Isolation of RNA, reverse transcription and quantitative PCR (qPCR) 101

de-scribed in the Material and Methods part of chapter 6. Target genes and the endogenous controls were analyzed using the following specific primer sets: 5’-AAGGTTCGTCGTTCCTCT-TCG-3’ and 5’-CGGAAGCACCGATAGCAATAGT-3’ for Lhcx1, 5’-CGCCATTACTCACCTCAACCAG-3’ and 5’-CAACCCAACCAATTTGAGCG-3’ forLhcx2, 5’-TCTTGAACGAGGACTACACCCC-3’ and 5’-TCCGTTCTG-GAGTTCCTTGGT-3’ for Lhcx3, 5’-ATGGCCGCGTTGCTATGTT-3’ and 5’-TATAGCAGGCCCCGAAACTTG-3’ for Lhcx4, 5’-GCCGATATCCCCAATGGATTT-3’ and 5’-CTTGGTCGAAGGAGTCCCATC-3’ for FcpB and 5’-TGCCCTTTGTACACACCGC-3’ and 5’-AAGTTCTCGCAACCAACACCA-3’ for 18S rDNA. Rela-tive transcript levels were calculated as described in [192] using the first sample of each light condition as calibrator and18S rDNA as endogenous control.

7.4. Results and Discussion

Cells of P. tricornutum were exposed to different light conditions to investigate the rela-tionship between irradiance and expression of theLhcx genes. Cultures that were grown to mid-logarithmic phase, were either transferred to complete darkness, moderate high light (750µmol·photons·m⁻²·s⁻¹) or remained under low light (45 µmol·photons·m⁻²·s⁻¹).

When illuminated, cells were subjected to a normal light/dark cycle. Samples were taken regularly for 33 h, RNA was extracted and transcript level determined by qPCR. In a second experminent, cells acclimated to low light for 6 h were transferred to high light (1500–2000 µmol·photons·m⁻² ·s⁻¹) and samples for transcript level analysis were har-vested after 15 min, 30min, 45 min, 60min and 120min. After the high light exposure, cells were transferred back to low light to recover and a final sample was taken 4 h after recovery. For comparison, the transcript level ofFcpB was measured in parallel. The gene FcpB encodes for an antenna protein and is known to be light induced and regulated in a circadian rhythm [143, 180]. Figure 7.1 shows the results of the relative transcript analy-sis. As already described in chapter 6, FcpB was up-regulated under low light conditions, showing the highest amounts of transcripts at 3 pm (after 7 h of light) in the afternoon (Fig. 7.1A). It showed a diurnal pattern and transcript levels decreased significantly under moderate high and high light (Fig. 7.1B).

Different expression patterns were observed for the four Lhcx genes. Lhcx2 and Lhcx3 showed a clear light induced expression and a strong up-regulation under moderate high light and high light conditions. At moderate high light, with the beginning of the light phase transcript levels that were about 45 and 35 times higher than in the calibrator sample (first sample, 0 am of the respective light condition) were measured for Lhcx2 and Lhcx3, respectively (see also table A.10, page 140). The amount of transcripts decreased slightly but remained high. ForLhcx3 the transcript level reached the initial level in the dark phase and increased again after the light was switched on. A similar effect was detected forLhcx2, although the decrease in the dark phase was not as strong as forLhcx3, so that the amount of transcripts was still higher than in the respective calibrator sample. When the cells were exposed to high light, a strong increase in transcripts could be measured for both Lhcx2 andLhcx3. Within 15minan increase of more than 50 fold and even 300 fold, respectively, could be measured. The amount of transcripts decreased during the following exposure, but was still very high. Strikingly, after recovery under low light for 4 h, transcript levels of both genes reached a value close to the one before the high light treatment, indicating a very sensitive, irradiance dependent regulation. At complete darkness the amounts of transcripts decreased forLhcx3 and slightly increased forLhcx2 at the time, where the light is usually switched on, indicating that this gene is regulated in a circadian rhythm. A different pattern was observed forLhcx1. A clear up-regulation was observed at darkness, peaking at 9 am with a transcript level about 14 fold higher than in the calibrator. The amount of transcripts

7.4. Results and Discussion

Dark Low Light Moderate High Light

Lhcx1

Figure 7.1.:Transcript level analysis of theLhcxgenes inP. tricornutumunder various light conditions. (A) Cells ofP. tricornutum grown for 4 days under 45µmol·photons·m⁻²·s⁻¹ and a dark/light cycle of8 h/16h either remained under those conditions (Low Light), were transferred to moderate high light with an intensity of 750µmol·photons·m⁻²·s⁻¹ (Moderate High Light) and the same dark/light cycle or remained in darkness for the experiment (Dark). Samples were taken every 3 hours over a time period of 33 hours and relative transcript levels analyzed by real time PCR. Dark periods are indicated by grey bars beneath the expression data, light periods are represented by white bars. (B)P. tricornutumcells grown to mid-logarithmic phase and adapted to low light for 6h were transferred to a light intensity of1500–2000µmol·photons·m⁻²·s⁻¹ (High Light). Samples for gene expression analysis were taken after 15min,30min,45min, 60minand 120minupon transfer to high light. After the high light exposure of2hthe cells were transferred to low light and a sample was taken4hafter recovery under low light. The color code indicates relative gene expression values as indicated by the scale bar. Relative gene expression was calculated with REST^r(Relative Expression Software Tool) as described in [192]. Samples were normalized to18S rDNA. Levels shown are relative to the first sample and average from 4 independent experiments. A grey star in the colored boxes indicates significant changes as given by the software. Detailed results as given by REST^rcan be found in table A.10, page 140.

also showed a peak at low light and moderate high light at the same time with levels that were about 85 and 65 times higher, respectively. The expression pattern was the same at all three light conditions, but to different extents. After the peak at 9 am the amount of transcripts decreased, reaching the same level in the dark phase or the time corresponding to the dark phase as in the respective calibrator sample. The fact, that for Lhcx1 under complete darkness the diurnal pattern is maintained in a similar way as under illumination indicates, that this gene is strongly regulated in a circadian rhythm and that light is only enhancing the expression but not the only factor inducing it. Lhcx4 was the only LHCX gene for which a more or less constant expression (independent of the light conditions) was measured. This suggests, that LHCX4 is possibly not in involved in photoprotection nor light harvesting, as the transcript level was constant for all light conditions used here.

Instead, it might be involved in stabilizing complexes in the thylakoid membrane or may

respond to other stress conditions not tested here.

The results presented here complement the microarray analysis of Nymark and co-workers who showed that transcripts of Lhcx2 and Lhcx3 drastically increase after exposure to HL [178] in P. tricornutum. As demonstrated here, no significant increase could be observed for Lhcx4 when comparing low light to moderate high light or high light samples. While Nymark and co could detect a slight increase inLhcx1 transcripts for the early phase of high light exposure in comparison to low light, our results indicate, that the expression is slightly higher under low light in comparison to moderate high light (750µmol·photons·m⁻²·s⁻¹), which is the irradiance most comparable to the ‘high light’ (500µmol·photons·m⁻²·s⁻¹) conditions of Nymark and co. Still, we also found that when cells ofP. tricornutum were exposed to irradiances of 1500–2000 µmol·photons·m⁻² ·s⁻¹ transcript levels of Lhcx1 indeed are slightly increased in comparison to low light. InC. cryptica three FCP proteins (FCP6, FCP7 and FCP12) can be found that share high similarity with LI818 proteins in green algae [55, 56]. The amount of mRNA ofFcp6,Fcp7 andFcp12 was increased at high light conditions compared to low light [179]. Immunoblot experiments showed, that the mRNA level ofFcp6 was up-regulated 4 to 5-fold at high light in comparison to low light.

Five LHCX proteins are found inT. pseudonana. For four of them an up-regulation at high light was shown, but to different extents [241]. During long-term high light stress a high level of one of the LHCX proteins (LHCX1) was maintained and high levels of NPQ were correlated with the elevated abundance of LHCX1 protein. An increase of transcript levels ofLI818 homologues in high light grown cells has not only been demonstrated for diatoms, but also for the green algaChlamydomonas reinhardtii and the primitive prasinophyte green algaOstreococcus tauri [144, 190] indicating, that the possible photoprotective involvement of these proteins is not limited to diatoms.

Conclusion

The expression patterns observed here support the hypothesis that the product of the LHCX genes play a role in the high light acclimation of diatoms. It was shown, that overexpression of Lhcx1, Lhcx2 and Lhcx3 in P. tricornutum leads to changes of NPQ in comparison to wildtype cells. Transformants develop higher values of NPQ when measured as a function of irradiance [Lavaud et al, unpublished], supporting the suggestion that LHCSR homologues might perform an equivalent role in diatoms as PSBS in higher plants [190, 242].

Acknowledgements

We thank Ansgar Gruber for help with the experiment. This work was supported by the University of Konstanz and grants from the Deutsche Forschungsgemeinschaft (project LA 2368/2-1) to JL.

8. Molecular Mechanisms of Light Stress Protection in the Diatom P. tricornutum, General Discussion

Sabine Sturm^*

Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany

*E-mail: sabine.sturm@uni-konstanz.de

About one-fifth of the photosynthesis on Earth is carried out by microscopic, eukaryotic phytoplankton known as diatoms [13]. The ecological success of these algae is partially due to their high photosynthetic flexibility which allows them to quickly adapt to a rapidly changing light climate. One of the most important photoprotective mechanisms that protect the cells from damage caused by an excess of light is the non-photochemical quenching of chlorophyll fluorescence (NPQ) [103, 175]. While extensively studied in higher plants, the molecular mechanisms of NPQ are only poorly understood in diatoms.

The role of Diatoxanthin in photoprotection

It is known, that NPQ can develop much higher values in diatoms than in higher plants and in cyanobacteria [133]. The development of NPQ in diatoms requires lumen acidification as well as accumulation of the xanthophyll diatoxanthin (DT). The actual point of view is that the amount of diatoxanthin is linearly correlated with the capacity to develop NPQ and that once NPQ development is started, DT alone is sufficient to maintain NPQ [133]. This linear relationship is found in different diatom species, although the quenching efficiency for the same amount of DT can differ among the species [138, 137]. By inserting a mutation within the D1 protein of photosystem II (PSII) we were able to impair the plastidic electron transport and consequently the generation of the transthylakoidal proton gradient which in-fluences the activity of the diadinoxanthin de-epoxidase (DDE) that converts diadinoxanthin (DD) into DT. The results show that the decrease of the electron transport rate (ETR) led to a decrease of NPQ (chapter 3). Interestingly, this was also the case for aP. tricornutum mutant that showed no decline in DT accumulation although ETR was lower compared to the WT. The amount of DT was comparable to the WT, but NPQ was lower. A possible explanation might be that the decrease in lumen acidification was not strong enough to disturb the activation of the de-epoxidase, which requires only minor acidification of the lumen [109]. On the other hand, the decrease was obviously strong enough to inhibit the protonation of LHC-sites, thus preventing the activation of the DT molecules. This hypoth-esis is supported by the data presented in chapter 5, showing that an increase in the amount of DT does not always lead to an increase in NPQ. This suggests that, although the kinetics of NPQ are solely correlated with the amount of DT, once NPQ has been formed, the in-duction of NPQ still requires sufficient lumen acidification for the protonation of LHC-sites as proposed previously [134]

One open remaining questions is, whether DT is able to protect the cells from photo-oxidative damage not only by dissipating excess energy but, for example also by preventing lipid peroxidation as proposed for zeaxanthin in C. reinhardtii [17]. Several studies have observed that significant amounts of DD/DT are not only found in isolated antenna proteins, but also in the fraction containing free pigments [20, 86, 135, 90]. Therefore, both pigments could also be present in the lipid phase of the thylakoid membrane. It was suggested, that

a pool of this pigments is not tightly attached to FCPs, because an increasing amount of DD and DT was lost in FCP preparations with enhanced purity [90].

Antenna proteins and their possible role in photoprotection

PSBS is known to be crucial for NPQ development in higher plants [146], but so far exper-imental evidence is missing for the presence of PSBS in diatoms [13, 25]. It is proposed that PSBS evolved by gene duplication of two-helix SEP [54], which can also be found in diatoms. We therefore investigated, whether two-helix SEPs and related proteins might play a role in photoprotection in the diatom P. tricornutum. SEPs belongs to the family LHC-like proteins which in higher plants consist of three helix ELIPs [2, 164], two helix SEPs (stress enhanced proteins) [100] and one helix proteins (OHPs) [112, 11]. The role of the members of the LHC-like family in light stress protection inA. thaliana is discussed widely, as the expression level is increased upon transfer to high light [100, 2]. As the increase of transcript levels comes along with carotenoid accumulation, it has been suggested that the original role of the LHC-like proteins was photoprotection rather than light harvesting [78, 164]. Homologues of the ELIPs were found in the genome of P. tricornutum [54] and analyzed with respect to their expression pattern under various light conditions (chapter 6).

In contrast to the findings for three-helix ELIPs in higher plants, the expression level of three helix REDCAP gene in P. tricornutum was not increased upon transfer of the cells to high light (Fig. 6.4, page 91). Interestingly,RedCAP showed an expression pattern more similar at FcpB, indicating that its functional role in diatoms might be light harvesting rather than photoprotection. Further investigations on the localization of REDCAP within the photosynthetic apparatus, possible interaction partners and pigment binding studies might help to elucidate the exact role of the protein in diatoms. RedCAP was not the only gene that showed a transcript pattern distinct from its homologue in higher plants. An increase in transcripts of Sepx could not be induced by high light treatment, the amount of transcripts was stable over time under low- , moderate high- and high light conditions (Fig. 6.4, page 91). This suggests that its function is independent of the light environment the cells are exposed to. It is therefore likely, that it is not involved in light harvesting, but could have important functions in stabilizing photosynthetic complexes as shown for HLIPs in cyanobacteria [228].

Another group of antenna proteins that were investigated during this work are the LHCX proteins, which are homologues of the LI818 proteins found in green algae [190]. The results presented in chapter 7 show that transcripts of three of the four Lhcx genes are clearly upregulated upon transfer of the cells to high light, suggesting that the respective proteins play a role in high light acclimation of the cells. An increase of the transcript levels of LI818 homologues upon high light treatment was observed in different species including the hap-tophyteEmiliania huxleyi [145] and the diatomsC. cryptica [179] andT. pseudonana [241].

It was proposed that the polypeptide might play the equivalent role of PSBS [190, 242], which is essential for NPQ development in higher plants [146]. If LHCX proteins are the site for NPQ in diatoms, they should bind DT and chlorophyll. Biochemical analyses of the diatom Cyclotella meneghiniana revealed that the LI818 homologues FCP6/7, which are part of the LHC sub-complex FCPa, are up-regulated after illumination in high light in comparison to low light. In parallel the xanthophyll pool size is increased in this isolated fraction and the chlorophyll fluorescence is quenched, suggesting that these polypeptides are involved in photoprotection [20]. A more recent study observed that the fluorescence yield of FCPa complexes, isolated by ion exchange chromatography or sucrose gradient, is down-regulated in parallel with an increase of DT [90] stressing the importance of FCPa in the thermal dissipation of excess light by accumulation of DT and up-regulation of FCP6/7.

Direct binding of pigments, especially DT to the complex has not been demonstrated so far. However, preliminary results show that when each of the LHCX1-3 are overexpressed in vivo in P. tricornutum, NPQ is enhanced [Lavaud et al, unpublished]. Taken together, the results from various studies indicate that members of the LHCX proteins are involved in photoprotection. Interestingly, LHCX1 fromT. pseudonana was also shown to be induced under iron deficiency. It was proposed that LHCX1 could be involved in the remodeling of PSI reaction center and may complement the photoprotection through thermal dissipation of PSI to prevent photooxidative stress under iron deficiency [241]. However, the exact location of LHCX1 remains unknown and needs further investigations to support this hypothesis.

Perspectives

Investigating the physiology of diatoms is important to understand how the algae may affect ocean ecology and biogeochemistry. Although diatoms are highly flexible concerning their photosynthetic capacity, some scientists predict that diatoms will have a reduced role in phytoplankton communities in the future due to global climate changes [13]. This could bring a dramatic reduction in the ability of phytoplankton to sequester CO₂ from the at-mosphere, deteriorating climate change [32]. Studying their photosynthetic capacity and their ability to adapt to various environmental conditions, including different light climates, might help to predict their role in the future. Diatoms are not only of particular interest due to their crucial role in the global carbon cycle but also as a source for alternative energies or for nanotechnology by using components of their silica cell wall. However, more detailed investigations on their physiology and metabolim is needed to make them applicable for these fields.

A. Supplementary Data

A.1. Supplementary Material, Chapter 2

Table A.1.:Pigment composition (inmol/100mol Chla) and photosynthetic properties of the wildtype (WT) and thepsbAmutants ofPhaeodactylum tricornutum. YSSis the concentration of active PSII reaction centers per Chla[138]; RC/CS0is the number of active PSII reaction center per PSII cross-section; 1/I1/2ofYSSis a measurement of the PSII light-harvesting antenna size [138];EKin µmol·photons·m⁻²·s⁻¹is the light intensity for saturation of photosynthesis; the DCMU resistance factor was calculated from the IC50PSII fluorescence reactivity to DCMU; J (* J phase is delayed to 9.7±2.5msin S264A) and I are the fluorescence levels of the J and I phases measured at2msand30ms, respectively (values normalized to the P peak = 1, see figure 2.2C on page 17, P corresponds to the concentration of QA−

QB2−

and PQH2);F0is the minimal level of fluorescence of dark-adapted cells;Fv/Fmis the maximum photosynthetic efficiency of PSII;ΦPSII [(Fm’−F’)/Fm’] is the effective PSII quantum yield for photochemistry measured at 50–100µmol·photons·m⁻²·s⁻¹(an irradiance for which there is no NPQ); ET0/CS0 is the steady-state electron transport in a PSII cross-section;µ is the growth rate. All measurements were performed on cells grown at 50 µmol·photons·m⁻²·s⁻¹, which was low enough to prevent the de-epoxidation of diadinoxanthin into diatoxanthin. n.d., not determined. Values are average±SD of three to four measurements. See the text for other details.

Pigment/Parameter WT V219I F255I S264A L275W

DCMU resistance – 3 150 3000 500

Chla (pg·cell⁻¹) 0.50 ±0.07 0.57± 0.08 0.53±0.17 0.54± 0.03 0.57 ±0.1 Materials and methods. DCMU resistance was evaluated measuring the inhibition of the PSII activity versus increasing DCMU concentrations using a self-made fluorometer [188]. The measure of inhibition is the increase in the amplitude of the I-45mspeak illustrating the accumulation of QA−

due to the electron QA-QB transfer being blocked by DCMU (see figure 2.2B). This way, the IC50, the DCMU concentration at which the PSII activity is inhibited by 50 % was measured and compared between WT and mutants to evaluate a resistance factor. Standard fluorescence nomenclature was

due to the electron QA-QB transfer being blocked by DCMU (see figure 2.2B). This way, the IC50, the DCMU concentration at which the PSII activity is inhibited by 50 % was measured and compared between WT and mutants to evaluate a resistance factor. Standard fluorescence nomenclature was