The fluorescence induction kinetics collected for this database are determined under the condition described below.
Cyanobacterial strain and growth condition
A glucose-tolerant wild-type strain Synechocystis sp. PCC 6803 was grown in BG-11 medium. 10 ml of cell culture, of which OD730 was adjusted to 0.5 by BG-11 liquid medium, was dropped on BG-11 agar plates. After incubation at 30oC under the light at 20 micromol m-2 s-1 for 72 h or at 200 micromol m-2 s-1 for 48 h, the plates were subjected to the measurements of chlorophyll fluorescence kinetics.
Chlorophyll fluorescence measurements
Cells on agar plates were dark adapted for 15 min before the measurements. The plates were put in fluorescence imaging system (FluorCam 700MF, Photon System Instruments, Czech Republic) and actinic light (200 micromol m-2 s-1) was applied for 45 s to monitor chlorophyll fluorescence. The actinic light was generated by two panels of orange light-emitting diodes (HLMP EH08, Agilent Technologies, USA) with peak wavelength at 615 nm and the spectral half-width of 18 nm. Fluorescence image was captured every 40 ms for 45 s by a CCD camera (ICX429AL, Sony, Japan) that produced 752 x 580 pixel images in an 12 bit gray scale. The sensitivity was adjusted to 20% on the FluorCam software. The fluorescence around 700 nm was detected by filtering the emission through a custom-made interference filter with a peak at 700 nm (the full-width at half-maximum is 30 nm) and a red blocking filter (RG697, Corion, USA). Fluorescence intensity was integrated over the area of each cyanobactrial patch for the fluorescence image at each time point. The fluorescence intensities were normalized with the initial value at the start of actinic light.
Similarity calculation of chlorophyll fluorescence kinetics
To correlate the gene function with chlorophyll fluorescence kinetics, it is essential to quantitatively compare the kinetics of different gene-disruptants. For this purpose, the fluorescence kinetics was first averaged for the quintuplicate cell patches of cyanobacteria, and then every 2, 4, 8, 16 and 35 data points were averaged for the data in the time range of 1-3, 3-7, 7-15, 15-31 and 31-45 s, respectively. The data in the time range of 0-1 s were directly used without averaging for the time resolution necessary to analyze the initial fast rise of the fluorescence. As a result, number of time points of the fluorescence kinetics was reduced from 1126 to 135. Two kinds of "similarity distance" were calculated for this database: Differential similarity distance and Vector similarity distance. The former compares the derivative of the trace of fluorescence kinetics. The difference of the two adjacent data points were used as "derivative" of the data. The square of difference of the derivatives between two cyanobacterial strains were calculated for each time point, and summed up for entire time range, and used for the differential similarity distance. The Vector similarity distance was defined based on the angle made by two phenotypic vectors. Since time sereis of 134 data points could be treated as one vector in 134 dimensions, the angle made by two vectors would represent the dissimilarity of the phenotype.
By either kind of similarity distances, the similarity of phenotypes of any two mutants could be calculated. Just as in the case of co-expression analysis of the genes in many organisms, one can list genes that might have similar function with any genes of the interest. The similarity list can be displayed from the information window of each gene.
More information can be obtained in:
For fluorescence measurements
Ozaki, H., Ikeuchi, M., Ogawa, T., Fukuzawa, H. and Sonoike, K. (2007)
Large scale analysis of chlorophyll fluorescence kinetics in Synechocystis sp. PCC 6803: Identification of the factors involved in the modulation of photosystem stoichiometry. Plant Cell Physiol. 48, 451-458.
For similarity calculations
Ozaki, H., and Sonoike, K. (2009)
Quantitative analysis of the relationship between induction kinetics of chlorophyll fluorescence and function of genes in the cyanobacterium Synechocystis sp. PCC 6803. Photosynth. Res. 101, 47-58.