Enhancement of carotenoid biosynthesis and antioxidant responses in microalgae by light ...

351 Pages·2012·9.76 MB·English

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Enhancement of Carotenoid Biosynthesis and
Antioxidant Responses in Diatoms by Light Modulation
Thesis
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Smerilli, Arianna (2018). Enhancement of Carotenoid Biosynthesis and Antioxidant Responses in Diatoms by
Light Modulation. PhD thesis The Open University.
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Enhancement of carotenoid biosynthesis and
antioxidant responses in microalgae by light
modulation

Arianna Smerilli
Master of Science in Biological Monitoring
University of Genova, Italy

Doctor of Philosophy
in Life and Biomolecular Science

The Open University – Milton Keynes, UK
Stazione Zoologica Anton Dohrn - Napoli, Italy

September 2017
Enhancement of carotenoid
biosynthesis and antioxidant responses
in microalgae by light modulation
Arianna Smerilli
Doctor of Philosophy in Life and Biomolecular Science

Director of Studies
Dr Christophe Brunet
Stazione Zoologica Anton Dohrn
Naples, Italy

External Supervisor
Prof Alexander Ruban
Queen Mary University
London, UK

September 2017

ABSTRACT
The ecological success of diatoms is mostly attributed to their ability to adjust biological
performances to the variable environmental conditions they experience in the water
column. While the efficient photoacclimative and photoprotective mechanisms in diatoms
are already reported, the involvement of the antioxidant network in lowering or repairing
light stress or damage is poorly understood.
My thesis consists of the concomitant investigation of the photoprotective and antioxidant
network functioning in the coastal diatom Skeletonema marinoi, and during an experiment
at sea in the Gulf of Naples. The aims of my work are to explore the antioxidant network in
diatoms to better understand its activation in coping with light variation, and the functional
link between antioxidant molecules synthesis/activity and photoprotection.
Results showed the spectral light dependent activity of antioxidant enzymes such as
ascorbate peroxidase, catalase, and superoxide dismutase. They act in complementarity of
the antioxidant molecules synthesis. A high concentration of ascorbic acid, phenolics
compounds and among them flavonoids were found in diatoms. These molecules respond
to light variations in terms of spectral composition, photon flux density, daily light dose,
light shape distribution and photoperiod duration. Also, in a natural microalgal community,
these molecules were found at high concentration following a dynamics that relied on light,
nutrient stress and photosynthetic regulation.
The photoprotective xanthophyll cycle, involving the pigments diatoxanthin and
diadinoxanthin, is activated by light and modulated in concordance with antioxidant
molecules synthesis. Indeed, under different light climates, a link between these two
defense processes was found. I also show that the xanthophyll cycle pigments have a high
antioxidant activity. This feature explains why they increase together with antioxidant
molecules during cell senescence. More generally, the physiological state of the cells
modulates the antioxidant and photoprotective network in diatoms.
I
II
ACKNOWLEDGMENTS
My acknowledgments go to the people that turned this PhD project into reality.
First of all, I would like to thank Stazione Zoologica Anton Dohrn which provided for the
financial and technical support to fulfill this project.
I am grateful to my director of studies Dr Christophe Brunet for introducing me to this
fascinating world of microalgae and photophysiology, and for trusting and supporting me
during these years.
My gratitude also goes to my external supervisor Prof. Alexander Ruban, who kept track of
my progress and provided precious comments and suggestions which improved the
research work.
I would like to thank also Federico Corato, the Master of Lights.
Indeed, one of the persons which greatly contributed to this work was Dr Ida Orefice,
without whom all of this would have been impossible. She gave me the basis for working
with these tiny and sometimes bossy organisms, we had fruitful discussions to improve the
work, from the smallest practical issues till the bigger theoretical ones, and above all
supported and comforted me during the harshest moments.
Then, surely I have to thank all the amazing people I had the chance to meet here in
Stazione, sometimes for their providential help in the work, but mostly for the fun, the non-
sense, the GOT evenings, the food, the affection, the hysterical crying and the big laughs.
So, thank you Laura, Laura, Greta, Eleonora, Maira, Vincenzo, Cecilia, Romain, Chetan,
and all the other friends. Yeah, even you Maria Paola and Katerina.
I must say thank you to Riccardo, for supporting and put up with me all these years, for the
invaluable advices and comfort, for the love, the travels, the Sundays on the couch.
Finally, I would like to express my infinite gratitude to my family. Firstly to my mother,
my rock, for her unconditional love, always ready to help me in all the ways possible and
even impossible.
Of course to my father, for occasionally faking some interest into my work.
And lastly to my brothers, without whom part of myself would be lacking.

III
IV
TABLE OF CONTENTS

ABSTRACT ......................................................................................................................................... I
ACKNOWLEDGMENTS .................................................................................................................III
TABLE OF CONTENTS .....................................................................................................................1
LIST OF FIGURES .............................................................................................................................9
LIST OF TABLES .............................................................................................................................21
LIST OF ABBREVIATIONS ............................................................................................................25
1. General introduction ......................................................................................................................31
1.1 The microalgal realm and the group of Bacillariophyceae ........................................ 33
1.1.1 Microalgal diversity ............................................................................................ 33
1.1.2 The Diatoms ........................................................................................................ 35
1.1.2.1 The rise of diatoms ....................................................................................... 35
1.1.2.2 Ecological success of diatoms ...................................................................... 39
1.1.2.3 Morphology, chloroplast structure and pigment content of diatoms ........... 40
1.2. Microalgae in biotechnology .................................................................................... 46
1.2.1 The state of art and potential of using microalgae in biotechnology .................. 46
1.2.2 Microalgal resources and challenges .................................................................. 48
1.3 Light-driven processes in the marine microalgae ...................................................... 51
1.3.1 Light in marine environment ............................................................................... 51
1.3.1.1 Light sensing in diatoms .............................................................................. 55
1.3.1.1.1 Photoreceptors ....................................................................................... 55
1.3.1.1.2 Indirect light signaling .......................................................................... 57
1.3.2 Photosynthesis ..................................................................................................... 58
1.3.2.1 The light-dependent reactions ...................................................................... 59
1.3.2.2 The light-independent reactions ................................................................... 63
1.3.3 Photoprotection ................................................................................................... 64
1.3.3.1 Photocclimation ........................................................................................... 64
1.3.3.2 Photoregulation ............................................................................................ 65
1.3.3.2.1 Non-photochemical quenching of chlorophyll a fluorescence (NPQ) .. 65
1
1.3.3.2.1.1 The proton gradient .........................................................................66
1.3.3.2.1.2 The xanthophyll cycle (XC) enzymes and the lipid phase ..............66
1.3.3.2.1.3 The antenna proteins .......................................................................68
1.3.3.2.1.4 The NPQ mechanism in diatoms ....................................................69
1.3.3.2.2 Other regulation mechanisms .................................................................70
1.3.3.2.2.1 Photorespiration and regulation of Rubisco ....................................70
1.3.3.2.2.2 The cyclic electron flow around PSII and PSI ................................71
1.3.3.2.2.3 The water-water cycle .....................................................................72
1.3.3.2.2.4 The chlororespiration ......................................................................72
1.4. Oxidative stress and antioxidants ...............................................................................73
1.4.1 Production of ROS in chloroplasts.......................................................................73
1.4.2 Production of ROS in other cell compartments ...................................................75
1.4.3 Scavenging of ROS ..............................................................................................76
1.4.3.1 The non-enzymatic antioxidants ...................................................................77
1.4.3.2 The antioxidant enzymes ..............................................................................78
1.4.3.3 The antioxidant network ...............................................................................79
1.5 Aims of the study ........................................................................................................81
2. Materials and methods ................................................................................................................... 87
2.1 The model species Skeletonema marinoi: biology and cultivation .............................89
2.2 Light system ................................................................................................................90
2.3 Cell density and growth rate .......................................................................................90
2.4 Determination of the photochemical efficiency of the photosystem II .......................91
2.5 Determination of the non-photochemical fluorescence quenching .............................93
2.6 Determination of the electron transport rate-light curves ...........................................93
2.7 S. marinoi absorption spectrum analysis.....................................................................95
2.8 Pigments analysis ........................................................................................................97
2.9 Cell pellet preparation .................................................................................................98
2.10 Ascorbic acid content determination .........................................................................99
2.11 Preparation of the methanolic extracts ......................................................................99
2.12 Total phenolic content .............................................................................................100
2.13 Total flavonoid content ...........................................................................................101
2.14 ABTS radical scavenging activity...........................................................................101
2