Download PBIO*3110 – Crop Physiology Lecture #8 Leaf Photosynthesis II

PBIO*3110 – Crop Physiology Lecture #8 Fall Semester 2008 Lecture Notes for Tuesday 30 September Leaf Photosynthesis II – The Dark Reactions How are photosynthetic rates determined at the level of chloroplast biochemistry? Learning Objectives 1. Understand how the products of the light reactions are utilized by the chloroplast to fix CO2 into organic molecules. 2. Be able to explain how photorespiration can decrease the efficiency of photosynthesis under certain environmental conditions. 3. Know how the C4 photosynthetic pathway circumvents the problem of photorespiration. 4. Understand how induction states of Calvin Cycle enzymes affect the ability of photosynthesis to respond to sudden changes in incident PAR. 5. Be able to explain how end product feedback mechanisms can inhibit photosynthesis. Introduction In the previous page of the notes, we saw how chloroplasts use light energy to create reductant (NADPH) and ATP. Now, we will review how these compounds are used in the process of carbon fixation in the so­called “dark reactions” of photosynthesis.
1 The Dark Reactions of Photosynthesis A simplified representation of the dark reactions of photosynthesis is shown below. This group of reactions, which takes place in the chloroplast stroma, has two phases: 1. The carboxylation / reduction phase, wherein carbon from CO2 dissolved in the chloroplast stroma is incorporated into the pool of substrates, and 2. The regeneration phase, wherein the substrate ribulose bisphosphate is produced. The regeneration phase is very complicated, involving 3, 4, 5, 6 and 7­carbon compounds (in fact, Melvin Calvin received the Nobel Prize in Chemistry in 1961 for elucidating these pathways, and the cycle now bears his name). Details of this phase have been mostly omitted from the first figure below, to better highlight those phases of the cycle which utilize the products of the light reactions. The Calvin Cycle (simplified). The steps after the carboxylation reaction are doubled, so there is a total of three ATP and two NADPH consumed per CO2 fixed by RubisCO. The first step in the cycle is the carboxylation of (i.e. adding of CO2 to) ribulose bisphosphate, in a reaction catalyzed by the enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). Ribulose bisphosphate is a 5­carbon sugar, and after carboxylation it splits into two 3­carbon sugars, called phospho­glycerate.
2 In subsequent reactions of the cycle, each with its own enzyme, the carbon compounds are reduced by (i.e. receive electrons from) NADPH produced by the light reactions, and phosphorylated by ATP from the light reactions. The main "output" from the cycle is a 3­carbon phosphorylated sugar called dihydroxyacetone­phosphate. Note that three molecules of ribulose bisphosphate must be carboxylated by RuBisCO (i.e. three "turns" of the cycle) before a single 3­carbon sugar can be output from the cycle. A more complete representation of the Calvin cycle is provided below. The Calvin Cycle, showing each intermediate molecule.
3 Photorespiration Besides the carboxylation reaction that is central to photosynthesis, RuBisCO can also catalyze the oxygenation of ribulose bisphosphate. That is, instead of the 5­carbon sugar being combined with CO2, it is combined with O2 to produce phosphoglycerate (3 carbons) and phosphoglycolic acid (2 carbons). The 2­carbon compound produced is then processed in a series of reactions involving the chloroplasts, peroxisomes and mitochondria. These reactions consume ATP and reducing electrons from the light reactions, but no net carbon fixation occurs. In fact, there is a net loss of carbon, since some of the carbon from phosphoglycolic acid (one C per two oxygenation events) is evolved as CO2 in the subsequent reactions (thus the term "photorespiration"). The carboxylation and oxygenation reactions catalyzed by RubisCO. The oxygenation reaction becomes favoured under high temperatures, and when the CO2:O2 ratio is low in the chloroplast. Photorespiration constitutes a net loss of carbon and energy for the plant. Fortunately, RuBisCO has a much higher affinity for CO2 than for O2, so that the carboxylation reaction occurs three to four times as often as the competing oxygenation reaction. This occurs even though the concentration of CO2 in the atmosphere is only about 0.04%, whereas the O2 concentration is approximately 20%.
4 However, under certain conditions, the oxygenation reaction can become a greater than normal portion of total RuBisCO activity. For instance, when temperatures are high, the oxygenation reaction becomes relatively more likely, for two reasons:
· as temperatures increase, the solubility of CO2 in the cell decreases relative to the solubility of O2
· as temperatures increase, the affinity of RuBisCO for O2 increases relative to its affinity for CO2. Also, when the concentration of CO2 inside the leaf drops relative to the O2 concentration, photorespiration increases relative to photosynthesis. This effect may be important during drought stress conditions, as we will see in a future lecture. 180 40 140 35 120 Ks CO2 / O2 solubility 160 30 100 80 60 25 40 20 20 0 0 10 20 30 40 50 60 0 Temperature (°C) 10 20 30 40 50 60 Temperature (°C) Effect of temperature on the ratio of CO2 to O2 solubility (left), and on the CO2 vs O2 specificity ratio of RubisCO (right) Consider the example of a leaf with an interior CO2 concentration of 300 ppm and a temperature of 25°C. The O2 concentration is assumed to be the same as atmospheric, which is 20%, or 200,000 ppm. CO2 and O2 compete for RubisCO in the chloroplast, according to their relative concentrations, and the specificity ratio of RubisCO. In the gas phase, the ratio of O2 to CO2 is 200,000 : 300 = 667 But, at 25°C, CO2 is approximately 27 times more soluble than O2. So, dissolved in the chloroplast the O2 : CO2 concentration ratio is 667 / 27 = 24.7 Then, at 25°C, the CO2 vs O2 specificity ratio of RubisCO is approximately 88:1. So, the ratio of oxygenation events (photorespiration) to carboxylation events (photosynthesis) is
5 24.7 / 88 = 0.28 For each oxygenation event, only 0.5 molecules of CO2 are released by photorespiration, so the ratio of photorespiratory CO2 release to photosynthetic CO2 fixation in this example will actually be 0.28 / 2 = 0.14 2.5 Ci = 50 ppm Ci = 150 ppm 2 Ci = 300 ppm RP / AG 1.5 1 0.5 0 0 10 20 30 40 50 Tem perature (°C) The calculated ratio of photorespiration (RP) to gross CO2 assimilation (AG) as affected by temperature and leaf internal CO2 concentration (Ci). Enzyme Induction Status as a Limitation to Photosynthesis In darkness, many of the enzymes of the Calvin cycle exist in an inactive state: before they can function they must be induced by light. When PPFD declines, the induction state decays. The PPFD level at which the induction begins to decay, as well as the rate at which it decays, depends on the particular enzyme in question. There are two components to the overall loss of induction of the Calvin cycle at low PPFD: 1) The fast component. This component decays relatively rapidly at low PPFD, but upon return to saturating PPFD it is always restored within 2 minutes. Decay of this component of induction state can occur at PPFDs as low as 300 :mol m ­2 s ­1 . Leaves within a fully expanded crop canopy commonly have an incident PPFD less than 300 :mol m ­2 s ­1 , even at solar noon. Recent evidence indicates that induction of fructose­1,6­bisphosphatase, an enzyme involved in the regeneration phase of the Calvin cycle, brings about the fast component of induction.
6 Photosynthetic induction of a soybean leaf under high PPFD (1500 µmol m ­2 s ­1 ) following dark periods of various lengths. (Sassenrath­Cole and Pearcy, 1992) Effect of time in darkness (closed symbols) or low PPFD (35 µmol m ­2 s ­1 ; open symbols) on the induction state of the “slow” and “fast” components of photosynthetic induction in a soybean leaf. Induction state was calculated as the photosynthetic rate 5 or 120 s after increasing the PPFD to a saturating level, divided by the fully induced rate prior to the imposition of the low light treatment. (Sassenrath­Cole and Pearcy, 1992)
7 2) The slow component. When leaves experience very low PPFD (i.e., below 100 :mol m ­2 s ­1 ), another induction component begins to decay. This decay is very slow relative to the loss of induction of fructose­1,6­bisphosphatase and decay is not reversed within 2 minutes after return to saturating PPFD. This component has been attributed to loss of induction of RubisCO. Because the slow component is only important during extended periods at very low PPFD, it probably does not constitute a significant limitation to CO 2 uptake under field conditions. Feedback Inhibition of Photosynthesis – Chloroplast Starch Accumulation Following the reduction phase of the Calvin cycle, the phosphoglyceraldehyde or dihydroxyacetonephosphate (or, more generally, the triose phosphate (TP)) produced is either recycled for the regeneration of RuBP or leaves the cycle for (i) starch formation in the chloroplast or (ii) sucrose synthesis after export to the cytosol. The export of TP from the chloroplast is linked with the import of P i which is released in the cytosol during sucrose synthesis. Accumulation of sucrose in the cytosol may lead to feedback inhibition of sucrose synthesis; hence, if sucrose accumulates in the cytosol due to reduced translocation out of the cell, return of P i to the chloroplast is reduced. A lack of Pi in the chloroplast has a strong feedback effect on the light reactions of photosynthesis, since it restricts ATP formation (photophosphorylation). Reduction of the activation state of RubisCO is also observed under these conditions, consistent with the reduced rate of the dark reactions. When such a Pi limitation occurs, more of the TP is diverted to starch production in the chloroplast. This releases Pi thereby allowing the light reactions and of course also the Calvin cycle to continue at higher rates than would otherwise be possible. These regulatory mechanisms are by no means simple, and are far from fully understood. For example, excessive starch accumulation in the chloroplast will also eventually inhibit photosynthesis, but the mechanism of that inhibition remains unknown. Also, over the longer term feedback inhibition of photosynthesis is associated with a whole host of changes in gene regulation leading to adaptive metabolic changes in the leaf (See Paul and Foyer (2001) J. Exp Bot. 52:1383­1400).
8 The linkage between sucrose synthesis in the cytosol and inorganic phosphate (Pi) availability in the chloroplast. A lack of chloroplastic Pi leads to feedback inhibition of the light reactions, and subsequent down regulation of the dark reactions. The C4 Photosynthetic Pathway Most important crop species exhibit the "normal" Calvin­cycle photosynthetic pathway. These plants are termed C3 photosynthetic types, since the first product of carboxylation is a 3­carbon sugar (phosphoglycerate). Examples of C3 crop species are wheat, barley, rice, soybean, cotton, peanut, and clover. Some crop species, mostly of tropical or sub­tropical origin, exhibit a different photosynthetic pathway, known as C4 photosynthesis, so named because the first product of carboxylation is a 4­carbon compound. Examples of C4 crop species are corn, sorghum, millet and sugarcane. The C4 photosynthetic pathway allows plants to virtually eliminate photorespiration. The C4 pathway includes an additional energy cost, but this is compensated for by the elimination of the energy and carbon losses associated with photorespiration in C3 species. Note that the C4
9 pathway is most advantageous under growing conditions that would tend to favour photorespiration (hot, dry). In C4 plants, the Calvin cycle occurs only in specialized cells, usually the bundle sheath cells. The rest of the C4 pathway provides a "CO2 concentrating mechanism", such that the CO2 concentration in the bundle sheath cells is maintained at a very high level; thus, RuBisCO in bundle sheath cell chloroplasts operates in a high CO2 environment, and therefore rarely catalyzes the oxygenation reaction. The initial carboxylation reaction (i.e. production of the C4 intermediate) occurs in the normal mesophyll cells, and is catalyzed by the enzyme PEP carboxylase. A partial cross section of a C4 leaf, showing both normal mesophyll and bundle­sheath cells, is shown below, followed by a schematic representation of the reactions of C4 photosynthesis. Partial cross­section of a leaf from the C4 grass species Panicum miliaceum. A single layer of large bundle sheath cells surrounds each vascular bundle. Production of the C4 intermediate malate occurs in the surrounding mesophyll cells.
10 Simplified outline of the C4 photosynthetic pathway. PEP = phosphoenolpyruvate. Photosynthetic pathways of major turfgrass species. Note that C4 turfgrasses are generally classified as “warm season” types, whereas C3 turfgrasses are “cool season” types. C3 Pathway Creeping bentgrass Colonial bentgrass Tall fescue Red fescue Perennial ryegrass Annual bluegrass Kentucky bluegrass Rough bluegrass C4 Pathway Buffalograss Bermudagrass Centipedegrass Bahaigrass St. Augustinegrass Zoysiagrass
11 CAM Photosynthesis Certain slow­growing, succulent species exhibit a further adaptation to arid environments, called Crassulacean acid metabolism (CAM). There are a few plants of commercial importance that exhibit this photosynthetic pathway, including pineapple, agave and aloe. In these plants, accumulation in cell vacuoles of 4­carbon acids, such as malic acid, occurs during the night, using atmospheric CO2 as the carbon source. Thus, CAM plants must open their stomates at night, to allow CO2 to diffuse into the leaves. Since the light reactions are not active during the night, the reductant required for malate production is provided by the respiration of starch. During the day, the stomates close (to prevent leaf desiccation due to excessive transpiration); thus CO2 can not diffuse into the leaves at rates sufficient to support Calvin cycle activity. Instead, malic acid stored in the vacuoles is decarboxylated to provide the necessary CO2, and NADPH and ATP required by the Calvin cycle are derived from the light reactions in the normal fashion. Thus, while in normal C4 plants the initial carboxylation reaction and the Calvin cycle are separated in space (mesophyll vs. bundle sheath cells), in CAM plants they are separated in time (day vs. night). In both cases, the 4­carbon acids serve as the temporary reservoir of carbon to be subsequently released as CO2 to be used by the Calvin cycle.
12