Time-resolved fluorescence measurements on leaves: principles and recent developments

Abstract

Photosynthesis starts when a pigment in the photosynthetic antennae absorbs a photon. The electronic excitation energy is then transferred through the network of light-harvesting pigments to special chlorophyll (Chl) molecules in the reaction centres, where electron transfer is initiated. Energy transfer and primary electron transfer processes take place on timescales ranging from femtoseconds to nanoseconds, and can be monitored in real time via time-resolved fluorescence spectroscopy. This method is widely used for measurements on unicellular photosynthetic organisms, isolated photosynthetic membranes, and individual complexes. Measurements on intact leaves remain a challenge due to their high structural heterogeneity, high scattering, and high optical density, which can lead to optical artefacts. However, detailed information on the dynamics of these early steps, and the underlying structure–function relationships, is highly informative and urgently required in order to get deeper insights into the physiological regulation mechanisms of primary photosynthesis. Here, we describe a current methodology of time-resolved fluorescence measurements on intact leaves in the picosecond to nanosecond time range. Principles of fluorescence measurements on intact leaves, possible sources of alterations of fluorescence kinetics and the ways to overcome them are addressed. We also describe how our understanding of the organisation and function of photosynthetic proteins and energy flow dynamics in intact leaves can be enriched through the application of time-resolved fluorescence spectroscopy on leaves. For that, an example of a measurement on Zea mays leaves is presented. Electronic supplementary material The online version of this article (10.1007/s11120-018-0607-8) contains supplementary material, which is available to authorized users.

Understanding the photophysics and structural organization of photosynthetic proteins using model lipid membranes assembled from natural plant thylakoids

“…This lifetime is in agreement with our ensemble spectroscopy data of the extracted thylakoids in solution (<τ> ~ 0.5 ns) and is in good agreement with previous reports of LHCII and PSII within intact chloroplasts and leaves. [58][59][60] This <τ> is much shorter than the lifetime known for isolated LH and PS proteins in detergent (~ 4ns), as expected, due to the quenching effect of protein-protein interactions present in thylakoids. Overall, this shows that our FLIM data on LH membrane samples agree nicely with standard spectroscopy and that the deposition of the membranes on glass surfaces has no apparent impact on the sample.…”

Observation of dissipative chlorophyll-to-carotenoid energy transfer in light-harvesting complex II in membrane nanodiscs

“…The in vitro environments, which employ detergent or crystallization, may introduce additional, non-native conformational changes that could alter or even denature the functional structure of membrane proteins [30][31][32] . In contrast, in vivo spectroscopy on whole leaves provides physiological information [33][34][35] . However, identifying the photophysical pathways in each of the homologous antenna complexes is not possible.…”

Chloroplast ultrastructure in plants

“…Consequently, spectroscopic techniques have been developed that allow measurements with intact leaves or leaf tissues (e.g. Sacksteder et al ., ; Klughammer et al ., ; Chukhutsina et al ., ). Nowadays, the functionalities of all energy‐converting protein complexes as well xanthophyll cycle activities and membrane energization can be measured with commercially available instruments in intact leaves.…”