Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms 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 (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms

Proteinimport in Plastiden von Kieselalgen

Diatoms, like many other algal groups, evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent reduction and specialisation to a “complex” plastid. Comparable to the evolution of primary plastids, targeting mechanisms had to be developed to reimport preproteins into the plastid. Seven putative subunits of the translocons at the inner envelope membrane of chloroplasts (Tic) are encoded in the genome of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Fusion proteins of Tic presequences or full length fusions to GFP show that the investigated Tics are plastid associated. Fusion proteins consisting of bipartite plastid targeting presequences from various algal groups show that they are also functional when heterologously expressed as GFP fusion proteins in the diatom P. tricornutum. Interestingly, also the modified signal peptide of a carbonic anhydrase from Arabidopsis thaliana, which apparently is targeted to A. thaliana plastids via the endoplasmic reticulum, is able to direct GFP into P. tricornutum plastids. This indicates that a conserved transport route is used for protein import into all secondary plastids, and that this route might be related to the signal peptide dependent route to plastids of higher plants. Colocalisation analyses suggest that native and artificial presequences from diatoms lead to an accumulation of GFP in a “blob”-like structure and that this structure is identical in both cases. At the moment, several models for the import of proteins into the “complex” plastids of diatoms are discussed. The models differ in the way they explain transport from the CER into the interenvelope space (ies). In the “pore model” it is proposed that a connection between the CER lumen and the ies might route nucleus encoded proteins across the periplastidic space. With the aid of a self-assembling GFP system we could show that proteins cannot reach the ies from the CER on a direct way. Furthermore from these results it can be concluded that nucleus-encoded plastid proteins from diatoms pass the four plastid envelope membranes via translocators and not via pores or vesicles which are also proposed

Protein targeting into complex diatom plastids: functional characterization of a specific targeting motif

Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence: GFP fusion proteins with or without modifications of the "ASAFAP"-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import

A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids

Background: Light, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages. Results: Here, we demonstrate that the recently discovered “red lineage chlorophyll a/b-binding-like proteins” (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities./nConclusions: The absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light shifts as regularly experienced by diatoms in nature. Remarkably, in plastids of the red lineage as well as in green lineage plastids, the phycobilisome based cyanobacterial light harvesting system has been replaced by light harvesting systems that are based on members of the extended LHC protein superfamily, either for one of the photosystems (PS I of red algae) or for both (diatoms). In their proposed function, the RedCAP protein family may thus have played a role in the evolutionary structural remodelling of light-harvesting antennae in the red lineage.This work was supported by the Universität Konstanz and by grants of the Deutsche Forschungsgemeinschaft (KR 1661/3 to PGK, AD 92/7-2 to IA, LA 2368/2-1 to JL). JE was supported by a grant (I/82 750) from the Volkswagenstiftung (“Förderungsinitiative Evolutionsbiologie”

A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids

Background: Light, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages. Results: Here, we demonstrate that the recently discovered “red lineage chlorophyll a/b-binding-like proteins” (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities./nConclusions: The absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light shifts as regularly experienced by diatoms in nature. Remarkably, in plastids of the red lineage as well as in green lineage plastids, the phycobilisome based cyanobacterial light harvesting system has been replaced by light harvesting systems that are based on members of the extended LHC protein superfamily, either for one of the photosystems (PS I of red algae) or for both (diatoms). In their proposed function, the RedCAP protein family may thus have played a role in the evolutionary structural remodelling of light-harvesting antennae in the red lineage.This work was supported by the Universität Konstanz and by grants of the Deutsche Forschungsgemeinschaft (KR 1661/3 to PGK, AD 92/7-2 to IA, LA 2368/2-1 to JL). JE was supported by a grant (I/82 750) from the Volkswagenstiftung (“Förderungsinitiative Evolutionsbiologie”

NPQ development in WT and the <i>Dde</i> transformants of <i>Phaeodactylum tricornutum</i>.

<p>Cells were grown at low light (45 µmol photons⋅m⁻²⋅s⁻¹) before the experiment. (A) and (B) show the respective NPQs as a function of light intensity for a 5 min light exposure, while in (C) and (D) NPQ was measured as a function of time at an irradiance of 450 µmol photons⋅m⁻²⋅s⁻¹. Values are averages ± standard deviation (SD) of three to four measurements.</p

Schematic vector maps of the silencing constructs used for transformation.

<p>Anti-sense constructs: <i>Dde</i> fragments of 198 or 523 bps were cloned in anti-sense orientation downstream of the <i>fcpA</i> promoter (pDDE-AS198 and pDDE-AS523). Inverted repeat constructs: <i>Dde</i> fragments of 293 and 523 bp lengths were cloned in sense and anti-sense orientation. The two fragments were linked with an <i>eGFP</i> fragment supposed to function as spacer. Amp: Ampicillin resistance; Zeo: Zeocin™ resistance; fcpA: Fucoxanthin Chlorophyll <i>a</i>/<i>c</i>-binding Protein A promoter; eGFP: enhanced green fluorescent protein.</p

Diadinoxanthin and diatoxanthin (DD and DT) content and de-epoxidation state (DES) of the WT and <i>Dde</i> transformants of <i>Phaeodactylum tricornutum</i> grown under low light (LL).

<p>Diadinoxanthin (DD) and diatoxanthin (DT) content (in mol/100 mol Chl <i>a</i>), and the de-epoxidation state (DES) of the WT (+/− dithiothreitol, DTT) and the <i>Dde</i> transformants of <i>P. tricornutum</i> cells (LL grown) after a 5 min 450 µmol photons⋅m⁻²⋅s⁻¹ light treatment. DES (in%)  =  DT/(DD+DT) × 100. Values are averages ± SD of three to five measurements.</p

Pigments and photosynthetic properties of WT and the <i>Dde</i> transformants of <i>Phaeodactylum tricornutum.</i>

<p>Pigment content (in mol/100 mol Chl <i>a</i>) and photosynthetic properties of the WT and the <i>Dde</i> transformants of <i>P. tricornutum</i> cells grown under low light (45 µmol photons⋅m⁻²⋅s⁻¹). Chl <i>a</i> is given in pg cell⁻¹, DD: diadinoxanthin, F_v/F_m: the maximum photosynthetic efficiency of photosystem (PS) II, rETR_max: is the relative maximal rate of linear electron transport (µmol photons⋅m⁻²⋅s⁻¹), α: the maximum light used efficiency (in µmol photons⋅m⁻²⋅s⁻¹), E_m: the light intensity needed to reach rETR_max in µmol photons⋅m⁻²⋅s⁻¹, μ: the growth rate in d⁻¹. Values are averages, ± SD of seven to nine measurements for the pigment data (except Chl <i>a</i>, three measurements) and three to four measurements for the other data.</p

Relative quantification of <i>Dde</i> transcripts in the WT and five <i>Dde</i> transformants of <i>Phaeodactylum tricornutum</i>.

<p>Transcript levels are relative to the WT and normalized to <i>Gapdh</i> expression. Values are averages of at least two replicates. Black bars: total RNA, untreated RNA used for qPCR; Grey bars: ssRNA, single stranded RNA (total RNA treated with RNaseIII).</p