2.1.5   C4 photosynthesis

Printer-friendly version

Most vascular plants perform C3 photosynthesis, including commercially significant cereals, broad-leafed herbaceous plants such as legumes and oil seeds as well as all tree species. However, some important modern groups that are phylogenetically derived from C3 plants perform C4 photosynthesis (see Hatch 1988 and Feature essay 2.1). Plants in this group include maize, sugar cane, sorghum, a wide variety of tropical pasture grasses and most of the world’s worst tropical weeds (such as crabgrass and nutgrass). C4 plants are generally characterised by high rates of photosynthesis and growth, particularly in subtropical/tropical environments.


Figure 2.3 C4 photosynthesis is an evolutionary development where specialised mesophyll cells initially fix CO2 from the air into four-carbon acids which are transported to the site of the PCR cycle in the bundle sheath. The bundle sheath cells are relatively impermeable to CO2, so that when the CO2 is released here from the four-carbon acids, it builds up to high levels. The C4 photosynthetic mechanism is a biochemical CO2 pump. The pathway shown here is overlayed on a micrograph of a C4 leaf, showing bundle sheath and mesophyll cells. Rubisco and the other PCR enzymes are in the bundle sheath cells while phosphoenolpyruvate (PEP) carboxylase is part of the CO2 pump in the mesophyll cells. In C4 plants, after radioactive labelling, 14C appears first in a four-carbon acid, rather than in 3-PGA. Scale bar = 10 µm. (Original drawings courtesy M.D. Hatch)

C4 plants have a competitive advantage over C3 plant at high temperature and under strong light because of a reduction in photorespiration plus an increase in absolute rates of CO2 fixation at ambient CO2. Such increase in photo-synthetic efficiency results in faster carbon gain and commonly higher growth rates. The C4 pathway (Figure 2.3) is ‘a unique blend of modified biochemistry, anatomy and ultra-structure’ (Hatch 1988). Initial and rapid fixation of CO2 within mesophyll cells results in formation of a four-carbon compound which is then pumped to bundle sheath cells for decarboxylation and subsequent incorporation into the PCR cycle in that tissue. This neat division of labour hinges on specialised anatomy and has even resulted in evolution of distinct classes of chloroplasts in mesophyll compared with bundle sheath cells. Three variants of C4 photosynthesis are known to have evolved from C3 progenitors (Section 2.2) and in all cases with a recurring theme where the C4 cycle of mesophyll cells is complemented by a PCR cycle in bundle sheath cells. In effect, a biochemical ‘pump’ concentrates CO2 at Rubisco sites in bundle sheath cells thereby sustaining faster net rates of CO2 incorporation and virtually eliminating photorespiration.

For this overall mechanism to have evolved, a complex combination of cell specialisation and differential gene expression was necessary. Figure 2.3 shows a low-magnification electron micrograph of a C4 leaf related to a generalised scheme for the C4 pathway.

By analogy with Calvin’s biochemical definition of the C3 pathway at Berkeley in the 1950s, the C4 pathway was also delineated with radioactively labelled CO2 (see Feature essay 2.1). Significantly, and unlike C3 plants, 3-PGA is not the first compound to be labelled after a 14C pulse (Figure 2.3b). Specialised mesophyll cells carry out the initial steps of CO2 fixation utilising the enzyme phosphoenolpyruvate (PEP) carboxylase. The product of CO2 fixation, oxaloacetate, is a four-carbon organic acid, hence the designation ‘C4’ photo-synthesis (or colloquially, C4 plant). A form of this four-
carbon acid, either malate or aspartate depending on the C4 variant, migrates to the bundle sheath cells which contain Rubisco and the PCR cycle. In the bundle sheath cells, CO2 is removed from the four-carbon acid by a specific decarboxylase and a three-carbon product returns to the mesophyll to be recycled to PEP for the carboxylation reaction. Thus, label appears in the four-carbon acid first, after 14C feeding, and then in 3-PGA and, finally, sucrose and starch (Figure 2.1b).


Figure 2.4 CO2 photosynthesis response curves show that C4 plants have a higher affinity for CO2. At common ambient levels of CO2, photosynthesis in a C4 leaf is already almost fully expressed, whereas a C3 plant is operating at only one—half to two-thirds maximum rate. This contrast is due to the CO2-concentrating function of C4 photosynthesis. More sophisticated measurement of leaf assimilation as a function of intercellular CO2 (Figures 1-3 in Case study 1.1) can be used to reveal component processes. (Original drawings courtesy M.D. Hatch)

A physical barrier to CO2 diffusion exists in the cell walls of the bundle sheath compartment (suberised lamellae), preventing CO2 diffusion back to the mesophyll and allowing CO2 build up to levels at least 10 times those of ambient air. Rubisco is thus exposed to a saturating concentration of CO2 which both enhances carboxylation due to increased substrate supply, and forestalls oxygenation of RuBP (hence photorespiration) by outcompeting O2 for CO2-binding sites on Rubisco (Figure 2.4).

In leaves of C3 plants, the PCR cycle operates in all mesophyll chloroplasts, but in C4 plants the PCR cycle is restricted to bundle sheath cells (Figure 2.3). Rubisco is pivotal in this cycle, and can be used as a marker for sites of photosynthetic carbon reduction. Rubisco can be visualised by localising this photosynthetic enzyme with antibodies via indirect immunofluorescent labelling (Figure 2.5a, b). In this pioneering method (Hattersley et al. 1977) ‘primary’ rabbit anti-Rubisco serum (from rabbits injected with purified Rubisco) is first applied to fixed transverse sections of leaves. Rabbit antibodies to Rubisco bind to the enzyme in situ. Then ‘secondary’ sheep anti-rabbit immunoglobulin tagged with a fluorochrome (fluorescein isothiocyanate) is applied to the preparation. This fluorochrome binds specifically to the rabbit antibodies and fluoresces bright yellow wherever Rubisco is located (blue light excitation using an epifluorescence light microscope).


Figure 2.5 Rubisco can be localised in transverse sections of leaves by indirect immunofluorescent labelling where treated sections are viewed in conjunction with autofluorescence controls. Tissues such as bundle sheath extensions and epidermes fluoresce naturally, and such emission has to be ‘subtracted’ from present images. Considering (a), all chloroplasts in this C3 grass leaf (Microlaena stipoides) show a strong yellow fluorescence, indicating general distribution of Rubisco, and hence operation of the PCR cycle. By contrast, in (b), the C4 grass (Digitaria brownii) has restricted Rubisco to bundle sheath cells. In that case, mesophyll cells are devoid of Rubisco, fixing CO2 via the action of phosphoenolpyruvate carboxylase into a four—carbon acid which moves to bundle sheath cells, there providing CO2 for subsequent relaxation via Rubisco and the PCR cycle. Scale bar = 100 µm. (Original light micrographs courtesy Paul Hattersley)

In the C3 grass Microlaena stipoides (Figure 2.5a), all chloroplasts are fluorescing bright yellow and this indicates wide distribution of Rubisco throughout mesophyll tissue. By contrast, only bundle sheath cells are equipped with Rubisco in the C4 grass Digitaria brownii (Figure 2.5b).

These two native Australian grasses co-occur in the ACT but contrast in relative abundance. M. stipoides (weeping grass) is common in dry sclerophyll woodlands throughout southeast temperate Australia, whereas D. brownii (cotton panic grass) in the ACT is at the southern end of its distribution, being far more abundant in subtropical Australia and, in keeping with its C4 physiology, especially prevalent in semi-arid regions.

C4 energetics

One disadvantage of the C4 pathway is that an energy cost is incurred by C4 plants to run the CO2 ‘pump’. This is due to the ATP required for recycling PEP (Figure 2.3 and Hatch 1988). Under ideal conditions five ATP and two NADPH are required for every CO2 fixed in C4 photosynthesis (two ATP are required to run the CO2 pump). From the previous section,the C4 pathway is obviously energetically more expensive than the C3 pathway in the absence of photorespiration. However, at higher temperatures the ratio of RuBP oxygenation to carboxylation is increased and the energy requirements of C3 photosynthesis can rise to more than five ATP and three NADPH per CO2 fixed in air (for these calculations see Edwards and Walker 1983, and Hatch 1988).


Figure 2.6 Generalised light response curves for leaf photosynthesis show that C4 plants assimilate comparatively faster at high temperature (35°C), but that C3 plants are advantaged at low temperature (10°C). Photorespiration increases with temperature, and is largely responsible for this contrast. C4 plants are equipped with a CO2-concentrating device in their bundle sheath tissue which both enhances Rubisco’s perforniance at that location, and forestalls photorespiratory loss
(Original drawings coustesy M.D. Hatch)

Representative light response curves for photosynthesis in C3 cf. C4 plants (Figure 2.6) can be used to demonstrate some of these inherent differences in photosynthetic attributes. At low temperature (10°C in Figure 2.6) a C3 leaf shows a steeper initial slope as well as a higher value for light-saturated photosynthesis. By implication, quantum yield is higher and photosynthetic capacity is greater under cool conditions. In terms of carbon gain and hence competitive ability, C3 plants will thus have an advantage over C4 plants at low temperature and especially under low light.

By contrast, under warm conditions (35°C, upper curves in Figure 2.6) C4 photosynthesis in full sun greatly exceeds that of C3, while quantum yield (inferred from initial slopes) remains unaffected by temperature. Significantly, C3 plants show a reduction in quantum yield under warm conditions (compare 10°C and 35°C curves; right side of Figure 2.6). At 35°C C3 plants also show lower rates of light-saturated assimilation compared with C4 plants. Increased photorespiratory losses from C3 leaves at high temperature are responsible (Section 2.3). C4 plants will thus have a competitive advantage over C3plants under warm conditions at both high and low irradiance.