PBIO*3110 – Crop Physiology Lecture #7 Fall Semester 2008 Lecture Notes for Thursday 25 September Leaf Photosynthesis I – The Light Reactions How do plants convert absorbed photons to biochemical energy? Learning Objectives 1. Be able to describe how the energy in photons is used by chloroplasts to produce NADPH and ATP. 2. Understand what is meant by the term Photosystem II efficiency, and know what factors can affect this efficiency. 3. Explain how Photosystem II regulates the balance between photochemical activity and energy dissipation.
1 Introduction Photosynthesis carried out by green algae and higher plants is a process of profound importance, for a number of reasons. First and foremost, photosynthesis is (essentially) the only means by which energy can enter the biosphere; thus, the chemical potential energy in all plant and animal tissues is ultimately derived from solar energy harvested by photosynthetic organisms. Secondly, oxygenic photosynthesis, the process by which green plants use light energy to split water into protons, electrons and molecular oxygen, is the source of almost all of the oxygen in the earth’s atmosphere. Therefore, the evolution of oxygenic photosynthesis permitted the evolution of all aerobically respiring organisms. Of course, from a crop physiologist’s point of view, photosynthesis is the process responsible for crop dry matter accumulation; thus, maximizing crop growth and yield is, to a great extent, simply a matter of maximizing total seasonal canopy photosynthesis. Much of the rest of this course will focus on how different abiotic factors affect a crop's ability to optimize photosynthesis and growth. However, understanding these plant­environment interactions requires at least a basic understanding of the photosynthetic process at the biochemical level. We will consider the topic of photosynthesis over several lectures. In this lecture and the next, we will discuss the biochemical basis of oxygenic photosynthesis, and some of the variations in photosynthetic processes among different crop species. Then, we will examine the effects of environmental factors on leaf photosynthesis. Finally, we will see how whole canopy photosynthesis is quantitatively determined by photosynthesis of individual leaves. Leaf and Chloroplast Structure A partial cross­section of a typical sun leaf is shown below. The leaf thickness is approximately 250 µm (0.25 mm). Locate the vascular bundle, and a guard cell pair. Note that chloroplasts are visible in the parenchyma cells, but not in the epidermal cells or in the vascular bundle. A schematic representation of a chloroplast is shown in the next figure. The chloroplast has a double membrane wall, called the chloroplast envelope. The interior of the chloroplast is divided into distinct regions by the thylakoid membrane. This membrane forms "sacks", called thylakoids, which are arranged in stacks, called grana (sing. granum). The interior of each thylakoid is called the lumen. The thylakoid membrane separates the lumen from the rest of the interior of the chloroplast, termed the stroma.
2 Partial cross­section of a typical sun­leaf. (H.R. Bolhar­Nordenkampf, University of Vienna). A real chloroplast is shown in the electron micrograph below. See if you can identify the chloroplast structures labelled in the drawing above. Note that the entire chloroplast is approximately 10 µm in length. A close­up view of a granum is shown in the second micrograph. Here, the lumen of individual thylakoids can be seen. In what follows, we will see that the separation of the interior of the chloroplast into the lumenal and stromal regions by the thylakoid membrane is critical to the photosynthetic process.
3 Electron micrographs of a spinach chloroplast (top) and an individual stack of thylakoids (bottom). (D.A. Greenwood) The Light Reactions of Photosynthesis The process of photosynthesis is often divided into two stages: the so­called "light reactions" and the "dark reactions". While the light and dark reactions both take place only in the light (with some exceptions ­ see CAM species), only the light reactions utilize light directly. The purpose of the light reactions is to provide substrate for the dark reactions. Specifically, the light reactions produce the energy (ATP) and the reductant (NADPH) required by the CO2 fixation process. A simplified schematic diagram of the light reactions is shown below. The main points to remember are as follows:
· The light reactions take place in the thylakoid membrane. The proteins that bind chlorophyll are imbedded in the membrane, as are some of the electron carriers and the various enzymes involved.
4 · Electrons are abstracted from water and passed along an electron transport chain in a series of reduction / oxidation reactions. The final electron acceptor in the chain is NADP, which is reduced to NADPH. Note that NADPH is produced on the stromal side of the membrane, where it is needed in the "dark reactions" of photosynthesis.
· Light energy is required to carry out the "splitting" (photolysis) of water into electrons, protons (H + ) and oxygen. This occurs at Photosystem II, which is surrounded by chlorophyll­binding proteins. This chlorophyll absorbs incident photons and passes the excitation energy to the photosystem reaction center, which carries out the photolysis reaction. The water is split on the lumen side of the membrane. The O2 produced can diffuse freely across the membrane and out of the lumen, but the protons are "trapped" inside the lumen, causing it to become acidified.
· The electrons abstracted from water are passed from Photosystem II to plastoquinone (an electron carrier that is mobile in the membrane). Along with the electrons from Photosystem II, plastoquinone picks up some protons from the stroma side of the membrane. When plastoquinone passes the electrons to the next carrier in the chain, these protons are deposited on the lumen side, which further acidifies the lumen.
· When the electrons reach Photosystem I, light energy is again required to "power" an energetically unfavorable redox reaction. Like Photosystem II, Photosystem I is surrounded by chlorophyll binding proteins that provide the required excitation energy. On the stromal side of Photosystem I, the electrons reduce NADP to NADPH.
· The accumulation of protons in the lumen results in an electrochemical gradient that can be used to do chemical work. As the protons diffuse out of the lumen into the stroma via the ATPase, ATP is generated. This ATP provides the chemical energy required by the dark reactions of photosynthesis.
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A simplified schematic representation of the light reactions of photosynthesis. The electron transport chain from water to NADP is shown in yellow. PQ = plastoquinone, PC = plastocyanin, Fd = ferredoxin. Efficiency of the Light Reactions So, what proportion of the photons that are absorbed by the leaf are actually used to drive photochemistry? That is, what is the efficiency of PSII? This question is of great interest for a number of reasons. First, recall that the photolysis of water requires the generation of enormous oxidizing potentials (in fact, that largest known to occur in nature, at approximately –1.0 eV). The mere presence of such high oxidizing potentials within a biological system creates a danger of unintentional redox reactions occurring and causing damage to the system. Indeed, PSII is known to regularly undergo such damage, necessitating replacement of the affected proteins. When a significant fraction of PSII centers become damaged, the overall efficiency of PSII photochemistry declines. To try to avoid such damage, plants have developed efficient mechanisms for safely dissipating excess excitation energy as heat when PPFD absorbed by chlorophyll is in excess of that which can be used to drive photochemistry. For instance, if the rate of photosynthesis is reduced by, say, chilling temperatures, but incident PPFD is high, there will be less need for the products of the light reactions (NADPH and ATP). In this case, feedback regulation of PSII will activate thermal dissipation mechanisms, excess energy will be dissipated, and efficiency of photochemistry will decline.
6 Also, whenever a photochemical event occurs at PSII, that particular reaction center becomes “closed” for a period of time (on the order of ms). So, when PPFD is high, a greater proportion of reaction centers are closed at any given moment in time, and the efficiency of photochemistry declines. So, efficiency of PSII is less when (a) incident PPFD is high, (b) factors limiting the rate of photosynthesis cause feedback regulation of the light reactions, and / or (c) PSII has sustained damage (known as photoinhibition). For healthy leaves under very low PPFD, this PSII efficiency may be as high as 0.84. It is never higher than this, since a minimum of about 16% of “excitons” is always released from the chlorophyll pigment bed as either heat or fluorescence. Recall from the instrumentation lab that chlorophyll fluorescence readings can provide an estimate of the efficiency of PSII (FII). This is a direct measure of the fraction of photons absorbed by PSII that are used to drive photochemistry, as opposed to being dissipated as either heat or fluorescence. The figure below shows the effect of both PPFD and water stress on FII of maize grown in the field. 0.8 control 0.7 drought 0.6 F II 0.5 0.4 0.3 0.2 0.1 0 0 500 1000 1500 ­2 ­1 PPFD (µmol m s )
Relationship between FII and incident PPFD for maize leaves in the field, under water­replete (control) or water stress (drought) conditions. (Data of H.J. Earl and R.F. Davis) In the above figure, one can clearly see that the efficiency of photon use at photosystem II declines as PPFD increases, and also when the rate of photosynthesis is limited by water stress. But what happens to those photons that are absorbed at PSII but not used to drive photochemistry? Lets look more closely at how PSII is regulated, and how its activity can be measured using the chlorophyll fluorescence signal.
7 Energy Dissipation at Photosystem II When a photon of light is absorbed by a chlorophyll molecule in the pigment bed, that molecule achieves the first excited singlet state (i.e., an electron jumps from the ground state to the next highest orbital). A red (680 nm) photon has just enough energy to bring about this state change. Shorter wavelength photons in the PAR range have more energy than is required, so when these are absorbed the additional energy is released as heat. The energetic states of chlorophyll. Regardless of the energy in the photon absorbed, the final energetic state is the first excited singlet state. The excitation energy can then be passed to adjacent chlorophyll molecules via inductive resonance energy transfer. Once the first excited state has been achieved, the excitation energy (also referred to as an “exciton”) has three possible fates: 1) being re­released from the chlorophyll pigment bed as a photon of red light when an excited chlorophyll molecule returns to ground state. This is chlorophyll fluorescence, and the wavelength of the photon released is always the same – i.e., it has an energy exactly equivalent to the difference in energy between the first excited state and the ground state of chlorophyll. 2) being released from the chlorophyll pigment bed as heat when an excited chlorophyll molecule returns to ground state. 3) being transferred to the PSII reaction center, where it can is used to drive photochemistry. These three possibilities can be considered as competing first order reactions, all occurring in the pigment bed surrounding Photosystem II.
8 Four possible fates of an exciton Photon F
D
N
P = photochemistry
F = fluorescence
D = basal dissipation
PQ N = active (variable) dissipation P P680 O.E.C. An exciton in the pigment bed can be used to drive photochemistry, released as heat (basal or active dissipation), or released as a photon of red light (fluorescence). While there is always a certain chance that the absorbed excitation energy will be released as heat (i.e., the reaction labelled as “basal dissipation” in the above figure), when PPFD is excessive relative to the ability of the chloroplast to utilize the absorbed energy to drive photosynthesis, active mechanisms are engaged to dissipate even more of the absorbed energy as heat. These can be considered as safety mechanisms that prevent the chloroplast from developing excessive oxidizing potentials that could be damaging. The main trigger for the activation of energy dissipation mechanisms is the acidification of the thylakoid lumen (refer to the previous diagram of the light reactions). When the rate of the light reactions exceeds the ability of the dark reactions to utilize ATP, the ATPase becomes “backed up”, and protons accumulate on the lumen side. Thus, excessive lumen acidification is a feedback signal, indicating that the light reactions need to be regulated by dissipating some additional excitation energy as heat. Although the specifics of this energy dissipation method are not well understood, we know that it depends on a family of pigments located in the light harvesting complex called the xanthophyll carotenoids. When excitation energy is excessive, lumen acidification triggers the conversion (specifically, the de­epoxidation) of the xanthophyll violaxanthin to antheraxanthin and zeaxanthin. The presence of zeaxanthin and antheraxanthin in the pigment bed greatly increases the probability of an exciton being dissipated as heat.
9 The xanthophyll cycle. Plants that are adapted or acclimated to sunny conditions tend to have smaller chlorophyll “antennae complexes” associated with each PSII reaction center, i.e., fewer total chlorophyll molecules “feed into” one reaction center. This helps to prevent the reaction center from receiving more excitation energy than it can process. Light-Harvesting Complexes of PSII Peripheral LHC (chl a / chl b)
Core LHC (chl a)
PQ PQ P680 P680 O.E.C. O.E.C. Sun-adapted PSII
Shade-adapted PSII Sun adapted plants tend to have smaller chlorophyll antennae complexes associated with each PSII reaction center.
10 In addition, sun­adapted plants also have larger total xanthophyll pools, and more of their xanthophylls in the de­epoxidized (energy­dissipating) forms – again, this reflects the stronger requirement for safe energy dissipation in such plants. This is also true of plants that are grown under conditions that reduce the rate of the dark reactions, thus increasing the requirement to dissipate energy “upstream” in the light reactions. Effect of growth environment on the total xanthophyll pool size (indicated by the size of the circle) and the xanthophyll deepoxidation state (fractions of violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z)). Demmig Adams et al., 1995 Because the reactions of PSII photochemistry, chlorophyll fluorescence and thermal (heat) dissipation all compete with one another for excitons in the pigment bed, a change in the rate of one of these reactions affects the other two. For example, when either photochemistry or thermal dissipation is reduced, the fluorescence signal increases. This property makes measurement of chlorophyll fluorescence a powerful probe of PSII chemistry at the leaf level. A more detailed description of the theory underlying this technique is beyond the scope of this course, but in the laboratory you will use one particular fluorescence parameter to measure the efficiency of photosystem II (FII, also annotated FP), in illuminated leaves. Many other fluorescence parameters have been devised. For example, the graphs below show how FP and
FN, the efficiency of active thermal dissipation, vary with PPFD in both maize and cotton leaves.
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30 45 0.8 40 0.7 35 0.6 25 0.5 20 0.4 15 0.3 F P 1 cotton
0.9 0.8 A G
0.7 ­1 ­2 ­ 1 AG (µmol m s ) 35 0.9 ­2 A G
40 AG (µm ol m s ) 45 50 30 0.6 FN
25 20 0.5 0.4 F P 15 0.3 10 0.2 10 0.2 5 0.1 5 0.1 0 0 0 0 500 1000 1500 ­2 2000 2500 0 0 ­1 PFPD (µmol m s ) quantum efficiency 1 maize quantum efficiency 50 500 1000 1500 ­2 2000 2500 ­1 PFPD (µm ol m s ) As PPFD increases, gross photosynthetic CO2 assimilation (AG) increases, photosystem II efficiency (FP) decreases, and active thermal dissipation (non­photochemical quenching, FN) increases. 12