, Streusand and Portis 1987); four other thioredoxin-dependent enzymes: d-fructose1,6-bisphosphatase, phosphoribulokinase, and sedoheptulose-1,7-bisphosphatase (Buchanan 1984; Scheibe 1990) and ATP synthase (Stumpp et al. 1999); and FNR (Carillo et al. 1981; Satoh 1981). These enzymes are active in the Necrostatin-1 molecular weight light, and during a light-to-dark transition, they gradually become inactive again. The half-time of inactivation of Rubisco under in vivo conditions is 2–4 min (Stitt et al. 1987; Laisk and Oja 1998). Inactivation of ATP synthase and the three other Calvin–Benson cycle enzymes is under control of the thioredoxin system (Scheibe 1990), and their

inactivation depends on the re-oxidation of stromal components such as ferredoxin and NADPH. FNR inactivation varies depending on the species: pea leaves need ~15 min for full inactivation (Schansker et al. 2006), whereas in a Pinus species, an hour is needed learn more (Schansker et al. 2008). Once inactivated, all of

these enzymes must first be activated again before steady state photosynthesis is induced, and this affects the fluorescence induction kinetics (see Papageorgiou et al. 2007; Papageorgiou and Govindjee 2011 for an in-depth discussion of the fluorescence kinetics beyond P or F M in a variety of photosynthetic organisms). In addition, active FNR (i.e., an activated acceptor side of PSI) has an effect on the IP phase of the OJIP transients and on the amplitude of the F M that can be reached by a strong pulse of light (Schansker

et al. 2008). In most fluorescence studies, many are not interested in the processes mentioned above, and in that case, it is best to make the dark-adaptation time long enough to allow at least FNR to become inactive again (a marker for this is a regeneration of the fluorescence IP phase and in addition a regeneration of 820 nm re-reduction phase paralleling the IP phase, see Schansker et al. 2006, 2008). As mentioned in Question 2 Sect. 3, several regulatory and stress-related processes that affect the fluorescence yield (quench F M) are induced in the light. Following a light-to-dark transition, i.e., on turning off the light, these processes are reversed. State Florfenicol transitions (the transfer of a part of the antenna system among PSII and PSI) and XC related processes may take a considerable amount of time to reverse (Fork and Satoh 1986; Ruban and Horton 1999) and the recovery of a plant from photoinhibition takes hours (Havaux 1989; Long et al. 1994). An answer to the question as to what a good dark-adaptation time is, depends on the information we want to obtain. If the aim is the study of the regulatory and photoinhibition-related processes, a dark-adaptation time of 15 min that allows FNR (at least in plants like pea) to become inactive again would be sufficient.