Stress resistance of polar hydro-terrestrial algae Zygnema spp.

160 Pages·2014·5.12 MB·English

Charles University in Prague
Faculty of Science

Ph.D. Program: Botany

Mgr. Martina Pichrtová

Stress resistance of polar hydro-terrestrial algae
Zygnema spp. (Zygnematophyceae, Streptophyta)

Stresová odolnost polárních hydro-terestrických řas
Zygnema spp. (Zygnematophyceae, Streptophyta)

Ph.D. Thesis

Supervisor: Doc. RNDr. Yvonne Němcová, Ph.D.
Consultant: Doc. Ing. Josef Elster, CSc.

Prague, 2014

Declaration

I hereby declare that I have written this thesis independently, using the listed references; or in
cooperation with other paper co-authors. I have submitted neither this thesis, nor any of its
parts, to acquire any other academic degree.

Prague, 4th September 2014
..........................................
Martina Pichrtová

This thesis is based on the following three papers:

I. PICHRTOVÁ, M., REMIAS, D., LEWIS, L. A. & HOLZINGER, A. (2013): Changes in
phenolic compounds and cellular ultrastructure of Arctic and Antarctic strains of
Zygnema (Zygnematophyceae, Streptophyta) after exposure to experimentally
enhanced UV to PAR ratio. – Microbial Ecology 65 (1): 68–83.

II. PICHRTOVÁ, M., HÁJEK, T. & ELSTER, J. (2014): Osmotic stress and recovery in
field populations of Zygnema sp. (Zygnematophyceae, Streptophyta) on Svalbard
(High Arctic) subjected to natural desiccation. – FEMS Microbiology Ecology.
DOI: 10.1111/1574-6941.12288.

III. PICHRTOVÁ, M., KULICHOVÁ, J. & HOLZINGER, A. (submitted): Nitrogen limitation
and slow drying induce desiccation tolerance in conjugating green algae
(Zygnematophyceae) from polar habitats. PLoS ONE.

Authors‘ contributions:

Paper I. Daniel Remias, Andreas Holzinger and Martina Pichrtová designed the study.
Martina Pichrtová cultivated the algae, performed the experiments and Daniel Remias helped
with HPLC. Martina Pichrtová wrote the major part of the manuscript and performed
statistical analyses. Andreas Holzinger was responsible for the transmission electron
microscopy and Louise A. Lewis for molecular phylogeny and they both wrote the respective
parts of the text. All co-authors participated in final improvements of the manuscript.

Paper II. Tomáš Hájek and Martina Pichrtová jointly planned the study, performed the
experiments and the statistical analyses. Josef Elster and Tomáš Hájek organized and planned
the field work. Martina Pichrtová took both light and fluorescence microscope images,
created the figures and wrote the manuscript. Tomáš Hájek and Josef Elster contributed to
improve the text of the manuscript.

Paper III. Martina Pichrtová planned the study together with Andreas Holzinger. Martina
Pichrtová cultivated the algae, performed the desiccation experiments, light microscope
observations and fluorescence measurements. Andreas Holzinger performed transmission
electron microsopy investigations. Martina Pichrtová performed the molecular work and
statistical analyses together with Jana Kulichová. Martina Pichrtová and Andreas Holzinger
jointly wrote the manuscript and Jana Kulichová helped with the parts concerning molecular
phylogeny.

On behalf of all the co-authors, we declare the keynote participation (as first author) of
Martina Pichrtová in completing the research and writing the papers, as described above.

........................................ ........................................
Josef Elster Andreas Holzinger

Acknowledgements

Yvonne Němcová, thank you very much for supervising me during all those many years
since my bachelor study. Thanks for your advice, support, care, understanding and
encouragement that came exactly when I needed them the most.
Josef Elster, thank you very much for the idea of studying Zygnema, for introducing me
to polar research and for the unique opportunity to participate in those great and unforgettable
Svalbard expeditions.
Tom Hájek, thanks a lot for the countless hours of private lessons in plant physiology
and statistics, for being always willing to help me and to answer my questions, for your
encouragement and simply for becoming a very good friend of mine.
Andreas Holzinger, thank you for sharing your passion for algae, for your hospitality
during my stays in Innsbruck, for all your help and above all, for urging me to write. I am
especially grateful for the cooperation we started because without you it would doubtlessly
take me much longer to finish.
Jana Kulichová, many thanks for introducing me to molecular methods, for your time
and patience.
Daniel Remias, thanks for inviting me to come and work in Innsbruck despite hardly
knowing me, for your help and support and also for your humor.
My colleagues from the phycology research group in Prague, thank you for creating a
friendly and inspiring atmosphere, but also for your severe criticism and doubts.
My dear family and friends, thank you so much for your continuous support, love and
understanding.
Special thanks go to all of you who read the previous versions of the thesis or at least its
parts. Your comments and ideas were really helpful.

Funding of this work was provided by the Charles University Science Foundation, project
GAUK 794413; by the Ministry of Education, Youth and Sports of the Czech Republic,
project ME 934, project INGO LA 341 and project LM2010009, by the long-term research
development project of the Academy of Sciences of the Czech Republic RVO 67985939 and
by the institutional resources of the Czech Ministry of education. The research stays at the
University of Innsbruck were supported by OeAD scholarship (program AKTION) and by the
Czech-Austrian project AKTION 65p5.

Abstract

Filamentous green microalgae of the genus Zygnema belong to the most common primary
producers in the polar hydro-terrestrial environment. In such unstable habitats, organisms are
subject to various stress factors, e.g., freeze–thaw cycles, desiccation and high irradiation
levels. However, the stress resistance mechanisms that enable Zygnema spp. to thrive in this
extreme environment are only partially understood. Therefore, polar Zygnema spp. were
examined under various stress conditions using both field samples and cultures. Moreover,
molecular phylogeny methods were applied that provided first insights into the diversity of
polar Zygnema. Sequencing of the chloroplast gene rbcL revealed several different Zygnema
genotypes and, surprisingly, one Zygnemopsis sp. with vegetative Zygnema sp. morphology.
First set of experiments examined the effects of UV exposure. It turned out that polar strains
of Zygnema produce phenolic substances as UV screens. These substances are most likely
stored in vacuoles and other vesicles at the cell periphery, providing protection for other
organelles. In the next study, Zygnema spp. were investigated under natural conditions in the
Arctic. At the end of summer, the cells gradually lose their typical vegetative appearance
(with large vacuoles and stellate chloroplasts) and form pre-akinetes, which are stationary-
phase-like cells filled with storage material and characterized by reduced chloroplast lobes
and thickened cell walls. While all natural populations consisted of pre-akinetes regardless of
their water status, significant differences were revealed in their osmotic stress tolerance.
These results indicated that formation of pre-akinetes was not triggered by desiccation, but
hardening during slow dehydration was required for the pre-akinetes to become stress-
resistant. Subsequent laboratory experiments showed that the formation of pre-akinetes was
induced by nitrogen starvation. In general, viability and recovery rate after experimental
desiccation depended on pre-cultivation conditions and drying rate. Moreover, the pre-
akinetes survived even rapid drying (at 10% relative air humidity) when hardened by mild
dehydration stress. Presented thesis contributes to our understanding of algal stress resistance
mechanisms in polar hydro-terrestrial environment. The results indicate that naturally
hardened pre-akinetes play a key role in survival under extreme conditions, while the
production of other types of specialized cells (e.g., zygospores) is largely suppressed.
Moreover, desiccation-tolerant cells derived from disintegrated filaments can act as airborne
propagules.