Chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant physiologists and ecophysiologists. This review aims to provide an introduction for the novice into the methodology and applications of chlorophyll fluorescence. After a brief introduction into the theoretical background of the technique, the methodology and some of the technical pitfalls that can be encountered are explained. A selection of examples is then used to illustrate the types of information that fluorescence can provide.

Chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant physiologists and ecophysiologists. This review aims to provide an introduction for the novice into the methodology and applications of chlorophyll fluorescence. After a brief introduction into the theoretical background of the technique, the methodology and some of the technical pitfalls that can be encountered are explained. A selection of examples is then used to illustrate the types of information that fluorescence can provide.

A number of useful photosynthetic parameters are commonly derived from saturation pulse-induced fluorescence analysis. We show, that qP, an estimate of the fraction of open centers, is based on a pure 'puddle' antenna model, where each Photosystem (PS) II center possesses its own independent antenna system. This parameter is incompatible with more realistic models of the photosynthetic unit, where reaction centers are connected by shared antenna, that is, the so-called 'lake' or 'connected units' models. We thus introduce a new parameter, qL, based on a Stern-Volmer approach using a lake model, which estimates the fraction of open PS II centers. We suggest that qL should be a useful parameter for terrestrial plants consistent with a high connectivity of PS II units, whereas some marine species with distinct antenna architecture, may require the use of more complex parameters based on intermediate models of the photosynthetic unit. Another useful parameter calculated from fluorescence analysis is ΦII, the yield of PS II. In contrast to qL, we show that the ΦII parameter can be derived from either a pure 'lake' or pure 'puddle' model, and is thus likely to be a robust parameter. The energy absorbed by PS II is divided between the fraction used in photochemistry, ΦII, and that lost non-photochemically. We introduce two additional parameters that can be used to estimate the flux of excitation energy into competing non-photochemical pathways, the yield induced by downregulatory processes, ΦNPQ, and the yield for other energy losses, ΦNO.

The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in Arabidopsis (Arabidopsis thaliana) and its salt-tolerant close relative Thellungiella (Thellungiella halophila). Plants cultivated on soil were challenged for 2 weeks with NaCl. Arabidopsis showed a much higher sensitivity to salt than Thellungiella; while Arabidopsis plants were unable to survive exposure to greater than 150 mM salt, Thellugiella could tolerate concentrations as high as 500 mM with only minimal effects on gas exchange. Exposure of Arabidopsis to sublethal salt concentrations resulted in stomatal closure and inhibition of CO 2 fixation. This lead to an inhibition of electron transport though photosystem II (PSII), an increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving PSII. This additional electron transport activity is oxygen dependent and sensitive to the alternative oxidase inhibitor n-propyl gallate. PSII electron transport in Thellungiella showed a reduced sensitivity to 2#-iodo-6-isopropyl-3-methyl-2#,4,4#-trinitrodiphenylether, an inhibitor of the cytochrome b 6 f complex. At the same time, we observed a substantial up-regulation of a protein reacting with antibodies raised against the plastid terminal oxidase. No such up-regulation was seen in Arabidopsis. We conclude that in salt-stressed Thellungiella, plastid terminal oxidase acts as an alternative electron sink, accounting for up to 30% of total PSII electron flow.

Cyclic electron flow is increasingly recognized as being essential in plant growth, generating a pH gradient across thylakoid membrane (ΔpH) that contributes to ATP synthesis and triggers the protective process of nonphotochemical quenching (NPQ) under stress conditions. Here, we report experiments demonstrating the importance of that ΔpH in protecting plants from stress and relating to the regulation of cyclic relative to linear flow. In leaves infiltrated with low concentrations of nigericin, which dissipates the ΔpH without significantly affecting the potential gradient, thereby maintaining ATP synthesis, the extent of NPQ was markedly lower, reflecting the lower ΔpH. At the same time, the photosystem (PS) I primary donor P700 was largely reduced in the light, in contrast to control conditions where increasing light progressively oxidized P700, due to down-regulation of the cytochrome bf complex. Illumination of nigericin-infiltrated leaves resulted in photoinhibition of PSII but also, more markedly, of PSI. Plants lacking ferredoxin (Fd) NADP oxidoreductase (FNR) or the polypeptide proton gradient regulation 5 (PGR5) also show reduction of P700 in the light and increased sensitivity to PSI photoinhibition, demonstrating that the regulation of the cytochrome bf complex (cyt bf) is essential for protection of PSI from light stress. The formation of a ΔpH is concluded to be essential to that regulation, with cyclic electron flow playing a vital, previously poorly appreciated role in this protective process. Examination of cyclic electron flow in plants with a reduced content of FNR shows that these antisense plants are less able to maintain a steady rate of this pathway. This reduction is suggested to reflect a change in the distribution of FNR from cyclic to linear flow, likely reflecting the formation or disassembly of FNR–cytochrome bf complex.