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.