3. High light photoacclimation of diatom psbA mutants 23
3.5.4. How do diatoms cope with high light on a genetic and physiological
A summary of how diatoms acclimate to a light intensity just exceeding their photosynthetic capacity is shown in Figure 3.7. The WT cells reacted to the increase in light intensity by drastically enhancing the amount of photoprotective xanthophylls DD and DT. An increase in the DD pool size as well as a constitutive DT synthesis is usually reported in diatoms
3.5. Discussion
when cells are acclimated to a higher light intensity [133, 178, 189] to increase the efficiency of photoprotection through the amplitude of NPQ [133]. On the other hand, increase in NPQ might also be due to the up-regulation of LHCX genes and the probable concomitant synthesis of the respective polypeptides [242]. LHCX proteins are involved in NPQ in green microalgae [190]; their up-regulation under high light coupled with higher DT synthesis and NPQ [20, 90, 178, 189] makes them a strong candidate for a similar functional role in diatoms.
The increase in the DD pool size was not correlated with an up-regulation of the Dde gene, a feature also reported earlier [178]. Similarly, in higher plants, an inverse relationship between the amount of xanthophylls and the quantity of the enzyme responsible for their epoxidation has been reported [28]. Consequently, although the pool size of the DD de-epoxidase (DDE) substrate doubles, it appears that the concentration of enzyme is already high enough to ensure the fast and optimal de-epoxidation of DD in high light acclimated cells. Hence, the increase in DT synthesis seems to be only due to the parallel increase in the quantity of DD and not the increase in the concentration of the DDE [133, 178].
The increase in NPQ extent was paralleled by a decrease in the efficiency of the PSII CET.
Both photoprotective mechanisms are tightly related to each other [140, 133]. Decrease in PSII CET is possibly due to the reduction of the PSII antenna absorption cross-section and excitation pressure on the PSII, generated by the parallel decrease in pigment content [178]
and the increase in the capacity for dissipation of excitation energy (NPQ). This observation confirms that in contrast to NPQ, the PSII CET first deals with the acclimation to fast light fluctuations, in order to optimize photosynthetic flexibility, instead of acclimation to prolonged high light exposure. There was no up-regulation of the gene encoding the superoxide dismutase (SOD), an enzyme known to help scavenging the reactive oxygen species (ROS) during oxidative stress generated by high light exposure [233]. Most probably the ‘high light’ conditions used here were not stressful enough to generate any reaction in the WT cells, as reported before [201], the combination of PSII CET, NPQ and DT is sufficient to reduce the production of ROS, thus preventing lipid peroxidation [209]. Ultimately, the number of active PSII slightly decreased and was accompanied by a slight up-regulation of the gene encoding the D1 protein (psbA). This illustrates a minor PSII photodamage confirming the ability of photoacclimatory and photoprotective mechanisms examined here to help the cells to cope with an exposure at just saturating light intensities.
The parallel examination on physiological and molecular level revealed new features of diatom photoacclimation and photosynthetic gene regulation. V219I reacted in a similar as the WT, although up-regulation of theDde,Sod andpsbAgenes was slightly (×2) enhanced.
It probably reflects the minor photosynthetic impairment of photosynthesis (about 5–10 %) in this mutant compared to the WT [156]. In mutant F255I, although the DD+DT amount reached the same level as in the WT, DT synthesis was lower because of the constitutive significant ETR impairment, a feature probably accentuated by the prolonged high light
Figure 6
Schéma résumant la relation ETR, XC, NPQ avec effet DCMU et NH4Cl ?
Figure 3.7.:Summary of the reaction of the psbA mutants of P. tricornutum upon transfer to high light in comparison to WT cells. Left: the electron pathways whithin the PSII reaction center (see [156]); symbols:
red star, mutation; size of QA−
/QB−
, concentration of QA−
/QB−
; thickness of the e− arrows, value of the ETRmaxand of the QB−
to QA back-transfer; dotted feature of the OEC arrow, proportion of the disturbance of the OEC operation. Abbreviations: arrow up, increased value (the exact value is given in brackets); arrow down, decrease; flat arrow, no change; DT, diatoxanthin; PSII CET, photosystem II cyclic electron transfer;
NPQ, non-photochemical quenching of fluorescence quenching.
3.5. Discussion
exposure in comparison to the light conditions described above (see table 3.1, page 31). To compensate this drawback, Dde was up-regulated. As a consequence of a low DT amount, NPQ development was strongly impaired (see Fig. A.6) and LHCX genes were up-regulated possibly to increase the number of NPQ loci. Because NPQ remained relatively low regard-ing the light conditions, the PSII CET extent did not decrease in contrast to WT and V219I and remained high. Sod and psbAgenes were significantly up-regulated, reflecting the lack of photoprotection which ultimately led to a high PSII photodamage, a drop down of PSII photochemical quantum yield and an altered growth (see Fig. A.8, page A.8). S264A reacted similar as F255I but to a larger extent due to the fact that the synthesis of DD and DT were strongly impaired in this mutant. This was partially compensated by the up-regulation of Dde. Nevertheless, NPQ was kept to a level higher than F255I probably thanks to a high synthesis of LHCX proteins as illustrated by the strong up-regulation of Lhcx genes. Addi-tionally, the amplitude of the PSII CET was multiplied by 1.5 at 250µmol·photons·m−2·s−1 which allowed the cells to partially compensate the lower NPQ ability. Sod was up-regulated, as well aspsbAwhich surprisingly allowed the cells to restrict the PSII photodamage at the WT level and to maintain a high number of active PS II. All in all, although the photosyn-thesis of S264A is highly impaired (by about 50%; [156]), its characteristics were identical to the low light conditions (see Fig. A.6) thanks to strong up-regulation of the transcrip-tion of specific photoacclimatory genes. L275W showed a different situatranscrip-tion although its photosynthesis ability was impaired at the same level than S264A [156]. Indeed, even if the DD pool was lower than in the WT, it remained higher than in S264A. Most of all, L275W showed a high capacity for de-epoxidation generating high amount of DT (1.5 more than in the WT) probably by an up-regulation ofDde. Consequently, NPQ increased, also partially supported by a strong up-regulation ofLhcx genes, which resulted in maintaining NPQ at a level close to S264A and higher than F255I although L275W showed the lowest NPQ ability under low light conditions (see above). In parallel, the PSII CET slightly decreased to the same extent as in WT but still remained the highest of all types of cells examined. Both Sod and psbA showed the highest up-regulation. Such up-regulation was sufficient enough to maintain the destruction of active PSII to a level similar to the one observed under low light (see Fig. A.6). Although the PSII photodamage in L275W was high and its growth rate was the lowest, it was still able to produce biomass even under high light conditions (see Fig. A.8).
Conclusion
The mutants of this work allow to study the regulation of fast photoprotective NPQ and PSII CET processes, a topic increasingly shifting into the focus of current research [133].
In addition, the mutants provide first insights into the D1 repair cycle in diatoms, which
represents a previously uncovered area of research. More thorough investigation might help to understand how diatoms can acclimate to a prolonged high light stress when the linear electron transport activity is extremely deficient and how the redox state of the PQ pool can influence the regulation of photosynthetic gene expression under such environmental conditions [193]. The present mutants can help to elucidate how linear and cyclic electron transport and carbon assimilation [226] are coordinated and controlled in diatoms, the most important primary producers of the contemporary oceans [60].
Acknowledgements
This work was supported by the University of Konstanz, the DFG (grant LA2368/2-1 to JL), the European network MarGenes (QLRT-2001-01226 to PGK) and the land Baden-Württemberg (‘Stipendium der Landesgraduiertenförderung’ to SS). We thank I. Adamska (University of Konstanz), C. Bowler (ENS Paris) and C. Wilhelm (University of Leipzig) for the access to some of the instruments used here and for helpful discussions, D. Ballert for technical assistance, B. Rousseau (ENS Paris) and T. Jakob (University of Leipzig) for the help with some of the experiments. This work is part of the PhD projects of SS and ACM.
4. Silencing of the diadinoxanthin de-epoxidase (DDE) gene in Phaeodactylum tricornutum and its
consequence on the physiology of the cells
Arne C. Materna*, 1, Johann Lavaud*, 2, Sabine Sturm, Sascha Vugrinec and Pe-ter G. Kroth†
Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany
*These authors contributed equally to this work.
†Author for correspondence. E-mail: peter.kroth@uni-konstanz.de
1present address: Alm Laboratory, Civil and Environmental Engineering, Massachusetts Insti-tute of Technology (MIT), 77 Massachusetts Ave., 48-208, Cambridge, MA 02139, USA.
2present 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.
4.1. Abstract
Diatoms are a major group of phytoplankton ubiquitous in all marine and freshwater ecosys-tems. To protect themselves from photooxidative damage in a fluctuating underwater light climate, they have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms.
NPQ depends on the conversion of diadinoxanthin into diatoxanthin by the diadinoxanthin de-epoxidase. To study the role of the diadinoxanthin de-epoxidase in control of NPQ we generated transformants ofP. tricornutum in which the gene encoding for the enzyme (Dde gene) is silenced. For this purpose, RNA interference was induced by genetic transforma-tion of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or with a plasmid mediating the expression of a self-complementary hair-pin (inverted repeat, IR) like construct with a 5’-sense-overhang. The silencing approaches generated transformants with a phenotype clearly distinguishable from wildtype cells. Real-time PCR based quantification of Dde transcripts showed differences in transcript levels between AS strains and wildtype cells but also between AS and IR strains, suggesting the presence of two different gene silencing mediating mechanisms in diatoms. The physiological results confirm the tight dependency between the ability to de-epoxidize diadinoxanthin and to develop NPQ in diatoms.
Keywords
Phaeodactylum tricornutum ·antisense ·diadinoxanthin de-epoxidase·diatom · non-photo-chemical quenching·dsRNA·RNA interference ·violaxanthin de-epoxidase
Abbreviations
AS: antisense; Chl a and Chl b: chlorophyll a and b; DD: diadinoxanthin; DEP: DD de-epoxidation; DT: diatoxanthin; IR: inverted repeat; NPQ: non-photochemical chlorophyll fluorescence quenching; PAM: pulse amplitude modulation; WT: wildtype; XC: xanthophyll cycle.
4.2. Introduction
4.2. Introduction
Diatoms, together with cyanobacteria belong to the most abundant phytoplanktonic organ-isms in the world’s oceans and are therefore of great ecological relevance. The ecological success of diatoms is more than partially owed to their ability to tolerate or quickly accli-mate to the stress of rapidly changing light cliaccli-mates. Photosynthetic growth in fluctuating light intensities requires a fast responding mechanism to protect the organism from poten-tial damage by excess energy absorption at saturating light intensities. Plants and algae have evolved a number of protecting mechanisms including NPQ [102, 104]. NPQ mediates thermal dissipation of light energy absorbed in excess by the light-harvesting antenna com-plex (LHC) of PSII. This dissipation of the excess radiant energy is partly controlled by the interconversions between the carotenoids violaxanthin, antheraxanthin and zeaxanthin during the so-called xanthophyll cycle [42, 43, 58, 72]. The xanthophyll cycle in diatoms differs from the xanthophyll cycle in plants. It is simpler and involves different pigments: di-adinoxanthin (DD) is converted into diatoxanthin (DT) by the didi-adinoxanthin de-epoxidase [92]. The accumulation of DT was shown to be crucial for NPQ [16, 183, 184, 138], the more DT is synthesized from DD by de-epoxidation, the higher the NPQ and vice versa, the relationship being especially true for P. tricornutum [138, 137, 134]. In higher plants, Niyogi and co-workers showed that, when the gene coding for the de-epoxidase is mutated or its transcription is inhibited, NPQ is much lower [175].
Since ‘antisense-mediated silencing’ has been discovered in 1995 [91, 63], double-stranded RNA (dsRNA) was proven to be an extremely potent activator of RNA interference [157].
DsRNA can originate from different sources. Endogenous sources include, for instance, short forms of fold-back dsRNA [18] which are the precursor molecules of micro RNAs (miRNAs) (reviewed by [10]). Another source of endogenous dsRNA is a class of enzymes called RNA-directed RNA polymerases (RDR). Abnormal or ‘aberrant’ transcripts are recognized by RDR and subsequently converted into endogenous dsRNA by primer-independent synthesis of complementary RNA [208, 46, 19, 153]. Both, dsRNA and miRNA precursors are pro-cessed by Dicer proteins to small interfering RNA (siRNA) or miRNA, respectively (reviewed by [157, 159]. Subsequently these small RNA products are rearranged to form the RNA-induced silencing complex (RISC) [95] or the miRNA containing effector complex (miRNP) [167], which both guide distinct protein complexes to the target RNAs. While RISC medi-ates the target mRNA degradation, miRNP can also guide translational repression of target mRNAs [157]. Since dsRNA triggers these RNAi processes the experimental introduction of dsRNA into target cells became a powerful tool for functional genomics specifically me-diating gene silencing. Experimental introduction of complementary RNA molecules into target cells to form dsRNA can be achieved via transgene transcription or micro injection of small interfering RNAs (siRNA).
Although little is known about the gene silencing mechanisms in diatoms, successful
sup-pression of the exsup-pression of two endogenous genes by gene silencing with inverted repeats was recently shown to be feasible in the diatom P. tricornutum [39]. We studied the role of the diadinoxanthin de-epoxidase (DDE) in fast photoprotective processes and the reg-ulation of photosynthesis in diatoms by testing techniques known to induce specific RNA interference. Cells were transformed with plasmids that allow the transcription of transgenic fragments designed to either hybridize with complementary Dde transcripts or to contain complementary inverted repeats folding back on themselves to form dsRNA hairpins [229].
4.3. Materials and Methods
4.3. Materials and Methods
4.3.1. Cell cultivation and preparation for physiological measurements
P. tricornutum wildtype and transformants cells were grown photoautotrophically in sterile 50 % artificial seawater f/2 medium [87]. Cultures of 200 mL were incubated at 21◦C in airlifts continuously flushed with sterile air. They were illuminated at a light intensity of 45 or 135µmol·photons·m−2·s−1 (respectively ‘low light-LL’ and ‘high light-HL’) with white fluorescent tubes (OSRAM) with a 16:8 h light:dark (L:D) cycle. Cells were harvested during the exponential growth phase, 2±0.5 h after the onset of light in the morning.
Specific growth rates,µ(d−1), were calculated from regression of natural logarithm of culture chlorophylla (Chl a) concentration or fluorescence during the exponential growth phase of acclimated cultures.
4.3.2. PCR and construction of plasmids
PCR was performed with a Master Cycler Gradient (Eppendorf, Hamburg, Germany) using recombinant Pfu polymerase (Fermentas, St.Leon-Rot, Germany) according to the manu-facturer’s instructions. Small letters in primer sequences indicate degenerated nucleotides (table A.4 on page 127). All antisense (AS) fragments were amplified from the N-terminal, less conserved part of the diadinoxanthin de-epoxidase (Dde) gene in order to avoid any unspecific hybridization of antisense transcripts with transcripts of the two de-epoxidase like or de-epoxidase related genes (see [33]). The fragments have been inserted into the Phaeodactylum tricornutum transformation vector pPha-T1 [12], which allows transforma-tion of the diatom selecting for positive transformants using Zeocin (Invitrogen, Carlbad, CA, USA). The 198 bpDdeantisense fragment was amplified using the primers DDE-198AS-HindIII-5’ and DDE-198AS-BamHI-3’, the 523 bp fragment was amplified using the primers DDE-523AS-HindIII-5’ and DDE-523AS-BamHI-3’. Inserts were ligated in antisense ori-entation into the plasmid downstream of the FcpA promoter. To construct the pPha-T1 IR (inverted repeat) plasmid several subsequent cloning steps were performed. First a 293 bp fragment of the Dde gene was amplified using the primers DDE-293AS-HindIII-5’
and DDE-523AS-BamHI-3’ and cloned in pPha-T1 in antisense orientation giving rise to pPha-T1 AS293. In the following steps a fragment containing the FcpA 5’ untranslated region (UTR) downstream of the FcpA promoter followed by eGFP was amplified from a pPha-T1 vector - which contains eGFP - using the primers SgfI-NcoI-pTV-MCS-5’ and pTV-MCS-BamHI-PmeI-3’. Further a 523 bp long fragment ofDde was amplified using the primers SgfI-EcoRI-DDE-523-5’ and DDE-523-NcoI-PmeI-3’. Both fragments were ligated in the pF1-A directional cloning vector (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The resulting vectors were termed pF1-A DDEnc S and pF-1A pTV-MCS. pF1-A DDEnc S was digested with Ecl136II and FspI, pF-1A pTV-MCS was
digested with PvuII and FspI. For both plasmids the half containing the respective insert was separated by horizontal gel-electrophoresis and eluted from the agarose gel. Both parts were ligated, thus giving rise again to complete pF1-A vector, which contains the 523 bp Dde fragment and the 5-UTR-eGFP construct, now ligated together. The resulting Dde (sense)-5’-UTR-eGFP insert was amplified from the plasmid using the primers DDE-523AS-HindIII-5’ and pTV-MCS-BamHI-PmeI-3’. The amplicon was subsequently digested with BamHI. After digesting the plasmid pPha-T1 AS293 withEcoRV andBamHI the digested amplicon was ligated into the plasmid, thus giving rise to the final transformation vector pPha-T1 IR.
4.3.3. 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 (0.7µmmedian diameter) were coated with 5µgof plasmid DNA in the presence of CaCl2 and spermidine, as described by the manufacturer. One hour prior to bombardment approximately 108 cells were spread in the center of a plate containing 20 mL 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 24hbefore being suspended in 1 mLof sterile 50 % artificial seawater medium. 250 µL of this suspension were plated onto solid medium containing 50µg/mLZeocin. The plates were incubated at 20◦C under constant illumination (40µmol·photons·m−2·s−1) for three weeks.
4.3.4. Isolation of RNA and cDNA synthesis
Cells were harvested by brief centrifugation (1 min at 2800 g) at room temperature. The pellet was immediately shock-frozen in liquid N2. Cell pellets were homogenized by grinding the frozen pellets under liquid N2. 1 mL Trizolr (Invitrogen, Carlsbad, CA, USA) was added to the deep frozen grinded powder, which was further homogenized in subsequent steps by vortexing and shaking the solution at room temperature. After adding 200µL of chloroform, rigorous mixing and centrifugation at 4◦C, the aqueous phase was transferred into pre-cooled tubes. After adding 1 volume of ethanol (70 %) the solution was transferred into RNeasyr RNA purification columns (Qiagen, Hilden, Germany). Subsequent RNA purification steps were performed according to the manufacturer’s instructions using the provided RW1 and RPE buffers. Although both Trizolrand the RNeasyr purification are designed to remove genomic DNA from the RNA extract, we additionally treated aliquots of the extracted RNA with TurboTM-DNase (Ambion, Woodward, TX, USA). The obtained genomic DNA-free RNA was reverse transcribed using the reverse transcriptase provided by the QuantiTectr reverse transcription Kit (Qiagen, Hilden, Germany). Complete removal of genomic DNA from RNA samples was verified by PCR amplification of intergenic regions
4.3. Materials and Methods
after cDNA synthesis. The resulting genomic DNA free cDNA was further used for real-time PCR assays.
4.3.5. Real-time PCR assays
Real-time PCR was performed using the Real-Time PCR System 7500 (Applied Biosys-tems, Lincoln, CA, USA). The following program was utilized for all genes: 10 min of pre-incubation at 95◦C followed by 40 cycles for 15 s at 95◦C and 1 min at 60◦C. Indi-vidual real-time PCR reactions were carried out in 20 µL volumes in 96-well plates using Power SYBRrGreen PCR Master Mix and optical covers by Applied Biosystems. For am-plification of the target (Dde) fragment the primers RT-DDE-629-fw and RT-DDE-729-rev (see table A.4) were used, the endogenous control fragment (Gapdh) was amplified using the primers RT-GapDH-775-fw and RT-GapDH-875-rev. All samples were analyzed in six replicates per experiment and each experiment was repeated independently at least twice.
At the end of each reaction, the cycle threshold (Ct) was manually set at the level that
At the end of each reaction, the cycle threshold (Ct) was manually set at the level that