Backward transitions in the analysis of oxygen production under flashing light were introduced by Packham et al., 1988, Photosynth. Res. 15: 221-232. In order to take backward transitions into account, a new method of analysis is presented: the 'eigenvalue method'. This method is based on the recurrence relation of oxygen production with four coefficients (also known as the four 'sigma' coefficients). It shows less susceptibility to round-off errors than other methods and permits the computation of double-hits directly from the coefficients, which was not possible before. With it we discovered that the inconsistent behaviour of double-hits observed previously under low flash intensities or low flash frequencies was mainly due to the inclusion of the backward transitions into the double-hit probability. In these conditions backward transitions seemed to be due either to the combination of an S-state deactivation and a miss, or to two S-state deactivations and a single-hit.In the presence of 3-(3, 4-Dichlorophenyl)-1, 1-dimethylurea (DCMU), the previous methods of 'sigma' analysis failed. In contrast, the new method resolved all four S-state probabilities; thus it has the further advantage of being more 'robust' (robustness being defined as the ability to yield a meaningful answer under difficult conditions).

No abstract

We present a model of the S-state mechanism, as well as an improved eigenvalue analysis, that integrate into a coherent ensemble several features found since the S-state model was initially developed. These features include the presence of S-1, deactivations in the dark interval between flashes, and the change in the number of active PS II centers by photoinhibition or photoactivation. A new feature is the capacity to predict the steady-state distribution of S-states under conditions of steady photoinhibition or photoactivation. The improved eigenvalue analysis allowed the calculation of the initial S-state distribution. In addition, the model resolved 'true' photochemical misses from apparent misses due to deactivations in the dark interval between flashes. The model suggested that most of the misses that are commonly reported are due to deactivations, and not to an intrinsic inefficiency of the photochemical mechanism of PS II. Because models that allow double-hits encompassing the S2 to S3 transition often predict negative initial quantities of S2 in cyanobacteria, our proposed model specifically prohibited them. The model accounts for inhomogeneous misses and a steady-state distribution of the type (S2)≈(S1)>(S3)≈(S0). This 5-step model uses only 4 probabilities, and is therefore easy to handle. The use of this model is critical for the analysis of several cyanobacterial strains, as well as for any species that show non-negligible deactivations in the dark interval between flashes.

No abstract

We investigated the slow signal of apparent O2 release under brief light flashes by using mutants of Synechocystis sp. PCC 6803 which lacked CP43 and D1. The slow signal was present at higher amplitudes in the mutants. It was inhibited by starving the mutants of glucose (>90%), by 10 mM NaN3 (85%) and by boiling samples for 2 min (100%). In the mutants and in the wild-type, the slow signal was 95% inhibited by the combination of DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). In the wild type, the addition of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) or CCCP (carbonylcyanide m-chlorophenylhydrazone) completely inhibited photosynthetic O2 evolution, yet failed to inhibit the slow signal. We explain the kinetics of the wild-type signal as a positive deflection due to the inhibition of respiration by PS I activity, and a negative deflection due to the stimulation of respiration by electrons originating from PS II. We found no evidence of a 'meta-stable S3' in Synechocystis sp. PCC 6803 that could contribute to the slow signal of apparent O2 release. We present a calculation which involves only averaging, division and subtraction, that can remove the contribution of the slow signal from the true photosynthetic O2 signal and provide up to a 10-fold improved accuracy of the S-state models.