3. High light photoacclimation of diatom psbA mutants 23
3.4.2. Response of the P. tricornutum psbA mutants to a prolonged increase
Under low light (50µmol·photons·m−2·s−1), all types of cells except L275W, show a similar growth rate and Fv/Fm (Fig. 3.3) [156]. When exposed to an irradiance five times higher, which just saturates the photosynthetic rate (EK) [156], all types of cells showed the same decrease in their growth rate of about −49 ± 4 % except L275W (−60 %). Nevertheless,
3.4. Results
Table 3.1.:Photosynthetic activity, NPQ and xanthophyll cycle operation in the wildtype (WT) and the psbA mutants ofPhaeodactylum tricornutum. The light conditions were5minat 450µmol·photons·m−2·s−1 (light intensity which is necessary to fully saturate the linear electron transport rate (ETR) for all cell lines [156]).
1−qP is a fluorescence parameter to estimate the reduction state of QA[27], ETR is inµmol·photons·m−2·s−1, NPQ is the non-photochemical Chl a fluorescence quenching, DT is diatoxanthin (inmol/100mol Chl a), DEP (DD de-epoxidation, in %) = DT/(DD+DT)× 100, DD is diadinoxanthin. Final DCMU and NH4Cl concentrations were 0.4 mM and 12.5 mM, respectively. They were added at the beginning of the dark-adaptation period,20minprior to the illumination. Values are average±SD of three to four measurements.
Methods: see Fig. A.7, page 122.
1−qP ETR NPQ DT DEP extent
WT 0.62 ±0.07 25.6± 1.2 1.54 ±0.09 3.4 ±0.1 36 ±1
Figure 3.2.:Photoinhibition kinetics (as measured by the decrease in the number of active PSII, YSS) in the wildtype (WT) and the fourpsbAmutants (V219I/F255I/S264A/L275W) of P. tricornutum. (A) YSS as a function of a light intensity gradient from darkness (0µmol·photons·m−2·s−1) to full sunlight in nature (2000µmol·photons·m−2·s−1, [150]). The illumination duration was5min; a new sample was used for each irradiance. (B) YSS as a function of time for an irradiance of 2000 µmol·photons·m−2·s−1. For L275W,YSSwas measured only after5minat this irradiance, as indicated by the arrow. Values are average
±SD of three to four measurements.
0.5
Figure 3.3.:Growth rate and the maximum PSII quantum yield for photochemistry (Fv/Fm) of the wildtype (WT) and the psbA mutants (F255I/S264A/L275W) ofP. tricornutum (V219I is not shown, it shows the same response than WT). The three light intensities are: 50µmol·photons·m−2·s−1, low light growth;
250µmol·photons·m−2·s−1, similar toEKthe intensity to just saturate ETR, the electron transport rate in all types of cells (212±29µmol·photons·m−2·s−1); 450µmol·photons·m−2·s−1, the intensity to reach maximal photosynthetic rate in the mutants. Data are from the exponential phase of growth (see Fig. A.8, page 125 and [156]). Values are average±SD of four to five measurements.
the growth of F255I and S264A showed peculiarities (Fig. A.8A on page 125): S264A showed a drastic increase in its growth after 48h acclimation, but finally reached the same maximal biomass as the WT while F255I was unable to do so. While WT cells were able to maintainFv/Fm, it slightly decreases (−6±1 %) in the four mutants;Fv/Fm in L275W being much lower. Surprisingly, S264A was able to maintainFv/Fm at a level close to WT especially during the first 48 h of acclimation (Fig. A.8C and E). When increased up to two times EK, all types of cells showed the same growth pattern (decrease of about 70 ± 3 % compared to low light) except F255I for which growth was even lower than L275W (Fig. A.8B). Compared to EK conditions, all mutants and the WT achieved to maintain Fv/Fm except F255I (Fig. 3.3). S264A behaved even better than the WT in maintaining Fv/Fm (Fig. A.8D and F).
The physiological state of the cells was examined during acclimation to EK conditions (250µmol·photons·m−2·s−1) (table 3.2). All photosynthetic pigments but diadinoxanthin (DD) decreased compared to low light conditions [156]. The content in Chl c, fucoxanthin and β-carotene remained the same in all cells, except in S264A for which fucoxanthin was significantly lower. DD increased in all cells but to a different extent: while it was multiplied by a factor of about 2 in WT, V219I and F255I, its increase was lower in L275W (+40 %) and S264A (+15 %). Also, a slight de-epoxidation of DD into diatoxanthin (DT) occurred generating a constitutive presence of DT (less than 1molDT/100molChla). The synthesis
3.4. Results
Table 3.2.:Pigment composition (in mol/100mol chlorophylla(Chla)) and photosynthetic properties of the wildtype (WT) and the psbA mutants of Phaeodactylum tricornutum acclimated to 250 µmol·photons· m−2 ·s−1. DEP (DD de-epoxidation) = DT/(DD+DT) × 100 where DD is diadinoxanthin and DT is diatoxanthin; YSS is the concentration of active PSII reaction centers per Chl a [138], YSS of low light (50µmol·photons·m−2·s−1) growing cells were WT: 92±8.4, V219I: 107±11, F255I: 126±8.7; S264A: 139
±10, L275W: 54±8.5 which is similar to the values observed before [156]; 1−qP is a fluorescence parameter to estimate the fraction of reduced QA[27]; NPQ is the non-photochemical chlafluorescence quenching. 1−qP was determined after having exposed the cells to 250µmol·photons·m−2·s−1 for5minto reach the steady-state of fluorescence yield. NPQmax was determined at a light intensity of 1200µmol·photons·m−2·s−1. Values are average±SD of three to four measurements. See the text for other details.
Pigment/Parameter WT V219I F255I S264A L275W
Fucoxanthin 69.0±4.2 70.1 ±3.4 73.3 ±3.1 61.2± 5.2 70.1±3.6
of DT was lower in S264A and higher in L275W (Fig. A.6, page 121) which was confirmed and even more pronounced in cells reaching the stationary phase of growth (72hacclimation) in which the DD amount and DEP were higher [16] than in 48hacclimated cells (see table A.3 on page 120). The number of O2evolving PSII (YSS) decreased in all cells but to a different extent; WT and V219I showing the lowest decrease (−13 ± 3 and −7 ± 1 %, respectively) while YSS drastically dropped down in L275W (−41.5 ±3.5 %, Fig. A.6). More surprising was the ability of S264A to maintainYSS (−9.5 ±4.5 %) compared to low light conditions, and the inability of F255I to do so (−36 ± 2 %, Fig. A.6). Hence, the distribution of the number of active PSII was: S264A>V219I>WT = F255I>L275W. These data were confirmed by western-blot analysis of the D1 protein (Fig. 3.4). At 50µmol·photons·m−2· s−1, the D1 content of S264A and F255I was higher than in WT cells which fits with the YSS data previously reported [156]. The 23 and 16 kDa products showing degradation of D1 were present in these two mutants, as well as in L275W [156], in high amounts even under low light. When acclimated to 250µmol·photons·m−2·s−1, the D1 content of WT decreased in parallel to YSS together with the appearance of a D1 degradation product.
The same was true for the D1 content of S264A and F255I. The proportion of reduced PSII reaction centers (evaluated from the fraction of reduced QA, 1−qP) was the highest in S264A and L275W as previously reported [156]. The extent of the PSII CET and NPQ followed the tendency already described in Fig. 3.1A and B, respectively. Noteworthy is the high PSII CET in S264A under these light conditions. Also, NPQ was higher in all type of cells reflecting the general increase in DD/DT amounts [138] compared to low light conditions
33
WT S264A F255I WT S264A F255I
50 250
10
Figure 3.4.:Western-blot of the D1 protein of the PSII reaction center ofP. tricornutumwildtype (WT) and the psbAmutants S264A and F255I cells (for L275W see [156]; V219I was similar to WT). Cells were grown at 50 and 250 µmol·photons·m−2·s−1 (16:8h light:dark cycle) and sampled during the exponential phase of growth,3h after initiating the light period. Bands representing D1 degradation products of23kDaand 16kDaresulting from D1 degradation [15] are highlighted.
[156]. The kinetics of NPQ development at 250 µmol·photons·m−2 ·s−1 (Fig. A.9 on page 125) revealed, as above (Fig. A.7B on page 122), a strong delay for both S264A and L275W so that it needed longer time to reach the maximal value compared to WT, V219I and F255I (Table 3.2, NPQmax). In cells acclimated for 72h (Fig. A.3, page 120), although the amount of both DD and DT were higher, NPQ extent did not increase in S264A and L275W in contrast to WT, V219I and F255I which followed the linear relationship usually observed between NPQ and DD/DT (see Fig. 3.5).
In parallel, we measured the transcript level of some photosynthetic genes: psbA which encodes for the D1 protein, Dde encodes for the DD de-epoxidase, Sod encodes for the superoxide dismutase, an enzyme responsible for the scavenging of reactive oxygen species, Lhcx1-Lhcx4 which are homologues of the LHCSR (formerly called LI818) proteins that are suspected to play a role in NPQ [190, 242], and three LHC-like genes (Ohp1-like1,Ohp2 and Sep) for which up-regulation in response to light increase was partly shown (see chapter 6).
Surprisingly, under LL conditions, the transcript level of some genes was already higher in the mutants compared to the WT. This was especially the case for L275W, where we could measure a much higher transcript level of psbA, Dde and Sod than in the WT (Fig. 3.6).
Transcript levels ofOhp1-like1,Ohp2 and Sepwere also increased in L275W, but to a much lower extent. The amount of psbA, Dde and Sod transcripts was also increased in S264A, but not as strong as for L275W. When grown at 250µmol·photons·m−2·s−1, no significant increase in the amount of transcripts was observed for all genes in the WT (Fig. A.11), indicating that the light intensity was not high enough to generate stressful conditions in WT cells. Under those conditions, the transcript level ofpsbAincreased in L275W, while it decreased forDdeandSodcompared to LL conditions. In comparison to the WT under high
3.4. Results
Supplementary File S6
Relationship between the non-photochemical fluorescence quenching (NPQ) and the amount of diadinoxanthin+diatoxanthin (DD+DT) in the WT and the four psbA mutants of Phaeodactylum tricornutum
Cells were acclimated at 250 µmol photons m-2 s-1 for 48 and 72 h, corresponding to the exponential phase of growth and the beginning of the stationary phase of growth (see Fig. S3).
When diatom cells reach the stationary phase of growth, their DD/DT content increases (Arsalane et al, 1994). Data were extracted from Table 1 (for 48 h grown cells) and Fig S5 (for 72 h grown cells).
Methods: see the Material and methods section for pigment analysis and the Fig. S1 for NPQ measurement.
Figure 3.5.:Relationship between the non-photochemical fluorescence quenching (NPQ) and the amount of diadinoxanthin+diatoxanthin (DD+DT) in the WT and the four psbAmutants of Phaeodactylum tricornu-tum. Cells were acclimated at 250µmol·photons·m−2·s−1for 48 and72h, corresponding to the exponential phase of growth and the beginning of the stationary phase of growth (see Fig. A.8). When diatom cells reach the stationary phase of growth, their DD/DT content increases [16]. Data were extracted from table 3.2 on page 33 (for48hgrown cells) and Fig. A.5 on page 120 (for 72hgrown cells). Methods: see the Material and Methods section (chapter 3.3) for pigment analysis and Fig. A.7 for NPQ measurement.
light, in this mutant a higher amount of transcripts was measured for the three LHC-like genesOhp1-like1,Ohp2 andSepand to a lower extent also forLhcx1,Lhcx2 andLhcx3. The transcript level ofLhcx4 did not change significantly in the mutants compared to the WT, supporting the suggestion that this gene is not irradiance regulated as shown in chapter 7.
In general, for most of the genes the transcript level was up-regulated in the following order:
WT<V219I<F255I<S264A<L275W. An exception were the genesLhcx1-Lhcx3,Ohp1-like1 and Sep under high light, for which transcript level was significantly higher for S264A in comparison to L275W. For both light conditions the amount of transcript of all genes in V219I was similar to the WT.
Figure 3.6.:Transcript level of photosynthetic genes in the four psbAmutants and the WT of P. tricornutum grown at 50µmol·photons·m−2·s−1(Low Light, upper graph) and 250µmol·photons·m−2·s−1(High Light, lower graph). Transcript levels of each gene in the mutants is shown relative to the corresponding gene in the WT which was set to 1 (continous black line in the graph). Values are average±SD of three measurements.
3.5. Discussion
3.5. Discussion
3.5.1. Mutations in or close to the QB pocket generate impairment in NPQ