Light Stress and Photoprotection in Green algae, Mosses and Diatoms

164 Pages·2017·21.79 MB·English

Thesis submitted to
Light Stress and Photoprotection in Green algae, Mosses
and Diatoms
Giulio R. Stella
Ph.D. thesis submitted to
University of Verona
and
University Pierre et Marie Curie
September 13th, 2016
Abstract
The molecular bases of responses to light excess in photosynthetic organisms having different
evolutionary histories and belonging to different lineages are still not completely characterized.
Therefore I explored the functions of photoprotective antennae in green algae, mosses and di-
atoms, together with the role of the two xanthophyll cycles present in diatoms.
IstudiedtheLightHarvestingComplexStress-Related(LHCSR/LHCX)proteinsindifferent
organisms. In the green alga Chlamydomonas reinhardtii, LHCSR3 is a protein important for
photoprotection. Iusedsite-specificmutagenesisinvivo andinvitro andidentifiedthreeresidues
of LHCSR3, exposed to the thylakoidal lumen, that can sense pH changes. These residues are
essential for Non-Photochemical Quenching (NPQ) induction in vivo and are responsible for
LHCSR3 activation in excessive light conditions (chapter 2).
InthemossPhyscomitrella patens Istudiedthe in vitro spectroscopicandquenchingcharac-
teristicsofdifferentpigment-bindingmutantsoftheproteinLHCSR1,tounderstandhowenergy
is transfer and thermally dissipate in this protein. I found that at least eight chlorophylls are
bound to LHCSR1 and I identified the pigments with the lowest energy levels that could be the
traps for energy transfer (chlorophylls A2 and A5, chapter 3).
LHCSRs in diatoms are named LHCXs, and in the model organism Phaeodactylum tricornu-
tum I found that multiple abiotic stress signals converge to differently regulate the four isoforms
of LHCXs, modulating proteins contents and providing a way to fine-tune light harvesting and
photoprotection (chapter 4).
Theothermaindriverofphotoprotectionindiatomsisthexanthophyllcycle. Twocyclesare
presentin diatoms, the violaxanthin-zeaxanthin(also presentin plants)and the diadinoxanthin-
diatoxanthin one (specific to diatoms). In addition to that, P. tricornutum have expanded the
protein families involved in this photoprotective process. I found that the the knock-down of
VDRandVDL2(putativeviolaxanthinde-epoxidaseenzymes)leadstotheaccumulationofviola-
and zeaxanthin, and that this accumulation have a negative effect in the development of NPQ.
ThissuggeststhatzeaxanthindoesnotparticipateintheenhancingofNPQindiatomsandmay
even interferes with the action of diatoxanthin (chapter 5).
Thanks to these studies done on different organisms, we gained a deeper knowledge on the
shared characteristics and on the peculiar features about photoprotection in green algae, mosses
and diatoms.

Université Pierre et Marie Curie
Ecole doctorale 515 Complexité du vivant
Laboratoire de Biologie Computationnelle et Quantitative
et
Università degli Studi di Verona

Light Stress and Photoprotection in Green Algae, Mosses
and Diatoms
Par Giulio Rocco Stella
Thèse de doctorat de Biologie
Dirigée par Dr. Angela Falciatore, Prof. Roberto Bassi et Dr. Fayza Daboussi
Présentée et soutenue publiquement le 13/09/2016

Devant un jury composé de :

Tomas MOROSINOTTO Professeur Univ. Padova Rapporteur
Maurizio RIBERA d'ALCALÀ Directeur de Recherche CNR Rapporteur
Benjamin BAILLEUL Chercheur CR2 CNRS Examinateur
Pierre CAROL Professeur UPMC Examinateur
Angela FALCIATORE Directrice de Recherche CNRS Directrice de thèse
Roberto BASSI Professeur Univ. Verona Directeur de thèse
Fayza DABOUSSI Directrice de Recherche INRA Directrice de thèse

Except where otherwise noted, this work is licensed under
http://creativecommons.org/licenses/by-nc-nd/3.0/

Università degli Studi di Verona
Department of
Biotechnology
Graduate School Of
Life and Health Sciences
Doctoral Program in
Molecular, Industrial and Environmental Biotechnology
With the Financial Contribution of
the European Commission

Cycle / year (1° year of attendance) 28th / 2013

Light Stress and Photoprotection in Green Algae, Mosses
and Diatoms

In co-tutelle de thése with the
Universitè Pierre et Marie Curie (Paris VI)

S.S.D. BIO/04*

Coordinator: Prof. Roberto Bassi

Signature __________________________

Coordinator: Dr. Angela Falciatore

Signature __________________________

Coordinator: Dr. Fayza Daboussi

Signature __________________________

Doctoral Student: Giulio Rocco Stella

Signature _______________________________

* For the list of S.S.D. please refer to the Ministerial Decree of 4th October 2000, Attachment A “Elenco
dei Settori Scientifico – Disciplinari” available at: http://www.miur.it/atti/2000/alladm001004_01.html
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Light Stress and Photoprotection in Green Algae, Mosses and Diatoms
Giulio Rocco Stella
Tesi di Dottorato

Università degli Studi di Verona
Area Ricerca – U.O. Dottorati di Ricerca Nazionali ed Internazionali
tel. [phone] - fax [phone] - Via Giardino Giusti n. 2 - 37129 Verona
Contents
Nomenclature II
Ph.D. context V
Résumé de la thèse (Français) VII
Riassunto della tesi (Italiano) X
1 Introduction 1
1.1 Oxygenic photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Photosynthetic pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Chlorophylls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.2 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.3 Excited state relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3 Photosystems and Antenna proteins . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4 Photoprotective mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.4.1 Short-term and long-term responses to excessive light . . . . . . . . . . . 12
1.4.2 PSBS, LHCSR and LHCX proteins . . . . . . . . . . . . . . . . . . . . . . 13
1.4.3 The Xanthophyll Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.5 My three working organisms: green algae, mosses and diatoms . . . . . . . . . . 16
1.5.1 The green lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.5.2 Chlamydomonas reinhardtii . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.5.3 Physcomitrella patens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.5.4 Diatoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.5.5 Phaeodactylum tricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . 24
1.5.6 Genetic manipulation of P. tricornutum . . . . . . . . . . . . . . . . . . . 25
Aims of the thesis 29
2 Identification of pH-sensing sites in the Light Harvesting Complex Stress-
Related 3 protein essential for triggering non-photochemical quenching in
Chlamydomonas reinhardtii 31
Summary of the work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Published paper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3 Invitro characterizationofchlorophyll-bindingsitesofLHCSR1fromPhyscomitrella
patens 47
Summary of the work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
I
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.3 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4 Multisignal control of expression of the LHCX protein family in the marine
diatom Phaeodactylum tricornutum 65
Summary of the work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Published paper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Supplementary information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5 The Xanthophyll Cycles in Phaeodactylum tricornutum 87
Summary of the work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Characterization of VDR and VDL2 proteins in Phaeodactylum tricornutum
provides new insights on the function of the two xanthophyll cycles in marine
diatoms 89
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.3 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.5 Supplementary information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
6 Conclusion and perspectives 109
Bibliography 115
Acknowledgments 143
Thesis summary (multilanguage) 144
Page II