Light is the most important source of energy for algae, mosses, and plants to perform photosynthesis. Nevertheless the excess of light is dangerous since can damage the photosynthetic apparatus. Non-Photochemical Quenching (NPQ) is one of the photo-protection mechanisms evolved by photosynthetic organisms to protect themselves from excess energy through heat. NPQ is a complex mechanism and involves different proteins. This thesis has been developed studying two proteins involved in the activation and relaxation of NPQ in the moss Physcomitrella patens: Light-Harvesting Complex Stress-Related (LHCSR) and Zeaxanthin Epoxidase (Zep). LHCSR is able to sense the excess of light through the presence of protonatable residues upon lumen acidification. Since LHCSR binds pigments (chlorophylls and carotenoids) is also able to dissipate the excess of energy into heat. Zep is involved in the xanthophyll cycle and catalyzes the conversion of zeaxanthin into violaxanthin. This protein is activated at neutral pH, in low light conditions and is involved in the relaxation of NPQ. To understand the NPQ molecular mechanisms, this thesis focused on two distinct projects: i) LHCSR site-directed mutagenesis of protonatable residues and chlorophyll-binding sites and ii) ZEP production in E. coli and purification by affinity chromatography and creation of zep knock out in the moss P. patens.
La luce è la fonte indispensabile di energia di alghe, muschi e piante superiori per svolgere la fotosintesi. Tuttavia l’assorbimento di una quantità di energia luminosa eccessiva può portare al danneggiamento dell’apparato fotosintetico. Il Non-Photochemical Quenching (NPQ) è uno dei meccanismi di foto-protezione evoluti dagli organismi fotosintetici per difendersi dall’eccesso di energia luminosa e consiste nella dissipazione di questa energia in eccesso sotto forma di calore. Si tratta di un meccanismo complesso costituito da diverse componenti e che vede coinvolti diversi partners proteici. Questo lavoro di tesi si è concentrato sullo studio di due proteine che partecipano all’attivazione e al rilassamento dell’NPQ del muschio Physcomitrella patens: LHCSR (Light-Harvesting Complex Stress-Related) e zeaxantina epossidasi (Zep). LHCSR attraverso la presenza di siti protonabili percepisce lo stress da alta luce in seguito all’acidificazione del lume tilacoidale e dissipa l’energia in eccesso grazie al legame con clorofille e carotenoidi. La Zep fa parte del ciclo delle xantofille e catalizza la conversione del carotenoide zeaxantina in violaxantina. Tale proteina è attiva a pH neutro e si attiva in condizioni di bassa luce. Al fine di comprendere il meccanismo molecolare dell’NPQ, questo elaborato di tesi ha sviluppato due progetti diversi: i) mutagenesi sito-specifica dei siti protonabili e dei residui di interazioni con le clorofille di LHCSR e ii) purificazione mediante cromatografia di affinità e caratterizzazione mediante knock out genico della proteina Zep.
Studio dei meccanismi molecolari dell’NPQ del muschio Physcomitrella patens
COLOMBO, CHIARA
2022/2023
Abstract
Light is the most important source of energy for algae, mosses, and plants to perform photosynthesis. Nevertheless the excess of light is dangerous since can damage the photosynthetic apparatus. Non-Photochemical Quenching (NPQ) is one of the photo-protection mechanisms evolved by photosynthetic organisms to protect themselves from excess energy through heat. NPQ is a complex mechanism and involves different proteins. This thesis has been developed studying two proteins involved in the activation and relaxation of NPQ in the moss Physcomitrella patens: Light-Harvesting Complex Stress-Related (LHCSR) and Zeaxanthin Epoxidase (Zep). LHCSR is able to sense the excess of light through the presence of protonatable residues upon lumen acidification. Since LHCSR binds pigments (chlorophylls and carotenoids) is also able to dissipate the excess of energy into heat. Zep is involved in the xanthophyll cycle and catalyzes the conversion of zeaxanthin into violaxanthin. This protein is activated at neutral pH, in low light conditions and is involved in the relaxation of NPQ. To understand the NPQ molecular mechanisms, this thesis focused on two distinct projects: i) LHCSR site-directed mutagenesis of protonatable residues and chlorophyll-binding sites and ii) ZEP production in E. coli and purification by affinity chromatography and creation of zep knock out in the moss P. patens.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/16089