A chloroplast “wake up” mechanism: Illumination with weak light activates the photosynthetic antenna function in dark-adapted plants

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Abstract

The efficient and fluent operation of photosynthesis in plants relies on activity of pigment-protein complexes called antenna, absorbing light and transferring excitations toward the reaction centers. Here we show, based on the results of the fluorescence lifetime imaging analyses of single chloroplasts, that pigment-protein complexes, in dark-adapted plants, are not able to act effectively as photosynthetic antennas, due to pronounced, adverse excitation quenching. It appeared that the antenna function could be activated by a short (on a minute timescale) illumination with light of relatively low intensity, substantially below the photosynthesis saturation threshold. The low-light-induced activation of the antenna function was attributed to phosphorylation of the major accessory light-harvesting complex LHCII, based on the fact that such a mechanism was not observed in the stn7 Arabidopsis thaliana mutant, with impaired LHCII phosphorylation. It is proposed that the protein phosphorylation-controlled change in the LHCII clustering ability provides mechanistic background for this regulatory process.

Introduction

Life on Earth is powered by the energy of light reaching our planet from the Sun but the conversion of energy of electromagnetic radiation to the forms which can be directly used to drive biochemical reactions is realized via the process of photosynthesis. The primary photochemical reactions take place in the photosynthetic reaction centers but their fluent and efficient operation is maintained by the activity of numerous, specialized pigment-protein complexes called antennas, absorbing light quanta and transferring electronic excitations towards the reaction centers. Light-harvesting pigment-protein complex II (LHCII) is the major antenna protein in plants and the most abundant membrane protein in the biosphere (Barros and Kuhlbrandt, 2009). Owing to its relatively high concentration in chloroplasts, the complex has a significant influence on structural and dynamic properties of the thylakoid membranes (Ruban and Johnson, 2015). Exceptionally high protein concentration in the thylakoids of plants, ranging up to 80 % of the surface of the lipid-protein membranes of grana structures, is referred to as a “molecular crowding” (Kirchhoff, 2008). One can expect that such a tight packing of pigment-protein complexes facilitate long-range excitation energy transfer, which is critically dependent on a donor-acceptor distance (Andrews and Demidov, 1999, Förster, 1959). On the other hand, photosynthetic antenna complexes and in particular LHCII, present an exceptionally strong tendency to form clusters in the lipid phase, leading to excitation quenching manifested by shortening of lifetimes of their excited states (Gruszecki et al., 1997, Moya et al., 2001, Natali et al., 2016). Obviously, a process of energy dissipation decreases the efficiency of excitation transfer and reduces a performance of the photosynthetic antenna function. This process seems to be particularly unfavorable under low light conditions, at which the photosynthetic apparatus essentially functions as a quantum counter, employing every single light quantum to drive electric charge separation. In the present work, we address the problem of this apparent paradox in experiments carried out on single chloroplasts with the application of the fluorescence lifetime imaging microscopy technique.

Section snippets

Plant material

Arabidopsis thaliana (L.) plants were grown under controlled conditions in a climate chamber with photosynthetic photon flux density (PPFD) of 120 μmol photons m−2s−1, 60% humidity and a 22/18 °C day/night temperature. The photoperiod was 8 h light and 16 h dark. Ecotype Columbia was used in all experiments. Wild-type Columbia (Col) and stn7 mutant (SALK 073254) used in the experiments were obtained from the Salk Institute. The stn7 mutant was characterized in (Bellafiore et al., 2005). Spinacia

Results

Fig. 1 presents a chloroplast of Spinacia oleracea imaged with the application of fluorescence lifetime imaging microscopy (FLIM), with a laser operating at different repetition frequencies. An increase in repetition frequency results in a linear rise in photon flux density directed to a sample. As can be seen, the increase in the photon flux density results in the alterations in an average chlorophyll a (Chl a) fluorescence lifetime. A detailed analysis of average fluorescence lifetime in this

Discussion

The results of the FLIM analyses of intact chloroplasts of S. oleracea reveal the increase in average Chl a fluorescence lifetime, observed locally at low light intensities (Figs. 1, 2, S2 and S3). Such a mechanism was associated with the thylakoid membrane remodeling, concluded on the basis of the analysis of the fluorescence signal-based chloroplast cross-sections (Fig. 3). Both phenomena were observed selectively in the low light intensity range. Owing to the fact that process of

Conclusions

We show that maximum phosphorylation of LHCII, the largest photosynthetic antenna complex of plants, which is observed at relatively low light intensities, substantially improves properties of the protein as a photosynthetic antenna. This is realized via disassembly of energy-dissipating molecular clusters of the complex and via the thylakoid membrane remodeling leading to the more homogeneous distribution of light-absorbing centers. It means that the photosynthetic antenna function in plants

Acknowledgements

The authors would like to thank Dr. Stefano Santabarbara for a discussion and for numerous valuable comments and suggestions regarding the interpretation of the results. National Science Center of Poland is acknowledged for a financial support within the project 2016/22/A/NZ1/00188. The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Development of Eastern Poland Operational Programme.

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      Citation Excerpt :

      As a clear difference to WT, vte1 lacks this rapid and temporary maximum, which suggests that LHCII antenna are at light-dissipative mode already at the onset of light. This is similar to the results obtained with stn7 mutant by Janik et al. (2017). In spite of that LHCII proteins are phosphorylated in vte1, they are, however, unable to reach the conformation necessary for an efficient quenching of excess excitation energy as heat, as envisaged by a high values of YNO and somehow restricted NPQ.

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    1

    These authors contributed equally to this work.

    2

    Present address: Department of Medicine, Imperial College London, London W12 0NN, UK.

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