As noted above, the fixation of carbon dioxide occurs downstream of the energy capture, where the reducing power inherent in NADPH is deployed in the synthesis of carbohydrate. The flow of reductant drives the Calvin cycle of RuBP consumption and regeneration, during which carbon dioxide is drawn in and glucose is discharged. The cycle is summarised in Fig. 3.2. In the algae and in many higher plants, RUBISCO-mediated carboxylation of RuBP yields the first stable product of so-called C3 photosynthetic carbon fixation, the 3-carbon glycerate 3-phosphate (G3P). (Note that in this, the process differs from those terrestrial C4 fixers that synthesise four-carbon malate or aspartate.)
After the further NADPH-reduction of G3P to glyceraldehyde 3-phosphate (GA3P), the metabolism proceeds through a series of sugar-phosphate intermediates to yield a hexose (usually glucose). In this way, one molecule of hexose may be exported from the Calvin cycle for every five of GA3P returned to the cycle of RuBP regeneration and, ideally, one for every six molecules of carbon dioxide imported. In this case of steady-state photosynthesis, the following equation summarises the mass balance through the Calvin cycle:
The Calvin cycle. Carboxylation by RUBISCO of RuBP at 1 is driven by ATP and NADPH generated by the light reactions of photosynthesis, and results ultimately in the synthesis of sugar precursors and the renewed availability of RuBP substrate, thus maintaining the cycle. The cycle is regulated at the numbered reactions, where it may be short-circuited as shown. Abbreviations: DHAP, dihydroxyaceton phosphate; E4P, erythrose 4-phosphate; FBP, fructose 1,5-biphosphate; F6P, fructose 6-phosphate; GA3P, glyceraldehyde 3-phosphate; GBP, glycerate 1,3-biphosphate; G3P, glycerate 3-phosphate; G6P, glucose 6-phosphate; Pi, inorganic phosphate; RuBP, ribulose 1,5-biphospharte; Ru5P, ribulose 5-phosphate; R5P, ribose 5-phospate; SBP, sedoheptulose 1,7-biphosphate; S7P, sedoheptulose 7-phosphate; Xu5P, xylulose 5-phosphate. Redrawn with permission from Geider and MacIntyre (2002).
According to demand, the glucose may be respired immediately to fuel the energy demands of metabolism, or it may be submitted to the amination reactions leading to protein synthesis. Excesses may be polymerised into polysac-charides (glycogen, starch, paramylon). In this way, the overall photosynthetic Eq. (3.1) is balanced, at the minimal energy cost of eight photons per atom of carbon fixed. Thus, the theoretical maximum quantum yield of photosynthesis (p) is 0.125 mol C (mol photon)-1 (D. Walker, 1992).
However, neither the cycle nor its fixed-carbon yield is immutable but it is subject to deviation and to autoregulation, according to circumstances. As stated at the outset, these have reverberations at successive levels of cell growth, community composition, ecosystem function and the geochemistry of the biosphere. The variability may owe to imbalances in the light harvest and carbon capture, or to difficulties in allocating the carbon fixed. To evaluate these resourcing impacts requires us to look again at the sensitivity of the Calvin-cycle reactions, beginning with the initial carboxylation and the action of the RUBISCO enzyme.
RUBISCO, the catalyst of the CO2-RuBP conjunction, is a most highly conserved enzyme, occurring, with little variation, throughout the photosynthetic carbon-fixers (Geider and Mac-Intyre, 2002). From the bacteria, through the 'red line' and the 'green line' of algae (see Section 1.3), to the seed-bearing angiosperms, present-day photosynthetic organisms have to contend with acknowledged catalytical weaknesses of RUBISCO. These are due, in part, to the fact that carbon-dioxide-based photosynthesis evolved under different atmospheric conditions from those that presently obtain. In particular, the progressive decline in the partial pressure of CO2 exposes the rather weak affinity of RUBISCO for CO2 (Tortell, 2000). According to Raven (1997), the maximum reported rates of carboxylation (80 mol CO2 (mol RUBISCO)-1s-1: Geider and MacIn-tyre, 2002) are low compared to those mediated by other carboxylases. Even these levels of activity are dependent upon a significant concentration of carbon dioxide at the reaction site (with reported half-saturation constants of 12-60 |M among eukaryotic algae: Badger et al., 1998). Supposing that the cell-specific rate of carbon fixation could be raised by elevating the amount of active RUBISCO available, the investment in its large molecule (~560 kDa) is relatively expensive. RUBISCO may account for 1-10% of cell carbon and 2-10% of its protein (Geider and MacIntyre, 2002).
Having more RUBISCO capacity is not necessarily helpful either, owing to the susceptibility of RUBISCO to oxygen inhibition: at low CO2 concentrations (<10 | M) and high O2 concentrations (>400 |M), RUBISCO functions as an oxidase, in initiating an alternative reaction that leads to the formation of glycerate 3-phosphate and phosphoglycolate. In the steady-state Calvin-cycle operation, the activity of RUBISCO serves to maintain the balance between NADPH generation and the output of carbohydrates. For a given supply of reductant from PSI, the rate of carbon fixation may be seen to depend upon an adequate intracellular carbon supply and upon the RUBISCO capacity or, at least, upon that proportion of RUBISCO capacity that is actually 'active'. To be catalytcally competent, the active site of RUBISCO has also to be carbamylated by the binding of a magnesium ion and a nonsubstrate CO2 molecule. Under low light and/or low carbon availability, RUBISCO is inactivated (decarbamylated), by the reversible action of an enzyme (appropriately known as RUBISCO inac-tivase), to match the slower rate of RuBP regeneration. The resultant down-cycle sequestration of phosphate ions and lower ATP regeneration brings about an increase in ADP : ATP ratio and, thus, a decrease in RUBISCO activity (for further details of Calvin-cycle self-regulation, refer to Geider and MacIntyre, 2002).
The action of RUBISCO inactivase is itself sensitive to the ADP:ATP ratio and to the redox state of PSI. Thus, RUBISCO activity responds positively to a cue of a light-stimulated acceleration in photosynthetic electron flow. With conditions of high-light-driven reductant fluxes and high CO2 availability at the sites of carboxylation, the limitation of photosynthetic rate switches to the rate of RuBP complexation and renewal, both of which become subject to the overriding constraint of the RUBISCO capacity (Tortell, 2000). However, the kinetics of RUBISCO activity impose a heavy demand in terms of the delivery of carbon dioxide to the carboxylation sites. Although many phytoplankters invoke biophysical mechanisms for concentrating carbon dioxide (see Section 3.4), the relatively high levels needed to saturate the carboxylation function of RUBISCO may frequently be overtaken. Circumstances that combine low CO2 with the high rates of reductant and oxygen generation possible in strong light are liable to effect the competitive switch to the oxygenase function of RUBISCO and the inception of photorespiration.
Photorespiration is a term introduced in the physiology of vascular plants to refer to the sequence of reactions that commence with the formation of phosphoglycolate from the oxygenation of RuBP by RUBISCO (Osmond, 1981). In the present context, the term covers the metabolism of reductant power and controlling photosynthesis at low CO2 concentrations. The manufacture of phosphglycolate carries a significant energetic cost through the altered ATP balance (see Raven et al., 2000), though this is partly recouped in the continued (albeit smaller) RUBISCO-mediated contribution of G3P to the Calvin cycle. Meanwhile, the phosphoglycolate is itself dephos-phorylated (by phosphoglycolate phosphatase) to form glycolic acid. In the 'green line' of algae (including the prasinophytes, chlorophytes and euglenophytes) and higher plants, this glycolate can be further oxidised, to glyoxalate and thence to G3P. The full sequence of reactions has been called the 'photosynthetic carbon oxidation cycle' (PCOC) (Raven, 1997). In the Cyanobacteria and in the 'red line' of algae, this capacity seems to be generally lacking. When experiencing oxidative stress at high irradiance levels, these organisms cells will excrete glycolate into the medium.
Excreted glycolate is sufficiently conspicuous outside affected cells for its production to have been studied for many years as a principal 'extracellular product' of phytoplankton photosynthesis (Fogg, 1971). It is now known that not only glycolate but also other photosynthetic intermediates and soluble anabolic products are released from cells into the medium. This apparent squandering of costly, autogenic products seemed to be an unlikely activity in which 'healthy' cells might engage (cf. Sharp, 1977). However, it is now appreciated that, far from being a consequence of ill health, the venting of unusable dissolved organic carbon (DOC) into the medium constitutes a vital aspect of the cell's homeo-static maintenance (Reynolds, 1997a). It is especially important, for example, when the producer cells are unable to match other growth-sustaining materials to the synthesis of the carbohydrate base. In natural environments, the DOC compounds thus released - glycolate, monosac-charides, carboxylic acid, amino acids (Sorokin, 1999, p. 64; see also Grover and Chrzanowski, 2000; Sondergaard et al., 2000) - are readily taken up and metabolised by pelagic microorganisms. The far-reaching ecological consequences of this behaviour are explored in later sections of this book (Sections 3.5.4, 8.2.1).
So far as the biochemistry of photosynthesis is concerned, these alternative sinks for primary product make it less easy to be precise about the yields and the energetic efficiency of photosyn-thetic carbon fixation. The basic equation (3.1) indicates equimolecular exchanges between carbon dioxide consumed and oxygen released (the photosynthetic quotient, PQ, mol O2 evolved/mol CO2 assimilated, is 1). In fact, both components are subject to partially independent variation. Oxygen cycling may occur within the photosyn-thetis electron transfer chain (the Mehler reaction), independently of the amount of carbon delivered through the system. The 'competition' between the carboxylation and oxidation activity of RUBISCO are swayed in favour of oxygen production, photorespiration and glycolate metabolism (Geider and MacIntyre, 2002). The PQ may move from close to 1.0 in normally photosynthesising cells (actually, it is generally measured to be 1.1 to 1.2: Kirk, 1994) to the range 1.2 to 1.8 under high rates of carbon-limited photosynthesis. Low photosynthetic rates under high partial pressures of oxygen may force PQ < 1 (Burris, 1981).
The effects on energy efficiency are also sensitive to biochemical flexibility. Taking glucose as an example, the energy stored and released in the complete oxidation of its molecule is equivalent to 2.821 kJ mol-1, or ~470 kJ per mol carbon syn-thesised. The electron stoichiometry of the synthesis cannot be less than 8 mol photon (mol C)-1 but, energetically, the photon efficiency is weaker. The interconversion of Morel and Smith (1974; see above; 1 mol photon ~218 kJ) implies an average investment of the energy of 12.94 photons mol-1. This coincides more closely to the highest quantum yields determined experimentally (0.07-0.09 mol C per mol photon: Bannister and Weidemann, 1984; D. Walker, 1992).
Clearly, even these yields are subject to the variability in the fate of primary photo-synthate. Moreover, the alternative allocations of the fixed carbon (whether polymerised and stored, respired, allocated to protein synthesis or excreted) need to be borne in mind. It is well accepted that about half the photosynthate in actively growing, nutrient-replete cells is invested in protein synthesis and in the replication of cell material (Li and Platt, 1982; Reynolds et al., 1985). However, this proportion is very susceptible to the physiological stresses experienced by plankters in their natural environments as a consequence of low light incomes, carbon deficiencies or severe nutrient depletion. These effects are explored in subsequent sections.
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