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Photosynthesis depends on the diffusion of CO2 from the air surrounding a leaf into the leaf and into the chloroplasts. As leaves are typically very thin, most plant physiologists think of this diffusion as a vertical one-dimensional flux of CO2, across one or both epidermes into the mesophyll, imagining a typical textbook cross-section of a leaf (see Fig. 1). However, the CO2 can diffuse in any direction inside the leaf. Leaves usually contain multiple air spaces, and there is clearly the possibility of lateral CO2 diffusion if these air spaces are well connected, and there is a gradient in concentration. A paper by Pieruschka et al. in this issue of New Phytologist (pp. 779–787), shows that such lateral diffusion can occur and can affect photosynthetic rates, and that there are substantial differences in rates of lateral CO2 diffusion depending upon leaf vein anatomy. The authors argue that these lateral fluxes may be important in enhancing photosynthesis and these fluxes may, in some situations, reduce possible photoinhibitory effects resulting from light and/or drought stress.
‘Studies exploring gaseous diffusion have rekindled interest in lateral diffusion and refocussed attention on the importance of leaf anatomy.’
Until recently, it has not been possible to determine spatial patterns of CO2 concentrations within leaves. However, because stomatal apertures and densities vary substantially across a leaf (Fig. 2), we must expect steep CO2 gradients, depending on photosynthetic rates. This can be the result of patchy light distribution in the natural environment, causing possible heterogeneous photosynthesis and stomatal behaviour. However, technological advances in chlorophyll a fluorescence imaging (Oxborough, 2004), combined with standard gas exchange analysis, now permit photosynthetic efficiency to be determined at the micrometre scale, and because photosynthetic efficiency is largely dependent on CO2 concentration, to thus infer information on the pattern of CO2 across the leaf. In a key paper, Meyers & Genty (1998) used this approach to map CO2 molar fractions across a photosynthesising leaf and showed that abscisic acid (ABA) primarily affects photosynthesis by decreasing stomatal conductance. This approach has been extended in recent papers that have determined internal leaf diffusion resistances and lateral CO2 diffusion in leaves with differing vascular anatomies. In this issue, Pieruschka et al. used this method to assess lateral diffusion of CO2 from shaded to illuminated areas of different leaves. When parts of leaves were artificially shaded, they measured an increase in PSII quantum efficiency in adjacent well-illuminated areas in Vicia faba and Nicotiana tabacum but not in Glycine max and Phaseolus vulgaris, which they attributed to differences in leaf venation. Clearly, such shading might occur in nature as a result of other leaves, but this point is also relevant to the artificial situation in which part of the leaf is enclosed in a cuvette. This and other studies (e.g. Morison et al., 2005) exploring gaseous diffusion have rekindled interest in lateral diffusion and refocussed attention on the importance of leaf anatomy.
Lateral CO2 diffusion in leaves depends on the anatomy of the leaf, and two extreme types are usually considered (Fig. 1). Lateral CO2 diffusion is considered to be totally restricted in ‘heterobaric’ leaves, in which the bundle sheath surrounding the vein extends to the upper and lower epidermis, compartmentalising the leaf into small areoles and restricting lateral movement of CO2 (Fig. 2). This is usually contrasted with ‘homobaric’ leaves in which such extensions are absent, which apparently allows significant CO2 movement within the leaf lamina. Therefore, if there is good connectivity in homobaric leaves, we should not expect to see spatially variable photosynthesis if the leaf is evenly illuminated, whereas it could occur in heterobaric leaves. However, we should be cautious when assigning vein status to a leaf, because no leaf is truly homobaric. The midrib always dissects a leaf into two halves, with further fragmentation by primary veins. Similarly, no leaf is truly heterobaric because a proportion of the vein extensions may not be continuous, resulting in joined-up areoles. It may therefore be more accurate to refer to a degree of heterobaricity or homobaricity (Lawson, 1997) or to define a scale of compartmentalisation when categorising the vein anatomy of leaves. It should also be obvious that the porosity of the leaf (measured as the proportion of leaf cross-sections that comprises air spaces) is not necessarily a good indicator of connectivity and permeability, and therefore whether large lateral CO2 gradients occur.
Leaf ‘patchiness’ or heterogeneity of function
Heterobaric leaf anatomy and the consequent gaseous compartmentalisation are believed under certain conditions to result in the development of ‘patchy stomatal behaviour’, in which stomata in adjacent regions exhibit significantly different mean apertures from each other (Weyers & Lawson, 1997). The discovery of patchy stomatal behaviour in the late 1980s received great attention because of its impact on the gas exchange calculations of intercellular CO2 concentration or Ci (e.g. Sharkey, 1990). Although homobaric leaves should have good internal diffusion because there is no compartmentalisation, they have been found to exhibit similar variability in stomatal apertures as in heterobaric leaves, but manifested in aperture trends rather than patches (Hashimoto et al., 1984; Smith et al., 1989; Weyers & Lawson, 1997). If the distribution of open stomata is uneven, this could lead to a significant gradient in Ci (Terashima, 1992; Lawson & Weyers, 1999). Confirming this, Terashima et al. (1988) reported patchy photosynthesis in homobaric leaves of V. faba, although this was not to the same extent and on a slightly larger spatial scale than that observed on the heterobaric leaves of Helianthus annuus, and they attributed this to areas of the leaf where stomata were closed. These observations reinforce the point that the heterobaric anatomy is not a prerequisite for nonuniform stomatal behaviour and/or photosynthesis.
The significance of variation in stomatal apertures and their impact on leaf photosynthesis have been highlighted in a recent paper by Morison et al. (2005). Using artificial ‘patches’ of grease on leaves, and chlorophyll fluorescence imaging, we established that, in moderate light, effective lateral CO2 diffusion in leaves of both homobaric and heterobaric species was restricted to less than 0.5 mm as a result of photosynthetic consumption of the CO2 along the diffusion path. Extending these observations with a diffusion model, we suggest that even if the diffusion effectiveness inside the leaf was as high as 50% of that in free air, the drawdown of CO2 in the leaf by photosynthesising leaves would be large, and lateral diffusion rates would only permit low rates of photosynthesis (Morison et al., 2005).
While neither our results nor those of Pieruschka et al. found any appreciable diffusion in the heterobaric Phaseolus leaves, the homobaric leaf results disagree with our own. Part of the reason may be other differences in anatomy beyond the simple description of heterobaric or homobaric. For example, our homobaric leaf was Commelina communis, which is a monocot, and V. faba used by Pieruschka et al. (2006) is a dicot. Another reason may be the slightly different scales considered, and the spatial resolution of the systems used.
The importance of lateral CO2 diffusion
In addition to the question of patchy leaf function, there are two situations where significant lateral CO2 diffusion could be important. Firstly, Pieruschka et al. show that when clamp-on leaf cuvettes are used, they inevitably shade an area of leaf around that being measured inside the cuvette. Thus, lateral diffusion of respiratory CO2 from shaded to illuminated areas in homobaric leaves can directly affect gas exchange measurement by increasing internal CO2 concentration, resulting in an underestimate of net CO2 exchange rate. If this is the case, smaller chambers will greatly influence the ratio of gasket shaded to illuminated area, increasing the edge-to-area ratio (Long & Bernacchi, 2003). This was highlighted by Jahnke & Krewitt (2002) in an earlier paper that demonstrated lateral diffusion of CO2 over a distance of 8 mm, a similar distance to that of a typical cuvette gasket.
Secondly, sun and shade flecks are an important aspect of the light environment experienced by plants in their natural habitat. Pieruschka et al. suggest that light flecks will therefore cause CO2 gradients in leaves so that in homobaric leaves the shaded areas may supply CO2 to the illuminated areas. Given that sun flecks and their consequent shade flecks (Pearcy, 1990) are usually transitory, this may be important. If stomata are shut, then the effect must be only temporary, as photosynthesis will rapidly mop up CO2 in the shaded leaf, given that respiration rates are usually an order of magnitude less than photosynthetic uptake rates. However, as leaves of shade plants, such as those on the forest floor, often keep stomatal aperture relatively high even in the shade (Barradas & Jones, 1996), it may be that appreciable lateral diffusion could act to increase the effective stomatal area supplying an area of illuminated mesophyll. It is therefore possible that lateral CO2 diffusion from shaded to illuminated areas of the leaf will enhance photosynthesis, and possibly protect against the damaging effects of excess light absorption by the leaf (Pieruschka et al.). Stomatal closure in shaded areas of the leaf will only occur after prolonged light reduction.
This leads to the question: what is the adaptive benefit of varying vein anatomy? As Pieruschka et al. point out, surveys have shown that leaves exhibiting bundle sheath extensions were dominant in Northern American deciduous plants, whereas those without were found mainly in evergreen broadleaved foliage of hotter climates. These authors also speculated that the presence of bundle sheath extensions in heterobaric leaves is possibly an adaptive mechanism to reduce the spread of disease and/or to provide structural support because these leaves tend to be thinner and more easily damaged by wind. Pieruschka et al. suggest that homobaric leaves found in hotter climates may have evolved to increase water use efficiencies, by allowing lateral CO2 movement. Although we may not fully understand the function of varying vein anatomy in relation to lateral gas movement and CO2 assimilation in leaves, technical advances in photosynthetic imaging, combined with traditional plant anatomy, are bringing us one step closer.