2.1 - C<sub>3</sub> photosynthesis

Oula Ghannoum, University of Western Sydney, Australia

Despite much diversity in life form and biochemical process, all of the photosynthetic pathways focus upon a single enzyme which is by far the most abundant protein on earth, namely ribulose-1,5-bisphosphate carboxylase/oxygenase, or Rubisco (Figure 2.1a). Localised in the stroma of chloroplasts, this enzyme enables the primary catalytic step in photosynthetic carbon reduction (or PCR cycle) in all green plants and algae. Although Rubisco has been highly conserved throughout evolutionary history, this enzyme is surprisingly inefficient with a slow catalytic turnover (Vcmax), a poor specificity for CO2 as opposed to O2 (Sc/o), and a propensity for catalytic misfiring resulting in the production of catalytic inhibitors. This combination severely restricts photosynthetic performance of C3 plants under current ambient conditions of 20% O2 and 0.039% CO2 (390 μL L-1). Furthermore, Rubisco has a requirement for its own activating enzyme, Rubisco activase, which removes inhibitors from the catalytic sites to allow further catalysis. Accordingly, and in response to CO2 limitation, C4, C3-C4 intermediate, CAM and SAM variants have evolved with metabolic concentrating devices which enhance Rubisco performance (Section 2.2).

2.1.1 - Photosynthetic carbon reduction


Figure 2.1 Photosynthetic carbon reduction (PCR cycle, also termed the Calvin-Benson cycle) utilises ATP and NADPH produced by thylakoid electron transport to drive CO2 fixation by Rubisco (a). CO2 is incorporated into a 5-carbon sugar phosphate to produce two 3-carbon sugar phosphates which can either be exported from the chloroplast for sucrose synthesis, be recycled to make more 5-carbon acceptors, or be used to make starch. The appearance of radioactive carbon in 3-carbon sugar phosphates and then in starch and sucrose following photosynthesis in 14CO2 was evidence for the pathway of photosynthesis. (b) (Original drawing courtesy Robert Furbank).

The biochemical pathway of CO2 fixation was discovered by feeding radioactively labelled CO2 in the light to algae and then extracting the cells and examining which compounds accumulated radioactivity. Figure 2.1(b) shows a typical labelling ‘pattern’ for a C3 plant. Here, a short burst of labelled CO2 was given to the plants, then the label was ‘chased’ through the photosynthetic pathway by flushing with unlabelled air. Atmospheric CO2 is initially incorporated into a five-carbon sugar phosphate (ribulose-1,5-bisphosphate or RuBP) to produce two molecules of the phosphorylated three-carbon compound 3-phosphoglycerate, often referred to as the acidic form 3-phosphoglyceric acid (3-PGA). Hence, plants which use Rubisco as their primary enzyme of CO2 fixation from the air are called C3 plants. Consequently, in C3 plants, 3-PGA is the first labelled sugar phosphate detected after a pulse of 14CO2 has been supplied (Figure 2.1b). In the PCR cycle, 3-PGA is phosphorylated by the ATP produced from thylakoid electron transport (see Chapter 1) and then reduced by NADPH to produce triose phosphate. Triose phosphates are the carbon backbones, produced by the PCR cycle, for the synthesis of critical carbohydrate for the maintenance of plant growth and the productive yield of stored carbohydrate in seed.

Newly synthesised triose phosphate faces three options. It can be (1) exported to the cytosol for sucrose synthesis and subsequent translocation to the rest of the plant, (2) recycled within the chloroplast to produce more RuBP or (3) diverted to produce starch (Figure 2.1a). This is shown by the time-course of the appearance of radioactivity in starch and sucrose after it has passed through 3-PGA (Figure 2.1b). The energy requirements of the PCR cycle are three ATP and two NADPH per CO2 fixed, in the absence of any other energy-consuming processes.

Sucrose and starch synthesis

Most of the triose phosphate synthesised in chloroplasts is converted to either sucrose or starch. Starch accumulates in chloroplasts, but sucrose is synthesised in the surrounding cytosol, starting with the export of dihydroxyacetone phosphate and glyceraldehyde phosphate from the chloroplast. A condensation reaction, catalysed by aldolase, generates fructose-1,6-bisphosphate, and this is converted to fructose-6-phosphate after an hydrolysis reaction catalysed by fructose-1,6-phosphatase. Sucrose-6-phosphate synthase then generates sucrose-6-phosphate from the reaction of fructose-6-phosphate and UDP-glucose. The phosphate group is removed by the action of sucrose-6-phosphatase. This Pi is transported back into the chloroplast where it is available for ATP synthesis. For each molecule of triose phosphate exported from a chloroplast, one Pi is translocated inwards.

Sucrose synthesised within the cytosol of photosynthesising cells is then available for general distribution and is commonly translocated to other carbon-demanding centres via the phloem (see Chapter 5).

By contrast, starch synthesis occurs within chloroplasts. The first step is a condensation of glucose-1-phosphate with ATP. Starch synthase then transfers glucose residues from this molecule to the non-reducing end of a pre-existing molecule of starch. Starch consists of two types of glucose polymer, namely amylose and amylopectin. Amylose is a long, unbranched chain of D-glucose units connected via (α1–4) linkages. Amylopectin is a branched form, with (α1–6) linkages forming branches approximately every 24–30 glucose residues.

2.1.2 - RuBP regeneration


Figure 2.2. A simplified (above) and detailed (below) description of the photosynthetic carbon reduction (PCR) cycle. The fixation of CO2 by Rubisco to the acceptor molecule RuBP initiates the cycle with the production of two molecules of PGA. The subsequent, enzyme catalysed, generation of cycle intermediates are cycled to either regenerate RuBP or produce triose phosphates which are precursors for carbohydrate synthesis. The cycle is powered by the co-factors NADPH and ATP that are synthesised from the chloroplast electron transport chain. Enzymes include: PGK, phosphoglycerate kinase; GAPDH, glycraldehyde-3- phosphate dehydrogenase; TIM, triose phoshphate isomerase; ALD, aldolase; FBPase, fructose-1,6-bisphosphatase; TKL, transketolase, SBPase, seduheptulose-1,7-bisphosphatase; RPI, ribose-5-phosphate isomerase; RPE, ribose-5-phosphate epimerase and PRK, phosphoribulose kinase. (Courtesy Robert Sharwood).


Figure 2.2b

Ribulose bisphosphate (RuBP) is consumed in the carboxylating step of carbon fixation. If such fixation is to continue, RuBP must be regenerated, and in this case via the PCR cycle. The PCR cycle operates within the stroma of chloroplasts, and consists of a sequence of 11 steps where a three-carbon compound (3-phosphoglycerate) is phosphorylated, reduced to glyceraldehyde 3-phosphate and isomerised to dihydroxyacetone phosphate. Condensation of this three-carbon compound with glyceraldehyde 3-phosphate yields a six- carbon compound (fructose bisphosphate). Following a series of carbon shunts, involving four-, five- and seven-carbon compounds, RuBP is regenerated.

Important features of the PCR cycle include: (1) for every step of the cycle to occur once, three carboxylations must occur via ribulose bisphosphate carboxylase thus generating six moles of phosphoglycerate (18 carbons); (2) for one turn of the cycle, three molecules of RuBP participate (15 carbons) and thus a net gain of three carbons has occurred for the plant; (3) in regenerating three molecules of RuBP, nine ATP and six NADPH are consumed.

2.1.3 - Properties of Rubisco

Photosynthetic carbon fixation in air is constrained by the kinetic properties of Rubisco. Form I Rubisco in higher plants is a large protein (approximately 550 kDa) comprised of eight large (approx. 50-55 kDa) and eight small subunits (approx. 13-18 kDa) to form an L8S8 hexadecamer. Rubisco synthesis and assembly in higher plants is a complex process whereby the large subunit gene (rbcL) is encoded in the chloroplast genome, while the small subunit genes (rbcS) are encoded as a multi-gene family in the nucleus. The Rubisco small subunits are translated as precursors in thc cytosol and are equipped with a transit-peptide to target them to the chloroplast. Upon import in the chloroplast the transit-peptide is cleaved by a stromal peptidase and the N-terminus modified by methylation of the n-terminal methionine. The large subunits  are synthesised within the chloroplast and also post-translationally modified through the removal of the the N-terminal methionine and serine amino acids and the subsequent acetylation of proline at the N-terminus and the methylation of lysine at position 14. The assembly of large and small subunits into functional hexadecameric Rubisco is reliant on the coordination of chloroplast-localised chaperones.

Despite selection pressure over evolutionary history, Rubisco remains an inefficient catalyst (Spreitzer and Salvucci 2002). Therefore, to achieve a productive maximum CO2 assimilation rate (Amax), plants must compensate for catalytic inefficiency by investing large amounts of nitrogen in Rubisco. Consequently, Rubisco comprises more than 50% of leaf soluble protein in C3 plants. On a global scale, this investment equates to around 10 kg of nitrogen per person!

More than 1000 million years of evolution has still not resulted in a ‘better’ Rubisco adapted for the current and future concentrations of CO2. Such a highly conserved catalytic protein is an outcome of thermodynamic and mechanistic difficulties inherent to this reaction. Rubisco requires carbamylation of the absolutely-conserved residue K201 that is then stabilised by the binding of Mg2+. Without this activation step Rubisco is unable to function. The fixation of CO2 to RuBP to form two molecules of 3-PGA is a five step catalytic process that produces highly reactive transition state intermediates that bind CO2. The highly reactive transition states make Rubisco prone to generating misfiring products, which generate inhibitors within the active site. Therefore, Rubisco requires its own catalytic protection enzyme Rubisco activase. Plants devoid of this enzyme fail to grow properly in air as the activation and subsequent activity of Rubisco is impeded (Portis and Salvucci 2002). Rubisco activase is an ATP-dependent process that removes inhibitors from the active site of Rubisco allowing for activation and catalysis to proceed. Recently, the crystal structure of Rubisco activase has been solved, which will provide key insight into the molecular interaction between Rubisco and Rubisco activase (reviewed by Portis et al. 2008).

Rubisco first evolved when the earth’s atmosphere was rich in CO2, but virtually devoid of O2. With the advent of oxygen-producing photosynthesis by land plants, and the resulting increases in atmospheric O2, one key deficiency of this enzyme became apparent. Rubisco would not only catalyse fixation of CO2 but would also permit incorporation of O2 into RuBP to produce, instead of two molecules of 3-PGA, just one molecule of 3-PGA with one molecule of a two-carbon compound, 2-phosphoglycolate (Section 2.3). Indeed, CO2 and O2 compete directly for access to the active sites of Rubisco. So feeble is Rubisco’s ability to distinguish between these two substrates that in air (20% O2) approximately one molecule of O2 is fixed for every three molecules of CO2.

Fixation of O2 and subsequent photorespiration (Section 2.3) is an energy-consuming process, due to competition between O2 and CO2 for RuBP, plus the energy cost of converting the phosphoglycolate product to a form which can be recycled in the PCR cycle. This energy cost is increased at higher temperatures because O2 competes more effectively with CO2 at the active site of Rubisco. Such sensitivity to temperature × O2 explains why CO2 enrichment, which reduces photorespiration, has a proportionally larger effect upon net carbon gain at higher temperatures than at lower temperatures (Section 13.3).


Figure 2.3. Mechanisms underlying CO2 fixation by Rubisco have changed very little during evolution but Rubisco efficiency has improved. The enzyme in more 'highly evolved' species such as C3 angiosperms is able to fix more CO2 and less O2 in air, reducing photorespiratory energy costs. A measure of this is the relative specificity of Rubisco for CO2, shown here for a range of photosynthetic organisms. (Based on Andrews and Lorimer 1987).

Notwithstanding a meagre catalytic effectiveness in present day Rubisco, more efficient variants would still have had a selective advantage, and especially during those times in the earth’s geological history when atmospheric CO2 concentration was decreasing. Indeed there has been some improvement (Figure 2.3) such that specificity towards CO2 as opposed to O2 has improved significantly. Recently evolved angiosperms show a relative specificity almost twice that of 'older' organisms such as photosynthetic bacteria.

Despite such improvement, Rubisco remains seemingly maladapted to its cardinal role in global carbon uptake, and in response to selection pressure for more efficient variants of CO2 assimilation, vascular plants have evolved with photosynthetic mechanisms that alleviate an inefficient Rubisco. One key feature of such devices is a mechanism to increase CO2 concentration at active sites within photosynthetic tissues. Some of these photosynthetic pathways are dealt with below.