Xanthophyll de-epoxidation in the WT and Dde transformants grown

To check whether it would be possible to increase the silencing effects without stressing the cells, we took advantage of the light dependent FcpA promoter driving the silencing constructs and performed the same experiments as before but at 3 times higher light inten-sities (135µmol·photons·m⁻²·s⁻¹). Similar irradiances were used in previous studies to growP. tricornutum in a number of photophysiological studies [183, 29, 218, 16] being close to the light intensity for saturating photosynthesis in diatoms [207, 133]. At these growth conditions, F_v/F_m is slightly decreased (−6 %) in the WT while the growth rate remained the same (table 4.3) indicating that the cells were not stressed. As in low light conditions, in theDde transformants F_v/F_m was very similar to that of the WT. The pigment content of the WT and theDde transformants did not change, except for the amounts of DD and DT (table 4.3). The DD pool size was increased in all types of cells, as usually reported for P. tricornutum when cells are switched to a higher light intensity [30, 209]. This reaction was shown to enhance the ability of the cells to produce DT [232, 166] thus increasing the efficiency of photoprotection, especially via increasing the amplitude of NPQ [138, 30, 209].

Interestingly, the increase of DD amounts was not the same in the different transformants.

While the amount of DD increased by a factor of ×1.7 ± 0.1 in the WT and AS transfor-mants, it was higher by a factor of ×2.1 in the IR transformants. As a consequence and in comparison to the low light growing conditions, the pool size of DD was the same in all the cells (13.5±0.6mol/100molChl a). Additionally and in contrast to low light growing conditions, DT molecules (0.68 ± 0.12 mol/100 mol Chl a) were ‘constitutively’ accumu-lated due to the higher light excitation pressure (as reported before [183, 30]) confirming the beginning of DD de-epoxidation between 100 and 150µmol·photons·m⁻²·s⁻¹(Fig. 4.5B).

When the cells were then exposed to a light intensity of 450 µmol·photons ·m⁻² · s⁻¹ for 4 min, the WT and the IR transformants showed a similar DD de-epoxidation (total de-epoxidation minus the constitutive one) as under LL conditions. This was not the case for the AS transformants for which it was slighly lower (−4.7 ± 1 %) (figure 4.4A).

The amount of DT synthesized during the light treatment was 61 % higher in the WT in comparison to the LL cells (an average of 4.2 and 2.6 DT molecules, respectively) which was similar to the increase (+53 %) in the total DD+DT pool size (figure 4.4B). This feature is common when the cells are shifted from low light to higher light conditions: more DD is synthesized so that the subsequent increase in DT is due only to an increase in the substrate quantity but not in the activity of the de-epoxidase [232, 166, 138, 209]. For the IR transformants the same observation was true even though the increase in both DD+DT and DT was higher: ×2.11 and ×2.18 respectively for IR-4, and×2.29 and ×2.30 respectively for IR-5 (figure 4.4B). Still, the amount of DT synthesized exclusively during the light treatment (total DT amount minus the constitutive one) in the IR transformants was about

4.4. Results and Discussion

Table 4.3.:Maximum photosynthetic efficiency of PSII (Fv/Fm), growth rate (µin d⁻¹), diadinoxanthin (DD) and diatoxanthin (DT) content (inmol/100molChla), and the de-epoxidation degree (DEP) of the WT and theDde transformants ofP. tricornutum cells grown under high light (135µmol·photons·m⁻²·s⁻¹). DD, DT and DEP were determined before and after a4min450µmol·photons·m⁻²·s⁻¹ light treatment (LT).

DEP degree (in %) = DT/(DD+DT)×100. Values are average,±SD of three measurements.

WT AS-198 AS-523 IR-4 IR-5

F_v/F_m 0.67± 0.02 0.64±0.03 0.63 ±0.03 0.65 ±0.04 0.64± 0.02 µ 1.25± 0.06 1.01±0.01 1.04 ±0.07 1.03 ±0.04 1.1± 0.03 Before LT

DD 13.0± 2.2 13.4±2.2 12.1 ±3.2 12.4 ±0.3 12.9 ±0.6 DT 0.7± 0.2 0.8±0.1 0.8±0.1 0.6 ±0.2 0.5± 0.2 DD+DT 13.9± 2.3 14.2±2.1 12.8 ±3.2 13.2 ±0.2 13.6 ±0.6 DEP 4.8± 0.3 5.6±1.0 5.2±2.0 5.3 ±0.2 4.3± 0.6 After LT

DD 8.6± 1.4 10.2±0.8 8.9±0.8 10.1 ±0.6 10.4 ±0.3 DT 4.9± 0.5 2.1±0.3 2.2±0.3 3.0 ±0.3 2.8± 0.1 DD+DT 13.6± 1.9 12.7±0.3 11.9 ±0.8 13.3 ±0.1 13.5 ±0.5 DEP 32.8± 2.4 17.0±2.8 18.8 ±2.7 22.0 ±2.8 22.0 ±1.8

two times less than in the WT, as for the LL cells, illustrating the stable inability for de-epoxidation in these transformants. Surprisingly the same relationship was not found in the AS transformants (figure 4.4B). While the size of the DD+DT pool was increased by 74.5± 0.5 % in AS-198 and AS-523, the corresponding increase in DT synthesized during the light treatment was only 18 % and 27 %, respectively. Consequently, for these two transformants there are in average 52±4 % molecules of DT which were not synthesized during the light treatment in comparison to what happened in the WT and the IR transformants. Indeed, the amount of DT molecules synthesized during the light treatment (1.3–1.4 molecules, table 4.3) was close to the one synthesized by the LL cells during the same light treatment (1.1 molecules, table 4.2, page 59). The reason might be, that the higher irradiance enhanced the silencing effect by inducing the FcpApromotor and therefore increasing the amount of transcripts. Surprisingly this effect was only visible in the AS transformants while the silencing effect was stable in the RNAi transformants independent of the irradiance. A possible explanation might be, that by enhancing theFcpApromotor, more doublestranded mRNA accumulated in the AS transformants compared to low light, increasing the silencing effect. Further experiments including transcript analysis for cells grown at higher light intensities are necessary to explain these findings.

Conclusion

Our results confirm the tight relationship between DT synthesis and NPQ development and highlight the fine regulation of the DDE concentration and activity to provide the

photo-synthetic apparatus with an efficient and fast photoprotective system whatever the light intensity is. This might explain the fast ability for DD de-epoxidation in diatoms [137, 74]

which is most probably essential to help the diatom cells to maintain an optimal photosyn-thetic productivity in fluctuating light conditions [133]. Transformation of P. tricornutum with constructs supposed to induce silencing of the DDE led to the expected NPQ phe-notype. The results indicate that we were able to successfully suppress expression of the DDE by targeted gene silencing. This was true for the anti-sense construct as well as for the ‘inverted repeat’ construct. By increasing the light intensity we could induce a different response in the AS and IR transformants. While for the latter a stable silencing effect was observed, for the AS transformants the silencing effect was enhanced with increasing irradi-ance, suggesting that there are two different gene silencing mediating effects in the diatom P. tricorntum. Investigating this mechanism might help to understand what molecules are involved in targeted downregulation of transcription and provide us with another tool to further investigate the biology and ecology of diatoms.

Acknowledgements

We thank D. Ballert for help with the transformation and cultivation of P. tricornutum and I. Adamska for access to some of the instruments used in this work. This study was supported by the University of Konstanz and grants of the European community (MarGenes, project QLRT-2001-01226) to PGK.

5. Investigations on transformants of Phaeodactylum tricornutum overexpressing the diadinoxanthin de-epoxidase (DDE)

Sabine Sturm^†, Katrin Leinweber, Peter G. Kroth and Johann Lavaud¹ Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany

†Author for correspondence. E-mail: sabine.sturm@uni-konstanz.de

1present address: UMR CNRS 6250 ‘LIENSs’, Institute for Coastal and Environmental Research, University of La Rochelle, 2 rue Olympe de Gouges, 17042 La Rochelle Cedex, France.

5.1. Abstract

Xanthophylls are oxygenated derivates of carotenoids that take part in light harvesting and that are essential in protecting the chloroplasts from photooxidative damage. In particular, they are involved in the dissipation of excess light energy absorbed as heat, a process that is also called non-photochemical chlorophyll fluorescence quenching (NPQ). In diatoms, it is known, that the amount of the xanthophyll diatoxanthin (DT) plays a crucial role in regulating the ability of the cells to perform NPQ. To investigate the roles of xanthophylls in photoprotection, we have generated transformants that overexpress the diadinoxanthin de-epoxidase, an enzyme which catalyzes the conversion of diadinoxanthin to diatoxanthin.

All investigated transformants showed an increase in Dde transcript level and a higher de-epoxidation degree, under darkness as well as under low light, in comparison to the WT.

Growth rates were comparable in all cell lines. Different NPQ patterns were observed in WT cells and in transformants, also depending on how the cells were light acclimated before.

We show, that an increase in DT does not necessarily lead to an increase in NPQ in the diatomP. tricornutum.

Keywords

Diatoms·Non-photochemical fluorescence quenching·Diadinoxanthin de-epoxidase·P. tri-cornutum·Diatoxanthin

Abbreviations

Chl a and Chl b: chlorophyll a and b; DD: diadinoxanthin; DEP: DD de-epoxidation;

DT: diatoxanthin; NPQ: non-photochemical chlorophyll fluorescence quenching; PAM: pulse amplitude modulation; WT: wildtype; XC: xanthophyll cycle.

5.2. Introduction

5.2. Introduction

Light is essential for all oxygenic photosynthetic organisms. Nevertheless, when absorption of light energy exceeds the capacity of photosynthesis, photoxidative damage might occur to the photosystems [66]. For the survival of the organisms it is therefore important to maintain an optimal balance between the capture and the use of light energy. When light energy is absorbed in excess, components of the photosynthetic linear transport chain might become overreduced which can result in the generation of highly reactive oxygen species (ROS). These singlet oxygen molecules ¹O₂^∗ can irreversibly damage lipids, proteins and pigments. In the photosystem II (PSII) reaction center, the formation of superoxide is thought to be the cause of direct damage to structural protein components, which need to be repaired byde novo protein synthesis [158]. To avoid such a situation, algae and plants have developed several mechanisms to either reduce formation of ROS or to detoxify super-oxide and other free radicals once they have been formed (reviewed in [175]). Xanthophylls can play an important role in both of these protective functions. It was suggested that zeax-anthin accumulation might prevent lipid peroxidation by quenching oxygen- and/or other free radicals under high light directly [98, 17]. On the other hand, zeaxanthin is known to be involved in the thermal dissipation of excess light energy also known as non-photochemical fluorescence quenching (NPQ) [169]. In higher plants and green algae, zeaxanthin evolves by de-epoxidation of violaxanthin under high light conditions in the so called xanthophyll cycle [42, 194]. Investigations in different zeaxanthin deficient or accumulating mutants show, that the amount of this xanthophyll can influence NPQ [175].

Diatoms are a major group of phytoplankton and are assumed to contribute to about 40 % of the aquatic primary production (i.e., ∼20 % of the annual global production) [60].

They tend to dominate in ecosystems with highly mixed water bodies, like coasts and estu-aries, where they have to cope with a fast changing underwater light climate. Depending on the rate of water mixing, diatoms can be exposed to punctual or chronic excess light, possibly generating stressful conditions that impair photosynthesis (i.e., photoinactivation/-inhibition) [150, 133]. Like in higher plants and green algae, diatoms use NPQ for regulating photosynthesis and to avoid photooxidative damage of the photosystems. In contrast to green plants different xanthophylls are involved in diatoms to regulate NPQ. The diadinox-anthin de-epoxidase is catalyzing the conversion of diadinoxdiadinox-anthin (DD) into diatoxdiadinox-anthin (DT) [92] and is normally activated under high light [218, 232, 183]. The accumulation of DT was shown to be crucial for NPQ [16, 183, 184, 138], the more DT is synthesized from DD de-epoxidation, the higher is the NPQ and vice versa. To investigate whether a con-stitutive accumulation of DT may lead to an enhanced level or rate of NPQ induction, we generated mutants ofP. tricornutum overexpressing the DD de-epoxidase and investigated their ability to perform NPQ.

5.3. Materials and Methods

5.3.1. Cell cultivation

Cells ofPhaeodactylum tricornutum Bohlin (University of Texas Culture Collection, strain 646) wildtype (WT) and transformant cells were grown in 200mLsterile f/2 50 % medium [87] using Tropic Marin^rartificial seawater at 21^◦C in airlift columns continuously flushed with sterile air. The cultures were illuminated at a light intensity of 45 µmol·photons· m⁻²·s⁻¹ with white fluorescent tubes (OSRAM) with a 16:8hlight:dark cycle. When used, solid media contained 1.2 % Bacto Agar (Difco Lab., Becton Dickinson and Co., Sparks, MD, USA).

5.3.2. PCR and construction of plasmids

Polymerase chain reaction (PCR) was performed in a Master Cycler Gradient (Eppendorf, Hamburg, Germany) using recombinant Pfu polymerase (Fermentas, St. Leon Rot, Ger-many) according to the manufacturer’s instructions. The gene coding for the diadinox-anthin de-epoxidase was amplified using the primers DDE EcoRV 5’ (5’-GGCCAGatAtcG-TTACCATGAAGTTTC-3’) and DDE HindIII 3’ (5’-GGTAagCttGTCATTTATTGCTGGGAGG-3’). Small letters in the primer sequences indicate nucleotide substitutions to introduce restriction sites for cloning. The amplified product was ligated into thePhaeodactylum tricornutum trans-formation vector pPha-T1 (GenBank AF219942, [239]) resulting in the vector PTV-DDE.

The construct was sequenced (GATC Biotech AG, Konstanz, Germany) to ensure correct cloning.

5.3.3. Biolistic transformation

Cells were bombarded using the Bio-Rad Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, Canada) fitted with 1350 psi rupture discs. Tungsten particles (0.7 µm median diameter) were coated with approximately 5µg of plasmid DNA in the presence of CaCl₂ and spermidine, as described by the manufacturer. 24 h prior to bombardment approximately 10⁸ cells were spread in the center of a plate containing 20mL of solid culture medium. The plate was positioned at the second level within the biolistic chamber for bombardment. Bombarded cells were allowed to recover for 24h before being suspended in 1 mL of sterile 50 % artificial seawater medium. 250 µL of this suspension were plated onto solid medium containing 50 µg/mLZeocin. The plates were incubated at 20^◦C under constant illumination (40µmol·photons·m⁻²·s⁻¹) for three weeks.

5.3. Materials and Methods

5.3.4. Isolation of RNA, cDNA synthesis and real-time PCR assays

Cells were harvested during exponential phase of growth by centrifugation (1min, 5000 g) and crushed in a mortar using liquid nitrogen. Total RNA was isolated using a combi-nation of phenol/chloroform extraction with Trizol^r reagent (Invitrogen, Carlsbad, CA, USA) and the RNeasy Kit by Qiagen (Hilden, Germany). Genomic DNA (gDNA) contam-inations were removed using Turbo DNase (Ambion, Woodward, TX, USA) according to the manufacturer’s instructions. 280 ng gDNA free RNA was reverse transcribed with the QuantiTect^r reverse transcription kit (Qiagen, Hilden, Germany). Resulting cDNA was diluted 3-fold in RNase/DNase free water and one µL of this cDNA template was used in a 20µLqPCR reaction containing primers and DNA polymerase master mix with SYBR^r Green (MESA Green MasterMix, Eurogentec, Belgium). The reaction was heated to 95^◦C followed by 40 cycles for 15sat 95^◦C and 1min at 60^◦C. The amount of amplified DNA was monitored by measuring fluorescence at the end of each cycle using the Real-Time PCR System 7500 (Applied Biosystems, Lincoln, CA, USA). The Dde gene was analyzed using the following specific primer sets: 5’-ACATCTCAGCCGGACAAAACA-3’ and 5’-CCAATTCAGTTTGCC-GAAGAAC-3’. 18S rDNA served as endogenous control and was analyzed using the primers 5’-TGCCCTTTGTACACACCGC-3’ and 5’-AAGTTCTCGCAACCAACACCA-3’. Relative transcript levels were calculated using REST^r as described in [192].

5.3.5. Pigment extraction and analysis

For growth curves the Chlaconcentration was determined spectroscopically using the extinc-tion coefficient described in [113]. For pigment analysis, 1mLof cells was sampled and the pigment extracted in a 4^◦C cold methanol+ammonium acetate (90/10, v/v):ethyl acetate (90:10, v/v) mix. HPLC measurements were performed as described by [108]. The xan-thophyll de-epoxidation degree (DEP degree in %) was calculated as (DT/DD+DT)×100 where DD is diadinoxanthin, the epoxidised form and DT is diatoxanthin, the de-epoxidised form.

5.3.6. Chlorophyll fluorescence yield

Chl a fluorescence yield was monitored with an Imaging-PAM (Walz, Effeltrich, Germany).

Fluorescence was excited by a very weak (non-actinic) modulated 470 nm light. After 15 min dark-adaptation, continuous actinic light of adjustable intensity was applied with gradually increasing intensity from 0 to 1050 µmol·photons·m⁻² ·s⁻¹ through 9 steps.

Saturating pulses of blue light were used to monitor the evolution of fluorescence during actinic light exposure at the end of each light step. The average fluorescence measured during the pulses was taken asF_m orF_m’. For each experiment, the cells were concentrated in the dark as a homogenized cell layer to a final Chl a concentration of 20 µg Chl a·

mL⁻¹ (see [177]) on a Millipore A20 paper prefilter. It was controlled that the prefilter was kept wet during the measurement. Standard fluorescence nomenclature was used. F₀ and F_m are defined as the minimum PSII fluorescence yield of dark-adapted cells and the maximum PSII fluorescence yield reached in such cells during a saturating pulse of blue light, respectively. Non-photochemical fluorescence quenching was NPQ =F_m/F_m’−1, whereF_m’ is the maximum PSII fluorescence yield of light-adapted cells.

5.4. Results and Discussion

5.4. Results and Discussion

5.4.1. Transcript analysis of the Dde in the WT and the transformants

The gene coding for the diadinoxanthin de-epoxidase (DDE) was cloned into the vector pPHA-T1 as described in the Material and Methods part (chapter 5.3) and biolistically transferred into cells of P. tricornutum. From the resulting zeocin-resistant colonies 24 transformants were selected and subjected to transcript level analysis of the Dde gene by real-time PCR (data not shown). Four transformants showing a significant higher amount of transcripts compared to the WT were selected and used for further analysis. The relative transcript levels of these transformants are shown in figure 5.1. Compared to WT cells, the transformants ddeOE1 and ddeOE22 showed an increase in transcripts of about 50 fold, while the transcript levels of ddeOE4 and ddeOE16 was elevated by a factor of about 130 and 110, respectively, compared to WT cells.

relative dde expression

ddeOE1

ddeOE4

ddeOE16 ddeO

E22

(fold increase vs WT=1)

0 20 40 60 80 100 120 140

Figure 5.1.:Relative transcript level ofDdein the WT andDdeoverexpressing transformants ofP. tricornutumas calculated by the Relative Expression Software Tool (REST) [192]. Four independent measurements were per-formed. Transcript levels are relative to the WT and normalized to18S rDNA.Ddeis significantly upregulated in all transformants as calculated by the software. For details of the data see table A.6 on page 129.

5.4.2. Growth rate and pigment content of the WT and the Dde overexpressing transformants

Growth was evaluated by measuring the amount of chlorophyll a (Chl a) each day. There were no differences in theDdeoverexpressing transformants compared to the WT (figure 5.2) when the cells were grown under low light conditions (45 µmol·photons·m⁻²·s⁻¹). All

cell lines had a lag phase of two days before the exponential phase of growth started. Cells reached the stationary phase after five days of growth and the same final biomass was reached for the WT and the transformants.

0 1 2 3 4 5

0 1 2 3 4 5 6 7

WT ddeOE1 ddeOE4 ddeOE16 ddeOE22

Biomass (µg chl a per mL)

days

Figure 5.2.:Growth curves of WT cells and the Dde overexpressing transformants of P. tricornutum. Growth rates were measured by determining the amount of Chla(inµg·mL⁻¹). Values are average,±SD of three to five measurements.

The amounts of pigments were measured in the WT cells and in the selectedDde overex-pressing transformants grown at two light conditions. Cells were either dark- or low light-adapted for several hours. Pigment analyses show that when cells were light-adapted to darkness, no significant differences between the WT and the transformants were measured concerning the light harvesting pigments Chl c and fucoxanthin. β-carotene varied little among the different cell lines, indicating that the amount of photosystems per Chl a was similar in the WT and the transformants [135]. Still, further measurements need to be done to verify this as these data are based on only one measurement. Interestingly, the amount of xantho-phyll was different in the WT cells and in the transformants. While ddeOE4 showed similar amounts of diadinoxanthin (DD) as the WT, the amount was slightly increased in ddeOE1 or decreased in ddeOE16 and ddeOE22, respectively (table 5.1). The amount of diatoxan-thin (DT) was significantly increased in all transformants leading to elevated de-epoxidation degrees (DEP) compared to WT. This was surprising as the de-epoxidase usually is acti-vated by a lumen acidification due to light dependent linear electron transport. Possibly the enzyme was activated by a proton gradient originating from chlororespiratory electron flow [108, 109]. Although the dark period was rather short (8h), the increased amounts of enzyme might have led to an increase in DT synthesis and thereby to an increase of DEP. At low light conditions no significant differences in the amount of Chlc and fucoxanthin were

5.4. Results and Discussion

measured between the WT and the transformants, although the amounts of fucoxanthin were slightly decreased in comparison to dark-adapted cells. The amount of β-carotene was increased in all transformants compared to the WT, but as for the dark-adapted cells only one measurement was performed so far especially for the WT, so that further measurements are necessary to support these findings. Having a closer look at the xanthophyll cycle

measured between the WT and the transformants, although the amounts of fucoxanthin were slightly decreased in comparison to dark-adapted cells. The amount of β-carotene was increased in all transformants compared to the WT, but as for the dark-adapted cells only one measurement was performed so far especially for the WT, so that further measurements are necessary to support these findings. Having a closer look at the xanthophyll cycle

Im Dokument Molecular Mechanisms of Light Stress Protection in the Diatom Phaeodactylum tricornutum (Seite 76-0)