Transcript analysis upon transfer to light

To investigate the influence of light on the expression of the LHC-like genes, cells ofP. tricor-nutum were exposed to different light intensities. For investigation of circadian regulation, three light conditions were applied: Cells grown at LL (45 µmol·photons·m⁻²·s⁻¹) (1) either remained under those conditions or (2) were transferred to moderate HL (750µmol· photons·m⁻²·s⁻¹) or (3) remained in complete darkness for the duration of the experiment.

Samples for transcript level analysis were taken every three hours for 33h, starting with the beginning of a dark period. The expression of LHC-like genes was investigated by real-time PCR using specific primers for the LHC-like genes and FcpB, a gene which is known to be light induced and regulated in a circadian rhythm [143, 180]. Relative transcript levels were calculated as described in [192] using the first sample of each light condition as calibrator and18S rDNAas endogenous control. Different gene expression patterns were observed for each of the LHC-like transcripts and are shown in figure 6.4A. In general, a decrease in tran-script level was measured when cells remained in darkness. The decrease was very strong forOhp1-like1. Four genes (Ohp1-like1,Ohp2,RedCAP, FcpB) showed a slight increase of transcripts between 6 am and noon, when the light is usually switched on, indicating that those genes undergo diurnal fluctuations. Under LL conditions only small changes in

tran-GFP Chlorophyll

DIC Chl

GFP (B)PtOhp2pre:GFP

(C)PtRedCAPfull:GFP

GFP Chlorophyll

DIC Chl

GFP (A)

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PtOhp2pre:GFP MRWTCAFLWCVVVPTLHAWVPSTTNPASRIGTRRWEALGDRELEEPRp:GFP

PtRedCAPfull:GFP MAPLRTTFALLLSLVSASAFAPVQNVARKQTSVSAFKID...180aa..GGAYFp:GFP

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BOLD: signal peptide predicted by SignalP’s hidden Markov models UNDERLINED: estimated transit peptide domain

ITALIC: mature protein lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site BLACK: enhanced green fluorescent protein

Figure 6.3.:Localization of OHP2 and REDCAP. (A)Ohp2 andRedCAP GFP fusion constructs, an additional proline (artificial sequence) is inserted during cloning of the constructs. (B) Expression of PtOHP2pre:GFP in P. tricornutum, GFP accumulates in the plastid. (C) expression of PtRedCAPfull:GFP in P. tricornutum, GFP accumulates within the plastids; Nomarski’s differential interference contrast (DIC), Chlorophyll autoflu-orescence, GFP fluorescence and merged images showing the respective channels in the indicated colours are shown from left to right.

script level were measured for Ohp1-like2 and Sepx, while an increase in transcripts with beginning of the light phase was observed for all other genes, but with different patterns.

For Ohp1-like1 the amount of transcripts rapidly increased to a level about 10–15 times higher than in the calibrator (0 am sample). The level stayed high for the remaining time of the light phase and decreased, when the light was switched off. Transcript levels ofOhp2 increased initially when the light phase began and decreased thereafter to the initial level to increase again when the next light phase began. RedCAP showed a very similar pattern in transcript level thanFcpB but with different extents. The transcript level of both genes steadily increased and peaked at 3 pm to a level 18 (RedCAP) and 5 (FcpB) times higher than the initial level and decreased afterwards. Both genes were extremely downregulated

6.4. Results

Dark Low Light Moderate High Light

Ohp1-like1

Figure 6.4.:Transcript level analysis of Ohp1-like1, Ohp1-like2,Ohp2, Sepx,RedCAP and FcpB. (A) Cells of P. tricornutumgrown for 4 days under 45µmol·photons·m⁻²·s⁻¹and a dark/light cycle of8h/16heither remained under those conditions (Low Light), were transferred to a light intensity of 750µmol·photons·m⁻²· s⁻¹ (Moderate High Light) and the same dark/light cycle or remained in darkness for the whole experiment (Dark). Samples were taken every 3 hours over a time period of 33 hours and relative transcript levels were analyzed by real time PCR. Dark periods are indicated by grey bars at the bottom of the expression data, light periods are represented by white bars. (B)P. tricornutum cells grown to mid-logarithmic phase and adapted to LL for 6 h were transferred to a light intensity of1500–2000 µmol·photons·m⁻²·s⁻¹ (High Light).

Samples for gene expression analysis were taken after15min,30min,45min,60min and120min upon transfer to high light. After the HL exposure of2hthe cells were transferred to LL and a sample was taken 4hafter recovery. The color code indicates relative gene expression values as indicated by the scale bar on the right. Relative gene expression was calculated with REST^r (Relative Expression Software Tool) as described in [192]. Samples were normalized to 18S 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^r can be found in table A.9, page 134.

under moderate HL. As under LL conditions no significant changes were measured forSepx at a light intensity of 750 µmol·photons·m⁻²·s⁻¹. An increase of transcripts in the late phase of the light period was detected for Ohp1-like2 suggesting, that this gene might be involved in long-term acclimation of the cells to moderate HL intensities. A strong increase in transcript level was observed for Ohp1-like1 which showed a 26-fold higher amount of transcripts at noon than the initial sample. The transcript level slightly decreased after-wards but was still high for the remaining time of the experiment. Ohp2 showed a similar pattern but with lower extents, transcript level was about 3 times higher at noon and in the afternoon compared to the respective calibrator (0 am sample).

To test whether LHC-like genes inP. tricornutumare upregulated at HL, we adapted cells to low light for 6hand transferred them to high light (1500–2000µmol·photons·m⁻²·s⁻¹).

Samples were taken after 0, 15, 30, 45, 60 and 120minas well as 4hafter recovery in LL for transcript analysis. For comparison, the transcript level of samples from LL cultures which were taken in parallel was also measured. As can be seen in figure 6.4B only small changes under LL were measured forOhp1-like1 and Ohp1-like2. Sepx,Ohp2 and RedCAP showed a lower amount of transcripts at the end of the experiment in comparison to the calibrator (0min), which can be explained by the normal diurnal changes that occur at this time of the day. The same explanation can be given for the observed increase in transcript level under LL forFcpB. Under HL almost all genes showed a decrease in transcripts. The decrease was very strong and fast for Ohp1-like2,RedCAP and FcpB. In contrast toRedCAP and FcpB the transcript level ofOhp1-like2 increased back to almost the same level as before the high light treatment after recovery under LL for 4h. The decrease in transcripts was slower and not as strong for Ohp2 and the amount of transcripts increased again during recovery. An increase was observed forSepx after 15 min, transcript level steadily decreased afterwards and increased again under low light recovery. The only gene for which a strong increase in transcripts under HL was observed wasOhp1-like1. Within 15 minthe transcript level increased strongly and a high amount of transcript was maintained for about 30minbefore the level began to sink slowly. After recovery in LL the amount of transcripts was lower than before the light treatment. The fact, thatOhp1-like1 shows an increase in transcript level upon transfer to moderate and HL suggests that it might play a role in protecting the cells from photooxidative damage.

6.5. Discussion

6.5. Discussion