AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles

Abstract

A blue light (BL) receptor was discovered in stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae). Two homologs were identified in Vaucheria; each has one basic region/leucine zipper (bZIP) domain and one light–oxygen–voltage (LOV)-sensing domain. We named these chromoproteins AUREOCHROMEs (AUREO1 and AUREO2). AUREO1 binds flavin mononucleotide via its LOV domain and forms a 390-nm-absorbing form, indicative of formation of a cysteinyl adduct to the C(4a) carbon of the flavin mononucleotide upon BL irradiation. The adduct decays to the ground state in ≈5 min. Its bZIP domain binds the target sequence TGACGT. The AUREO1 target binding was strongly enhanced by BL treatment, implying that AUREO1 functions as a BL-regulated transcription factor. The function of AUREO1 as photoreceptor for BL-induced branching is elucidated through RNAi experiments. RNAi of AUREO2 unexpectedly induces sex organ primordia instead of branches, implicating AUREO2 as a subswitch to initiate development of a branch, but not a sex organ. AUREO sequences are also found in the genome of the marine diatom Thalassiosira pseudonana (Bacillariophyceae), but are not present in green plants. AUREOCHROME therefore represents a BL receptor in photosynthetic stramenopiles.

Blue light (BL) (350–500 nm) plays an essential role in the lives of many organisms. For land plants, BL is an important environmental factor that serves to optimize their photosynthetic potential through responses such as phototropism. Of all plant BL responses examined to date, phototropism is essential for the orientation of growing organs toward or away from the source of light. Indeed, there is a long history of the study of phototropism. However, it was not until the discovery of phototropin (phot), the photoreceptor for phototropism (1), that our knowledge of the photoperception processes underlying plant BL responses was greatly enhanced. It is now evident that not only phototropism (2) but also chloroplast movements (3) and stomatal opening (4) in land plants are mediated by phot (5, 6).

In contrast, little is known regarding the photodetection mechanisms found in plants living in aquatic environments. Photosynthetic stramenopiles such as Vaucheria (3, 7–10), brown algae (11–13), and diatoms (14) are known to use BL for light regulation of their behavior and life cycle. In the marine environment, BL is predominant because light of shorter and longer wavelengths cannot penetrate the thick water mass (15). This may be the reason that these species respond mainly to BL.

Photosynthetic stramenopiles are phylogenetically different from green plants because they originated from red algal symbionts and nonphotosynthetic eukaryotic hosts through secondary endosymbiosis (16). Photosynthetic stramenopiles are found in widely diverse aquatic environments, ranging from oceans, brackish lakes, ponds, and rivers to moist soil. These organisms are important not only from an ecological perspective but also as energy and biomass resources. Despite their importance, only limited groups of stramenopile species have been investigated, probably owing to the difficulties associated with culturing these organisms under laboratory conditions. Moreover, the presence of extracellular mucilage on many of them has impeded their study.

Among photosynthetic stramenopiles, the xanthophycean multinucleate algae in the genus Vaucheria is exceptionally suitable for cell biology studies and gene cloning. Vaucheria can be cultured easily and lacks extracellular mucilage. The body of Vaucheria consists of a sparsely branched, multinuclear tube, and there is usually no segregation wall unless they have been injured. Active tip growth and positive or negative phototropic bending occurs at the apex of each branch (7). Phototropism in Vaucheria was first described by Oltmanns as early as 1892 (17) and subsequently analyzed in detail by Kataoka (7–9). BL-induced chloroplast movement of Vaucheria was also described by Senn in 1908 (3) and later studied extensively by Blatt and Briggs (18). BL-induced branching in Vaucheria has also been reported (8). Light-mediated branching in Vaucheria is an example of photo(cyto)morphogenesis in stramenopiles and appears to be of primary importance for understanding the spatial control of plant development. Irradiation of a narrow region of the cylindrical Vaucheria cell with a moderate intensity of BL can induce a new center of tip growth in the irradiated region (8). Branch induction requires the accumulation of nuclei in the irradiated region from adjacent shaded region (10). Expression of an as yet unidentified set of gene targets in the BL-irradiated nuclei is hypothesized to occur in advance of branch initiation (10).

Whether these BL responses of Vaucheria are mediated by phototropin-related receptors or other photoreceptor systems is an important question, not only for surveying the occurrence of BL receptors among photosynthetic organisms but also for investigating further the biochemical mechanisms underlying phototropism and photomorphogenesis in plants. Here we report that Vaucheria frigida, Fucus distichus, and the marine diatom Thalassiosira pseudonana possess a class of BL receptors that is distinct from but related to the phototropin photoreceptors and discuss their role in BL-induced photomorphogenesis.

Results

Structure of AUREOCHROMEs.

Light–oxygen–voltage (LOV) domains are associated with many BL receptor molecules other than the phototropins, including circadian clock regulators found in both plants (ZTL, FKF1, LKP2) and fungi (WC-1, VVD), that are known to bind flavin as a chromophore (19). We thus searched for LOV-domain-encoding sequences within the genomes of the yellow–green alga V. frigida and the brown alga F. distichus by means of RT-PCR, by using degenerate primers (see Materials and Methods). We isolated four LOV fragments from V. frigida and one LOV fragment from Fucus, and isolated full-length cDNAs containing the two most abundantly cloned fragments in V. frigida by using 3′- and 5′-RACE. The sequences obtained encoded similar proteins composed of 348 and 343 aa, respectively, each having one putative basic region/leucine zipper (bZIP) transcription-regulation domain located at the center of the protein and a single LOV domain near the carboxyl end (Fig. 1A). We named these encoded proteins AUREOCHROMEs (AUREO1 and AUREO2), because stramenopile species are typically golden-yellow in color.

Fig. 1.

Structure and aligned sequences of AUREOCHROMEs from V. frigida and their orthologs. (A) bZIP and LOV domains are colored red and blue, respectively. Green vertical lines with asterisks indicate the position of introns. (B) Sequence alignment of AUREO1 and AUREO2. Red and blue frames indicate bZIP and LOV domains, respectively. Basic amino acids are in orange, and heptad leucine residues of bZIP domains are in red. The asterisks indicate identical amino acids between AUREO1 and AUREO2. Amino acid residues that are 100% identical and 100% similar to all 37 LOV domains thus far identified (19) are in black and blue backgrounds, respectively. Residues identical and similar to Arabidopsis thaliana phot1–LOV2 (AAC01753) are in green and yellow, respectively. The exclamation marks denote the 11 conserved amino acid residues that are associated with flavin binding (19). Note that all of these residues are perfectly conserved in AUREO1; but in AUREO2, only 9-aa residues are conserved, i.e., I and F are replaced by L and V, respectively. Marks E and K are the position of amino acid residues that are conserved in all other LOV domains identified to date that form a surface salt bridge (19). (C) Alignment of AUREO orthologs found in cDNA of F. distichus (Fd) and in the Thalassiosira pseudonana (Tp) genome (21). Upper rows are for the bZIP region (AUREO1Z, AUREO2Z, FdZ, Tp1Z, Tp2Z, and Tp3Z); lower rows are for the LOV region (AUREO1L, AUREO2L, FdL, Tp1L, Tp2L, and Tp3L). AUREO orthologs of Thalassiosira were found by a BLAST search (http://genome.jgi-psf.org/thaps1/thaps1.home.html). Amino acid residues that are 100% identical have a red or blue background; those that are 80% similar have a yellow background. Note that TpAUREO1 has 11 identical amino acids necessary for binding with FMN.

The LOV domains of AUREOs are similar to the LOV1 and LOV2 domains of plant phots. AUREO1 and AUREO2 conserve respectively 11- and 9-aa residues necessary for the flavin mononucleotide (FMN) binding and cysteinyl adduct formation (19) (Fig. 1B, red exclamation points), suggesting that these proteins may also function as BL receptors. However, no homolog of a phototropin was found in V. frigida. Although circadian clock regulators such as WC-1 from the filamentous fungus Neurospora crassa contain a characteristic insertion of 11 aa in the middle of LOV domain, AUREOs lack this insertion, as do all phototropin LOV domains (19). AUREO LOV domains also lack a Glu-Lys salt bridge that is conserved in all other known LOV domains (19).

We found through a separate analysis of V. frigida genomic DNA that the AUREO1 gene lacks introns, whereas AUREO2 contains two introns, 61 base pairs from the N-terminal end and 840 base pairs from the N-terminal end in the junction between the bZIP domain and the LOV domain. The complete absence of introns in the AUREO1 gene suggests that it may have arisen by retrotransposition, as was proposed to be the case for the fern NEOCHROME gene (20). Southern blot analysis suggests that four copies of AUREO are present in the V. frigida genome (data not shown).

We found putative orthologs of AUREO in mRNA isolated from zygotes of F. distichus. Although the full-length sequence is not shown here, bZIP and LOV domains (FdZ and FdL) can be clearly seen in an ortholog at the corresponding positions found in V. frigida AUREOs (Fig. 1C). Of the 11 aa residues of the LOV domains required for FMN-binding, nine are identical and two are conservative substitutions in F. distichus. In addition, the connecting domain between the bZIP and LOV domains of V. frigida and F. distichus AUREOs contains many identical or similar amino acids indicating that this region is also highly conserved (data not shown). Furthermore, from blast searches, we found three AUREO-like genes in the genome of the marine centric diatom Thalassiosira pseudonana (21) (Fig. 1C). Orthologs of AUREOs are also seen in genome of the marine pennate diatom Phaeodactylum tricornutum (http://genome.jgi-psf.org/Phatr2/Phatr2.home.html). However, PHOT-encoding genes are absent in the T. pseudonana genome (21). Likewise, no phot ortholog was found in mRNA of V. frigida or F. distichus zygotes suggesting that phots are not present in stramenopiles.

AUREOCHROME as a BL Photoreceptor.

To determine the ability of AUREOs to bind flavin, fusion proteins of a calmodulin-binding peptide (CBP) and the LOV domains of V. frigida AUREO1 and AUREO2 were expressed in Escherichia coli. E. coli expressing LOV of AUREO1, but not AUREO2, became yellow in color, suggesting that AUREO1 bound flavin. The purified LOV domain of AUREO1 showed an absorption spectrum typical of a flavoprotein (Fig. 2A). FMN was chromatographically confirmed to be the flavin species attached to the LOV domain (Fig. 2B). After irradiation with strong BL (470 nm, 500 μmol m⁻² s⁻¹, 1 min) absorption peaks of the LOV domain of AUREO1 between 410 and 500 nm greatly decreased, whereas the absorption ≈390 and <340 nm increased (Fig. 2C). However, this change in absorbance decayed within 20 min, indicating the light-activated formation of a C(4a) cysteinyl adduct and its slow return to the ground state in darkness (22). As the ground state is fluorescent but the adduct state is not, in vitro photochemical analysis of the excitation-relaxation cycling of the LOV domain of AUREO1 was performed by measuring the recovery of fluorescence after photoexcitation. Fig. 2D clearly indicates that the rate of dark reversion (t½) from an excited state was ≈4.9 min at room temperature. In contrast, E. coli expressing the LOV domain of AUREO2 did not become yellow. Changes of the highly conserved Ile and Phe in AUREO1 to Leu to Val, respectively in AUREO2, might affect the binding capacity of AUREO2 for FMN. However, the possibility of AUREO2-FMN binding in the V. frigida cell cannot be excluded, as the heterologously produced peptide may simply not fold properly.

Fig. 2.

AUREOCHROME is an FMN-binding photoreceptor. (A) Absorption spectrum of purified CBP–LOV_AUREO1. (B) Thin-layer chromatography showing that the chromophore of AUREO1 is FMN. Positions of authentic flavin mixture (Left) and the LOV domain of AUREO1 (Right) are shown. The asterisks indicate the baselines. (C) Absorbance difference spectrum after a 1-min BL pulse (460 nm; 500 μmol m⁻² s⁻¹). From top to bottom: before the BL pulse (−1 min); 0 min after the pulse; 3 min after the pulse in darkness; 30 min in darkness. (D) Kinetics for the dark recovery of the LOV domain of AUREO1 after irradiation. The log percentages of the difference between initial and second fluorescence peaks that correspond to the amount of remaining photoproduct produced by the first pulse of light are plotted vs. the length of the dark period before the second pulses. The line best fitting the data points intersects the ordinate close to log 2 (= 100%). As indicated with dashed lines, the half-life of the photoexcited state is ≈4.9 min at room temperature.

AUREOCHROME as Transcription Regulator.

To determine the role of AUREOCHROME as a transcription factor, its nuclear localization and its ability to bind DNA were first examined (Fig. 3). A cauliflower mosaic virus (CaMV) 35S promoter driven GFP–AUREO1 fusion protein expressed in onion epidermal cells was found localized in both the nucleus and the cytoplasm (Fig. 3A). In contrast, GFP–AUREO2 was localized exclusively in the nucleus. Nuclear localization in combination with their bZIP domain therefore supports a role for AUREOCHROMEs as light-sensitive transcription factors. Unfortunately, no expression of these GFP fusion proteins could be detected in V. frigida, suggesting either that the CaMV 35S promoter is not functional in stramenopiles or that the codon usage in the GFP was not optimized.

Fig. 3.

AUREOCHROME is a BL-activated transcription factor. (A) GFP–AUREO fusion proteins heterologously expressed in onion epidermis are partially or entirely located in the nucleus. Representative differential interference contrast images (Left) and GFP fluorescence images (Right) are demonstrated. Arrowheads indicate the nuclei. (Scale bar, 50 μm.) Fluorescence of GFP–AUREO2 was emitted only from the nucleus, whereas that of GFP–AUREO1 was found both in the nucleus and cytoplasm. (B) A random oligonucleotide-binding selection assay indicated that AUREO1 (GST–AUREO1N) binds to the target sequence TGACGT. (C) Gel-shift assay of the GST–AUREO1N using a WT probe and six single-point-mutated oligonucleotides (M1–M6) (bottom of the photograph) as the competitors. The upper bands (double arrowhead) are probably dimeric forms of bZIP protein, and the lower bands (single arrowhead) are probably monomeric forms of bZIP proteins. Only in the WT lane is fluorescent WT oligonucleotide competed away by the cold WT oligonucleotide. (D) Representative data of gel-shift assay indicating that binding of AUREO1 and the target DNA sequence TGACGT is activated by BL. Binding and irradiation timings are shown at Left. The AUREO1 and the DNA probe was mixed at time 0 (upward arrowhead), and the mixture was either kept in darkness throughout (lane 1) or exposed to a 5-min BL pulse (470 nm, 100 μmol m⁻² s⁻¹, lanes 2 or 3). After incubation for 30 min, the mixture was electrophoresed. Stronger binding to the DNA probe (arrow) was detected when the mixture was exposed to BL just before the end of incubation (lane 2). The binding was slight, however, when BL was followed by a 25-min dark period (lane 3). This may reflect the dark decay of the cysteinyl adduct with consequent loss of capacity to bind the DNA.

To determine whether AUREOs bind DNA directly, a random oligonucleotide-binding assay was used to evaluate the DNA binding activity and specificity of AUREO1 and AUREO2. The N-terminal region of AUREO1, containing the bZIP domain, was able to recognize the consensus sequence TGACGT (Fig. 3B), which is the typical binding site for S-type or D-type bZIP proteins (23). The target sequence for AUREO1 was confirmed by using a gel-shift assay with a probe harboring the TGACGT sequence (WT) and competitors each having a single base pair mutation (Fig. 3C). Gel-shift experiments with full-length AUREO1 expressed and purified in darkness demonstrate that a short pulse of BL activates the binding of AUREO1 to the target sequence TGACGT (Fig. 3D). (Similar activation of binding was shown after 100–200 μmol m⁻² s⁻¹ BL for from 3 to 10 min, data not shown) This may indicate that AUREO1 is a BL-regulated transcription activator. The activation seemed to be decayed when a 25-min long dark period was inserted after the short pulse of BL (Fig. 3D, lane 3), suggesting that this BL activation may be mediated by photo-excited LOV domain (Fig. 2 C and D). By contrast, no consensus sequences were obtained that were bound by the N-terminal region of AUREO2 by using the random oligonucleotide-binding assay. This negative result could indicate that AUREO2 does not function as a transcription factor, or again, as with flavin binding, improper folding may prevent its binding to a specific DNA sequence.

AUREOCHROME Is a Photoreceptor for BL-Induced Photomorphogenesis: RNAi Experiment.

The above biochemical analysis implies that AUREOs may function as BL-sensitive transcription factors in V. frigida. The question now arises as to the nature of the BL responses that are controlled by these photoreceptors. Forward mutational analysis was not possible by using Vaucheria because this organism is a coenocyte and the gene expression system is poorly understood. Therefore, to determine the functional roles of V. frigida AUREOs, a loss-of-function experiment was performed by using RNAi. Vaucheria is particularly suitable for RNAi experiments because a large amount of double-stranded (ds)RNA can be introduced into a giant multinucleate cell and the dsRNA immediately spreads out over the entire intracellular lumen.

dsRNAs encoding the 5′ and 3′ portions of AUREO1 and AUREO2 mRNA sequences were synthesized in vitro and microinjected singly or mixed simultaneously into V. frigida segments. After several days of culture (12 h light/12 h dark), a few growth points regenerated usually from the ends of the segment. The regenerated thalli, which consist of a coenocytic continuum, gradually grew and branched to form a group of thalli. Most of the RNAi lines showed very retarded tip growth and abnormal morphology, such as constrictions and kinks (Fig. 4A). Such constrictions (likely resulting largely from AUREO1 suppression; Fig. 4D) were indicative of daily growth arrest and were not observed in noninjected thalli. The effect of RNAi appeared to last for longer than 6 months because the abnormal morphology persisted over this time period. After 6 months, when many thalli had developed from the original dsRNA-injected fragments, the capacity for BL-induced branching was tested. Upon local irradiation with BL, either no branching was induced in the RNAi lines or if any branching was observed, it was extraordinarily delayed (Fig. 4B). By using RT-PCR, we confirmed the reduction of mRNA encoding both AUREO1 and AUREO2 to undetectable levels in the cells (“ramets”) that did not produce any branches within 24 h of BL irradiation (Fig. 4C). These RNAi ramets, however, exhibited normal chloroplast accumulation, indicating that AUREOs do not regulate this response in V. frigida.

Fig. 4.

AUREO knockdown experiment by using RNAi. (A) Abnormal morphology of tubes (aureo1&aureo2) regenerated from segments into which a dsRNA mixture was microinjected. Constrictions, a sign of daily growth arrest, are not formed in the control cell. Photographs were taken 37 days after injection. (Scale bar, 200 μm.) (B) BL-induced branch induction inhibited by simultaneous RNAi of AUREO1 and AUREO2 (aureo1&aureo2). After 5–6 months of culture, a 200-μm-wide region of the coenocytic tube was illuminated with BL for 10 h (gray) or 24 h (black), and the proportion of branch induction in the irradiated tubes (%) are shown. Control, buffer-injected cells; aureo1&aureo2, dsRNA mixture-injected cells. In the control condition, 52.6% of the specimen (n = 19) produced branches after 10 h, and 91.7% (n = 24) produced branches when irradiated for 24 h. In the dsRNA-injected cells, only 4.3% (n = 46) and 28.6% (n = 34) of tubes produced branches after 10 and 24 h, respectively. (Inset) The normal course of the BL-induced branching is shown. A 200-μm region of the proximal part of the Vaucheria tube was irradiated with BL with safe, dim, red background light. (Scale bar, 100 μm.) (C) RT-PCR demonstrating that loss of AUREOCHROME mRNAs (#1A1 and #1B3) resulted in the loss of branching ability (blind test adopted). (D) Early phenotypes of either AUREO1 or AUREO2 separately inactivated by RNAi. Images were photographed 1 month after the injection. Many immature sex organs developed from aureo2 cells. The phenotype of aureo1 was similar to that of aureo1&aureo2 (see A). (Scale bar, 200 μm.)

Whether or not AUREOCHROME mediates the phototropic response in V. frigida is inconclusive because tip growth is greatly retarded by RNAi, so that no detectable phototropic bending can be observed after 1 hour of unilateral BL irradiation. Nevertheless, we could conclude that AUREOCHROMEs are the principal photoreceptors for the BL-induced branching. We next found a striking phenomenon when only the expression of AUREO2 was suppressed: namely, many sex organs were initiated several weeks after the injection (Fig. 4D). Control cells and cells in which the expression of AUREO1 was suppressed did not produce sex organs under the same conditions. The sex organs initiated from aureo2 cells did not mature, but in some cases, vegetative thalli grew through from oogonia, or in other cases, the cell died. These findings therefore indicate that AUREO1 and AUREO2 play different roles in BL-induced branch induction: AUREO1 may serve as the main switch from nonbranch to branch development, whereas AUREO2 may act as a subswitch and cause the branch primordium to develop into a branch and not develop into a sex organ [supporting information (SI) Fig. 5]. To test this hypothesis, we need more detailed analyses to determine the correlation of AUREO1 and AUREO2 mRNAs remaining in the cell with the phenotype. Nevertheless, this hypothesis seems plausible because a bulge at the surface of the coenocytic cell initiates both sex organs and branches.

Because bZIP proteins typically bind DNA by forming heterodimer (24), AUREO1 (and possibly AUREO2) and some other as yet undetermined bZIP molecule (not necessarily a bZIP–LOV molecule) may cooperatively regulate different kinds of BL responses by forming homo- and heterodimers. This hypothesis requires further investigation. In a previous study (10), we suggested that light-induced branching requires not only chloroplast accumulation in the irradiated region of the cell but also nuclear accumulation and the expression of a still unknown set of genes by the irradiated nuclei. However, chloroplast accumulation by itself is insufficient for inducing branch primordia, indicating that chloroplast accumulation is of only secondary importance. We observed that chloroplast accumulation occurred normally when AUREO1 and AUREO2 were suppressed by RNAi (data not shown). This result indicates that AUREOCHROME is not the photoreceptor of the chloroplast accumulation.

Perspectives.

AUREOCHROME, to our knowledge, represents the first BL photoreceptor to be identified in photosynthetic stramenopiles. We are finding AUREOCHROMEs in other stramenopiles, such as Ochromonas (Chrysophyceae), but not in Saprolegnia (Oomycetes), Haptophyceae, and Cryptophyceae. AUREOCHROMEs may therefore be widely distributed in all photosynthetic stramenopiles and serve as BL receptors, as do phots in green plants. Because stramenopiles contain nonphotosynthetic families, such as oomycetes or labyrinthulas, our present findings not only contribute to the photobiology of Vaucheria and Fucus but should also shed light on the evolution and phylogeny of stramenopiles and eukaryotes.

Materials and Methods

Cloning the DNA Encoding the LOV Proteins.

Primers used in the present study are listed in SI Table 1. DNA sequences for the LOV proteins were isolated by using degenerate primers (LOV-F and LOV-R). PCR products were cloned by the pGEM-T Easy system (Promega) and sequenced. Full-length cDNAs were obtained by using 5′ and 3′ RACE. (For further details, see SI Materials and Methods.)

Photochemical Analysis.

The method for heterologous expression of the CBP–LOV fusion protein is described in SI Materials and Methods. Absorption spectra of CBP–LOV fusion protein were acquired by using a spectrophotometer [DU-65 (Beckman Instruments) and Biospec-mini (Shimadzu)]. A light-emitting diode (LED) illuminator (450 nm, ISL-150 × 150-BB, CCS) was used for BL-induced photoadduct formation. Kinetic analysis of dark recovery of the LOV domain of AUREO1 (CBP–LOV_AUREO1) was performed as described in ref. 25. The LOV suspension was excited at 450 nm and the emission was monitored at 520 nm by using a fluorescence spectrophotometer (850, Hitachi). The LOV solution was first excited with a 6-min pulse of BL. After an initial burst, fluorescence declined sharply and almost reached steady state at the end of the pulse (data not shown). After leaving the sample in darkness for 2–10 min, the suspension was excited with a second 4-min pulse. The magnitude of the immediate fluorescence increased with increasing dark period. The difference (percentage) between initial and second fluorescence peaks corresponds to the remaining photoproduct. This value exponentially decreased with increasing dark interval (Fig. 2E).

Intracellular Localization of AUREOCHROMEs in Onion Epidermis Cells.

GFP fusion proteins GFP–AUREO1 and GFP–AUREO2 were used to observe the intracellular localization of AUREOCHROMEs in onion epidermal cells (26). (For details see, SI Materials and Methods.) Onion epidermal cells were bombarded with gold particles coated with plasmids by using a PDS-1000/He particle delivery system (Bio-Rad). The bombarded epidermis was incubated at 25°C for 12 h before observation by fluorescence microscopy (Axioskop, Zeiss).

Random Oligonucleotide-Binding Assay.

Random oligonucleotide-binding assays were performed as described in ref. 27 by using the GST fusion proteins and oligonucleotide mixture. For purification procedures of the GST fusion proteins, see SI Materials and Methods. For constructing random oligonucleotide mixtures, Random1, a forward primer (5′-AATCTTAGTATCATCGACATC-3′) and a reverse primer (5′-AATCTTGATCTAGAGTAGTAG-3′) were used. The double-stranded 68-bp oligonucleotide (dsDNA) was generated by PCR by using Random1 as a template. A 100-ng Random1 probe was mixed with 100 ng of the GST–bZIP domain (GST–AUREO1-N or GST–AUREO2-N) in 100 μl of binding buffer {20 mM Tris·HCl (pH 8.0), 50 mM KCl, 0.5 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 20 μg/ml BSA, 2.0 mM phenylmethansulfonylfluoride, 10 μg/ml poly[d(I-C)], 10% glycerol}. After gentle mixing of the mixture for 10 min, glutathione–Sepharose 4B beads were added to bind with the DNA–protein complex. The beads were collected by centrifugation and were washed three times with 200 μl of wash buffer [20 mM Tris·HCl (pH 8.0), 50 mM KCl, 0.5 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 20 μg/ml BSA, 2.0 mM phenylmethansulfonylfluoride, 10% glycerol]. The pellet resuspended in wash buffer was heated at 94°C for 3 min and then centrifuged. A 5-μl aliquot of supernatant was used as a template for PCR with the primers for Random1. The PCR mixture was precipitated with ethanol and sodium acetate and resuspended in water. A portion of the PCR product was used for the next DNA–protein binding reaction, and this process was repeated four times.

Gel-Shift Assay.

Gel-shift assay was performed with a digoxigenin (DIG) gel-shift kit (2nd Generation; Roche) according to the protocols described in SI Materials and Methods. BL activation of binding between AUREO1 and the TGACGT-containing DNA probe (Fig. 3D) was performed as follows. A GST–AUREO1 full-length protein was expressed in E. coli (Rosetta) by using the vector pGEX6P1 in darkness. The protein was purified, and its GST was excised by PreScission protease (GE Healthcare) under very dim red light (<0.02 μmol·m⁻²·s⁻¹). The AUREO1 protein and DIG-labeled oligonucleotide probe was mixed and either placed in darkness or exposed to a short BL pulse (470 nm LED, 100 μmol·m⁻²·s⁻¹ for 5 min). After 30 min of binding response, the AUREO1–target nucleotide complex was separated by electrophoresis in darkness (with brief exposure to dim red light for observation).

RNAi Experiments.

Four dsRNA fragments (LOV_AUREO1, bZIP_AUREO1, LOV_AUREO2, and bZIP_AUREOO2) were synthesized by using PCR (for primer sets, see SI Table 1) according to the instruction manual (Ambion). Each fragment was ≈500 bp in length. dsRNAs were suspended in TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0) at 5 μg·μl⁻¹ and stored in a freezer until use. Either two or four dsRNA mixtures were microinjected into a V. frigida segment ≈10 mm in length, and the injected individual segments were cultured separately in an incubator (12 h light/12 h dark, 20°C) for up to 6–7 months, with renewal of the culture medium several times. The injection solution contained 7–10 μg·μl⁻¹ of either two or four (i.e., all four dsRNAs or LOV_AUREO1 plus bZIP_AUREO1 or LOV_AUREO2 plus bZIP_AUREO2) dsRNA mixture (20–30 mM K₂SO₄, 17–25 mM MgSO₄, 0.07–0.1 mM EGTA, 40–60 mM sorbitol, and 17–25 mM Pipes–K, pH 7.0).