and Its Applications
|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 (firstname.lastname@example.org). The web page for "The World & I" is at http://www.worldandi.com/).|
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.
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.
|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.|
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.|
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.
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.
|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.|
|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 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 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.
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. It is likely that this can be improved upon by introduction of genetic changes. However, a remaining problem is that 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. 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.
updated Tuesday, June 23, 1998
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