The mutant strain was further analyzed with respect to fluorescence kinetics. The fluorescence curve demonstrates that the fluorescence yield is constant and equal to FM (step 3); the results suggest that the mutant RG-7388 in vitro exhibits essentially no photochemical or non-photochemical quenching. Furthermore, analysis of the carotenoid electrochromic shift (a measure of the electrochemical

gradient generated from electron flow through PSI and PSII; step 4) indicates that DCMU has no effect on the membrane potential. Considering the overall information, the results suggest that PSII activity in the cgl28 mutant is severely compromised, although further spectroscopic and biochemical analyses are required. Fig. 3 Analyses of mutants defective for genes encoding GreenCut Adavosertib ic50 proteins. Step 1: Mutants are grown at varying light intensities on medium containing acetate or in minimal medium supplemented with CO2. In this example, a strain with a mutation in the CGL28 gene (red box, step 1) grew slower than wild-type cells (blue box) on acetate-containing medium, and did not

grow at all on minimal medium supplemented with CO2. Step 2: Fv/Fm values, shown as a false color image, are determined for colonies grown on solid medium containing acetate. In this case, the cgl28 mutant (red box) was determined GDC-0068 supplier to have a markedly reduced Fv/Fm relative to wild-type cells (blue box). Step 3: The mutants are further analyzed after growth in the dark in liquid medium containing

acetate for photochemical and non-photochemical quenching using fluorescence assays. This strain (blue curve) has no variable ID-8 fluorescence (which can be observed in the pink curve of wild-type [WT] cells). When the horizontal bar at the top of the image is unfilled (white, outlined in black), the sample is being exposed to actinic light, while the black-filled region of the bar indicates that the sample is in the dark. All downward arrows are the times at which the sample is exposed to a pulse of saturating light, which allows for the determination of maximal fluorescence yield. Step 4: Samples are further analyzed for the contribution of each of the reaction centers to the generation of the electrochemical gradient across the thylakoid membranes by measuring the electrochromic band shift (carotenoid band shift at 520 nm) induced by illumination in the presence and the absence of the PSII inhibitors DCMU and hydroxylamine (HA). The upward arrow indicates light on, while the downward arrow indicates light off. PSII inhibitors have no effect on the electrochemical gradient generated in the cgl28 mutant in the presence of illumination, indicating that PSII cannot perform a charge separation. Step 5: In order to verify that the mutation is linked to the observed phenotype, the mutant is backcrossed with wild-type cells to determine whether the mutant phenotype is linked to the insertion (drug-resistant marker gene).