Index
Part I
1. General Indroduction
1.1 Chlamydomonas reinhardtii, unicellular green alghe. 2 1.2 The cell cycle and circadian clock in Chlamydomonas 3 1.3 Daily oxygen limitations experienced by Chlamydomonas in 5
nature.
1.4 The fermentative metabolism in Chlamydomonas 7
1.5 Chlamydomonas and hydrogen production 10
Part II
2. Alcohol dehydrogenase and hydrogenase transcript fluctuations during a day-night cycle in Chlamydomonas reinhardtii: the role of anoxia.
2.1 Summary 13
2.2 Introduction 15
2.2 Materials and methods 20
2.3.1 Strains and growth conditions 20
2.3.2 Oxygen readings. 21
2.3.3 RNA extraction and real-time reverse-transcription
polymerase chain reaction (qPCR). 21
2.4 Results 24
2.4.1 Anoxia tolerance is influenced by the time of day 24 2.4.2 The concentration of O2 dissolved in the media varies 26
rhythmically over 24 h.
2.4.3 ADH1 expression in Chlamydomonas depends on time 26 of day, not on O2 concentration.
circadian expression pattern
2.4.5 Effects of continuous light on circadian and cell-cycle 32 related genes
2.4.6 HYD1, HYD2, HYDEF, HYDG, but not ADH1 are 36 induced by anoxia
2.5. Discussion
Part III
3. Characterization of other fermentative genes in chlamydomonas reinhardtii in relation to their response to light/dark cycles and oxygen availability. First results on proteins and metabolites.
3.1 Summary 43
3.2 Introduction 44
3.2.1 The role of fermentation in the absence of oxygen. 44 3.2.2 Genes in the ethanol producing pathway 45 3.2.3 Characterizing the PFL1, PFR1, ACK2 & PAT2
expression pattern. 46
3.3 Materials and Methods 48
3.3.1 Strains and growth conditions. 48
3.3.2 Western Blot 49
3.3.3 RNA extraction and real-time reverse-transcription
polymerase chain reaction (qPCR). 50
3.3.4 Ethanol quantification in the media. 51
3.4 Results 53
3.4.1 ADH2 and PDC3 – two genes involved in ethanol
production, show expression patterns similar to ADH1. 53 3.4.2 ADH2 and PDC3 are not oxygen responsive. 54 3.4.3 Current transcript abondance modifies response profile to
dark, but not to anoxia in ADH1 and ADH2, but not PDC3 58 3.4.4 Protein level of PDC3 does not correlate with the observed
transcript fluctuations. 59
3.4.5 Metabolite level of ethanol in the media does not correlate with the observed transcript fluctuations. 60
3.4.6 Daily expression profiles of PFL1, PFR1, ACK2 and PAT2. 61 3.4.7 PFR1 is a circadian gene that also responds to oxygen
Levels 64
3.5Discussion 66
Reference 69
A c k n o w l e d g e m e n t s 8 1
