Frequently asked questions about chlorophyll fluorescence, the sequel.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121-158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V /F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

High temperature specifically affects the photoprotective responses of chlorophyll b-deficient wheat mutant lines.

The effects of high temperature on CO2 assimilation rate, processes associated with photosynthetic electron and proton transport, as well as photoprotective responses, were studied in chlorophyll b-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). Despite the low chlorophyll content and chlorophyll a-to-b ratio, the non-stressed mutant plants had the similar level of CO2 assimilation and photosynthetic responses as WT. However, in ANK mutant plants exposed to prolonged high temperature episode (42 °C for ~10 h), we observed lower CO2 assimilation compared to WT, especially when a high CO2 supply was provided. In all heat-exposed plants, we found approximately the same level of PSII photoinhibition, but the decrease in content of photooxidizable PSI was higher in ANK mutant plants compared to WT. The PSI damage can be well explained by the level of overreduction of PSI acceptor side observed in plants exposed to high temperature, which was, in turn, the result of the insufficient transthylakoid proton gradient associated with low non-photochemical quenching and lack of ability to downregulate the linear electron transport to keep the reduction state of PSI acceptor side low enough. Compared to WT, the ANK mutant lines had lower capacity to drive the cyclic electron transport around PSI in moderate and high light; it confirms the protective role of cyclic electron transport for the protection of PSI against photoinhibition. Our results, however, also suggest that the inactivation of PSI in heat stress conditions can be the protective mechanism against photooxidative damage of chloroplast and cell structures.

Effect of photosystem I inactivation on chlorophyll a fluorescence induction in wheat leaves: Does activity of photosystem I play any role in OJIP rise?

Interpretation of the fast chlorophyll a fluorescence induction is still a subject of continuing discussion. One of the contentious issues is the influence of photosystem I (PSI) activity on the kinetics of the thermal JIP-phase of OJIP rise. To demonstrate this influence, we realized a series of measurements in wheat leaves subjected to PSI photoinactivation by the sequence of red saturation pulses (15,000 µmol photons m(-2) s(-1) for 0.3 s, every 10 s) applied in darkness. Such a treatment led to a moderate decrease of maximum quantum efficiency of PSII (by ~8%), but a strong decrease of the number of oxidizable PSI (by ~55%), which considerably limited linear electron transport and CO2 assimilation. Surprisingly, the PSI photoinactivation had low effects on OJIP kinetics of variable fluorescence. In particular, the amplitude of variable fluorescence of IP-step (ΔVIP), which has been considered to be a measure of PSI content, was not decreased, despite the low content of photooxidizable PSI. On the other hand, the slower relaxation of chlorophyll fluorescence after saturation pulse as well as the results of the double-hit method suggest that PSI inactivation treatment led to an increase of the fraction of QB-nonreducing PSII reaction centers. Our results somewhat challenge the mainstream interpretations of JIP-thermal phase, and at least suggest that the IP amplitude cannot serve to estimate reliably the PSI content or the PSI to PSII ratio. Moreover, these results recommend the use of the novel method of PSI inactivation, which might help clarify some important issues needed for the correct understanding of the OJIP fluorescence rise.

Repetitive light pulse-induced photoinhibition of photosystem I severely affects CO2 assimilation and photoprotection in wheat leaves.

It was previously found that photosystem I (PSI) photoinhibition represents mostly irreversible damage with a slow recovery; however, its physiological significance has not been sufficiently characterized. The aim of the study was to assess the effect of PSI photoinhibition on photosynthesis in vivo. The inactivation of PSI was done by a series of short light saturation pulses applied by fluorimeter in darkness (every 10 s for 15 min), which led to decrease of both PSI (~60 %) and photosystem II (PSII) (~15 %) photochemical activity. No PSI recovery was observed within 2 days, whereas the PSII was fully recovered. Strongly limited PSI electron transport led to an imbalance between PSII and PSI photochemistry, with a high excitation pressure on PSII acceptor side and low oxidation of the PSI donor side. Low and delayed light-induced NPQ and P700(+) rise in inactivated samples indicated a decrease in formation of transthylakoid proton gradient (ΔpH), which was confirmed also by analysis of electrochromic bandshift (ECSt) records. In parallel with photochemical parameters, the CO2 assimilation was also strongly inhibited, more in low light (~70 %) than in high light (~45 %); the decrease was not caused by stomatal closure. PSI electron transport limited the CO2 assimilation at low to moderate light intensities, but it seems not to be directly responsible for a low CO2 assimilation at high light. In this regard, the possible effects of PSI photoinhibition on the redox signaling in chloroplast and its role in downregulation of Calvin cycle activity are discussed.

Low PSI content limits the photoprotection of PSI and PSII in early growth stages of chlorophyll b-deficient wheat mutant lines.

In vivo analyses of electron and proton transport-related processes as well as photoprotective responses were carried out at different stages of growth in chlorophyll b (Chl b)-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). In addition to a high Chl a-b ratio, ANK mutants had a lower content of photo-oxidizable photosystem I (PSI, P m), and several parameters indicated a low PSI/PSII ratio. Moreover, simultaneous measurements of Chl fluorescence and P700 indicated a shift of balance between redox poise of the PSII acceptor side and the PSII donor side, with preferential reduction of the plastoquinone pool, resulting in an over reduced PSI acceptor side (high Φ NA values). This was the probable reason for PSI inactivation observed in the ANK mutants, but not in WT. In later growth phases, we observed partial relief of "chlorina symptoms," toward WT. Measurements of ΔA 520 decay confirmed that, in early growth stages, the ANK mutants with low PSI content had a limited capacity to build up the transthylakoid proton gradient (ΔpH) needed to trigger non-photochemical quenching (NPQ) and to regulate the electron transport by cytochrome b 6/f. Later, the increase in the PSI/PSII ratio enabled ANK mutants to reach full NPQ, but neither over reduction of the PSI acceptor side nor PSI photoinactivation due to imbalance between the activity of PSII and PSI was mitigated. Thus, our results support the crucial role of proper regulation of linear electron transport in the protection of PSI against photoinhibition. Moreover, the ANK mutants of wheat showing the dynamic developmental changes in the PSI/PSII ratio are presented here as very useful models for further studies.