Analysis of Moss Light-Harvesting Complex Stress-Related (LHCSR1) Protein Function Upon Heterologous Expression in Arabidopsis thaliana

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is a process essential for the regulation of photosynthesis and plant protection from light stress. In vascular plants this process is triggered by a luminal pH sensor, the PSBS protein, which transduces chloroplast lumen acidification, induced by excess light, into a quenching reaction occurring within specific interacting chromophore-bound lightharvesting proteins (LHC). In algae, such as Chlamydomonas reinhardtii, stress-related light-harvesting proteins (LHCSR) fulfill both pH sensing and quenching reactions, due to their capacity of binding chlorophylls and xanthophylls. The moss Physcomitrella patens, an evolutionary intermediate between algae and plants, has both PSBS and LHCSR active in quenching with LHCSR working in a direct zeaxanthin-dependent manner. Plants and mosses have a very similar organization of thylakoid membranes thus suggesting LHCSR might be active in plants. To verify this hypothesis, we overexpressed lhcsr1 gene into Arabidopsis thaliana PSBS mutant, npq4, and screened transformants by fluorescence video-imaging, resulting to the isolation of A. thaliana plants, which accumulate a pigment-binding, NPQ-active LHCSR1 in thylakoid membranes. In the context of functional and structural analysis of LHCSR1 protein, a series of in vivo transformations was performed using A. thaliana mutants altered in xanthophyll content or lacking specific LHC subunits. For this reason the double mutant npq1npq4 - unable to convert violaxathin into zeaxanthin - was complemented in order to verify the direct dependence of LHCSR1 on zeaxanthin, mutant lut2npq4 was used due to its complete lack of lutein and antenna mutants NoMnpq4 and ch1lhcb5 were used due to their lack of either minor antennas or the complete antenna system respectively; all of them overexpressing LHCSR1 in different levels. Finally, a first approach for the in vivo mutational analysis of P. patens LHCSR1 has been initiated, using A. thaliana as a tool for heterologous protein expression

A systems-wide understanding of photosynthetic acclimation in algae and higher plants

The ability of phototrophs to colonise different environments relied on the robust protection against oxidative stress in phototrophs, a critical requirement for the successful evolutionary transition from water to land. Photosynthetic organisms have developed numerous strategies to adapt their photosynthetic apparatus to changing light conditions in order to optimise their photosynthetic yield, crucial for life to exist on Earth. Photosynthetic acclimation is an excellent example of the complexity of biological systems, in which highly diverse processes, ranging from electron excitation over protein protonation to enzymatic processes coupling ion gradients with biosynthetic activity interact on drastically different timescales, ranging from picoseconds to hours. An efficient functioning of the photosynthetic apparatus and its protection is paramount for efficient downstream processes including metabolism and growth. Modern experimental techniques can be successfully integrated with theoretical and mathematical models to promote our understanding of underlying mechanisms and principles. This Review aims to provide a retrospective analysis of multidisciplinary photosynthetic acclimation research carried out by members of the Marie Curie Initial Training Project “AccliPhot”, placing the results in a wider context. The Review also highlights the applicability of photosynthetic organisms for industry, particularly with regards to the cultivation of microalgae. It aims to demonstrate how theoretical concepts can successfully complement experimental studies broadening our knowledge of common principles in acclimation processes in photosynthetic organisms, as well as in the field of applied microalgal biotechnology

Functional analysis of LHCSR1, a protein catalyzing NPQ in mosses, by heterologous expression in Arabidopsis thaliana

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