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Figure 1.1 A scanning electron micrograph of an uncoated and rapidly frozen piece of tobacco leaf showing a hairy lower leaf surface and cross-sectional anatomy at low magnification. Notional values for resistances to CO₂ diffusion are given in units of m² s mol^-1. Corresponding values for CO₂ concentration are shown in µL L^-1. C_i is routinely inferred from gas exchange measurements and used to construct A:C_i curves for leaf photosynthesis. Scale bar = 100 µm. (Image courtesy J-W. Yu and J. Evans)

In a typical herbaceous dicotyledon (Figure 1.1) lower leaf surfaces are covered with epidermal outgrowths, known to impede movement of small insects, but also contributing to formation of a boundary layer. This unstirred zone of air immediately adjacent to upper and lower epidermes varies in thickness according to surface relief, area and wind speed. Boundary layers are significant in leaf heat budgets and feature in the calculation of stomatal and mesophyll conductances from measurements of leaf gas exchange.  

The diffusion of CO₂ into leaves can be modelled using an analogue with electrical resistance (R) and conductance (the reverse of resistance), as in Figure 1.1, right hand side. This shows a series of resistances (r) that would be experienced by CO₂ molecules diffusing from outside (ambient) air, through the boundary layer (b), the stomata (s), the intercellular airspaces (i), the cell walls and liquid phase (w) to fixed sites inside chloroplasts. These values emphasise the prominence of stomatal resistance within the series.

Corresponding values for CO₂ concentration in ambient air (a), the leaf surface (s), the substomatal cavity (i), the mesophyll cell wall surface (w) to the sites of carboxylation with the chloroplasts (c) reflect photosynthetic assimilation within leaves generating a gradient for inward diffusion.

In transverse fracture as shown below in Figure 1.2(A) the bifacial nature of leaf mesophyll is apparent with columnar cells in the palisade layer beneath the upper surface and irregular shaped cells forming the spongy mesophyll below. Large intercellular airspaces, particularly in the spongy mesophyll, facilitate gaseous diffusion. The lower surface of this leaf is shown in Figure 1.2(B). On the left-hand side, the epidermis is present with its irregular array of stomata. Diagonally through the centre is a vein with broken-off hair cells and on the right the epidermis has been fractured off revealing spongy mesophyll cells beneath. Light micrographs of sections cut parallel to the leaf surface (paradermal) through palisade (C) and spongy (D) tissue reveal chloroplasts lying in a single layer and covering most of the internal cell wall surface adjacent to airspaces. Significantly, chloroplasts are rarely present on walls that adjoin another cell. Despite the appearance of close packing, mesophyll cell surfaces within the palisade layer are generally exposed to intercellular airspace. Inward diffusion of CO₂ to chloroplasts is thereby facilitated. 

Leaves that develop in sunny environments and have high photosynthetic capacities are generally thicker than leaves from shaded environments. This is achieved with more elongate cells within the palisade layer and/or several layers of cells forming the palisade tissue. Thicker leaves in a sunny environment enable more Rubisco to be deployed which confers a higher photosynthetic capacity. Fitting more Rubisco into a unit of leaf area with good access to intercellular airspace requires an increase in mesophyll cell surface which is possible by increasing the thickness of the mesophyll tissue and hence leaf thickness. A thicker leaf in sunny environments is energy effective because enough photons reach chloroplasts in lower cell layers to keep their Rubisco gainfully employed. By contrast, in a shaded habitat, less Rubisco is required for a leaf with lower photosynthetic capacity and this can be fitted into thinner leaves.

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Figure 1.2 A scanning electron micrograph of an uncoated and rapidly frozen piece of tobacco leaf fractured in (A) to reveal columnar mesophyll cells of the palisade layer beneath the upper leaf surface and spongy mesophyll in the lower half. Chloroplasts can be clearly seen covering the inner faces of cell walls. Looking onto the lower surface (B), the epidermis and stomata are present on the left side of the vein, whereas the epidermis was fractured away on the right side, revealing spongy mesophyll tissue. Light micrographs (C, D) of sections cut parallel to the leaf surface are shown for palisade (C) and spongy mesophyll (D) with solid lines showing where the paradermal sections align with (A). Chloroplasts form a dense single layer covering the cell surfaces exposed to intercellular airspace, but are rarely present lining walls where two cells meet. Scale bar in (A) = 50 µm and (B) = 200 µm.  (C) and (D) have same magnification as (A). (Images courtesy J. Evans and S. von Caemmerer)

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Figure 1.3 Light absorption by pigments in solution and by leaves. Absorbance (A) refers to attenuation of light transmitted through a leaf or a solution of leaf pigments, as measured in a spectrophotometer, and is derived from the expression A = log I₀/I where I₀ is incident light, and I is transmitted light. The solid curve (scale on right ordinate) shows absorbance of a solution of pigment—protein complexes equivalent to that of a leaf with 0.5 mmol Chl m^-2. The dotted curve shows absorptance (scale on left ordinate), and represents the fraction of light entering the solution that is absorbed. Virtually all light between 400 and 500 nm and around 675 nm is absorbed, compared with only 40% of light around 550 nm (green). The dashed curve with squares represents leaf absorptance, which does not reach 1 because the leaf surface reflects part of the incident light. Leaves absorb more light around 550 nm than a solution with the same amount of pigment (75 versus 38%, respectively) because leaves scatter light internally. This increases the pathlength and thereby increases the probability of absorption above that observed for the same pigment concentration in solution. (Based on K.J. McCree, Agric Meteorol 9: 191-216, 1972; J.R. Evans and J.M. Anderson, BBA 892: 75-82, 1987)

Pigments in thylakoid membranes of individual chloroplasts (Figure 1.7) are ultimately responsible for strong absorption of wavelengths corresponding to blue and red regions of the visible spectrum (Figure 1.3). Irradiated with red or blue light, leaves appear dark due to this strong absorption, but in white light leaves appear green due to weak absorption around 550 nm, which corresponds to green light. Ultraviolet (UV) light (wavelengths below 400 nm) can be damaging to macromolecules, and sensitive photosynthetic membranes also suffer. Consequently, plants adapt by developing an effective sunscreen in their cuticular and epidermal layers. 

Overall, absorption of visible light by mesophyll tissue is complex due to sieve-effects and scattering. Sieve-effect is an outcome from packaging pigments into discrete units, in this case chloroplasts, while remaining leaf tissue is transparent. This increases the probability that light can bypass some pigment and penetrate more deeply. A regular, parallel arrangement of columnar cells in the palisade tissue with chloroplasts all vertically aligned means that about 80% of light entering a leaf initially bypasses the chloroplasts, and measurements of absorption in a light integrating sphere confirm this. Scattering occurs by reflection and refraction of light at cell walls due to the different refractive indices of air and water. Irregular-shaped cells in spongy tissues enhance scattering, increasing the path length of light travelling through a leaf and thus increasing the probability of absorption. Path lengthening is particularly important for those wavelengths more weakly absorbed and results in nearly 80% absorption, even at 550 nm (Figure 1.3). Consequently, leaves typically absorb about 85% of incident light between 400 and 700 nm; only about 10% is reflected and the remaining 5% is transmitted. These percentages do of course vary according to genotype × environment factors, and especially adaptation to aridity and light climate.

Sunlight entering leaves is attenuated with depth in much the same way as light entering a canopy of leaves shows a logarithmic attenuation with depth that follows Beer’s Law (Section 12.4). Within individual leaves, the pattern of light absorption is a function of both cell anatomy and distribution of pigments. An example of several spatial profiles for a spinach leaf is shown in Figure 1.4. Chlorophyll density peaks in the lower palisade layer and decreases towards each surface. The amount of light declines roughly exponentially with increasing depth through the leaf. Light absorption is then given by the product of the chlorophyll and light profiles. Light absorption initially increases from the upper surface, peaking near the base of the first palisade layer, then declines steadily towards the lower surface. Because light is the pre-eminent driving variable for photosynthesis, CO₂ fixation tends to follow the light absorption profile (see ¹⁴C fixation pattern in Figure 1.4). However, the profile is skewed towards the lower surface because of a non-uniform distribution of photosynthetic capacity. Chloroplasts near the upper surface have ‘sun’-type characteristics which include a higher ratio of Rubisco to chlorophyll and higher rate of electron transport per unit chlorophyll. Chloroplasts near the lower surface show the converse features of ‘shade’ chloroplasts. Similar differences between ‘sun’ and ‘shade’ leaves are also apparent. Chloroplast properties do not change as much as the rate of absorption of light. Consequently, the amount of CO₂ fixed per quanta absorbed increases with increasing depth beneath the upper leaf surface. The lower half of a leaf absorbs about 25% of incoming light, but is responsible for about 31% of a leaf’s total CO₂ assimilation.

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Figure 1.4 Profiles of chlorophyll, light absorption and photosynthetic activity through a spinach leaf. Cell outlines are shown in transverse section (left side). Triangles represent the fraction of total leaf chlorophyll in each layer. The light profile (dotted curve) can then be calculated from the Beer—Lambert law. The profile of absorbed light is thus the product of the chlorophyll and light profiles (solid curve). CO₂ fixation, revealed by ¹⁴C labelling, follows the absorbed light profile, being skewed towards slightly greater depths. (Based on J.N. Nishio et al., Plant Cell 5: 953-961, 1993; J.R. Evans, Aust J Plant Physiol 22: 865-873, 1995)

Leaves are covered with a barrier or ‘cuticle’ on the outer walls of epidermal cells that is impermeable to both water and CO₂. To enable CO₂ entry into the leaf for photosynthesis, the epidermis is perforated by pores called stomata (Figure 1.5). As CO₂ molecules diffuse inwards they encounter an opposite flux of H₂O molecules rushing outwards that is three to four orders of magnitude stronger. This problem of transpirational water loss is a particular problem for plants in hot, dry climates, such as in most of Australia. Leaves control this gas exchange by adjusting the aperture of stomata which can vary within minutes in response to changes in several environmental variables including light, humidity and CO₂ concentration (see Chapter 15 for more details). Air-spaces inside leaves are effectively saturated with water vapour (equivalent to 100% relative humidity at that leaf temperature) and because air surrounding illuminated leaves is almost universally drier, water molecules diffuse outwards down this concentration gradient from leaf to air. 

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Figure 1.5 Diagram of a transverse section through an isolateral Eucalyptus pauciflora leaf which is normally pendulant. Palisade tissue occurs beneath both surfaces with spongy tissue and oil glands (not shown) in the middle. Putative pathways for diffusion of H₂O out of substomatal cavities are shown by the solid curved arrows. CO₂ diffuses inwards and H₂O diffuses outwards in response to concentration differences between the leaf and air. Such gas exchange is restricted by a boundary layer (the unstirred layer of air at the leaf surface) and by stomata. One stoma is shown on each surface. CO₂ diffusion continues inside the leaf mesophyll through airspaces between cells (curved dashed arrows) to reach cell walls adjacent to each chloroplast where CO₂ dissolves and then diffuses into the chloroplast to reach the carboxylating enzyme Rubisco. Bundle sheath extensions (bottom of diagram) reach both epidermis and create an internal barrier to lateral diffusion. (Based on J.R. Evans et al., Planta 189: 191-200, 1993)

The diffusion pathway for H₂O out of a leaf is usually divided into two parts, namely the boundary layer of still air at the leaf surface and stomatal pores (Figure 1.5). Boundary layer thickness depends on windspeed, leaf dimensions and the presence of surface structures (e.g. hairs in Figure 1.1). Positioning of stomata also varies between species. Leaves of terrestrial plants always have stomata on their lower (abaxial) surface but many species have stomata on both surfaces, especially if they have high photosynthetic rates and are in sunny locations such as pendulant leaves of eucalypts. Adaptations for arid environments include having surface structures like hairs and waxes, which increase the thickness of the boundary layer, and leaf rolling and encryption of stomata by placing them in crevices in the leaf surface. While these features restrict water loss, they also impose an increased resistance (decreased conductance) to CO₂ uptake. 

The flux of water escaping from a leaf, called transpiration rate, can be understood from Fick’s law. It depends on the product between conductance and the gradient in water vapour from the inside of the leaf to the surrounding air. The vapour pressure gradient depends on both the humidity of the surrounding air and leaf temperature. Dry air (low humidity), or hotter leaf temperatures will result in greater transpiration rates for a given conductance. Maximum leaf conductance depends on the number and size of stomata per unit leaf area which is a leaf property that becomes fixed during development. However, the aperture of stomata can be varied, so stomatal conductance can vary over the timescale of minutes. Stomatal conductance responds to light, CO₂ and humidity. The sensitivity of a leaf to these variables is not fixed but can change over time in response to, for example, drought. Transpiration rate can be measured by a variety of means. With the availability of portable instruments, it is now most commonly obtained by measuring the increase in water vapour content of air from a leaf enclosed in a chamber. Stomatal conductance can then be calculated from Fick’s law by dividing the transpiration rate by the vapour pressure gradient between the leaf and the air.

CO₂ molecules diffusing inwards from ambient air to chloroplasts encounter restrictions additional to boundary layer and stomata (Figure 1.5). CO₂ must also diffuse from substomatal cavities throughout the mesophyll, dissolve in wet cell walls, cross the plasma membrane to enter the cytosol, diffuse into chloroplasts across a double membrane (outer envelope in Figure 1.7) and finally reach fixation sites within the stroma of those chloroplasts. The combination of these restrictions from intercellular airspace to the sites of fixation within chloroplasts has been termed mesophyll conductance.

There is considerable variation in leaf anatomy and hence potential restriction to CO₂ diffusion, but in general leaves with high rates of photosynthesis tend to have more permeable leaves (e.g. tobacco in Figure 1.2) and this complex anatomy ensures a greatly enlarged surface area for diffusion across interfaces. Indeed the total mesophyll cell wall area can be 20 times that of the projected leaf surface.

Chloroplasts tend to be appressed against cell walls adjacent to intercellular spaces (Figure 1.2 C, D) which improves access to CO₂, and they contain carbonic anhydrase which speeds up diffusion of CO₂ by catalysing interconversion of CO₂ and bicarbonate within the stroma of chloroplasts. Although CO₂ rather than HCO₃^– is the substrate species for Rubisco, the presence of carbonic anhydrase enables bicarbonate ions, which are more abundant under the alkaline conditions (pH 8.0) that prevail inside chloroplasts, to diffuse to Rubisco in concert with diffusion of CO₂. By sustaining a very rapid equilibration between CO₂ and HCO₃^– immediately adjacent to active sites on Rubisco, carbonic anhydrase enhances inward diffusion of inorganic carbon.

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Figure 1.6 Photosynthetic response to photon irradiance for a Eucalyptus maculata leaf measured at three ambient CO₂ concentrations, 140, 350 and 1000 µmol mol^-1. Irradiance is expressed as µmol quanta of photosynthetically active radiation absorbed per unit leaf area per second, and net CO₂ assimilation is inferred from a drop in CO₂ concentration of gas passing over a leaf held in a temperature-controlled cuvette. CO₂ evolution in darkness is shown on the ordinate as an extrapolation below zero. The irradiance at which net CO₂ exchange is zero is termed the light compensation point (commonly 15-50 µmol quanta m^-2 s^-1, shade to sun species respectively). The initial slope of light-response curves for CO₂ assimilation per absorbed quanta represents maximum quantum yield for a leaf. (Based on E. Ögren and J.R. Evans, Planta 189: 182-190, 1993)

Light impinging on plants arrives as discrete particles we term photons, so that a flux of photosynthetically active photons can be referred to as ‘photon irradiance’. Each photon carries a quantum of electromagnetic (light) energy. In biology the terms photon and quantum (plural quanta) tend to be used interchangeably.

CO₂ assimilation varies according to both light and CO₂ partial pressure. At low light (low photon irradiance in Figure 1.6) assimilation rate increases linearly with increasing irradiance, and the slope of this initial response represents maximum quantum yield (mol CO₂ fixed per mol quanta absorbed). Reference to absorbed quanta in this expression is important. Leaves vary widely in surface characteristics (hence reflectance) as well as internal anatomy and chlorophyll content per unit leaf area. Therefore, since absorption of photosynthetically active quanta will vary, quantum yield expressed in terms of incident irradiance does not necessarily reflect the photosynthetic efficiency of the mesophyll. In the case of comparisons between sun and shade leaves, it has led to a widely held but mistaken belief that shade leaves (thinner and with higher chlorophyll content) are more efficient. Expressed in terms of absorbed quanta, sun and shade leaves have virtually identical quantum efficiencies for CO₂ assimilation. 

Assimilation rate increases more slowly at higher irradiances until eventually a plateau is reached where further increases in irradiance do not increase the rate of CO₂ assimilation (Figure 1.6). Chloroplasts are then light saturated. Absolute values for both quantum yield and light-saturated plateaux depend on CO₂ concentration. Quantum yield increases as CO₂ concentration increases as it competes more successfully with other species such as oxygen, at the binding site on Rubisco. Leaf absorptance has a hyperbolic dependence on chlorophyll content. For most leaves, 80–85% of 400–700 nm light is absorbed and it is only in leaves produced under severe nitrogen deficiency where there is less than 0.25 mmol Chl m^–2 that absorptance falls below 75%. 

The plateau in Figure 1.6 at high irradiance is set by maximum Rubisco activity. With increasing CO₂ partial pressure, the rate of carboxylation increases. The transition from light-limited to Rubisco-limited CO₂ assimilation as irradiance increases becomes progressively more gradual at higher CO₂ partial pressures. In part, this gentle transition reflects the fact that a leaf is a population of chloroplasts which have different photosynthetic properties depending on their position within that leaf. As discussed above, the profile of photosynthetic capacity per chloroplast changes less than the profile of light absorption per chloroplast (Figure 1.4). This results in an increase in CO₂ fixed per quanta absorbed with increasing depth. A transition from a light to a Rubisco limitation therefore occurs at progressively higher incident irradiances for each subsequent layer and results in a more gradual transition in the irradiance response curve of a leaf compared to that of a chloroplast. 

Photosynthetic capacity of leaves varies widely according to light, water and nutrient availability and these differences in capacity usually reflect Rubisco content. Leaves in high light environments (‘sun’ leaves) have greater CO₂ assimilation capacities than those in shaded environments and this is reflected in the larger allocation of nitrogen-based resources to photosynthetic carbon reduction (PCR cycle; Section 2.1). Sun leaves have a high stomatal density, are thicker and have a higher ratio of Rubisco to chlorophyll in order to utilise the larger availability of photons (and hence ATP and NADPH). Shade leaves are larger and thinner, but have more chlorophyll per unit leaf dry weight than sun leaves. They can have a greater quantum yield per unit of carbon invested in leaves, but with a relatively greater allocation of nitrogen-based resources to photon capture, shade leaves achieve a lower maximum rate of assimilation.

Despite such differences in leaf anatomy and chloroplast composition, leaves sustain energy transduction and CO₂ fixation in an efficient and closely coordinated fashion. Processes responsible are discussed below (Section 1.2).