 4). The use of small leaf pieces for the edge labelling procedure resulted in compression of the tissue during freeze clamping. Consequently, it was necessary to assume that the 40-µm-thick sections actually represented a layer of fresh tissue that was 48.6 µm thick. The profile (Fig. 2) shows a maximum at a depth of 170 µm with another slight rise again near the abaxial surface. For comparison, we have shown a profile of Rubisco measured on spinach leaves grown under similar conditions (Nishio et al. 1993). The two profiles closely resemble one another, apart from slight deviations at each surface.
The profile of light absorption was obtained by imaging chlorophyll fluorescence from a transversely cut leaf (Fig. 1, arrow 1) receiving light to the adaxial or abaxial surface (Fig. 3). When oriented normally, so that light fell on the adaxial surface and entered the mesophyll through the palisade tissue, light absorption increased to a maximum at a depth of 65 µm (blue) or 110 µm (green). For blue light, 50% was absorbed by the initial 120 µm of leaf tissue whereas it took 230 µm to capture 50% of green light. When the leaf was inverted so that light entered via the spongy mesophyll (Fig. 3b), maximum absorption occurred closer to the surface (39 and 65 µm for blue and green light, respectively) and in general, light did not penetrate as deeply. For blue light, 50% was absorbed by the first 85 µm of leaf tissue and by 155 µm for green light, with a reduced difference between the profiles for the two wavelengths.
Eight profiles of CO2 fixation were measured using two leaf orientations (adaxial versus abaxial), blue or green light and 600 or 150 µmol quanta m−2 s−1 (Fig. 4). As would be expected from the profiles of light absorption, CO2 fixation under blue light was mainly adjacent to the surface receiving the light. Under green light, CO2 fixation continued throughout the leaf, steadily declining at depths greater than 200 µm. Profiles obtained under 600 µmol quanta m−2 s−1 differed from those under 150 µmol quanta m−2 s−1, by having their maximum displaced to greater depth and an increased proportion of fixation occurring in the half of the leaf opposite the surface receiving the light. For inverted leaves receiving light on their abaxial surface, CO2 fixation was concentrated nearer to the surface, again consistent with more rapidly declining absorption of light. The curves drawn on each panel represent the CO2 fixation profiles predicted by the model using the chlorophyll fluorescence profiles as a measure of light absorbed by each layer and the 14CO2 fixation profile of edge-labelled spinach as a measure of photosynthetic capacity. There was good agreement between the measured and modelled CO2 fixation profiles in all eight conditions.
To examine the sensitivity of the model, the measured CO2 fixation profiles were used to predict the profiles of light absorption and photosynthetic capacity (Fig. 5). Under 150 µmol quanta m−2 s−1, the profile of CO2 fixation mainly reflects that of light absorption. Consequently, light absorption profiles were calculated first and are shown against the individual and mean leaf profiles (Fig. 5a & b). Overall, the predicted values fell within the spread of observed values. The only notable difference was seen with adaxial blue or green light, where less light appears to have been absorbed in the first 100 µm than expected from chlorophyll fluorescence. Light absorption profiles derived from 14CO2 labelling under 150 µmol quanta m−2 s−1 were placed in the model and the four 14CO2 profiles measured under 600 µmol quanta m−2 s−1 were then simultaneously analysed to seek the best solution for the profile of photosynthetic capacity (Rubisco). The derived profile (solid line) was remarkably close to the profile obtained by edge-labelling spinach (Fig. 5c). Model predictions were recalculated using the fitted profiles of light absorption and photosynthetic capacity to ascertain the internal consistency of the eight measured CO2 fixation profiles (Fig. 6). The only deviation between 14CO2 data and model prediction occurred in the two layers adjacent to the abaxial surface, under abaxial lighting. To account for this, either the variability between leaves was underestimated or the profile of light absorption varied depending on irradiance (e.g. chloroplast movement).
The observed pattern of 14C fixation through the leaf could be predicted by the model. To demonstrate how the pattern changes as irradiance varies for a given wavelength and orientation, a series of profiles are superimposed (Fig. 7). The upper bound is set by the profile of photosynthetic capacity. For a leaf that is oriented normally, green light results in a fairly uniform proportion of photosynthetic capacity being utilized at all depths (Fig. 7a). By contrast, in blue light 90% saturation of the upper layers is reached by 500 µmol m−2 s−1 so that subsequent increases in irradiance mainly increase photosynthesis in the spongy mesophyll (Fig. 7b). Due to the mismatch between photosynthetic capacity and light absorption for an inverted leaf, the increasing contribution by the layers opposite the light source is more evident with abaxial orientation (Fig. 7c & d). The proportion of 14C label fixed in the first eight layers nearest the light source is shown in Table 1. The observed proportion decreased as irradiance increased with the exception of green light applied to the adaxial surface (i.e. normal orientation).
Table 1. Percentage of 14C label fixed in the eight layers (320 µm) of the spinach leaf nearest the light source
Mean leaf thickness was 680 µm. Green or blue light was applied to the adaxial or abaxial surface at either 150 or 600 µmol quanta m−2 s−1. Mean percentage ± SE (n = 5–9).
58 ± 3
58 ± 3
78 ± 4
56 ± 2
87 ± 5
73 ± 2
94 ± 1
83 ± 4
As an additional test of the multi-layer leaf model, gas exchange and fluorescence measurements were made under monochromatic light. Rates of electron transport were calculated from CO2 exchange measurements and are shown as a function of absorbed irradiance (Fig. 8). Quantum yield was 22% lower under blue compared with green light. Inverting the leaf so that light was applied to the abaxial surface resulted in a depression of the rate of electron transport at higher irradiances. The rate under white light (1600 µmol quanta m−2 s−1) applied to the abaxial surface was 35% lower than when applied to the adaxial surface (101 and 155 µmol e– m−2 s−1 for the abaxial and adaxial orientation, respectively). The curves in Fig. 8 were generated by the multi-layer leaf model. The model was parameterized using the edge labelling (Fig. 2) for the profile of photosynthetic capacity and chlorophyll fluorescence profiles (Fig. 3) as a measure of light absorption through the leaf. It was also necessary to assume a quantum yield of 0.27 or 0.21 mol e– (mol absorbed quanta)−1 for green and blue light, respectively (McCree 1971; Evans 1987). The reduction in electron transport rate for a given absorbed irradiance when the leaf was inverted occurs because of the mismatch between light absorption and photosynthetic capacity, rather than any difference in quantum yield. Abaxial surface layers quickly reach light saturation and thus waste excess absorbed light as heat.
To assess the mismatch between photosynthetic capacity and absorbed light, we monitored chlorophyll fluorescence at 680 and 730 nm elicited by a blue LED (Fig. 9). The modulated light eliciting fluorescence was changed from the standard 650 nm to a 470 nm LED in an attempt to sample more closely to the leaf surface. Fluorescence was monitored at two wavelengths to capture different layers beneath the surface. Fluorescence emission is most intense near 680 nm but is strongly reabsorbed by chlorophyll. Therefore 680 nm fluorescence escaping from the leaf is likely to have arisen close to the leaf surface. Longer wavelength fluorescence (730 nm) avoids re-absorption by chlorophyll and may represent layers deeper within the leaf. Because the absorption profile for green light applied to the adaxial surface is quite similar to the profile of photosynthetic capacity, the relative quantum yield of all layers is quite similar (Fig. 9a). Inverting the leaf places maximum light absorption in layers that have lower photosynthetic capacity. Consequently, the relative quantum yield differs between layers for a given irradiance (Fig. 9b). The situation is further exaggerated when blue light is used as the mismatch between light absorption and photosynthetic capacity is greater than for green light (Fig. 9c & d). The quantum yields measured by chlorophyll fluorescence tend to follow the curves predicted by the model for the layers adjacent to the surface receiving the light. Longer wavelength fluorescence tended to have higher relative quantum yields (b730 > b680), consistent with the fluorescence signal originating from deeper layers. The quantitative agreement was very close for green light applied to either surface and for blue light applied to the adaxial surface.
However, for blue light applied to the abaxial surface (Fig. 9d), quantum yields measured by chlorophyll fluorescence were considerably greater than expected from the model, corresponding to a layer 140 µm beneath the surface. Although this may indicate that the profiles of light absorption and photosynthetic capacity were incorrect, the overall rate of electron transport predicted for the leaf closely matched that measured by gas exchange (Fig. 8). Under blue light, the relative quantum yield for the leaf as a whole is also shown, as this would be equivalent to that calculated from gas exchange (Fig. 9c & d). The relationship between quantum yield calculated from chlorophyll fluorescence and whole leaf gas exchange would clearly depend on how fluorescence was sampled through the leaf (i.e. the wavelength of the modulated fluorescence excitation light compared to the actinic light and the waveband used to sample fluorescence will influence the quantitative relationship).
The amount of light absorbed per unit chlorophyll declines with increasing depth into a leaf (Fig. 10). The decline is more rapid for a strongly absorbed wavelength (blue) than for green light. For an inverted leaf, the difference between the wavelengths is less pronounced. When oriented normally, the profile of photosynthetic capacity per unit chlorophyll is quite close to that of green light absorbed per unit chlorophyll. However, in white light, the profile of light absorbed per unit chlorophyll would be expected to fall between the blue and green curves. Therefore, the profile of photosynthetic capacity was not closely matched to the profile of light absorption expected under the growth lighting conditions.
In order to model the photosynthetic properties of a leaf, one needs the profiles of photosynthetic capacity and light absorption, both expressed per unit leaf area for sequential layers through a given leaf. Both of these have proved difficult to obtain directly, which has led to difficulty in validating models. Our comprehensive set of measurements with spinach leaves now enable us to infer both of these profiles. We first discuss the problems and assumptions associated with deriving each of these parameters. By combining the information of both profiles into a multi-layer photosynthesis model of a single leaf, we could explore the properties of a given leaf. We validate the robustness of the model by considering how well it can account for the range of data that we collected. Extensive comparisons can be made with the 14CO2 fixation profiles measured under eight different combinations of leaf orientation, wavelength and irradiance and the gas exchange measurements made concurrently with conventional fluorescence. Finally, we consider the consequences that the dynamic characteristics of the profiles of photosynthetic capacity have on the properties of whole leaf gas exchange.
Profile of photosynthetic capacity
The irradiance response function (Eqn 2) contains a term that defines the maximum rate, Pm. For uncoupled electron transport, this corresponds to the maximum capacity for electron transport. However, when coupled to the Calvin cycle, the maximum rate depends on the activity of Rubisco as it varies with the partial pressure of CO2 (Ögren & Evans 1993). At the level of the whole leaf, there is a close correlation between the electron transport capacity and Rubisco activity (von Caemmerer & Farquhar 1981; Terashima & Evans 1988), so that with appropriate scaling, either parameter could be used as the estimate of Pm. It is also apparent from paradermal sectioning, that both the ratio of electron transport rate to Rubisco and cytochrome f to Rubisco are approximately constant through a spinach leaf (Terashima & Inoue 1985b). Therefore, it is acceptable to use profiles of either electron transport capacity or Rubisco as a measure of the profile of Pm. The profiles of Rubisco have been measured from paradermal sectioning of spinach leaves (Terashima & Inoue 1985b; Nishio et al. 1993). They revealed a linear relationship between Rubisco per unit chlorophyll and cumulative chlorophyll, declining from the adaxial surface.
We took a different approach to obtain the profile of Pm, by examining the profile of 14CO2 labelling when light was applied to the transversely cut face of a vertically aligned leaf piece. Using fluorescence imaging, we first established that light penetrated equally through palisade and spongy mesophyll when light was applied to a transversely cut face (Vogelmann & Evans 2002). Near to the cut surface, absorbed light would saturate photosynthesis so that 14CO2 labelling should vary in proportion to the photosynthetic capacity of the tissue. At some depth, when much of the light had been attenuated, CO2 fixation would revert to being proportional to light absorption. Fortunately, the measured distribution of chlorophyll through our spinach leaves was nearly constant for depths greater than 150 µm so that one would expect attenuation of light to be similar for different layers. However, near the surface where chlorophyll content was less, one would expect light to penetrate deeper and thus lead to greater fixation, or an overestimation of the photosynthetic capacity in these layers. One can also calculate that the amount of CO2 fixation should be curvilinearly related to the photosynthetic capacity, leading to a small underestimation of the capacity in layers with the greatest capacity.
Despite these potential problems, the profile of 14CO2 fixation obtained by edge labelling (Fig. 2) was strikingly similar to the Rubisco profile published by Nishio et al. (1993). The main differences between the two profiles were near the surfaces where our relative 14C fixation data did not decline as greatly as the Rubisco profile measured by Nishio et al. (1993). The relative differences between the maximum and minimum values for a leaf were the same for the two profiles. Bearing in mind the potential to overestimate the capacity near the adaxial surface and underestimate the maximum mentioned above, it is important to note that a very similar profile was obtained by two different approaches. By reversing the model, 14CO2 fixation data obtained with normal and inverted leaf orientations under 600 µmol quanta m−2 s−1 was also used to estimate the profile of photosynthetic capacity (Fig. 5c). The two methods produced very similar profiles. For spinach at least, it seems that we can be confident of having correctly defined the profile of photosynthetic capacity as several independent approaches have all produced very similar results. As discussed below, the profile of photosynthetic capacity reflects the light environment during growth and can change in response to changes in the light environment.
Profile of light absorption
The technique of imaging chlorophyll fluorescence from a transversely cut face of a leaf while applying light to one surface (Takahashi et al. 1994; Koizumi et al. 1998; Vogelmann & Han 2000) is potentially able to provide a good spatially averaged estimate of light absorption through a leaf. Because the leaf needs to be cut in order to view the transverse face, cutting might alter the internal reflection of light, the light profile and hence the fluorescence profile. The fluorescence images measured using a 680 nm interference filter, sample fluorescence that has not passed through much chlorophyll-containing tissue. This is because this wavelength is very strongly absorbed by chlorophyll. Longer wavelength fluorescence (730 nm) is weakly absorbed by chlorophyll and thus can travel considerable distances through the leaf. The shorter the distance that fluorescence is able to move through the leaf, the more certain is the spatial resolution. It was for this reason that we collected fluorescence images at 680 nm (Vogelmann & Evans 2002). The images do reveal places where fluorescence is high and yet are devoid of chlorophyll (e.g. epidermal cells, vascular tissue), indicating that fluorescence can travel into such tissues and then be reflected out to the cut surface where it escapes. Therefore, to equate fluorescence intensity to light absorption, one has to be able to define the chlorophyll-containing zone of the image. This is relatively easy to do either by measuring fluorescence induced by epi-illumination, or capturing an image of the leaf cross-section. Good spatial averaging with depth is simple to achieve by averaging across an image, while retaining the spatial resolution set by the microscope and charge-coupled device camera optics.
Fluorescence images were obtained by briefly illuminating the leaf piece (around 0.5 s). Previous work investigated whether errors would be introduced during the Kautsky induction curve and demonstrated that they were negligible for the short illumination times used here (Vogelmann & Han 2000). Another source of error is potentially introduced by measuring dark-adapted leaves whereas 14CO2 fixation was measured on leaves pre-treated with 1000 µmol quanta m−2 s−1. It is possible that chloroplast movement within the mesophyll cells in response to the lighting conditions altered the profile of light absorption, (for example, see Park, Chow & Anderson 1996; Kagawa & Wada 1999). The thick and optically dense spinach leaves would not be very amenable to investigate this aspect, however. Given that the cell walls were densely covered with chloroplasts in these leaves, there may have been little scope for significant chloroplast rearrangement. During the 14CO2 labelling, transpiration from the leaf sometimes condensed on the upper chamber window, which would have progressively diffused the light to a greater degree. Since diffuse light does not penetrate as deeply into a leaf as collimated light, e.g. (Richter & Fukshansky 1998), this possibly altered the light absorption profile during the 10 s labelling period. Given the variability from leaf to leaf, it is unlikely that this source of error could be detected in the 14CO2 labelling. Instead, assessment of this error requires the use of optical models; for example, that of Richter & Fukshansky (1998). Increasing the zenith angle from 25 to 65° resulted in much more rapid attenuation of light with depth (Ustin et al. 2001). Although the effect of zenith angle could be examined by fluorescence imaging, the present chamber design does not readily enable large zenith angles to be measured because of reflection from the window and shadowing from the sides of the chamber.
Extensive tests of the optics of the system confirmed that the intensity of the captured fluorescence image was proportional to the amount of light absorbed (Vogelmann & Evans 2002). The profiles of light absorption were shown to be consistent with monochromatic light obeying the Beer–Lambert law. The apparent extinction coefficient depended on the wavelength of light, mesophyll anatomy (palisade versus spongy) and could be decreased by infiltrating the leaf with water, which reduced the scattering of light within the leaf. Consequently, we are confident that the fluorescence images provide a quantitatively accurate measure of the pattern of light absorption within the leaf. Most importantly, it provides the parameter that is needed, namely light absorbed, rather than space irradiance.
Complex and sophisticated treatments of leaf optics and photosynthesis have been developed to investigate the relationship between photosynthetic properties of a leaf and the spatial patterns in light absorption and photosynthetic capacity through the leaf (Fukshansky & Von Remisowsky 1992; Richter & Fukshansky 1996a). Until now, it has been necessary to make assumptions about one or another profile. This results in cautious conclusions that depend on these assumptions. For example, Ustin et al. (2001) state that ‘since no hypotheses have been made on the Rubisco distribution, our simulations show that (Rubisco) is not the only factor controlling carbon fixation’. However, their model leaf defines a profile of chlorophyll through the leaf and assumes two values for photosynthetic capacity per unit chlorophyll for the palisade and spongy tissue and their photosynthetic profiles strongly reflect this assumed profile. In the absence of direct measurements, it has not been possible to assess the authors’ assertions. The profiles that we have derived from spinach leaves should now be inserted into these models to enable them to be validated against the 14CO2 fixation profiles presented here and by others (Nishio et al. 1993; Sun 1996; Sun et al. 1998; Sun & Nishio 2001).
Multi-layer model of a single leaf
Terashima & Saeki (1985) published a 10-layer leaf model in which they calculated the attenuance of 680 and 550 nm light through a leaf. The optical properties of the layers could be changed and the photosynthetic capacity of the palisade layers differed from that of the spongy tissue layers. Using this concept, Evans (1995) created a model to re-analyse the data of Nishio et al. (1993). In Evans (1995), the profile of light absorption was calculated from the profile of chlorophyll using the Beer–Lambert law. The profile of photosynthetic capacity was calculated using a linearly declining function with cumulative chlorophyll from the adaxial surface, based on Rubisco profiles measured from paradermal sectioning of spinach leaves (Terashima & Inoue 1985b; Nishio et al. 1993). The model explained the observed profiles of 14CO2 fixation under white light at two irradiances for both sun and shade leaves of spinach (Evans 1995). Additional 14CO2 fixation profiles measured under monochromatic light at different irradiances and leaf orientations were published by Sun et al. (1998). These were used to further test the multi-layer model, which was able to quantitatively predict the observed profiles of 14CO2 fixation (Evans 1999). However, there can be several grounds for concern with this approach. First, the model relied on applying profiles extracted from various papers to 14CO2 fixation data obtained on different leaf material. Second, it has been implied that it is not valid to apply the Beer–Lambert law to calculate light absorption in leaves (Richter & Fukshansky 1996b).
The data presented in this paper address both of these concerns. The model was parameterized and tested against 14CO2 fixation data, all generated with equivalent leaf material, using light absorption inferred from chlorophyll fluorescence profiles rather than calculated from chlorophyll profiles and the Beer–Lambert law. As it turned out, the measured profile of photosynthetic capacity was very similar to the profile of Rubisco published by Nishio et al. (1993) and the simple linear function previously used. We have also shown (Vogelmann & Evans 2002), that the profile of light absorption through the leaf is consistent with the Beer–Lambert law. However, in the present test, profiles of absorbed light measured by chlorophyll fluorescence were used. A rigorous test of the model was made by measuring 14CO2 fixation profiles under both blue and green light and by measuring profiles when light was applied to either the upper or lower leaf surface. The predictions matched the observed profiles with high fidelity, given the experimental uncertainties. The model also successfully predicted the dependence on irradiance. Although our 14CO2 data do not give an absolute rate of fixation, we used them to derive the best fit for the profile of photosynthetic capacity (Fig. 5c). The observed profile was remarkably similar to that predicted from the four profiles measured under 600 µmol quanta m−2 s−1. The greatest uncertainty in the present results is a small discrepancy in light absorption in the first few adaxial layers in which the fluorescence imaging suggested slightly more blue and green light was absorbed than expected from the 14CO2 fixation. The uncertainty in the 14CO2 data given by the standard errors perhaps gives a false impression of the scatter. The difference between the highest and lowest value at any layer was between 30 and 50%, so the deviations are quite possibly due to experimental error in the 14CO2 data.
Additional tests of the model were made by combining gas exchange and chlorophyll fluorescence measurements (Figs 8 & 9). These confirmed predictions made by the model. Measuring irradiance response curves with inverted leaves reduces the rate of CO2 assimilation for a given absorbed irradiance due to the mismatch of photosynthetic capacity and light absorption. Chlorophyll fluorescence confirms this as it effectively only samples chloroplasts near the surface of the leaf. Using very similar fluorescence methodology with sunflower leaves, Peterson, Oja & Laisk (2001) showed that at a given irradiance and rate of photosynthesis, photochemical efficiency of far red fluorescence was greater than that of red fluorescence. Peterson et al. (2001) interpreted this both in terms of non-uniform profiles of non-photochemical quenching through the leaf and the relative absorption cross-section of photosystem II. They concluded that far red fluorescence would provide a more reliable measure of whole leaf quantum yield than red fluorescence.
Although the multi-layer model used here is quite simple, it can account for all observed features of 14CO2 fixation profiles in spinach leaves measured under a wide range of conditions. The model can be driven either by functions defining the profiles of chlorophyll and Rubisco (Evans 1999), or using profiles of fluorescence and edge labelling, as in this paper.
Irradiance response curves of leaves
The rate of photosynthesis per unit leaf area for a given irradiance depends on the orientation of the leaf with respect to the light source and the wavelength. Generally, the rate obtained for an inverted leaf at intermediate irradiances is less than for a normally oriented leaf, for example, see Moss (1964), Oja & Laisk (1976) and Evans, Jakobsen & Ögren (1993). The difference in rates depending on orientation, reflects the fact that there is a gradient in photosynthetic capacity per unit chlorophyll through a leaf. Spongy mesophyll tends to have lower photosynthetic capacity than palisade mesophyll (Terashima & Inoue 1985a, b). Consequently, with an inverted leaf, spongy mesophyll approaches light saturation at a lower irradiance and the light absorbed there is dissipated as heat rather than photosynthesis. The gradient in photosynthetic capacity through a leaf can change in response to changes in the light environment. For example, the irradiance response curves of a leaf changed when it was inverted after mesophyll differentiation had been fixed (Terashima 1986; Ögren & Evans 1993). Although the optical properties differ between palisade and spongy mesophyll, leaf inversion experiments show that photosynthetic capacity adapts to the amount of light within each cell and is not simply a result of the cell's position within a bifacial leaf. Just as leaf inversion alters the distribution of light absorption with respect to photosynthetic capacity through a leaf, so too does changing wavelength.
For spinach leaves, we now have comprehensive data on the profile of photosynthetic capacity with respect to light absorption (Fig. 10). Photosynthetic capacity per unit of chlorophyll declined with depth away from the adaxial surface, almost following the curve of absorbed green light per unit chlorophyll. The photosynthetic capacity increased again as it approached the abaxial surface. This may indicate that under our growth conditions, a proportion of light was intercepted by the abaxial surface of the leaf protruding beyond the edge of the pot, due to reflection and scattering within the growth cabinets. Evidence that photosynthetic capacity adapts to the light absorption gradient is clearly shown in the data of Sun & Nishio (2001). There, 14CO2 fixation per unit Rubisco was relatively constant throughout the leaf when white light was applied to the adaxial surface, achieving an even closer match to the profile of absorbed light than we observed.
How does the spatial distribution of resources within a leaf affect the photosynthetic return on that investment? One can calculate the return by analysing photosynthetic irradiance response curves generated by a multi-layer model of the leaf which incorporates the profiles of light absorption and photosynthetic capacity. To approximate white light, the fluorescence profiles in Fig. 3 for blue and green light were averaged. For a leaf with a capacity of 200 µmol e– m−2 s−1 and a Θ-value for each layer of 0.85, the curve fitted to the leaf with light entering the adaxial surface had Pm = 192 and Θ= 0.69, whereas for abaxial orientation, Pm = 187 and Θ = −0.32. The lower Θ-value reflects the mismatch between light absorption and photosynthetic capacity because each layer approaches light saturation at different external irradiances. By integrating the irradiance response curve to estimate daily photosynthesis, the observed spinach leaf achieved 90% of the maximum daily photosynthesis when photosynthetic capacity was distributed in direct proportion to the light absorption profile (Pm/Iabs for each layer was a constant). An inverted leaf gained only 72% of the potential photosynthesis. A leaf can only optimize the distribution of photosynthetic capacity for a given spectrum of light and a given light environment (i.e. the proportion of light received by each surface and the balance between collimated and diffuse light). Since the light environment changes daily depending on the weather and on even shorter time intervals due to wind and time of day, it is not possible to remain perfectly adapted. However, leaves have been shown to acclimate to changes in the light environment with a half-time of several days through changes in their biochemistry (Chow & Anderson 1987; Leverenz 1988; Evans 1993; Ögren & Evans 1993). During leaf development, it is possible to alter the mesophyll anatomy in response to the light environment. It is rare for mature leaves to be able to do this. However, when mature leaves of ivy were transferred from 40 to 400 µmol quanta m−2 s−1 for at least 40 d, the mesophyll thickness increased by 50% due to the formation of an additional palisade cell layer (Bauer & Thöni 1988). Mesophyll thickness has also been found to increase following decapitation of Mulberry trees due to elongation of palisade cells (Satoh, Kriedemann & Loveys 1977). More commonly, it is the flexibility of their biochemistry that enables mature leaves to respond to changes in their light environment.
Data presented in this paper enable a multi-layer analysis of spinach photosynthesis based on measured profiles of light absorption and photosynthetic capacity. Our model was comprehensively validated against 14CO2 fixation profiles obtained under a range of conditions in which leaf orientation, wavelength and irradiance were varied. Our model was further validated by comparison with gas exchange and chlorophyll fluorescence measurements on intact leaves. Photosynthetic capacity scales through a leaf approximately in proportion to green light absorption when the leaf is oriented normally. Sampling fluorescence signals from intact leaves as a surrogate estimate of photosynthetic electron transport will not necessarily give the correct estimate for the whole leaf because the detected fluorescence emanates from a small part of the mesophyll. The relationship between fluorescence and electron transport will depend on leaf anatomy and the actinic, modulated excitation and fluorescence sampling wavelengths used.
A remaining challenge is to be able to measure the profile of photosynthetic capacity more easily. Fluorescence imaging enables the relatively easy collection of spatially resolved profiles of light absorption and chlorophyll distribution. If the saturation pulse methodology could be applied through epi-illumination microscope optics, it would be possible to spatially resolve the profile of photosynthetic capacity. While equipment exists to measure this through fine fibre-optic probes (Schreiber et al. 1996), it has not yet been developed for this approach. If it became available, all of the leaf profile data could be readily obtained sequentially on a given leaf sample using a single microscope.
We would like to thank Susanne von Caemmerer and Murray Badger for lending us some of the equipment used in this experiment and to Paul Kriedemann for his comments. This research was supported by NSF DBI-9724499.
Received 3 July 2002; received in revised form 20 September 2002; accepted for publication 25 September 2002