Introduction to "Fluorome" Project

Photosynthesis and chlorophyll fluorescence

Oxygenic photosynthesis is the process by which plants, algae and some bacteria produce carbohydrate from carbon dioxide and water, accompanying the evolution of oxygen as a byproduct. Driving force of this process is light energy that absorbed by photosynthetic pigments such as chlorophylls. There are two photosystems, Photosystem I (PSI) and Photosystem II (PSII) that bind chlorophylls and driving photosynthetic electron transfer. Under normal condition, the light energy absorbed by chlorophylls is mainly used for driving photosynthesis. Thus, only a small fraction of the absorbed energy is "wasted". In the case of chlorophyll solution in organic solvents (where the energy could not be used for photosynthesis), the yield of fluorescence reaches to 30%. Light energy absorbed by chlorophyll molecules in vivo can be directed to one of three fates: it can be used to drive photosynthesis, excess energy can be dissipated as heat or the energy can be re-emitted as chlorophyll fluorescence. These three processes occur in competition, so that any increase in the efficiency of one will result in a decrease in the yield of the other two. Thus, by measuring the yield of chlorophyll fluorescence, we can obtain information on the changes in the efficiency of photosynthesis and heat dissipation. Chlorophyll can be regarded as an intrinsic fluorescent probe of the photosynthetic systems.

Changes in the yield of chlorophyll fluorescence were first observed by Kautsky and Hirsch in 1930s. They found that, when dark-adapted photosynthetic organisms are illuminated with continuous light, chlorophyll fluorescence displays characteristic changes in intensity accompanying the induction of photosynthetic activity. The change in the intensity of chlorophyll fluorescence is usually called induction kinetics of chlorophyll fluorescence. When a dark-adapted plant is exposed to continuous light, initial increase in the yield of chlorophyll fluorescence occur that reflects reduction of Q_A, the primary electron acceptor of PSII. Following the increase, the fluorescence level typically starts to fall again. This reflects the activation of enzymes involved in the downstream of electron transfer leading to the partial re-oxidation of Q_A^-. There are several steps in photosynthetic electron transfer that could be light-activated: ferredoxin-NADPH oxidoreductase (FNR) or several enzymes in Calvin cycle. The activation time course of these enzymes affects the induction kinetics of the chlorophyll fluorescence. Furthermore, proton gradient formed by the electron transfer suppresses the rate of electron transfer itself. This feed-back regulation also affects the induction kinetics. Thus, the intensity of chlorophyll fluorescence from photosynthetic apparatus shows very complex changes reflecting the condition of many components of photosynthetic metabolism.

Cyanobacteria as model organisms for photosynthesis study

Cyanobacteria are prokaryotes that have an ability of oxygenic photosynthesis. During the evolution of the modern biosphere, cyanobacteria have played a central role by elevating the oxygen level in the atmosphere of the Earth that starts 2.7 billion years ago. Cyanobacteria are the progenitors of chloroplasts in green plants, and the basic machinery of photosynthesis in cyanobacteria is quite similar to that of plants. From the photosynthetic point of view, cyanobacteria are different from the photosynthetic bacteria such as green sulfur bacteria or purple bacteria, and more related to algae and higher plants. As a prokaryote, cyanobacteria have simple cell structure without organelle, and homogeneous cell culture can be prepared for both physiological and biochemical study. Thus, cyanobacteria are widely used for the study of photosynthesis. Among several cyanobacterial strains, the unicellular, naturally transformable cyanobacterium Synechocystis sp. PCC 6803 is the first photosynthetic organism with a completely sequenced genome. This organism offers an excellent experimental system to investigate the photosynthetic apparatus, especially when the molecular biological approach is taken.

The main target of the biological research in post-genomic era

One of the main goals in the post-genomic era is to predict the biological function of each gene in the genome. Many methods have been developed for this purpose, among which the most straightforward method is to apply PSI-blast and FASTA to the sequence of the target gene and to find homologs of the gene. In this case, the function of the target protein is assumed to be the same with that of its homologs. Generally, this kind of sequence-based method works well. However, it does not work at all when the sequence similarity between the target protein and known proteins is very low or when the target sequence has homology only to proteins with unknown function. Furthermore, even if the "function" of the target gene could be predicted by these methods, the "role" of the gene in the biological system cannot necessarily be determined. For example, when the gene of interest has homology with a kinase, we can predict the product of the gene would have kinase activity. However, this is not the information we really want to know. We want to know, for example, in which signal transduction pathway the gene is working, or in what kind of biological phenomenon the gene is involved. These pieces of information cannot be obtained through examination of sequence homology.

One of the most powerful techniques for attributing functions to genes in organisms is the analysis of phenotype in gene-disrupted mutants. In this case, the "biological phenomenon" could be directly assigned to the gene of interest. However, this approach also has disadvantages itself. First, it cannot provide any information about the function of genes from mutants that do not exhibit distinguished phenotypes. On the other hand, if we employ several kinds of different phenotypes to collect more information, we confront another problem, i.e. how to integrate the different types of information to elucidate the function of the gene. Secondly, although we must first decide what kind of phenotype should be observed in the experiments, it is not easy to determine a suitable method when the function of the gene of interest is totally unknown. Only after we get some information such as, for example, that the gene may be involved in respiration, we could observe the respiration rate as a phenotype. Thirdly, in most cases, the phenotypes of the mutants must be checked one by one. It is not usually easy to observe the phenotype of the mutants on a large scale. Thus, three problems must be cleared for the effective analysis of the mutant phenotype. Requirement 1: The phenotype should be the one that can be observed in high throughput. Simultaneous observation of the phenotype of many mutants should be possible. Requirement 2: We must choose as simple phenotype as possible. The phenotype should be the one that can be treated quantitatively. Nevertheless, the phenotype of as many mutants as possible should be different from that of wild type. Requirement 3: We must choose appropriate phenotype that gives as much information as possible. The phenotype should reflect wide range of cellular processes.

The Fluorome Project

As stated above, there are three requirements for the elucidation of the gene function: 1) high throughput phenotype analysis, 2) simple and quantitative phenotype, and 3) phenotype that reflects a wide range of cellular processes. Here, we employ chlorophyll fluorescence as a phenotype to analyze the gene function for the following reasons. First, chlorophyll fluorescence is easily monitored by optical devices, and simultaneous measurements of many mutants are possible for high throughput analysis. Secondly, induction kinetics of the chlorophyll fluorescence can be expressed as simple one-dimensional numerical values that are easy to handle. Finally, chlorophyll fluorescence reflects the condition of photosynthetic electron transport. In plant cells, photosynthesis takes place in chloroplasts, and consequently, chlorophyll fluorescence principally reflects the condition of photosynthesis. In cyanobacteria, however, photosynthetic and other metabolic pathways are not separated into organelles. Thus, chlorophyll fluorescence of cyanobacteria has the potential to detect the effects of mutation not only on photosynthesis but also in a wide variety of metabolic systems. In fact, we observed that the induction kinetics were affected in about half of the mutant observed, suggesting that half of the genes on the genome have some interaction, directly or indirectly, with photosynthetic metabolism. If a broad range of effects from mutation is detected by one phenotype, then many kinds of mutants can be characterized simply by phenotype. We believe that we can realize such ideas in this Fluorome Project.

Acknowledgment

This project is partly supported by Grant-in Aid for Genome Biology from the Ministry of Education, Culture, Sports, Science and Technology.