Physcomitrella patens at the crossroad between algal and plant photosynthesis: a tool for studying the regulation of light harvesting

Through photosynthesis plants use solar energy for producing reduced compounds from CO2 and finally biomass. Photosystems (PSI and PSII) are multisubunit pigment-binding complexes responsible for light harvesting, charge separation and play an essential role in electron transport from water to NADPH. Coupled to photosynthetic electron transport is the formation of a transmembrane pH gradient that sustains ATPase activity to produce ATP. PSI and PSII represent extraordinary machines for solar energy exploitation and yet they have a weak point in being univalent reductants which leads to production of reactive oxygen species (ROS) in the present day oxygen-rich environment that photosynthetic organisms have been creating. Moreover, chlorophyll is an excellent sensitizer and its triplet excited state reacts with molecular oxygen to yield singlet oxygen. This is why excess light is harmful and algae have evolved photoprotective mechanisms, which plants have extended and improved for survival in the even more challenging land environment. Of particular interest is Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence which rapidly (within seconds) reacts to enhancement of the chlorophyll excited states. Quenching leads to the thermal dissipation of the energy absorbed in excess, is triggered by the ΔpH gradient generated across thylakoid membrane and requires specific members of the Light Harvesting Complexes (LHCs) protein family. LHCs form a large superfamily of chlorophyll-xanthophyll-binding proteins associated to PSII and PSI playing a direct role in light harvesting and/or energy quenching. Two LHC-like proteins, PSBS and LHCSR, are indispensable for NPQ respectively in vascular plants and green algae together with the xanthophylls lutein and/or zeaxanthin which are ligands for LHC proteins. Of particular interest is zeaxanthin because it is synthesized in excess light only from pre-existing violaxanthin in the so called xanthophyll cycle. Zeaxanthin plays a central role in photoprotection by scavenging of ROS quenching triplet states of chlorophyll (3Chl*) and, most interesting for my work, enhancing NPQ. During my PhD, I used the moss Physcomitrella patens as model organism to study the mechanism of NPQ with particular reference to the role of zeaxanthin. P. patens has a strategic position in the tree of life: it is an evolutionary intermediate between green algae and higher plants and was among the first organisms emerging from water to colonize the stressful land environment through the evolution of new photoprotective mechanisms. PSBS first appeared in P. patens and yet LHCSR proteins are still active yielding the possibility of studying both algal and plant NPQ in the same genetic and biochemical background. This opportunity can be exploited due to a further unique property of P. patens among eukaryotic photosynthetic organisms, i.e. its ability to perform Homologous Recombination (HR) at high efficiency, making gene targeting a standard procedure. Understanding the modulation of NPQ during acclimation to abiotic stress is essential for the full comprehension of its role. I started my work after the observation that P. patens responds to moderate salt and osmotic stress by increasing its NPQ activity. Surprisingly, NPQ enhancement was not due to over-accumulation of PSBS and/or LHCSR proteins as in the case of high light and cold acclimation. I could correlate NPQ enhancement under salt and osmotic stress with the over accumulation of zeaxanthin. When trying to verify the role of zeaxanthin we identified the unique VDE gene in P. patens genome and we knocked it out. vde KO plants were unable to produce zeaxanthin and showed a dramatic reduction in NPQ as well as an enhanced photoinhibition under excess light conditions. The introduction of the VDE mutation into LHCSR-only and PSBS-only genotypes showed that LHCSR-dependent NPQ is far more dependent on zeaxanthin than the PSBS-dependent NPQ with an activation ratio close to 10. In this work for the first time, I isolated LHCSR in the form of native chlorophyll a/b–xanthophyll-binding protein and found that the NPQ enhancement actually occurs through the direct binding of zeaxanthin to the LHCSR protein, different from the case of PSBS. Absorption spectrum and pigment binding properties of native LHCSR closely fit previously data reported for recombinant Chlamydomonas reinhardtii LHCSR3 whose activity, however, is zeaxanthin independent. Previous studies have identified two essential functions associated to essential proteins triggering NPQ: i) the pH detection function (also found in PSBS) and ii) the quenching function (also found in other LHCB proteins) such as LHCB4. In plants these two functions are carried out by distinct proteic subunits, thus making difficult in vitro studies. The recent finding of LHCSR protein has made the perspective of elucidating the molecular basis of NPQ possible: in fact, this protein is the only protein so far known to comprise the whole set of functions needed for NPQ into the same structural unit. Along the last part of my PhD work, I decided to move new steps towards the understanding of the mechanism of action of LHCSR by focusing on one side on the sub-organelle localization of this protein together with the study of the localization of PSBS in thylakoid membranes. P. patens thylakoid membranes are organized into well-defined grana stacks and stroma membranes which are differentially exposed to the stromal soluble compartment as in vascular plants. I exploited the possibility to fractionate grana and stroma-lamellae membranes to verify their localization using detergents and by mechanical fractionation. Surprisingly, I found that PSBS is localized in grana membranes while LHCSR is localized in stroma exposed membranes suggesting a different action mechanism on NPQ. Here on these basis I am proposing a tentative model for the activation of LHCSR-dependent quenching, specifically located at the periphery of grana stacks. LHCSR is rich in acidic residues in its lumen-exposed surface, acidification under excess light conditions would neutralize these charges and allow diffusion towards the grana partition domains thanks to a reduced repulsion with PSII-LHCII supercomplexes. The results reported in Chapter 2 (isolation of zeaxanthin-binding LHCSR) and Chapter 3 (localization of LHCSR in the margins/stroma fraction of thylakoid membranes) encouraged me to initiate the ambitious task of optimizing and scaling up these preparations. Although I was conscious about the difficulty of this work, I decided to try the purification of LHCSR +/- zeaxanthin from WT P. patens because the differential study of LHCSR in its quenched vs unquenched conformation is an ambitious but essential target for photosynthesis research. As for any long term project, I have conceived several strategies for the isolation of LHCSR from either WT P. patens or overexpressed using WT sequence or tagged versions of the protein using a poly-Histidine tail (His-tag) to facilitate its purification. Alternatively I also have attempted overexpressing LHCSR in tobacco. The potential advantages and pitfalls of this project are described and discussed in PhD thesis together with preliminary results.