Figure 1.
Vector map of the aureochrome 1a silencing construct.
The sequence of the NR promoter controlled hairpin construct is given. The sequence is colour coded corresponding to the vector map: NR promoter (black), aureochrome 1a sense (intense green), NTT1 intron (yellow) and aureochrome 1a antisense (light green). Active and inactively fused restriction sites are given in small letters in colour coding: KpnI (light blue), HpaI (purple), inactive fusion of StuI and HpaI (grey) and XbaI (dark red).
Table 1.
Incident light intensities and cellular parameters.
Figure 2.
Phylogenetic analysis of putative aureochromes from different stramenopiles.
The maximum likelihood tree was calculated using PhyML 3.0 [29] and incorporates 32 putative aureochrome sequences of twelve different stramenopiles identified by the unique bZIP/LOV domain setup. Numbers at nodes of subtrees correspond to bootstrap values greater than 45. The accession numbers correspond either to Protein IDs (only digits) from the Joint Genome Institute database (JGI; http://www.jgi.doe.gov/) or to the accession numbers (two letters and digits) from the database of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). Putative aureochromes of Phaeodactylum tricornutum (Pt), Fragilariopsis cylindrus CCMP 1102 (Fc), Pseudo-nitzschia multiseries (Pm), Thalassiosira pseudonana (Tp), Thalassiosira oceanica (To), Ectocarpus siliculosus (Es), Fucus distichus subsp. Evanescens (Fd), Aureococcus anophagefferens (Aa), Chattonella marina var. Antiqua (Cm), Ochromonas danica (Od), Nannochloropsis gaditana CCMP526 (Ng) and Vaucheria frigida (Vf) were taken into account. Four distinct groups of aureochromes could be identified highlighted by coloured markings. The different groups were designated aureochromes 1, 2, 3 and 4 with aureochromes 1 and 2 corresponding in homology to the aureochromes 1 and 2 of V. frigida [23], respectively.
Figure 3.
Localisation of GFP-fusion proteins of P. tricornutum aureochromes.
Maximum intensity z-projections of LSM analyses are shown. From left to right: GFP fluorescence (green), nucleus staining Hoechst 33342 dye (cyan), chlorophyll autofluorescence (red) and a merge of all channels with a representative DIC single plane. The white scale bars correspond to 10 µm. All aureochromes feature a distinct nuclear localisation. AUREO1a fusion proteins often exhibit additional cytosolic signals (Figure S6), as can be seen here for AUREO1a. GFP_control is a transformed cell line of P. tricornutum featuring the enhanced GFP protein (GenBank Accession number AAB08060.1), which is missing any targeting sequence. It serves as reference for cytoplasmatic localisation. Here, the z-projections imply a co-localisation in the nucleus as well, but orthoview analysis of the LSM data revealed that the GFP fluorescence was only accumulating around the nucleus, while it co-localises in case of the aureochromes (Figure S2).
Figure 4.
A) Exemplary relative quantification of AUREO1a concentration in protein extracts of P. tricornutum WT, aureo1a-15 and aureo1a-50 by an immunoblot using an antiserum specific for P. tricornutum AUREO1a.
Cultures were grown with either nitrate or ammonium as sole nitrogen source. Nitrate activates the promoter of the applied silencing construct resulting in a decreased amount of AUREO1a protein. Several dilutions of the protein extract of the ammonium grown aureo1a-15 culture were loaded on the gel in order to assess the efficiency of AUREO1a downregulation. Two co-regulated bands are visible, one at the expected size of AUREO1a, 41.5 kDa (indicated by arrows), and a weaker band at about 47 kDa, which possibly reflects a post-translational modification of the protein. B) Loading control of protein extracts used for immunoblotting. The gels used for immunoblotting and as loading control were loaded with identical amounts of protein. The proteins of the loading control gels were stained with colloidal Coomassie. +: Purified heterologously expressed AUREO1a with His-tag.
Figure 5.
Photosynthesis rates of aureo1a-15 (grey triangles) and aureo1a-50 (white circles) P. tricornutum cultures depending on the incident light intensity in the measuring cuvette; corresponding WT data of Schellenberger Costa et al.
[28] (black squares) are included for better comparison. Algae were cultivated at a QPhar of 10 µmol absorbed photons m−2 s−1 (LL) under illumination with blue (A) and red light (B) and further at a QPhar of 30 µmol absorbed photons m−2 s−1 (ML) under illumination with blue (C) and red light (D). Mean values are shown with standard deviation (n = 9). Maximum photosynthesis rates of each culture condition were tested for the occurrence of significant differences between WT and aureochrome 1a silenced strains using a one-way ANOVA followed by a Holms-Sidak pair-wise comparison test with the WT as control group. Significant differences are marked with asterisks (p<0.05).
Figure 6.
Non-photochemical quenching (NPQ) of aureo1a-15 (grey triangles) and aureo1a-50 (white circles) P. tricornutum cultures depending on the incident light intensity in the measuring cuvette; corresponding WT data of Schellenberger Costa et al.
[28] (black squares) are included for better comparison. Algae were cultivated at a QPhar of 10 µmol absorbed photons m−2 s−1 (LL) under illumination with blue (A) and red light (B) and further at a QPhar of 30 µmol absorbed photons m−2 s−1 (ML) under illumination with blue (C) and red light (D). Mean values are shown with standard deviation (n = 9). The maximum NPQ of each culture condition was tested for the occurrence of significant differences between WT and aureochrome 1a silenced using a one-way ANOVA followed by a Holms-Sidak pair-wise comparison test with the WT as control group. Significant differences are marked with asterisks (p<0.05).
Table 2.
Diadinoxanthin (Ddx) concentration and de-epoxidation state (DES).