Arizona State University College of Liberal Arts and Sciences

An Introduction to Photosynthesis
and Its Applications

By Wim Vermaas
Professor, School of Life Sciences, and
Center for the Study of Early Events in Photosynthesis
Arizona State University
This introduction to photosynthesis has appeared in condensed form in the magazine "The World & I" (March 1998 issue, pages 158-165) and was written by Wim Vermaas (wim@asu.edu). The web page for "The World & I" is at http://www.worldandi.com/).

The Basics.

Sunlight plays a much larger role in our sustenance than we may expect: all the food we eat and all the fossil fuel we use is a product of photosynthesis, which is the process that converts energy in sunlight to chemical forms of energy that can be used by biological systems. Photosynthesis is carried out by many different organisms, ranging from plants to bacteria (Figure 1). The best known form of photosynthesis is the one carried out by higher plants and algae, as well as by cyanobacteria and their relatives, which are responsible for a major part of photosynthesis in oceans. All these organisms convert CO2 (carbon dioxide) to organic material by reducing this gas to carbohydrates in a rather complex set of reactions. Electrons for this reduction reaction ultimately come from water, which is then converted to oxygen and protons. Energy for this process is provided by light, which is absorbed by pigments (primarily chlorophylls and carotenoids). Chlorophylls absorb blue and red light and carotenoids absorb blue-green light (Figure 2), but green and yellow light are not effectively absorbed by photosynthetic pigments in plants; therefore, light of these colors is either reflected by leaves or passes through the leaves. This is why plants are green.

Figure 1. Examples of photosynthetic organisms: leaves from higher plants flanked by colonies of photosynthetic purple bacteria (left) and cyanobacteria (right).

Figure 2. Absorption spectrum of isolated chlorophyll and carotenoid species. The color associated with the various wavelengths is indicated above the graph.

Other photosynthetic organisms, such as cyanobacteria (formerly known as blue-green algae) and red algae, have additional pigments called phycobilins that are red or blue and that absorb the colors of visible light that are not effectively absorbed by chlorophyll and carotenoids. Yet other organisms, such as the purple and green bacteria (which, by the way, look fairly brown under many growth conditions), contain bacteriochlorophyll that absorbs in the infrared, in addition to in the blue part of the spectrum. These bacteria do not evolve oxygen, but perform photosynthesis under anaerobic (oxygen-less) conditions. These bacteria efficiently use infrared light for photosynthesis. Infrared is light with wavelengths above 700 nm that cannot be seen by the human eye; some bacterial species can use infrared light with wavelengths of up to 1000 nm. However, most pigments are not very effective in absorbing ultraviolet light (<400 nm), which also cannot be seen by the human eye. Light with wavelengths below 330 nm becomes increasingly damaging to cells, but virtually all light at these short wavelengths is filtered out by the atmosphere (most prominently the ozone layer) before reaching the earth. Even though most plants are capable of producing compounds that absorb ultraviolet light, an increased exposure to light around 300 nm has detrimental effects on plant productivity.

Reaction Centers and Antennae.

Photosynthetic pigments come in a huge variety: there are many different types of (bacterio)chlorophyll, carotenoids, and phycobilins, differing from each other in their precise chemical structure. Pigments generally are bound to proteins, which provide the pigment molecules with the appropriate orientation and positioning with respect to each other. Light energy is absorbed by individual pigments, but is not used immediately by these pigments for energy conversion. Instead, the light energy is transferred to chlorophylls that are in a special protein environment where the actual energy conversion event occurs: the light energy is used to transfer an electron to a neighboring pigment. Pigments and protein involved with this actual primary electron transfer event together are called the reaction center. A large number of pigment molecules (100-5000), collectively referred to as antenna, "harvest" light and transfer the light energy to the same reaction center. The purpose is to maintain a high rate of electron transfer in the reaction center, even at lower light intensities.

Many antenna pigments transfer their light energy to a single reaction center by having this energy "hop" to another antenna pigment, and yet to another, etc., until the energy is "trapped" in the reaction center. Each step of this energy transfer must be very efficient to avoid a large loss in the overall transfer process, and the association of the various pigments with proteins ensures that transfer efficiencies are high by having appropriate pigments close to each other, and by having an appropriate molecular geometry of the pigments with respect to each other. An exception to the rule of protein-bound pigments are green bacteria with very large antenna systems: a large part of these antenna systems consists of a "bag" (named chlorosome) of up to several thousand bacteriochlorophyll molecules that interact with each other and that are not in direct contact with protein.

In many systems the size of the photosynthetic antenna is flexible, and photosynthetic organisms growing at low light (in the shade, for example) generally will have a larger number of antenna pigments per reaction center than those growing at higher light intensity. However, at high light intensities (for example, in full sunlight) the amount of light that is absorbed by plants exceeds the capacity of electron transfer initiated by reaction centers. Plants have developed means to convert some of the absorbed light energy to heat rather than to use the absorbed light necessarily for photosynthesis. However, in particular the first part of photosynthetic electron transfer in plants is rather sensitive to overly high rates of electron transfer, and part of the photosynthetic electron transport chain may be shut down when the light intensity is too high; this phenomenon is known as photoinhibition.

Photosynthetic Electron Transfer.

The initial electron transfer (charge separation) reaction in the photosynthetic reaction center sets into motion a long series of redox (reduction-oxidation) reactions, passing the electron along a chain of cofactors and filling up the "electron hole" on the chlorophyll, much like in a bucket brigade. All photosynthetic organisms that produce oxygen have two types of reaction centers, named photosystem II and photosystem I (PS II and PS I, for short), both of which are pigment/protein complexes that are located in specialized membranes called thylakoids. In eukaryotes (plants and algae), these thylakoids are located in chloroplasts (organelles in plant cells) and often are found in membrane stacks (grana) (Figures 3 and 4). Prokaryotes (bacteria) do not have chloroplasts or other organelles, and photosynthetic pigment-protein complexes either are in the membrane around the cytoplasm or in invaginations thereof (as is found, for example, in purple bacteria), or are in thylakoid membranes that form much more complex structures within the cell (as is the case for most cyanobacteria) (Figure 5).

Figure 3. Artist's rendition of a leaf (bottom), thylakoids within a chloroplast (middle), and a photosystem in thylakoids (top).

Figure 4. Electron micrograph of a thin section of an algal cell. The cup-shaped structure around the edge of the cell (open near the top) is the chloroplast. The structures resembling mostly parallel lines in the chloroplast are the thylakoid membranes. Courtesy of Dr. Ken Hoober.

Figure 5. Freeze-fracture electron micrograph of a cyanobacterial cell, showing exposed thylakoid membrane surfaces (upper right). Thylakoids are stacked like folded pancakes, and this image represents a surface-cut through these thylakoids.

All chlorophyll in oxygenic organisms is located in thylakoids, and is associated with PS II, PS I, or with antenna proteins feeding energy into these photosystems. PS II is the complex where water splitting and oxygen evolution occurs. Upon oxidation of the reaction center chlorophyll in PS II, an electron is pulled from a nearby amino acid (tyrosine) which is part of the surrounding protein, which in turn gets an electron from the water-splitting complex. From the PS II reaction center, electrons flow to free electron carrying molecules (plastoquinone) in the thylakoid membrane, and from there to another membrane-protein complex, the cytochrome b6f complex. The other photosystem, PS I, also catalyzes light-induced charge separation in a fashion basically similar to PS II: light is harvested by an antenna, and light energy is transferred to a reaction center chlorophyll, where light-induced charge separation is initiated. However, in PS I electrons are transfered eventually to NADP (nicotinamid adenosine dinucleotide phosphate), the reduced form of which can be used for carbon fixation. The oxidized reaction center chlorophyll eventually receives another electron from the cytochrome b6f complex. Therefore, electron transfer through PS II and PS I results in water oxidation (producing oxygen) and NADP reduction, with the energy for this process provided by light (2 quanta for each electron transported through the whole chain). A schematic overview of these processes is provided in Figure 6.

Figure 6: Overview of photosynthetic processes as they occur in plants, algae, and cyanobacteria.

Carbon Fixation.

Electron flow from water to NADP requires light and is coupled to generation of a proton gradient across the thylakoid membrane. This proton gradient is used for synthesis of ATP (adenosine triphosphate), a high-energy molecule. ATP and reduced NADP that resulted from the light reactions are used for CO2 fixation in a process that is independent of light. CO2 fixation involves a number of reactions that is referred to as the Calvin-Benson cycle. The initial CO2 fixation reaction involves the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which can react with either oxygen (leading to a process named photorespiration and not resulting in carbon fixation) or with CO2. The probability with which RuBisCO reacts with oxygen vs. with CO2 depends on the relative concentrations of the two compounds at the site of the reaction. In all organisms CO2 is by far the preferred substrate, but as the CO2 concentration is very much lower than the oxygen concentration, photorespiration does occur at significant levels. To boost the local CO2 concentration and to minimize the oxygen tension, some plants (referred to as C4 plants) have set aside some cells within a leaf (named bundle-sheath cells) to be involved primarily in CO2 fixation, and others (named mesophyll cells) to specialize in the light reactions: ATP, CO2 and reduced NADP in mesophyll cells is used for synthesis of 4-carbon organic acids (such as malate), which are transported to bundle sheath cells. Here the organic acids are converted releasing CO2 and reduced NADP, which are used for carbon fixation. The resulting 3-carbon acid is returned to the mesophyll cells. The bundle sheath cells generally do not have PS II activity, in order to minimize the local oxygen concentration. However, they retain PS I, presumably to aid in ATP synthesis. Even though C4 plants have reduced amounts of photorespiration, the amount of ATP they need per amount of CO2 fixed is a little higher than in other plants, and therefore their total production rate is similar to that of plants with higher rates of photorespiration.

Some plants living in desert climates, such as cacti, keep their stomates closed during the day to minimize evaporation (stomates are openings in the leaf surface to enhance gas exchange). These plants take up CO2 during the night when the stomates are open, and temporarily bind the CO2 to organic acids in the leaf. During the day the CO2 is released from the acids and used for photosynthesis. Plants using this mechanism of CO2 fixation are called CAM (Crassulacean Acid Metabolism) plants (Figure 7).

Figure 7. The light reactions of photosynthesis stop when the sun goes down. However, CO2 fixation can continue as long as ATP and NADPH is available. In cacti and other succulents CO2 uptake by the plant occurs primarily at night.

Increasing CO2 levels.

The amount of overall CO2 fixation in plants growing under optimal conditions is limited primarily by the amount of CO2 available. Therefore, the increase of CO2 in the atmosphere will lead to somewhat higher rates of plant growth in environments where the CO2 concentration limits growth rates. This is usually the case in an agricultural setting, where nutrients and water availability are not limiting. However, also in natural conditions, where limitations other than the CO2 concentration will generally limit plant productivity, plant productivity has been found to often increase upon increasing the CO2 concentration.

Photosynthesis and respiration.

Virtually all oxygen in the atmosphere is thought to have been generated through the process of photosynthesis. Obviously, all respiring organisms (including plants) utilize this oxygen and produce CO2. Thus, photosynthesis and respiration are interlinked, with each process depending on the products of the other. The global amount of photosynthesis is on the order of a trillion kg of dry organic matter produced per day, and respiratory processes convert about the same amount of organic matter to CO2. A large part (probably the majority) of photosynthetic productivity occurs in open oceans, mostly by oxygenic prokaryotes. Without photosynthesis, the oxygen in the atmosphere would be depleted within several thousand years. It should be emphasized that plants respire just like any other higher organism, and that during the day this respiration is masked by a higher rate of photosynthesis.

Diversity of Photosynthetic Organisms.

Even though plants are the most visible representatives of photosynthetic organisms, it should be emphasized that many other types of photosynthetic organisms exist. All photosynthetic bacteria other than the cyanobacteria and their relatives use only one photosystem, and for thermodynamic reasons they cannot use water as the ultimate electron donor. Instead, they can use reduced compounds such as H2S as donor. However, CO2 fixation occurs in these organisms. Some of these photosynthetic bacteria appear to have retained an evolutionary ancient arrangement of their photosynthetic apparatus, and are of interest for the analysis of evolutionary relationships of photosynthetic systems.

An extensive group of these photosynthetic bacteria, the heliobacteria, was discovered rather recently in the 1980s. The first representative of this group was isolated by the group of Dr. Howard Gest from a soil sample collected on the campus of Indiana University, and this isolation was the result of a fortunate coincidence of serendipitous events. Analysis of the heliobacterial reaction center has helped to lay the basis for the current concept that all photosynthetic reaction centers from the large variety of photosynthetic organisms are related to each other. The majority of bacteria cannot be maintained in pure culture (that is, without other oganisms). This has essentially limited analysis of photosynthetic prokaryotes to the relatively small group of organisms that can be grown in pure culture. It is likely that the actual diversity of photosynthetic organisms is much larger than is known thus far. Indeed, species with novel photosynthetic properties are reported virtually every year. For example, recently an organism was reported that has chlorophyll d (a chlorophyll that is very rare in nature) as the main pigment. Moreover, several years ago, previously undetected and very small chlorophyll a/b-containing prokaryotes were recognized to be the major contributors to photosynthetic production in the open ocean. This emphasizes that much relating to biodiversity and photosynthesis is still to be discovered, and that these discoveries are not limited to tropical rainforests and other ecological settings of large popular interest.

Evolution.

In eukaryotes, photosynthesis takes place in the chloroplast, which has long been known to have prokaryotic features. Chloroplasts are thought to have evolved from a cyanobacterium (or close relative) that was in a symbiotic relationship with a eukaryotic, non-photosynthetic cell and was engulfed inside this cell. The cyanobacterium and the eukaryotic cell presumably were in a mutually beneficial relationship (endosymbiosis), with the photosynthetic organism sharing some of its produced carbohydrates with the host, and the host providing the photosynthetic bacterium with other compounds. The prokaryote slowly gave up its independence as well as its cell wall, and some of its genetic information was transferred to the nucleus of its eukaryotic host. The resulting chloroplast maintains a small, prokaryote-like circular DNA of its own (DNA is material carrying genetic information); this DNA contains the genetic blueprint to make many of the membrane proteins needed in the chloroplast, which apparently are not easily targeted to and/or transported into the chloroplast. Occasionally, photosynthetic organisms are found where the chloroplast has retained a little more of the original cyanobacterial features. For example, in algae such as Cyanophora paradoxa plastids (called cyanelles) are found that resemble cyanobacteria in their overall morphology as well as in the fact that they are surrounded by a cell wall.

Not all chloroplasts have resulted from a single endosymbiotic event, but apparently from multiple events that occurred independently. Chloroplasts from higher plants and many green algae probably all result from the same endosymbiotic event, whereas chloroplasts from red and brown algae and from diatoms are the result of one or more other events. The situation is even more complicated in cryptomonads, a type of algae, and chlorachniophytes, photosynthetic amoebae, which apparently are the result of an endosymbiotic event of a eukaryotic alga in a eukaryotic host. The nucleus of the endosymbiont has been mostly degraded, resulting in a chloroplast enveloped by four membranes.

Early Events.

Chlorophyll is used by all photosynthetic organisms as the link between excitation energy transfer and electron transfer. Of particular note is the rate with which these transfer reactions need to occur. As the lifetime of the excited state is only several nanoseconds (1 nanosecond (ns) is 10-9 s), after absorption of a quantum, energy transfer and charge separation in the reaction center must have occurred within this time period. Energy transfer rates between pigments are very rapid, and charge separation in reaction centers occurs in 3-30 picoseconds (1 picosecond (ps) is 10-12 s). Subsequent electron transfer steps are significantly slower (200 ps - 20 ms) but, nonetheless, the electron transport chain is sufficiently fast that at least a significant part of the absorbed sunlight can be used for photosynthesis. However, in the presence of excess light, damage may occur, which may originate from the formation of chlorophyll in "triplet state". In a triplet state two electrons in the outer shell have identical rather than opposite spin orientation. This triplet chlorophyll readily reacts with oxygen, leading to the very reactive singlet oxygen, which can damage proteins. To counter this damaging reaction, carotenoids are usually present in close vicinity to chlorophylls. Many carotenoids efficiently "quench" triplet states of chlorophyll, thus avoiding formation of singlet oxygen. Chlorophyll in its free form is very toxic in the light in the presence of oxygen, because a close interaction with carotenoids is not always available under such circumstances. Therefore, all chlorophyll in a cell in aerobic organisms is bound to proteins, generally with carotenoids bound to the same protein.

Structure Determinations.

Because of the strict requirements of positioning of pigments and electron transfer intermediates to allow efficient electron transfer and minimal damage, the structure of pigment-protein complexes involved in photosynthesis is critical. With the exception of specific antenna complexes (such as phycobilisomes in cyanobacteria and chlorosomes in green bacteria), pigment-binding proteins are usually hydrophobic membrane proteins. This initially hampered attempts to elucidate the structure of these complexes, as membrane proteins do not readily form the well-ordered crystals that are needed for high-resolution X-ray diffraction studies. However, in the 1980s the first structure of a membrane protein complex, the photosynthetic reaction center from a purple bacterium, was determined at high resolution (about 3 Å; in comparison, the distance between neighboring atoms in a molecule is about 1 Å). Investigators from the Max Planck Institute in Martinsried (Germany) who were involved with this work, most notably Hartmut Michel and Johann Deisenhofer, received a Nobel Prize in chemistry for this research. Since then, the structure of various reaction centers and antenna complexes has been determined at resolutions between 2 and 4 Å. Figure 8 presents the structure of the photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides.

Figure 8. Molecular structure of a bacterial reaction center. Cofactors are indicated in red. The three proteins making up the reaction center are in blue, yellow, and green. Ribbons in the protein represent helices through the membrane. Wire-like protein regions represent domains outside of the membrane. Courtesy of Dr. Jim Allen.

Similarities Between Reaction Centers.

Surprisingly, structural comparison of reaction centers from different photosynthetic systems showed that these reaction centers are basically similar to each other in terms of their overall three-dimensional structure. The basic reaction center unit consists of a protein complex with 10 transmembrane helices originating from either two identical protein subunits or from two similar polypeptides of common evolutionary origin. Each of these polypeptides contribute five membrane-spanning helices, and bind 2-3 chlorophylls (or, in the case of anoxygenic bacteria, bacteriochlorophylls). The fourth membrane spans from each subunit are held together by two chlorophylls, which are the chlorophylls in the reaction center that can be oxidized (give up an electron) upon excitation. Directly associated with the reaction center are proteins that bind antenna pigments. In the case of PS I and similarly organized reaction centers from green and heliobacteria, the antenna portion, with six transmembrane helices, is attached to the N-terminal end of the reaction center proteins.

Implications of Photosynthesis Studies.

Photosynthesis has been studied in significant detail and photosynthetic systems are used frequently for development and application of advanced technologies, because photosynthetic systems are fairly well understood, are complex, and often undergo rather unusual biochemical reactions. Some examples are provided below.

Rapid electron transfer reactions.

A major difficulty in measuring enzyme kinetics at relatively short time scales (less than 1 ms) is that "traditional" enzyme reactions require a mixing of substrate and enzyme, which usually takes a relatively long time. Kinetic analysis of light-driven reactions such as photosynthetic electron transport have a great advantage in this respect: reactions can be triggered simply by a light pulse, which can be even shorter than 1 ps. Moreover, many of the components participating in electron transfer have different absorption spectra depending on whether they are in the oxidized or reduced form. Using laser spectroscopy methods or more standard optical spectroscopy, it is relatively simple to follow the electron around on a timescale between 1 ps and several ms. The primary charge separation occurs in several ps, and reactions become gradually slower as they involve components that are further away from the reaction center. Because of the fast speed of early reactions, the electron and the "electron hole" are physically separated rapidly by a large distance (the electron generally has traveled about 2 nm to the other side of the membrane within 1 ns after charge separation), so that back reactions (charge recombinations) are not favorable anymore. Unpaired electrons on reactants that are transiently formed during redox reactions involving transfer of a single electron in many instances can be detected using electron paramagnetic resonance (EPR) and derived techniques (including ENDOR, electron nuclear double resonance, and ESEEM, electron spin echo envelope modulation). Many of these techniques can be used to kinetically follow redox reactions, and provide detailed information regarding electron spin distributions etc. Therefore, photosynthetic membranes and reaction centers have a prominent place as experimental systems in biochemistry and biophysics.

Organic molecules mimicking reaction centers.

Reaction centers are essentially an assembly of cofactors, held in the appropriate position and orientation by the protein environment. Several groups have used the natural system as a model to design organic molecules where the equivalents of the different cofactors are linked together by covalent bonds of various lengths. The result is the creation of a number of sophisticated molecules that serve as "artificial reaction centers". The more advanced molecules consist of two chlorophyll-type molecules linked together (one serves as the electron donor, the other as the acceptor), with the electron-accepting molecule linked to two quinones, which serve as electron acceptors in the natural system. The electron donating chlorophyll analog is linked covalently to a carotenoid, which can donate an electron to the oxidized chlorophyll. Upon excitation of the chlorophyll, a charge separation occurs resulting in an oxidized carotenoid and a reduced quinone. This charge-separated state is formed with high efficiency. An example of such a molecule is presented in Figure 9.

Figure 9. Molecular model of an artificial reaction center. The two bulky structures in the middle are chlorophyll-like components, flanked by a carotenoid (left) and quinones (right). Courtesy of Dr. Devens Gust.

Such molecules can be introduced into liposomes (artificial membrane vesicles) in a specific orientation, and when these are excited by light, a charge separation will occur across the liposome membrane. This results in an electric potential or proton gradient across the liposome membrane, which may be used for a variety of purposes, including ATP synthesis (the latter requires introduction of the ATP synthesizing enzyme into the liposome membrane). The groups of Ana and Tom Moore and Devens Gust at Arizona State University are leaders in developments in this area.

Genetic modification and protein engineering.

Because of the ease of detailed functional analysis of reactions and their rates in photosynthetic systems, reaction center complexes are frequently used to determine the consequences of small alterations in the polypeptides on the functional characteristics of the cofactors. Changes at single amino acid residues in the reaction center complex are sufficient to introduce large changes in the properties of cofactors, which in turn leads to altered electron transfer rates and efficiencies. Single amino acid changes at specific sites in the protein are easily introduced by genetic modification techniques, and resulting functional changes can be studied. An elegant example of such an approach is the modification of the midpoint redox potential of the bacteriochlorophyll in the reaction center of purple bacteria. The midpoint redox potential is correlated with the ease with which an electron is given off after excitation and is taken up by the oxidized bacteriochlorophyll. Jim Allen, JoAnn Williams and coworkers at Arizona State University found that creating or deleting hydrogen bonds between the protein and the bacteriochlorophyll changed the midpoint redox potential of this bacteriochlorophyll in a rather predictable manner. In this way, reaction center complexes can be built with different oxidizing strengths, and effects on reaction rates and ultimately the effectiveness of alternate electron donors can be determined. Mutational analysis of photosynthesis proteins is simple in several bacterial systems. The reasons why this is so in selected cyanobacteria and purple and green bacteria are that (1) foreign DNA is taken up by the cell spontaneously or is introduced easily by other means such as electroporation ("electric shock"), (2) once the DNA is inside it is incorporated into the organism's genome at one predictable and specific site by means of a process named homologous double recombination, and (3) the organism can be propagated without relying on photosynthesis, for example using an added carbohydrate source.

Genetic approaches involving directed mutagenesis as described above have proven to be very useful in studying photosynthetic electron transfer and will be of increasing relevance for the design of photosynthetic organisms for biotechnological uses (see below). By this method the function of a large number of genes has been probed, and the role of individual domains and residues has been determined. Genomic sequencing projects are very useful in this respect, and the complete DNA sequence of one photosynthetic organism is already known. From the DNA sequence, the potential of the organism can be determined. The entire 3,573,470 nucleotide-long genomic DNA sequence of the transformable cyanobacterium Synechocystis 6803 was determined by Satoshi Tabata and coworkers at the Kazusa DNA Research Institute in Japan. This organism is used by several researchers, including in the Vermaas group at Arizona State University, to elucidate the role of many proteins thought to be involved in photosynthetic or other physiological processes. Meanwhile, other groups are working on the genomic sequence of two purple bacteria. With the genomic sequence in hand, the role of specific genes can be found by amplifying the gene of interest by means of polymerase chain reaction, cloning it into a plasmid, replacing the gene by a selectable marker (i.e., a piece of DNA coding for a protein inactivating a particular antibiotic, thus conferring antibiotic resistance), and analyzing the functional characteristics of resulting mutants. A website, CyanoBase (http://www.kazusa.or.jp/cyano/cyano.orig.html), has been established to facilitate searching of the genomic sequence of Synechocystis 6803, and a related site, CyanoMutants (http://www.kazusa.or.jp/cyano/mutants/), has been developed that accommodates information regarding targeted mutations and their effects in this organism.

Genetic modification of higher plants.

Globally, improving plant productivity by genetic means has been an important goal for many years. Even though initially it was hoped that crop productivity could be boosted by "improving" the photosynthesis process, it has become increasingly apparent that improved productivity will be best accomplished by genetic modification of crop plants to introduce insect or pathogen resistance, or to yield improved vitality under marginal conditions (for example, at high salinity, which has become a significant factor in many agricultural areas because of continued irrigation). A pertinent example is the development of varieties of cotton and other crops that express a pro-toxin from a bacterium, Bacillus thuringiensis, that is converted to a toxin in the midgut of particular insects such as caterpillars but not in other life forms. This allows efficient biological control as long as the insects do not develop resistance to this compound.

Genetic modifications to intrinsically and significantly improve the photosynthesis process have not yet been successful. The reason for the apparent inability to "improve" the photosynthesis process itself presumably is related to the fact that photosynthetic systems have evolved over a relatively long period of time, and that the selection factors have not changed significantly in recent history. This has led to the emergence of a very effective photosynthetic apparatus that is difficult to improve upon by simply changing some amino acid residues or by introducing or deleting some genes. If relatively simple changes could have significantly improved the photosynthesis process per se, Mother Nature would have already found these as natural mutations are rather frequent. However, it is possible that significant progress in this area can made in the future, if new design paradigms for enzyme function and specificity can be developed. For example, if protein structures (particularly the structure of the active site) can be better modeled and predicted, one should be able to further improve upon the RuBisCO specificity of CO2 vs. oxygen. It is also important in this respect to determine what the rate-limiting step in the process is under natural conditions. Even though more effective light capture by crop plants might be considered by introduction of antenna pigments that absorb in the green and yellow region of the light spectrum, light capture and the light reactions usually are not limiting plant productivity in agricultural settings. Therefore, such modifications in a plant will result in increased productivity only in light-limiting settings.

Future Directions.

Light energy is cheap, clean, and essentially inexhaustible. With limited supplies of fossil fuel and increasing concern about CO2 emissions, further development of technologies that make use of solar energy is inevitable. Current silicon-based technologies for the harvesting of solar energy require a very energy-intensive production process and even though they have improved significantly over the years in their efficiency, further development of photosynthesis-based technologies for energy collection is certainly warranted. However, photosynthesis and related processes can be applied to many more areas than just solar energy conversion. Realizing that novel designs and applications of light-mediated processes have enormous promise in the next decade and beyond, Arizona State University has started an initiative, Project Ingenhousz, named after the 18th century physician who discovered that light is needed for oxygen evolution by plants, and that only green parts of the plant carry out this process. The goal of the initiative is to capitalize on research progress and ideas in this area, and to more effectively interface academia, where many of the discoveries are made, with the private sector, where such discoveries are worked out further and applied.

For example, there are a myriad of possible applications of artificial reaction centers and related molecules in nanotechnology. Many synthetic pigments also have found biomedical uses in tumor detection, as they -for unknown reasons- tend to accumulate preferentially in tumors and are highly fluorescent and thus easily detectable in a patient whom is being operated on to surgically remove a tumor.

In the biotechnology field, photosynthetic organisms are likely to play an increasing role in (over)production of enzymes, pharmaceuticals, nutraceuticals, etc., which until now are produced primarily by genetically modified heterotrophic microorganisms such as yeast and selected bacteria. A major advantage of photosynthetic organisms is that no fixed-carbon source needs to be added for growth and, therefore, production costs are lower and the chances of contamination with other microorganisms are less. There are several ways to modify organisms to have them (over)produce useful compounds. One is to introduce specific genes under a strong promoter, leading to high expression of these genes and to synthesis of a "new" enzyme. Another way is to delete genes so that substrates will accumulate. For this to be optimally successful the metabolic pathways of an organism need to be fairly well understood, and genomic DNA sequences are an important step in this direction. A third way to produce a new compound is to utilize an existing enzyme and to modify the site where the substance to be converted (the substrate) binds, so that a different substrate can be bound and a different product can be formed. With any of these approaches, selection for randomly generated combinatorial mutants with specific properties also has been proven to be effective. With an increasing arsenal of genomic sequences, and with improving knowledge regarding determinants of protein structure and cofactor binding, any of these three approaches are very promising.

Another potential application of photosynthetic organisms is in bioremediation. Bioremediation is the clean-up of environmental (soil or water) pollutants by biological means. An example is the biological breakdown of toxic organic compounds into innocuous products. Also, remediation of nitrate from drinking water supplies is becoming an increasingly pressing issue. The advantage of using photosynthetic organisms is that no external energy source needs to be provided for growth of the organism if it is in the light, making these organisms very suitable for remediation of aqueous surface environments.

Another utilization of photosynthetic organisms is to have these organisms use solar energy to produce clean-burning fuels. Even under natural conditions some photosynthetic systems such as algae can produce hydrogen, which probably is the cleanest fuel as it reacts with oxygen to produce water. However, the cheapest and most universal electron donor of all, water, upon oxidation in PS II forms oxygen, which is not very safe in combination with hydrogen. For this reason methods have been sought to temporally separate oxygen evolution and hydrogen production in algae; in the laboratory this can be achieved by sulfur deprivation, which preferentially inhibits photosynthesis. Another option would be to use photosynthetic organisms for methane production. Even though methane upon combustion will form CO2, the overall atmospheric CO2 balance would not be disturbed as an equal amount of CO2 will have been taken out of the atmosphere upon methane production by the photosynthetic organism.

Research in photosynthesis in all its facets has proven to have opened many doors in a variety of disciplines, ranging from biophysics to plant physiology. Progress has been driven by an interdisciplinary approach to this complex, yet fascinating, spectrum of problems, challenges, and opportunities. Photosynthesis is the basis of our food and energy supply, and innovative utilization of solar energy is likely to be of increasingly critical importance in the future. This, together with novel uses of photosynthetic principles for other purposes, make it likely that photosynthesis and its applications will help to shape an increasingly broad area of exciting discoveries and innovative ideas.

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