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- Materials and Methods
- Note added in proof
Carbon dioxide is trapped mainly by the mesophyll cells of leaves, where it is converted into organic compounds by photosynthesis. This creates the driving force for the subsequent delivery of CO2 into leaves from the ambient air (Nobel, 1991). The gradient in CO2 concentration determines both the direction and the rate of net CO2 transport. Consequently, gas exchange is generally studied in vertical (anticlinal) directions of leaves, including boundary layers, stomata, intercellular air spaces, cell walls, membranes and liquid phases of mesophyll cells and, finally, the envelope and matrix of chloroplasts (Parkhurst, 1994).
In addition to this vertical transport, CO2 can also move in lateral (paradermal) directions through the intercellular air spaces of leaves. Lateral gradients in CO2 concentration are needed, as well as the absence of barriers, to enable internal lateral gas movement. This is a trait of homobaric leaves in which intercellular air spaces are laterally interconnected. Heterobaric leaves, on the other hand, have bundle sheath extensions that span the gap between the upper and the lower epidermis, forming physical barriers for lateral gaseous diffusion (Neger, 1918). Jahnke & Krewitt (2002) demonstrated that the lateral diffusion of CO2 inside homobaric leaves is effective over distances of at least 8 mm (the width of leaf chamber gaskets) and can cause artefacts in gas exchange measurements performed using clamp-on leaf chambers. Literature on lateral gas diffusion inside leaves is still rather scarce and, in general, restricted to short diffusion distances. The uneven distribution of closed stomata over a leaf blade may result in different intercellular CO2 concentrations (ci) in different mesophyll compartments of heterobaric leaves, causing nonuniformly distributed photosynthesis; this is unlikely to occur in homobaric leaves because of internal lateral CO2 diffusion (Terashima, 1992). It was found, using high-resolution fluorescence imaging, that the photosynthetic activity of guard cells was influenced by the lateral diffusion of CO2 when only a small area of the leaf was illuminated; respiration in the surrounding (shaded) area overrode any influence of changes in atmospheric CO2 concentration (ca) (Lawson et al., 2002). In addition, recent findings confirm that conductivities of homobaric leaves can be even larger in the lateral than in the vertical (anticlinal) directions (Pieruschka et al., 2005).
In the present work, we posed the question of whether lateral CO2 exchange inside leaves could play a role in partially shaded leaves. Generally, even under sunny conditions, only the outermost leaves of a canopy are fully exposed to light, while the others are at least partly or temporarily shaded. Only the upper five ‘layers’ of a canopy are above light compensation while, in lower layers, respiration may exceed assimilation (Nobel et al., 1993). In particular, understory plants demonstrate an extreme example where light fluctuations over time contribute largely to the carbon gain of the plants (Pearcy & Pfitsch, 1994; Pearcy et al., 1996). However, not only temporal fluctuations, but also spatial heterogeneities in the distribution of light, may contribute to net carbon gain; when some parts of a leaf are illuminated and others are shaded, internal lateral gradients in CO2 concentration can develop across light/shade borders (LSB). Shaded leaf sections may then act as an internal CO2 source, whereas illuminated areas are sinks for CO2 owing to photosynthetic activity. Shading or illuminating a leaf outside a clamp-on leaf chamber might then influence the leaf internal CO2 concentration gradients in lateral directions and alter the net CO2 exchange rates (NCER) of the leaf part enclosed in the leaf chamber. Furthermore, the leaf chamber gaskets of a leaf chamber artificially seal the stomata, and (respiratory) CO2 released underneath has to escape laterally. Such artificial closure of stomata, together with partial shading of a leaf, was simulated here by fixing a nontranslucent and gas-impermeable tape on both sides of a leaf. The effect of laterally diffusing CO2 was then visualized in the adjacent illuminated leaf areas by using chlorophyll fluorescence imaging, which is widely applied to measure heterogeneities in leaf photosynthesis (Oxborough, 2004).
Photosynthesis is progressively diminished under drought stress, while the exact mechanisms of this reduction are still under debate (Pankovic et al., 1999; Medrano et al., 2002a; Parry et al., 2002; Tezara et al., 2002; Kitao et al., 2003). However, it has been found recently that stomatal conductance represents an integrative basis for the overall effects of drought, and photosynthetic responses are understood to be a direct adjustment of photosynthetic metabolism to CO2 availability (Flexas et al., 2002; Medrano et al., 2002b; Bota et al., 2004). Low intercellular CO2 concentrations occurring under stomatal closure may cause light stress, even at low light intensities (Long et al., 1994; Ort & Baker, 2002). To avoid excess light, plants have developed different mechanisms. For example, changes in leaf orientation, relative to direct solar irradiance, affect the amount of light absorbed by a leaf and, consequently, photosynthetic activity, transpiration rate and temperature (Cornic & Massacci, 1996). In order to protect the photosynthetic apparatus from photoinhibitory damage, excess light energy is consumed by photorespiration in C3 plants (Osmond et al., 1997; Wingler et al., 1999; Ort, 2001; Cornic & Fresneau, 2002; Medrano et al., 2002a; Ort & Baker, 2002) and thermal energy dissipation of absorbed light is associated with the light-induced formation of zeaxanthin (Demmig-Adams & Adams III, 1992; Horton et al., 1996). Heat dissipation may also provide tolerance to rapidly fluctuating excitation pressure (Külheim et al., 2002). When stomatal conductance decreases at an advanced stage of drought stress, down-regulation of photosystem II activity was observed, resulting in reduced electron transport rates and an increase in thermal energy dissipation (Flexas et al., 2002; Medrano et al., 2002b; Omasa & Takayama, 2003; Souza et al., 2004), which may be mediated by cycling electron transport (Cornic et al., 2000; Golding & Johnson, 2003).
We hypothesize that lateral CO2 fluxes inside homobaric leaves have beneficial effects under conditions when parts of the leaves are under high light while others are shaded: partial shading of a leaf may cause lateral CO2 diffusion to adjacent illuminated leaf areas, thereby enhancing overall leaf photosynthesis. This must be especially important in plants under drought stress where stomata are largely closed and CO2 uptake from ambient air is hindered. Refixation of respiratory CO2, supplied from shaded (remote) parts inside a leaf, can then help to increase photosynthetic efficiency in illuminated areas and attenuate the effects of drought stress by reducing potential damage of the photosynthetic apparatus arising from overexcitation. The goal of the present work was to test whether the hypothesized effect of lateral CO2 diffusion on photosynthesis across LSBs does exist and to evaluate whether it provides an appreciable contribution to leaf photosynthesis.
- Top of page
- Materials and Methods
- Note added in proof
In the standard setup of gas exchange measurements with clamp-on leaf chambers, only the leaf part enclosed in the chamber is considered. The leaf parts outside a chamber are not taken into account, and their potential contribution to processes inside the chamber is ignored. Different CO2 concentrations inside and outside a leaf chamber, however, may substantially affect measurements of respiration in the dark when homobaric leaves are investigated (Jahnke & Krewitt, 2002). These observations led us to question whether changing the light intensities on leaf areas outside a chamber may cause similar artefacts in measurements of photosynthesis. In principle, such a treatment should have no effect at all. However, when a homobaric leaf of V. faba was shaded outside the chamber, the apparent NCER became smaller (cf. Fig. 1). This response can be explained by higher ci values in the (shaded) leaf part outside the leaf chamber as a result of CO2 released by respiration leading to a net lateral CO2 flux towards the illuminated clamped leaf part.
When gas exchange measurements are performed on fully illuminated leaves, the very use of clamp-on leaf chambers leads to shading by both the gaskets and the handle. Respiratory CO2, originating from the leaf areas below the gaskets, can only escape laterally, thereby affecting gas exchange measurements performed inside the leaf chamber. This reasoning is supported by the chlorophyll fluorescence experiments in which leaf chamber gaskets were simulated with black adhesive tape (see Fig. 3a–d). The stomata in the shaded area were effectively sealed by this treatment, and an increase in quantum yield within the illuminated leaf area adjacent to the shade was observed for homobaric V. faba leaves. We interpret this result by an increase in ci within the shaded leaf area, which causes lateral CO2 transport and, as a consequence, higher photosynthetic rates in the illuminated area along the LSB. The gas exchange measurements, on the other hand, indicated lower apparent photosynthetic NCER inside the leaf chamber when leaves were shaded outside (cf. Fig. 1b). The results obtained by gas exchange measurements and chlorophyll fluorescence imaging therefore appear to be in conflict. However, lateral fluxes of CO2 across the LSBs can easily explain this apparent contradiction. On the one hand, additional CO2 is available for photosynthesis in the illuminated areas, which is supported by the chlorophyll fluorescence data presented above (higher ΦPSII values close to the LSBs). On the other hand, an increase in ci lowers the CO2 gradient, and thus CO2 fluxes, between ambient air and the leaf mesophyll, resulting in a decrease in the measured NCER. This, however, is an experimental artefact: gas exchange measurements only detect changes in leaf external CO2 and cannot reflect true NCER when there is an internal supply of CO2 into a clamped leaf region.
Stomatal conductance is the main mechanism by which plants control gas exchange and leaf temperature (Farquhar & Sharkey, 1982). Stomatal conductance decreases under mild or moderate drought stress, which leads to a reduction in ci, thereby affecting photosynthesis (Lawlor, 2002). The data presented here show that the potential effect of lateral diffusion on NCER is also dependent on stomatal conductance (Fig. 2a). Lateral CO2 fluxes depend on the CO2 supply from ambient air (i.e. in a vertical direction) determined by stomatal opening: the impact of lateral diffusion on photosynthetic NCER in illuminated leaf areas near LSBs was large when the gleaf was low and declined with higher gleaf values (cf. Fig. 2a). The contribution of stomatal conductance became apparent also in the chlorophyll fluorescence imaging experiments. When stomata were artificially sealed by adhesive tape, ΦPSII gradients near the LSBs were large at the beginning because the experiment started with dark-adapted leaves in which the stomatal conductance was low (Fig. 3d); the gradients in ΦPSII declined with time as a result of the gradual reopening of stomata after illumination (cf. Pearcy et al., 1996). The interplay between vertical and lateral fluxes becomes even clearer when a single leaf of V. faba is observed under a changing water supply. During drought stress, respiratory CO2 released in the shaded leaf part augmented CO2 availability in the illuminated leaf area near the LSB (indicated by the ΦPSII gradient across the different ROIs in Fig. 6). Rewatering caused two major changes as a consequence of stomatal reopening: the ΦPSII gradient decreased, indicating a diminishing impact of lateral CO2 transport, while (absolute) ΦPSII values increased as a result of the re-establishment of CO2 supply from ambient air. We therefore conclude that lateral CO2 supply across LSBs may contribute to photosynthesis of homobaric leaves under drought stress, but cannot fully substitute ‘normal’ CO2 supply via the stomata.
When stomatal conductance decreases, photoinhibitory damage through excess light poses a threat to the photosynthetic apparatus of leaves. In C3 plants, the photosynthetic apparatus is protected from photoinhibition by various mechanisms, such as photorespiration (Osmond et al., 1997; Ort, 2001; Cornic & Fresneau, 2002; Medrano et al., 2002a; Ort & Baker, 2002) and heat dissipation through light-induced formation of zeaxanthin (Demmig-Adams & Adams III, 1992). At natural stands, most leaves are only partially illuminated owing to self-shading inside a canopy and, at LSBs, have to cope with high (or even extreme) differences in light intensity. The increased quantum yield near LSBs, observed in the present study, may then provide an additional, yet-unknown mechanism to reduce light stress in homobaric leaves. The increase in ci owing to lateral CO2 fluxes from shaded to illuminated leaf parts causes higher quantum yield and ΦPSII, and can also explain the observed decrease in NPQ (cf. Fig. 5). Interestingly, the relative decrease in NPQ was larger than the increase in photosynthetic efficiency. We interpret this as a first indication that protection from overexcitation through lateral CO2 fluxes across LSB could be even more beneficial for leaves under drought stress than the higher yield in CO2 assimilation. Plants that can withstand drought stress are more effective in conserving tissue hydration than drought-susceptible plants (Grzesiak et al., 1999). They reduce water loss by stomatal closure but then have to cope with a diminished supply of CO2. Considerable gas transport and, consequently, refixation of respiratory CO2 from remote parts of the leaves, however, are only possible if intercellular space is open for gas transport in the lateral directions. This is a characteristic trait of homobaric leaves for which lateral gas conductivities have been reported to be even larger than gas conductivities in vertical directions (Pieruschka et al., 2005). When stomatal conductance decreases and lateral conductance remains constant in such leaves, more respiratory CO2 is internally available for refixation, and the effects of lateral CO2 diffusion on photosynthesis and energy dissipation become pronounced (cf. Fig. 5). With rising temperatures, which cause an increase in VPD and force stomatal closure to prevent water loss (Mott & Parkhurst, 1991), the effects described might become even larger.
These observations may lead to the hypothesis that the homobaric leaf anatomy is an adaptation to the specific environmental conditions in which the evaporative demand of plants is high. Refixation of respiratory CO2 released from (remote) shaded leaf parts could result in higher water use efficiency (WUE). These processes cannot occur in plants that have heterobaric leaves. Plants with high WUE generally grow in relatively dry habitats (Larcher, 2003) and one may then speculate whether homobaric leaf anatomy may prevail in plant species native to such areas. Wylie (1952) presented a survey on 348 plant species with respect to the occurrence of bundle sheath extensions, which are the main barriers for lateral gas movement. Plants with homobaric leaves (c. 40% of the species) were from warmer regions, whereas those with heterobaric leaves were mostly from northern (temperate) areas. In particular, the investigated woody species featuring homobaric leaves favoured evergreen habitats and many showed xeromorphic leaf modifications (Wylie, 1952). In warmer habitats, plants may at least temporarily face low relative humidity (i.e. high VPD), which is one of the key factors mediating changes in stomatal sensitivity to CO2 (Monteith, 1995; Talbott et al., 2003). Thus, lateral gas conductance and internal CO2 refixation may also favour the efficient use of water under unfavourable conditions.
The proposed hypothesis, that the potential to use CO2 from remote leaf areas is beneficial for plants with homobaric leaves, has yet to be evaluated under natural conditions.