2. Photosynthesis: Dark cycle reactions, variation in the dark cycle system, protection of the photosynthesis system, control of photosynthetic rate. Objectives of the lecture: 1. Describe the dark cycle reactions of photosynthesis. 2. Illustrate field measurements of photosynthesis. 3. Discuss how dark cycle reactions can limit the rate of photosynthesis. 4. Describe photorespiration. 5. Describe C4 and CAM photosynthesis. Text book pages: 213-219.

3. Recap and importance: The photochemical reactions produce ATP and NADH at sites in the stroma. The Dark Cycle (Calvin Cycle), or more descriptively, the carbon reactions of photosynthesis ~200 billion tons of CO2 are converted to biomass each year The enzyme ribulose biphosphate carboxylase/oxygenase, Rubisco, that incorporates CO 2 is 40% of the protein in most leaves.

4. The Calvin cycle proceeds in three stages: carboxylation, reduction, and regeneration Carboxylation of the CO 2 acceptor, ribulose-1, 5-biphosphate, forming two molecules of 3-phosphoglcerate. Reduction of 3-phosphoglycerate to form glyceraldehyde-3-phosphate which can be used in formation of carbon compounds that are translocated. Regeneration of the CO 2 acceptor ribulose-1, 5-biphosphate from glyceraldehyde-3-phosphate Rubisco – the enzyme ribulose biphosphate carboxylase/oxygenase

5. The affinity of Rubisco for CO 2 is sufficiently high to ensure rapid carboxylation at the low concentration of CO 2 found in photosynthesizing cells The negative change in free energy associated with carboxylation of RuBP is large so the forward reaction is favored. RuBP Rubisco will also take O 2 rather than CO 2 and oxygenate RuBP – called photorespiration. The rate of operation of the Calvin Cycle can be enhanced by increases in the concentration of its intermediates. That is the cycle is autocatalytic. Also, if there are insufficient intermediates available, for example when a plant is transferred from dark to light, then there is a lag, or induction period, before photosynthesis reaches the level that the light can sustain. (There can also be enzyme induction.) Rubisco is notoriously inefficient as a catalyst for the carboxylation of RuBP and is subject to competitive inhibition by O 2, inactivation by loss of carbamylation, and dead-end inhibition by RuBP. These inadequacies make Rubisco rate limiting for photosynthesis and an obvious target for increasing agricultural productivity. Really?

6. Field measurement of photosynthesis and its control by environmental conditions

7. Infra-red Gas Analyzer measures the concentration of CO 2 in the air stream before and after it flows across the leaf in the chamber The chamber is enclosed over the leaf. Light and temperature are measured while photosynthesis is being measured. Photosynthesis rate calculated from gas flow rate and CO 2 concentration difference LI-COR 6400

8. Basics of foliage photosynthesis 0 0 Saturation level. sometimes called photosynthetic capacity. Compensation point The irradiance at which CO uptake is zero 2 Photosynthetic efficiency: Increase in photosynthesis per increase in irradiance Any questions? Increasing CO2 concentration in the atmosphere can increase the maximum rate of photosynthesis in the short term Light Reaction Limiting Dark Reaction Limiting

9. Measured light response curve of Abies amabilis first year foliage. Shade foliage with low maximum value and low compensation point.

10. Observed assimilation rates (µmolCO 2 /m 2 s) of Tsuga heterophylla and Abies amabilis in response to periods of 10 minutes high light (1500µmol/m 2 s PPFD), with 5 minutes intervals of darkness (shaded parts in the diagram) in between. Values measured using 200 mol/s flow rate.

11. Species differences in leaf photosynthesis A has the highest photosynthetic rate at light saturation B has the highest photosynthetic efficiency and the lowest compensation point. Another important measure is called Water Use Efficiency: the ratio of photosynthesis achieved per unit of water lost. Units: mmol/mol milli mols of CO per mol of water transpired 2 Units: μmol/m /s micro mols of CO per square meter foliage per second 2 2 milli [m] 0.001 (a thousandth) micro [µ] 0.000 001 (a millionth) Any questions?

12. Wind River Canopy Crane Research Facility

13. Thuja plicata Abies grandis Pseudotsuga menziesii Tsuga heterophylla Old-growth species: Douglas-fir Pseudotsuga Western hemlock Tsuga Upper CanopyLower Canopy Ph ot. Cap. 13.1 9.0 μmol/m /s 2 Water Use Eff. 6.2 4.9 mmol/mol Phot. Cap. 8.8 3.2 Water Use Eff. 3.5 4.8 Notice the difference in branch structure between the species

14. The problem of photorespiration and the evolution of photosynthesis When the enzyme Rubisco uses oxygen to breakdown carbohydrate to CO 2 rather than using CO 2 to synthesize carbohydrate How some grasses have evolved a C4 metabolic process and some desert plants have evolved Crassulacean Acid Metabolism

15. Although Rubisco acts like a carboxylase in photosynthesis, it can also act as an oxygenase when O 2 is available. O 2 and CO 2 compete for the same active site! This is called Photorespiration 3-phospho glycerate 2-phospho glycerate This becomes a problem when photosynthesis rates are high, i.e. photosystem II produces lots of O 2. P P P P C-C-C-C-C C-C-C + C-C RuBisCO Ribulose 1, 5-biphosphate Enzyme O2O2 CO 2

16. It is believed that photorespiration in plants has increased over geologic time due to increasing atmospheric O 2 concentration -the product of photosynthetic organisms themselves. In the presence of higher O 2 levels, photosynthesis rates are lower. The inhibition of photosynthesis by O 2 was first noticed by the German plant physiologist, Otto Warburg, in 1920, and called the "Warburg effect". 275 ppm CO 2 73 ppm CO 2

17. Decarboxylation of malate (CO 2 release) creates a higher concentration of CO 2 in bundle sheath cells than found in photosynthetic cells of C3 plants. The first product of CO 2 fixation is malate (C4) in mesophyll cells, not PGA as it is in C3 plants. This is transported to bundle sheath cells CO 2 is released from malate in bundle sheath cells, where it is fixed again by Rubisco and the Calvin cycle proceeds. PEP is recycled back to mesophyll cells. This enables C4 plants to sustain higher rates of photosynthesis. And, because the concentration of CO 2 relative to O 2 in bundle sheath cells is higher, photorespiration rates are lower. C4 Photosynthesis

18. Xylem Bundle sheath cells filled with chloroplasts. CALVIN REACTION SITE Phloem Parenchyma filled with chloroplasts C4 acids synthesized in the parenchyma move to the bundle sheath Carbon skeleton compounds return to parenchyma Anatomical separation of the C4 photosynthesis component processes

19. Crassulacean Acid Metabolism (CAM) Uses C4 pathways, but segregates CO 2 assimilation and Calvin cycle between day and night CAM plants open their stomates at night. This conserves H 2 O. CO 2 is assimilated into malic acid and stored in high concentrations in cell vacuoles During the day, stomates close, and the stored malic acid is gradually recycled to release CO 2 to the Calvin cycle First discovered in succulents of the Crassulacea: e.g.,sedums

20. C3, majority of species C4, e.g., sugar cane, corn CAM, e.g., cacti Leaf structure Typical habitat characteristics Productivity Optimum Temperature Efficiency in light Bundle sheath cells have chloroplasts Bundle sheath cells lack chloroplasts Mesophyll cells have large vacuoles Can be sun or shade plants Ineffective in shade CO 2 capture at night Requires relatively moist habitats Arid or tropical regions Arid environments Moderate HighLow15-25 o C 30-40 o C 35 o C

21. Things you need to know... 1. The basic reactions of the Calvin Cycle with the names and basic structures of the principal reactants but not their detailed chemical formulea. 2. The characteristics of Rubisco. 3. The method of field measurement of photosynthesis by gas exchange 4. The light saturation curve of leaf photosynthesis and its important features 5. Water use efficiency. Calculation and value as a physiological measure 6. Why photorespiration is important and the processes of C4 and CAM photosynthesis.