Abstract
Non-photochemical quenching, NPQ, of chlorophyll fluorescence regulates the heat dissipation of chlorophyll excited states and determines the efficiency of the oxygenic photosynthetic systems. NPQ is regulated by a pH-sensing protein, responding to the chloroplast lumen acidification induced by excess light, coupled to an actuator, a chlorophyll/xanthophyll subunit where quenching reactions are catalyzed. In plants, the sensor is PSBS, while the two pigment-binding proteins Lhcb4 (also known as CP29) and LHCII are the actuators. In algae and mosses, stress-related light-harvesting proteins (LHCSR) comprise both functions of sensor and actuator within a single subunit. Here, we report on expressing the lhcsr1 gene from the moss Physcomitrella patens into several Arabidopsis thaliana npq4 mutants lacking the pH sensing PSBS protein essential for NPQ activity. The heterologous protein LHCSR1 accumulates in thylakoids of A. thaliana and NPQ activity can be partially restored. Complementation of double mutants lacking, besides PSBS, specific xanthophylls, allowed analyzing chromophore requirement for LHCSR-dependent quenching activity. We show that the partial recovery of NPQ is mostly due to the lower levels of Zeaxanthin in A. thaliana in comparison to P. patens. Complemented npq2npq4 mutants, lacking besides PSBS, Zeaxanthin Epoxidase, showed an NPQ recovery of up to 70% in comparison to A. thaliana wild type. Furthermore, we show that Lutein is not essential for the folding nor for the quenching activity of LHCSR1. In short, we have developed a system to study the function of LHCSR proteins using heterologous expression in a variety of A. thaliana mutants.
Highlights
The need for a balance between light harvesting and photoprotection is one of the key driving forces that shaped adaptation of photosynthetic eukaryotic organisms on Earth (Genty et al 1990; Müller et al 2001; Baker 2008)
Non-photochemical quenching (NPQ) detected in complemented plants can be attributed to LHCSR1 based on the following observations: (i) quenching was only observed in LHCSR-complemented genotypes (Fig. 3); (ii) quenching was proportional to the level of LHCSR1 accumulation (Fig. 4); (iii) no LHCSR1dependent quenching activity was observed in the npq1npq4 background, lacking Zea (Fig. 7a, b), in agreement with the observation that the vde ko mutant in P. patens lost 95% of quenching activity (Pinnola et al 2013); (iv) higher and fast-developing quenching was observed upon expression in the npq2npq4 background lacking Vio and constitutively accumulating Zea (Fig. 7c, d)
Future research will need to devise new methods for assessing the xanthophyll composition of LHCSR1 in real time as well as other LHC proteins essential for quenching reactions in plants, mosses and algae since the exchange might be fast and reversible in minutes. It remains to be explained why the npq2npq4 + LHCSR1 or npq4 + LHCSR1 show an NPQ kinetic rapidly climbing to a peak and relaxing or remaining constant during the remaining light period. We suggest this depends on the ΔpH +Δψ gradient through the thylakoid membrane that appears to be different in A. thaliana versus P. patens. 9AA quenching showed a lower ΔpH contribution in P. patens and yet LHCSR1 might respond to Δψ as well
Summary
The need for a balance between light harvesting and photoprotection is one of the key driving forces that shaped adaptation of photosynthetic eukaryotic organisms on Earth (Genty et al 1990; Müller et al 2001; Baker 2008). NPQ includes components with different induction and relaxation kinetics: the fastest (1–2 min) and rapidly reversible type, qE, depends on a trans-thylakoid ΔpH promoted by excess light (Horton et al 1996; Kramer et al 1999; Kanazawa and Kramer 2002) which protonates specific residues on pH sensitive trigger proteins (Li et al 2004; Ballottari et al 2016); qZ, is activated in 8–10 min and depends on low luminal pH through the activation of violaxanthin (Vio) de-epoxidase (VDE), a lumenal enzyme-converting zeaxanthin (Zea) from pre-existing Vio. The slowest component, called qI, for photoInhibitory quenching, comprises components from the slow and reversible inactivation of Photosystem II (PSII) reaction centers as well as other long-term processes involved in acclimation to the light environment (Brooks et al 2013). In some organisms, such as Chlamydomonas reinhardtii an additional component, qT, is due to the displacement of LHCII from PSII to PSI upon phosphorylation (Allorent et al 2013). qE
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