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. 2024 Feb 12;195(1):326–342. doi: 10.1093/plphys/kiae060

Poaceae plants transfer cyclobutane pyrimidine dimer photolyase to chloroplasts for ultraviolet-B resistance

Momo Otake 1, Mika Teranishi 2, Chiharu Komatsu 3, Mamoru Hara 4, Kaoru Okamoto Yoshiyama 5, Jun Hidema 6,7,b,✉,c
PMCID: PMC11060685  PMID: 38345835

Abstract

Photoreactivation enzyme that repairs cyclobutane pyrimidine dimer (CPD) induced by ultraviolet-B radiation, commonly called CPD photolyase (PHR) is essential for plants living under sunlight. Rice (Oryza sativa) PHR (OsPHR) is a unique triple-targeting protein. The signal sequences required for its translocation to the nucleus or mitochondria are located in the C-terminal region but have yet to be identified for chloroplasts. Here, we identified sequences located in the N-terminal region, including the serine-phosphorylation site at position 7 of OsPHR, and found that OsPHR is transported/localized to chloroplasts via a vesicle transport system under the control of serine-phosphorylation. However, the sequence identified in this study is only conserved in some Poaceae species, and in many other plants, PHR is not localized to the chloroplasts. Therefore, we reasoned that Poaceae species need the ability to repair CPD in the chloroplast genome to survive under sunlight and have uniquely acquired this mechanism for PHR chloroplast translocation.

Poaceae plants have acquired a mechanism of translocation to chloroplasts—regulated by phosphorylation of cyclobutane pyrimidine dimer photolyase—to avoid ultraviolet-B-induced chloroplast damage.

Introduction

Plants use sunlight as an energy source for photosynthesis and are inevitably exposed to harmful ultraviolet-B (UV-B, 280 to 315 nm) radiation. Although UV-B is a minor fraction of the UV radiation that reaches the Earth's surface, it causes substantial damage to plant macromolecules such as lipids, proteins, and membranes, especially DNA (Teramura 1983). The cyclobutane pyrimidine dimer (CPD) represents a major type of DNA damage induced by UV-B radiation (Britt 1996), which induces CPD on DNA, and hence in all organelles containing DNA (Stapleton and Walbot 1994; Takahashi et al. 2011). The accumulation of DNA damage induces mutations or cell death by impeding replication and transcription, thereby inhibiting plant growth and development. CPDs are the principal cause of UV-B-induced growth inhibition in rice (Oryza sative) plants grown under supplementary UV-B radiation (Hidema et al. 2007; Teranishi et al. 2012; Mmbando et al. 2021).

Plants have developed mechanisms to cope with UV-B-induced CPDs, including photoreactivation and nucleotide excision repair (NER) (Britt 1996). In vascular plants, photoreactivation is the primary mechanism for repairing CPDs because the rate of photoreactivation is faster than that of NER. Photoreactivation is mediated by photolyase (PHR), which absorbs ultraviolet-A (UV-A, 315 to 400 nm) and blue radiation as energy to monomerize dimers (Britt 1996; Sancar et al. 2004). CPD PHR-deficient Arabidopsis (Arabidopsis thaliana) (Landry et al. 1997; Dündar et al. 2020) or rice (Hidema et al. 2007) plants show UV-B hypersensitive phenotype. We previously demonstrated that the difference in UV-B sensitivity among rice cultivars depends on PHR activity in the cell (Hidema et al. 2000; Teranishi et al. 2004; Mmbando et al. 2020) and that elevated PHR activity can markedly alleviate UV-B-induced growth inhibition in rice (Hidema et al. 2007; Teranishi et al. 2012; Mmbando et al. 2021). Thus, PHR is an essential protein for plants grown under sunlight.

PHR is widely distributed among species ranging from eubacteria and archaebacteria to eukaryotes, apart from eutherian mammals (Yasui et al. 1994). Eukaryotic cells have at least 2 organelles containing DNA: nuclei and mitochondria. In addition to the nuclear and mitochondrial genomes, photosynthetic eukaryotic cells possess chloroplasts as organelles that contain DNA. Yeast (Saccharomyces cerevisiae) PHR is transported into each organelle via a signal sequence for the nuclei or mitochondria, and efficiently photorepairs CPDs on both nuclear and mitochondrial DNA (Yasui et al. 1992). In rice plants, Oryza sativa PHR (OsPHR), which is encoded by a single-copy gene and not a splice variant, is expressed and targeted not only to the nucleus and mitochondria but also to chloroplasts (Takahashi et al. 2011). This protein repaired UV-B-induced CPDs in all 3 genomes. In addition, we have previously identified the nuclear and mitochondrial target signal sequences retained in the C-terminal region of OsPHR and found that each OsPHR target sequence was highly conserved among plant species (Takahashi et al. 2014). However, the location and nature of the chloroplast-targeting information contained within OsPHR remained unknown.

In this study, we searched for sequences required for OsPHR to be transferred to chloroplasts using a systematic deletion analysis. We found that the sequence is located within the N-terminal region and that the 7th serine residue and neighboring proline residues of OsPHR are important for its translocation through the endoplasmic reticulum (ER) and Golgi to the chloroplast. In addition, the phosphorylation of serine at position 7 of OsPHR (Teranishi et al. 2008, 2013) would be involved in the regulation of OsPHR chloroplast translocation via the ER-Golgi system. However, the amino acid sequence of the N-terminal region of PHR varies widely among plant species, and the sequences identified in this study were not highly conserved among plant species. We examined the chloroplast localization of PHR in various plants using transient expression analysis and found that PHR was not localized to the chloroplasts in many plants, except for Poaceae. It is thought that Poaceae plants have uniquely acquired a mechanism to protect chloroplasts from UV-B-induced damage by transferring PHR to chloroplasts on their own during evolution.

Results

N-terminal region sequence is required for chloroplast translocation of OsPHR

We have previously reported the possibility that the sequence required for OsPHR transfer to the chloroplast is located in the N-terminal region of OsPHR (Takahashi et al. 2014). Using TargetP (https://services.healthtech.dtu.dk/services/TargetP-2.0/) (Armenteros et al. 2019), a popular protein localization prediction algorithm, we analyzed the presence of sequences predicted to be transferred to the chloroplasts within the N-terminal region of OsPHR (amino acids 1 to 60) (Supplementary Table S1). Within the amino acid sequence of the N-terminal region of OsPHR, a signal peptide that is likely to be transported to the ER (score >0.4) was speculated but is unlikely to have a signal peptide that is transported to chloroplasts. The score value for the possibility of a signal peptide being transported to the ER was 0.55 for the amino acid region 1 to 52 of OsPHR, while it increased to 0.73 for 1 to 47 and further to 0.87 for 1 to 14, indicating the possibility of the presence of a signal peptide at the N-terminal tip that is transported to the ER. Recently, it has been reported that a part of the nuclear-encoded chloroplast protein is imported from the ER-Golgi system to the chloroplast through the secretory pathway (Friso et al. 2004; Kleffmann et al. 2004; Asatsuma et al. 2005) or is incorporated into chloroplasts without cleavage of the peptide within the N-terminal region of the protein (Armbruster et al. 2009). Thus, we first focused on the amino acids 1 to 47 of OsPHR and generated the transgenic rice plants stably expressing citrine fused to the C-terminus of a fragment of the N-terminus OsPHR (amino acids 1 to 47, N1 to 47-OsPHR) under the cauliflower mosaic virus (CaMV) promoter in OsPHR gene-targeting (GT) rice plant to determine whether the sequence of the N-terminal region of OsPHR can be transferred to chloroplasts. The generated OsPHR GT rice plants showed higher sensitivity to UV-B as well as antisense transgenic rice plants with little PHR activity (AS-D) (Hidema et al. 2007), confirming that endogenous OsPHR is deficient in the OsPHR GT rice plants (Supplementary Fig. S1). In transgenic rice stably expressing citrine fused to the C-terminus of full-length (FL) OsPHR under the CaMV promoter (FL-OsPHR) in the GT rice plant, the citrine fluorescence of FL-OsPHR was clearly detected in the chloroplasts of leaf cells, compared with the localization of citrine using a control vector (Fig. 1A). The citrine fluorescence of N1 to 47OsPHR was also clearly detected in the chloroplasts of leaf cells (Fig. 1A). Therefore, to examine whether OsPHR is translocated to chloroplasts using the sequence of the N-terminal region, transgenic rice plants expressing citrine fused to the C-terminus of partial OsPHR without amino acids 1 to 14 (Δ1 to 14OsPHR), which were predicted to be the likely signal peptide to the ER, were generated. When citrine fluorescence of Δ1 to 14OsPHR was observed in rice cells, citrine fluorescence was not detected in the chloroplasts, whereas it was detected in the nuclei and mitochondria (Fig. 1B). In addition, blue light (BL)-dependent repair activities of CPD on chloroplast DNA in the transgenic lines expressing Δ1 to 14OsPHR (Lines 11 and 13) were compared with that in transgenic rice overexpressing FL-OsPHR. After irradiating rice leaves of each transgenic line with UV radiation to induce CPD in DNA, the leaves were irradiated with BL, and the repair of CPD was analyzed by Southern blotting using T4 endonuclease (Hanawalt 1989; Chen et al. 1996; Stapleton et al. 1997). In transgenic rice overexpressing FL-OsPHR, CPDs on nuclear, mitochondrial, and chloroplast DNA were fully repaired after 1 h of BL irradiation (Supplementary Fig. S2). In contrast, the CPDs on chloroplast DNA fragments in the leaves of each Δ1 to 14OsPHR transgenic line were not removed after 1 h of BL exposure, although the CPDs on nuclear or mitochondria DNA fragments were notably removed (Supplementary Fig. S2). Furthermore, when the UV-B sensitivity of the transgenic lines expressing Δ1 to 14OsPHR (Lines 11 and 13) was compared with that of plants expressing FL-OsPHR, obvious leaf browning was observed in the transgenic lines expressing Δ1 to 14OsPHR. Both transgenic rice lines showed a UV-B-sensitive phenotype compared with the transgenic line with chloroplast-translocated OsPHR (FL-OsPHR) (Fig. 1C). Therefore, it is strongly suggested that the sequence required for OsPHR to translocate to chloroplasts in rice cells is located within the N-terminal region and that the translocation of OsPHR to chloroplasts is important for UV-B sensitivity in rice.

Figure 1.

Figure 1.

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The N-terminal 1 to 14 amino acid sequences are critical for chloroplast translocation of OsPHR and affect ultraviolet-B (UV-B) sensitivity. A and B) Confocal microscopic images of the transgenic rice plants expressing citrine fused to the C-terminus of full length (FL) OsPHR (amino acids 1 to 506, FL-OsPHR) and a fragment of OsPHR (N1 to 47 or Δ1 to 14 OsPHR) in each case under the control of a CaMV 35S promoter. Arrowheads indicate the fluorescence of citrine in chloroplast A) and mitochondria B). Bars = 10 μm. C) Effects of UV-B radiation on rice strains (FL-OsPHR, and lines 11 and 13 of Δ1 to 14 OsPHR). The images shown in the bottom row are enlargements of images in the top row. The rice plants were grown in the growth cabinet for 30 d with (+UV-B: 1.0 W m−2) or without (−UV-B) supplementary UV-B radiation. Bars = 5 cm. N1-47OsPHR or Δ1-14OsPHR in the Figure indicates N1 to 47OsPHR or Δ1 to 14OsPHR, respectively.

Chloroplast translocation of OsPHR is inhibited by brefeldin A (BFA) treatment

As mentioned previously, the N-terminal region of OsPHR contains sequences that are likely to be transported to the ER and required for OsPHR to translocate to chloroplasts in rice cells. Therefore, to deduce the transport pathway of OsPHR to chloroplasts, we analyzed the effects of brefeldin A (BFA), a fungal antibiotic that inhibits Golgi-mediated vesicular traffic (Ritzenthaler et al. 2002), on the translocation of OsPHR to chloroplasts. We constructed an expression vector expressing FL-OsPHR and performed a transient expression assay using protoplasts prepared from shoots of rice seedlings. When citrine alone was introduced into rice protoplasts as a control, citrine fluorescence was observed not only in the cytoplasm but also in the nucleus (Kaiser et al. 2009) (Fig. 2A). Relatively small proteins often enter the nucleus passively. When an FL-OsPHR expression vector was introduced into rice protoplasts, citrine fluorescence was detected in the nuclei and chloroplasts. In particular, within the chloroplasts, a strong nucleoid-like speckled signal merging with fluorescence stained by DNA-staining reagent Kakshine PC3 (Uno et al. 2021) was observed (Supplementary Fig. S3). When the protoplasts were treated with 0.1% dimethyl sulfoxide (DMSO) as a control, citrine signals were detected in the nuclei and chloroplasts of approximately 76% of the transformed protoplasts (Fig. 2, B and C). In contrast, BFA treatment reduced the number of protoplasts with a strong speckled signal of citrine fluorescent in chloroplasts to approximately 12%: we attempted to analyze the distribution of citrine fluorescence intensity within the chloroplasts of BFA-treated protoplasts, but no obvious signal was detected and was at background levels (Fig. 2, C to E). In order to confirm that BFA exclusively affects the ER-Golgi secretory pathway, the effect of BFA on chloroplast localization of the Rubisco small subunit (RBCS) was examined. RBCS has a chloroplast transit peptide and is translocated to chloroplasts without passing through the ER-Golgi system (Shen et al. 2017). The protoplasts transfected with constructs expressing RBCS showed a clear signal of citrine fluorescence in chloroplasts with or without BFA treatment (Supplementary Fig. S4). These results indicate that OsPHR is translocated to chloroplasts via the ER-Golgi system through the secretory pathway.

Figure 2.

Figure 2.

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Chloroplast translocation of OsPHR is inhibited by BFA treatment. A and B) Confocal microscopic images of rice protoplasts expressing citrine (citrine control) or FL-OsPHR (OsPHR) under the control of the CaMV 35S promoter A), and of the OsPHR-expressing protoplasts treated with DMSO as a control (without (No treatment) or with DMSO (+DMSO)) or with BFA (+BFA) B). Arrowheads indicate citrine fluorescence in chloroplasts. Bars = 5 µm. C) The percentage of protoplasts in which citrine fluorescence was detected in chloroplasts among all transformed protoplasts treated without (No treatment or + DMSO) or with BFA (+BFA) after expressing OsPHR (±se, n ≥ 11 images, total of transformed protoplasts ≥55). Different letters in each graph denote significant differences from each other based on Tukey’s test (P < 0.05). D) Confocal 2D and 2.5D images of rice protoplasts without (no treatment) or with BFA (+BFA) after expressing OsPHR. Bars = 5 μm. Chloroplast images framed by white squares in the 2D images are used for citrine fluorescence distribution analysis E). E) Fluorescence intensity along the white dotted lines (a to b) in the enlarged image of the area enclosed by the white square in the 2D image D). The intensity is shown relative to the maximum intensity of citrine fluorescence in chloroplast in protoplast without BFA treatment, which is represented as 1. Strong nucleoid-like speckled signals along the white dotted line are indicated by arrowheads. Bars = 1 μm. Bright-field microscopy image, Differential Interference Contrast (DIC).

Proline residues are important for chloroplast translocation of OsPHR

Certain proteins are transported to the mitochondria via the ER-Golgi transport system, which is characterized by the presence of a hydrophobic region (H-region) within the N-terminal region (Martoglio and Dobberstein 1998; Kanaji et al. 2000; Kida et al. 2000). In addition, proline residues preceding the H-region are critical for passage through the ER (Ritzenthaler et al. 2002). The N-terminal region of OsPHR is rich in hydrophobic amino acids, particularly proline residues (Fig. 3A). Hence, to investigate whether proline residues in the N-terminal region of OsPHR are involved in chloroplast translocation, a transient expression vector encoding mutated OsPHR was constructed by replacing each proline residue with an alanine residue in the 1 to 14 amino acid region (Fig. 3A), which is essential for the chloroplast translocation (Fig. 1B). The results showed that replacing the 13th proline residue with an alanine residue (P13A-OsPHR) did not alter the chloroplast translocation of OsPHR, whereas replacing the 2nd, 3rd, or 8th proline residues (P2, 3A- or P8A-OsPHR) slightly inhibited chloroplast translocation (Fig. 3, B and C). In contrast, when the 9th proline residue was converted to alanine residue (P9A-OsPHR), the chloroplast translocation of OsPHR was inhibited to a greater extent than when proline residue at other positions was converted to alanine residue, and the degree of inhibition increased further when both the 8th and 9th proline residues were converted to alanine residues (P8, 9A-OsPHR) (Fig. 3, B and C). These results suggested that the 8th and 9th proline residues of OsPHR play notable roles in the translocation of OsPHR to the chloroplasts.

Figure 3.

Figure 3.

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Proline residues are important for chloroplast translocation of OsPHR. A) Proline residues were mutated into alanine residues in 1 to 14 amino acid region of OsPHR as indicated. WT, wild type. B) Confocal microscopic images of rice protoplasts expressing FL-OsPHR (OsPHR) and a mutated OsPHR by replacing a proline residue with an alanine residue (P2, 3A, P8A, P9A, P8, 9A, and P13A) under the control of the CaMV 35S promoter. Bright-field microscopy image, Differential Interference Contrast (DIC). Arrowheads indicate citrine fluorescence in chloroplasts. Bars = 5 μm. C) The percentage of protoplasts in which citrin fluorescence of OsPHR or OsPHR by replacing a proline residue with an alanine residue (P2, 3A, P8A, P9A, P8, 9A, and P13A) was detected in chloroplasts among all transformed protoplasts (±se, n ≥ 10 images, total of transformed protoplasts ≥73). Different letters in each graph denote significant differences from each other based on Tukey’s test (P < 0.05).

Phosphorylated OsPHR suppresses chloroplast translocation

We have previously shown that phosphorylated and nonphosphorylated forms of the 7th serine residue of OsPHR are present in vivo (Teranishi et al. 2008, 2013) and that there is a difference in the quantity of phosphorylated OsPHR in the chloroplasts, mitochondria, and nuclei of rice cells (Takahashi et al. 2011). However, when, where, and by what kinase OsPHR is phosphorylated and the biochemical and physiological functions of phosphorylated PHR are unclear. These results raise the possibility that the phosphorylation state of OsPHR might be associated with its targeting of different organelles. Therefore, to confirm whether the difference in the phosphorylation state of the 7th serine residue of OsPHR affected the chloroplast translocation of OsPHR, an expression vector expressing FL-OsPHR with the 7th serine residue replaced by alanine residue (pseudo-unphosphorylated state: S7A-OsPHR), aspartic acid residue (pseudo-phosphorylated state: S7D-OsPHR), or glutamic acid residue (pseudo-phosphorylated state: S7E-OsPHR) under the control of the CaMV promoter was introduced into rice protoplasts. When pseudo-unphosphorylated S7A-OsPHR was introduced into rice protoplasts, the percentage of protoplasts in which citrine fluorescence was detected in the chloroplasts was approximately 70%, the same as that in unmutated OsPHR (Fig. 4, A to C). In contrast, when pseudo-phosphorylated S7D-OsPHR or S7E-OsPHR was introduced into rice protoplasts, the percentage of protoplasts in which citrine fluorescence was detected in the chloroplasts was dramatically reduced to approximately 25% compared to that of OsPHR or pseudo-unphosphorylated S7A-OsPHR (Fig. 4, A to C). Furthermore, when expression vectors expressing S7A-OsPHR were introduced into rice protoplasts and treated with DMSO or BFA, the percentage of protoplasts in which the fluorescent signal of S7A-OsPHR was detected in the chloroplasts was reduced by BFA treatment (Fig. 4, D and E). These results indicate that OsPHR with an unphosphorylated serine residue at position 7 is more clearly translocated to the chloroplasts via the ER-Golgi system than phosphorylated OsPHR. In addition, to confirm whether OsPHR in the unphosphorylated state of the 7th serine residue is more actively translocated to chloroplasts in vivo than phosphorylated OsPHR, we generated transgenic rice plants stably expressing S7A-OsPHR or S7D-OsPHR under the CaMV promoter. In transgenic rice plants introduced with S7A-OsPHR (Lines 7 and 9), a strong signal of citrine fluorescence was clearly detected in the chloroplasts and nuclei of rice leaf cells, similar to the OsPHR-transgenic rice (Fig. 4F). In contrast, in transgenic rice plants transformed with S7D-OsPHR (Lines 3 and 4), citrine fluorescence was detected in the nucleus but not in the chloroplasts (Fig. 4F). Thus, the phosphorylation of serine residue at position 7 of OsPHR is involved in the regulation of OsPHR chloroplast translocation.

Figure 4.

Figure 4.

Figure 4.

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Phosphorylation state of the 7th serine is involved in the chloroplast translocation of OsPHR. A) The phosphorylation site of the 7th serine residue was mutated into alanine, aspartic acid, or glutamic acid residues of OsPHR as indicated. WT, wild type. B) Confocal microscopic images of rice protoplasts expressing FL-OsPHR (OsPHR), pseudo-unphosphorylated S7A-OsPHR (S7A-OsPHR), pseudo-phosphorylated S7D- or S7E-OsPHR (S7D-OsPHR or S7E-OsPHR) in each case under the control of the CaMV 35S promoter. Bars = 5 μm. C) The percentage of protoplasts in which citrine fluorescence of OsPHR, S7A-OsPHR, S7D-OsPHR, or S7E-OsPHR was detected in chloroplasts among all transformed protoplasts (±se, n ≥ 9 images, total of transformed protoplasts ≥70). D) Confocal microscopic images of rice protoplasts treated with DMSO or BFA expressing S7A-OsPHR. Bars = 5 μm. E) The percentage of protoplasts in which citrine fluorescence of S7A-OsPHR was detected in chloroplasts among all transformed protoplasts treated with DMSO and BFA (±se, n ≥ 12 images, total of transformed protoplasts ≥119). F) Confocal microscopic images of the transgenic rice plants expressing citrine fused to the C-terminus of OsPHR, S7A-OsPHR, or S7D-OsPHR under the control of CaMV 35S promoter. Bars = 10 μm. Bright-field microscopy image, Differential Interference Contrast (DIC). Arrowheads indicate citrine fluorescence in chloroplasts B, D, F). Different letters in each graph denote significant differences from each other based on Tukey’s test (P < 0.05) C, E).

Chloroplast translocation of PHR differs among plant species

OsPHR was shown to translocate to chloroplasts using the sequence of the N-terminal region. However, the amino acid sequence of the N-terminal region of PHR varied widely among plant species, as shown in Fig. 5A and Supplementary Fig. S5. Interestingly, only plants belonging to the Poaceae family, such as wheat (Triticum aestivum) and barley (Hordeum vulgare), have CPD photolyases (PHRs) that show similarity with the amino acid sequence of the N-terminal region, which is important for the transfer of OsPHRs to chloroplasts. On the other hand, the sequences of amino acids 1 to 14 of OsPHR are missing from the Arabidopsis thaliana PHR (AtPHR) or Lotus japonicus PHR (LjPHR), etc., except plants belonging to the Poaceae family. This implies that most plant PHRs are not translocated to chloroplasts or that the sequence required for OsPHR to transfer to chloroplasts may differ among plant species. Therefore, we investigated chloroplast translocation of PHR in various plants. We first constructed an expression vector that expressed citrine fused to the C-terminus of AtPHR or LjPHR and performed a transient expression assay using rice protoplasts (Fig. 5B). Citrine fluorescence of OsPHR was detected in the nuclei and chloroplasts of protoplasts. In particular, strong citrine signals of OsPHR in chloroplasts were detected as nucleoids. In contrast, citrine fluorescence of AtPHR or LjPHR was not detected in the chloroplasts. The failure to detect citrine fluorescence in AtPHR or LjPHR in chloroplasts may have resulted from its transient introduction into rice protoplasts rather than into the host protoplasts. The AtPHR vector was transiently introduced into protoplasts prepared from Arabidopsis leaves (Fig. 5C). However, no citrine fluorescence of AtPHR was detected in the chloroplasts. In addition, to confirm whether AtPHR is translocated to chloroplasts in Arabidopsis cells, we transfected citrine fused AtPHR into PHR-deficient Arabidopsis mutant (uvr2-1) and generated transgenic Arabidopsis plants stably expressing AtPHR. Compared to the localization of citrine using a control vector, the citrine fluorescence of AtPHR was not detected in the chloroplasts of leaf cells (Fig. 5D), whereas fluorescence was detected in the nuclei of leaf cells (Fig. 5D) and in the mitochondria of root cells (Fig. 5E). Furthermore, we analyzed the BL-dependent repair of CPD in chloroplast DNA of Arabidopsis leaves of wild type (Landsberg erecta) with UV radiation by Southern blotting using T4 endonuclease (Hanawalt 1989; Chen et al. 1996; Stapleton et al. 1997). The CPDs on chloroplast DNA fragments in Arabidopsis leaves were not removed after 24 h of BL exposure, although the CPDs on nuclear and mitochondrial DNA fragments were repaired (Fig. 5, F and Supplementary Fig. S6). These results strongly indicate that in Arabidopsis, AtPHR cannot be translocated to the chloroplasts.

Figure 5.

Figure 5.

Figure 5.

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Chloroplast translocation of PHR varies among plant species. A) Alignments of the deduced amino acid sequences at the N-terminal from Oryza sativa, Arabidopsis thaliana, and Lotus japonicus. Color display is shown in order to emphasize amino acid properties. B) Confocal microscopic images of rice protoplasts expressing FL-OsPHR (OsPHR), FL-AtPHR (AtPHR), or FL-LjPHR (LjPHR). Bars = 5 µm. C) Confocal microscopic images of Arabidopsis protoplasts expressing citrine (Citrine control) or AtPHR. Bars = 10 µm. D, E) Confocal microscopic images in leaf D) and root cells E) of the transgenic Arabidopsis plants expressing citrine fused to the C-terminus of AtPHR in each case under the control of CaMV 35S promoter. Bars = 10 µm. F) Repair of CPD in Arabidopsis genomic DNA. Detached Arabidopsis leaves are harvested (no UV-C (−UV)) or were exposed to UV-C radiation (+UV) for 15 min. Leaves were then harvested immediately and exposed to BL for 10, 18, and 24 h or kept in a light-tight box for 24 h. Genomic DNA was isolated and digested with restriction enzymes, and then the digested DNA are incubated with (+) or without (−) T4 endo V. The membranes were incubated with 32P-labeled probes for the nuclear (Nc)-encoded genes NRPB1, the mitochondrial (Mt)-encoded genes COX1, and the chloroplast (Cp)-encoded gene PSBA. G) Schematic drawing of the constructs used for the analysis. H) Confocal microscopic images of rice protoplasts expressing citrine fused TaOsPHR, HvOsPHR, ZmOsPHR, GmOsPHR, AmOsPHR, SmOsPHR, and CrOsPHR. Bars = 5 µm. Bright-field microscopy image, Differential Interference Contrast (DIC). Arrowheads indicate citrine fluorescence in chloroplasts.

We further examined chloroplast localization in various land plants belonging to Poaceae, Fabaceae, Amborellaceae, and Selaginellaceae, and green algae, Chlamydomonas (Chlamydomonas reinhardtii), to understand the difference in the chloroplast localization of PHR among plant species. To conduct this experiment, the DNA sequences encoding the various plant N-terminal fragments of PHR, excluding the sequences corresponding to the amino acid sequence after the 52nd amino acid of OsPHR, were synthesized because the amino acid sequence after the 52nd amino acid of OsPHR was similar among plant species (Supplementary Fig. S5): amino acids 1 to 52 from Triticum aestivum PHR (N1 to 52TaPHR), 1 to 52 from Hordeum vulgare PHR (N1 to 52HvPHR) and 1 to 39 from Zea mays PHR (N1 to 39ZmPHR) belonging to Poaceae, 1 to 42 from Glycine max PHR (N1 to 42GmPHR) belonging to Fabaceae, 1 to 44 from Amborella trichopoda PHR (N1 to 44AmPHR) belonging to Amborellaceae, 1 to 74 from Selaginella moellendorffii Hieron PHR (N1 to 74SmPHR) belonging to Selaginellaceae, and 1 to 79 from Chlamydomonas reinhardtii PHR (N1 to 79CrPHR) belonging to Chlamydomonadaceae. Expression vectors encoding the protein chimeras of each N-terminal fragment prepared from various plants were fused to the N-terminus of OsPHR, in which amino acids 1 to 51 were deleted (Δ1 to 51OsPHR-Citrine). The constructs used in this analysis are as follows: N1 to 52TaPHR-Δ1 to 51OsPHR-Citrine (TaOsPHR), N1 to 52HvPHR-Δ1 to 51OsPHR-Citrine (HvOsPHR), N1 to 39ZmPHR-Δ1 to 51OsPHR-Citrine (ZmOsPHR), N1 to 42GmPHR-Δ1 to 51OsPHR-Citrine (GmOsPHR), N1 to 44AmPHR-Δ1 to 51OsPHR-Citrine (AmOsPHR), N1 to 74SmPHR-Δ1 to 51OsPHR-Citrine (SmOsPHR), or N1 to 79CrPHR-Δ1 to 51OsPHR-Citrine (CrOsPHR) (Fig. 5G). These expression constructs were transformed into rice protoplasts. In protoplasts transformed with TaOsPHR, HvOsPHR, or ZmOsPHR, citrine fluorescence was detected in the nucleoids and chloroplasts (Fig. 5H). In contrast, the citrine fluorescence of GmOsPHR, AmOsPHR, SmOsPHR, and CrOsPHR was not detected in the chloroplasts (Fig. 5H). We also examined whether BFA treatment inhibited the detection of citrine fluorescence in plants in which citrine fluorescence was detected in chloroplasts, such as TaOsPHR, HvOsPHR, and ZmOsPHR. In these Poaceae species, the percentage of protoplasts in which citrine fluorescence was detected in the chloroplasts among the transformed protoplasts was significantly reduced by BFA treatment, similar to OsPHR (Supplementary Fig. S7). Moreover, to investigate whether proline residues in the N-terminal region of PHR are involved in chloroplast translocation, a transient expression vector encoding mutated TaOsPHR or HvOsPHR was constructed by replacing the 7th or 9th proline residue with an alanine residue (P7A-TaOsPHR, P9A-TaOsPHR, P7A-HvOsPHR, or P9A-HvOsPHR). The results showed that in both TaOsPHR and HvOsPHR, the conversion of the 9th alanine residue was inhibited to a greater extent than that of the 7th alanine residue (Supplementary Fig. S8). These results suggested that other Poaceae plants also have a PHR chloroplast translocation via the ER-Golgi system similar to that of rice.

Discussion

Several chloroplast-localized proteins without predicted transit peptides are transported to chloroplasts through the secretory pathway of the ER-Golgi system (Friso et al. 2004; Kleffmann et al. 2004; Asatsuma et al. 2005). Our results showed that OsPHR, which is transported and functions in the nucleus, mitochondria, and chloroplasts, is transported to chloroplasts via the ER-Golgi system because BFA treatment inhibited OsPHR translocation to chloroplasts. Proteins transported to chloroplasts via the ER-Golgi system are characterized by (i) possessing a vesicular signal peptide in the N-terminal region and (ii) an N-terminal region rich in hydrophobic amino acids (Villarejo et al. 2005; Buren et al. 2012). Furthermore, amino acids with low helical propensities, such as proline, in the N-terminal hydrophobic amino acid region have been reported to be important for N-terminal vesicle transport (Kanaji et al. 2000; Kida et al. 2000). The N-terminal hydrophobic region of OsPHR contains numerous proline residues, and when the 8th and 9th proline residues were converted to alanine residues with a high helical propensity, translocation to the chloroplasts was significantly suppressed (Fig. 3). In addition, the N-termini of TaPHR, HvPHR, and ZmPHR, which were speculated to be translocated to chloroplasts by subcellular localization analysis (Fig. 5H), are also rich in hydrophobic amino acids, among which proline residues are abundant (Supplementary Fig. S5). Conversion of the 7th and 9th proline residues of TaPHR and HvPHR to alanine residues markedly suppressed chloroplast translocation, similar to OsPHR (Supplementary Fig. S8). These results indicate that proline residues in the N-terminal region of PHR are important amino acid residues for N-terminal vesicular transport.

Furthermore, we showed that the phosphorylation state of the 7th serine residue adjacent to the 8th and 9th proline residues is strongly involved in the chloroplast translocation of OsPHR (Fig. 4). Phosphorylation has been reported to not only cause changes in protein conformation and conformational dynamics (Johnson 2009; Kast et al. 2010; Park et al. 2010) but also affect the localization of proteins in the cell (Kuriyan and Eisenberg 2007; Ewens et al. 2010; Gelvin 2010; Muñoz-García and Kholodenko 2010; Pooler and Hanger 2010) because amino acid phosphorylation changes the structure and physicochemical properties of the amino acid itself. Thus, changes in the structure and physicochemical properties of the N-terminal region owing to serine-phosphorylation may inhibit its passage through the ER. In this experiment, the 7th serine residue of OsPHR was converted to alanine residue in the pseudo-unphosphorylated state, and the serine residue was converted to aspartic acid residue or glutamic acid residue in the pseudo-phosphorylated form. Alanine is slightly more hydrophobic than serine, whereas aspartic and glutamic acids are highly hydrophilic. The resulting suppression of chloroplast translocation of the pseudo-phosphorylated form of OsPHR may be due to the increased hydrophilicity of the 7th amino acid, which inhibits the chloroplast translocation of OsPHR through the ER. According to the molecular hydrophobicity potential analysis, phosphorylation is predicted to cause protein surface rearrangements and a local decrease in hydrophobicity near the phosphorylation site (Polyansky and Zagrovic 2012). Thus, there is a possibility that phosphorylation of the 7th serine residue affects not only the polarity of the serine itself, but also the neighboring proline residues. The resulting increase in hydrophilicity may inhibit chloroplast translocation through the ER. However, it is not clear what changes in the structure and properties of OsPHR are caused by phosphorylation of the 7th serine residue. These are important future issues in elucidating the molecular mechanisms of vesicular transport systems.

Previous analyses have shown that most OsPHRs are present in a phosphorylated state (Teranishi et al. 2008). This may indicate that PHR is phosphorylated immediately after synthesis to inhibit its transport from its unphosphorylated state through the ER to the chloroplast. Phosphorylated PHRs, whose transport to chloroplasts is inhibited by phosphorylation are then transported to the nucleus or mitochondria using C-terminal nuclear or mitochondrial transport signal sequences (Takahashi et al. 2014), respectively. However, when PHR is transported to chloroplasts, it is important that phosphorylated serine is dephosphorylated to a nonphosphorylated state, which allows PHR to be rapidly transported to chloroplasts via the ER. Thus, we propose that the distribution of PHR in the nucleus, mitochondria, and chloroplasts is regulated by the phosphorylation state of serine residue at position 7 (Fig. 6).

Figure 6.

Figure 6.

Open in a new tab

A speculative model of vesicle-mediated endoplasmic reticulum (ER) to Golgi to chloroplast PHR transport under the control of the sequence characteristics of N-terminal part of PHR. In Poaceae plants with proline-rich N-terminal sequences, the nuclear-encoded phr, in an unphosphorylated or dephosphorylated state, is transported in vesicle-mediated ER to Golgi then to chloroplast, according to the amino acid residues critical for N-terminal chloroplast translocation. On the other hand, PHR is not transported to the chloroplast when it is phosphorylated after translation initiation on the ribosome but is transported to the mitochondria and nucleus. Furthermore, in plants other than the Poaceae family, PHR is not transported to the chloroplast because it does not have the amino acid sequences important for translocation to the chloroplast on the N-terminal side and is transported to the nucleus and mitochondria according to the nuclear localization signal and mitochondrial targeting signal on the C-terminus. The N-terminal sequence enriched for proline residues is shown in red. Phosphorylated serine is shown by a yellow circle with the letter P.

No transfer to chloroplasts was observed in the PHRs of the plants used in this study, except for Poaceae plant PHRs (Fig. 5). Several studies have examined the CPD repair activity in the chloroplasts and mitochondria of vascular plants under visible light or dark conditions. Young Arabidopsis seedlings (5 days old) displayed no photorepair activity in their chloroplasts (Chen et al. 1996). Conversely, the leaves of Arabidopsis (14 days old) show BL-dependent removal of CPDs from chloroplast DNA (Draper and Hays 2000). However, it remains unclear whether this effect is mediated by PHR. Based on our results of (i) transient expression analysis of AtPHR using protoplasts prepared from rice or Arabidopsis seedlings (Fig. 5, B and C), (ii) analysis of the subcellular localization of AtPHR in transgenic Arabidopsis plants transformed with AtPHR (Fig. 5, D and E), and (iii) measurement of the activity of UV-B-induced CPDs loss on chloroplast DNA (Fig. 5F and Supplementary Fig. S6), we concluded that AtPHR is not translocated or localized to chloroplasts in Arabidopsis. Kaiser et al. (2009) transiently transformed a green fluorescent protein (GFP) fusion of AtPHR (2 × CaMV 35S::AtPHR1::GFP) into green protoplasts from an Arabidopsis mesophyll cell culture to investigate the subcellular localization of AtPHR1. They demonstrated that the AtPHR1::GFP fusion protein was exclusively found in the nucleus and was not transported to the chloroplasts. These results support our findings.

In studies on the chloroplast localization of PHR in plants other than Arabidopsis thaliana, the number of CPDs in the chloroplast DNA of maize (Zea mays) leaves (Stapleton et al. 1997) was reduced in response to visible light. In contrast, no photoreactivation activity was detected in isolated Spinacia oleracea (spinach) chloroplasts (Hada et al. 2000). Although previous studies have suggested that plant species differ with respect to chloroplast localization of PHR, our results indicate that the ability of PHR to localize to chloroplasts may be specific to Poaceae plants. Is this because non-Poaceae plants simply do not have the sequence necessary for transfer to chloroplasts via the ER that the Poaceae plant PHR has, or do they lack a system of chloroplast transfer via the ER itself that utilizes this sequence? An interesting result here is that no citrine fluorescence was detected in chloroplasts when FL-OsPHR was transiently introduced into Arabidopsis protoplasts (Supplementary Fig. S9). Thus, our results show that the differences in chloroplast translocation of PHR among plant species are due to differences in the presence or absence of a system that utilizes the specific N-terminal sequence of Poaceae plant PHR to translocate it to chloroplasts via the ER.

Conclusions

When transgenic rice plants, in which PHR do not function in chloroplasts, were grown under UV-B-supplemented conditions, leaf browning, typical UV-B-induced chloroplast damage, was observed, which apparently caused growth defects (Fig. 1C). Therefore, it is necessary for PHR to function in the chloroplasts of rice plants to grow in the presence of UV-B, and rice plants may have acquired the ability to repair CPD in chloroplast DNA by transferring PHR to chloroplasts on their own during evolution. However, when Arabidopsis was grown under UV-B-added conditions, no browning of the leaves was observed, although the leaves showed UV-B damage that caused them to turn white (Izumi et al. 2017; Dündar et al. 2020; Otake et al. 2021). Thus, the UV-B damage phenotype is not necessarily uniform. These phenotypic differences indicate that the mechanisms of defence and adaptation to UV-B differ among plant species. For example, in Arabidopsis, whole chloroplasts damaged by UV-B irradiation are actively removed into vacuoles by chloroplast-targeted autophagy, also known as chlorophagy. Autophagy-defective atg mutants exhibit UV-B-sensitive phenotypes (Izumi et al. 2017; Dündar et al. 2020). However, such chlorophagic removal of impaired chloroplasts has not been observed in rice leaf cells with large chloroplasts, although rice also retains the chlorophagy mechanism, as plastids are removed by chlorophagy by vacuolar-type H+-ATPase inhibitor concanamycin A treatment in the roots of nongreen tissues in rice (Izumi et al. 2015). Thus, in plants without PHR translocation to chloroplasts, such a mechanism of chlorophagy may be activated to reduce UV-B-induced chloroplast damage. These differences in UV-B defence and adaptation mechanisms may also be due to the light environment in which each plant grows. In Poaceae plants grown under intense sunlight, maintaining mechanisms to repair chloroplast DNA damage may be more important than in plants grown under low sunlight. Nevertheless, the differences in the chloroplast localization of PHRs will be an interesting challenge in the future, as it is expected to lead to the elucidation of uniquely UV-B defense/adaptation strategy mechanisms for growth in various light environments.

Materials and methods

Generating the expression constructs

The cDNA sequences of OsPHR (accession No. AB096003) and AtPHR (accession No. AF053365), LjPHR (accession No. XP_057449869) were inserted into the pENTR/D-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA). To generate truncated or mutated forms of OsPHR, site-directed mutagenesis by inverse PCR was performed using the KOD-Plus Mutagenesis Kit (TOYOBO, Osaka, Japan) with OsPHR-inserted pENTR/D-TOPO as a template, according to the manufacturer's instructions. Nucleotide sequences encoding the N-terminal positions of various plants were synthesized. The amino acid positions and accession numbers for each plant are as follows: TaPHR (amino acids 1 to 52, AK330529), HvPHR (amino acids 1 to 52, AK372010), ZmPHR (amino acids 1 to 39, NP_001130580), GmPHR (amino acids 1 to 42, NP_001238710), AmPHR (amino acids 1 to 44, ERN13316), SmPHR (amino acids 1 to 74, EFJ26958), and CrPHR (amino acids 1 to 79, AAD39433). The synthesized sequences were ligated to an expression construct encoding a truncated form of OsPHR (Δ1 to 51OsPHR) using restriction enzyme cloning or InFusion cloning (InFusion HD Cloning kit, Takara Bio, Shiga, Japan). The modified PHR was cloned into the binary vector pMpGWB106 (Ishizaki et al. 2015) via gateway cloning using LR Clonase II (Thermo Fisher Scientific). The expression constructs were driven by the CaMV 35S promoter with citrine fused to the C-terminus of the inserted sequence. The primers used for all plasmid constructs are listed in Supplementary Table S2.

Production of transgenic plants

To produce transgenic rice (Oryza sativa) plants, each prepared construct was transformed into the Agrobacterium strain EHA101. Transformation was performed as described previously (Hidema et al. 2007; Teranishi et al. 2012) using OsPHR GT rice plants generated as follows. For making the OsPHR GT rice, a 4.4 kb fragment containing the first exon from the promoter sequence and a 4.4 kb fragment starting from the first intron were amplified by PCR using the primers shown in Supplementary Table S2 (Supplementary Fig. S10A). The amplified fragments were cloned into the binary vector, pKOD4 (Saika et al. 2015). The construct contained neomycin phosphotransferase II (nptII) as a positive selection marker and the diphtheria toxin A subunit gene (DT-A) as a negative selection marker. The construct was then transformed into the Agrobacterium strain EHA105. Secondary calli induced from mature seeds of the rice cultivar Nipponbare (Oryza sativa L. ssp. japonica) were used for transformation (Teranishi et al. 2012; Saika et al. 2015). The transformed calli were selected by using G418 (35 μg mL−1). The plants were regenerated and then self-fertilized for 2 generations to obtain T2 plants. To confirm the GT, PCR was performed using the primers listed in Supplementary Table S2, flanking the nptII insertion region (Supplementary Fig. S10B). Growth under supplementary UV-B radiation was tested to confirm the presence of GT plants (Supplementary Fig. S1). Three independent lines were obtained using GT-c as the host in this experiment.

To produce transgenic Arabidopsis (Arabidopsis thaliana), the expression constructs were transferred into the Agrobacterium strain GV3101. Agrobacterium was infected with the AtPHR-deficient mutant, uvr2-1, using the floral dipping method. Transgenic seeds were selected by using hygromycin B (50 μg mL−1). The plants were self-fertilized for 2 generations to obtain T2 plants.

Plant materials and growth conditions

Transgenic rice plants were grown and treated with UV-B radiation as follows: seeds of each plant, soaked in water at 30 °C for 2 days, were sown in pots (15 cm wide × 6 cm deep × 10 cm high) containing fertilized soil in a large growth cabinet (Tabai Espec Ltd., Osaka, Japan), with a 12 h/12 h photoperiod and temperatures at 28/20 °C, as described previously (Mmbando et al. 2020). Photosynthetic photon flux density (PPFD) was recorded using a data logger (LI-1000; Li-Cor Inc., Lincoln, NE, USA) and an L1-190SA sensor (Li-Cor Inc.). PPFD was adjusted to approximately 350 μmol m−2 s−1 at the top of the plants. To assess UV-B sensitivity, plants were grown under visible radiation supplemented with UV-B radiation for 30 days using 3 UV-B bulbs (FL20SE; Toshiba, Tokyo, Japan) located above the plants. Plants receiving UV-B irradiation were subjected to the same photoperiod as the plants grown under visible radiation. Under UV-B bulbs, a UV29 glass filter (Toshiba Glass Co., Shizuoka, Japan) reduced 290 nm radiation by 50% (Kang et al. 1998). UV-B intensity was measured using a data logger (LI-1000) and an SD-104B sensor (Li-Cor Inc.). The UV-B intensity at the plant level is 1.0 W m−2. The spectral distribution was measured using a spectroradiometer (USR-45DA; Ushio Inc., Tokyo, Japan). Biologically effective UV-B radiation (14.7 kJ m−2 d−1) was calculated using the Caldwell plant action spectrum and normalized to unity at 300 nm (Caldwell 1971).

To observe subcellular localization, the seeds were soaked in water at 30 °C for 2 days, were sown in pots, and were grown for 10 to 14 days, as described previously (Takahashi et al. 2014).

Arabidopsis plants were grown in soil in chambers with a 16 h/8 h photoperiod and temperatures at 23 °C, under white-fluorescent lamps (100 μmol m−2 s−1), as described previously. Twelve to fourteen-day-old seedlings were used in all experiments.

Preparation of protoplast from rice and Arabidopsis plants

Rice and Arabidopsis protoplasts were isolated, as described by Kuroha et al. (2018), with minor modifications. Rice (O. sativa L., Nipponbare) plants were grown in plastic boxes with 1/2 Murashige and Skoog medium (Wako, Osaka, Japan) and 0.35% gellan gum (Wako) under white light conditions at 30 °C for 10 days. Arabidopsis (Arabidopsis thaliana Columbia-0) plants were grown in pots containing fertilized soil under white light condition at 23 °C for 14 days. Rice leaf sheaths were used to isolate rice protoplasts, and Arabidopsis leaves were used to isolate Arabidopsis protoplasts. After enzymatic digestion of each strip, the protoplasts were released by filtering through a 57 µM nylon mesh, followed by adjusting approximately 4×106 cells mL−1 with 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.7) containing 0.4 M Mannitol and 15 mM MgCl2 for polyethylene glycol (PEG)-mediated transformation.

Protoplast transformation

Transformation was performed as described by Kuroha et al. (2018) with minor modifications. One hundred μL of isolated rice protoplasts were transferred into a 1.5-mL microfuge tube containing 3 μg of plasmid DNA, and 100 µL of isolated Arabidopsis protoplasts were transferred into a 1.5-ML microfuge tube containing 15 μg of plasmid DNA. After 10 min incubation on ice, 100 μL of PEG solution (40% PEG 4000, 0.2 M mannitol, and 0.1 M CaCl2) was added, followed by incubation at room temperature for 30 min in the dark. After the incubation, 750 μL of MES buffer (ph 5.7) containing 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl was added, mixed, and incubated under darkness at 25 °C overnight.

Brefeldin A treatment

Brefeldin A (BFA) treatment was performed as described by Villarejo et al. (2005) and Isayenkov et al. (2011) with minor modifications. Stock solution of BFA (Sigma-Aldrich, Burlington, MA, USA) was prepared at 36 mM by dissolving BFA in DMSO. Protoplasts were treated with BFA (final concentration of 180 μM) by adding a stock solution 30 min after transformation. In this experiment, 0.1% DMSO was used as a control. After incubation under darkness overnight at 25 °C, the signal of Citrine fluorescence in the BFA-treated or untreated protoplasts was observed using laser-scanning confocal microscopy.

Percentage of number of protoplasts with citrine fluorescence detected in chloroplasts

Protoplast transformation experiments were performed at least 3 times per construct. The solution in which the constructs were introduced into the protoplasts was placed in a glass-based dish (AGC TECHNO GLASS Co., Shizuoka, Japan) and observed under a confocal microscope. Then, 9 to 12 images of the solution containing at least 10 protoplasts per image were randomly acquired. For each image, protoplasts in which citrine fluorescence was observed in the nucleus were defined as transformed protoplasts, and the number of protoplasts in which citrine fluorescence was detected in chloroplasts among the transformed protoplasts was measured. The percentage of the number of protoplasts in which citrine fluorescence was detected in chloroplasts among the number of transformed protoplasts per image was then calculated. The value of the percentages per image was used to calculate the average and standard error (se). The transformation efficiency per transformation experiment was at least 40%. The total number of transformed protoplasts measured per experiment was at least 55.

Laser-scanning confocal microscopy imaging

Thin layers were peeled from the young leaves of the transgenic rice plants using needles and forceps. To detect mitochondria, the root was incubated with 0.5 µM MitoTracker Red (Thermo Fisher Scientific) for 15 min at 25 °C and washed twice with H2O. Fluorescence was observed using a confocal laser-scanning microscopy (LSCM) performed using a C-apochromat LD63× water-immersion objective (numerical aperture = 1.15; LSM800, Carl Zeiss, Oberkochen, Germany). Citrine was excited using a 488-nm laser, chlorophyll autofluorescence was excited using a 640-nm laser, and MitoTracker Red was excited using a 561-nm laser, and fluorescent images were acquired.

Southern blot analysis

Southern blot analysis of organelle-specific DNA repair was based on a quantitative comparison of the intensity of T4 endonuclease V-treated and untreated DNA (Hanawalt 1989; Chen et al. 1996; Stapleton et al. 1997). T4 endonuclease V cleaves single DNA strands at the CPD sites. The fully expanded fourth leaves of rice grown on soil were irradiated for 25 min at a distance of 15 cm from the germicidal lamp (GL-15, Toshiba Electronic Co., Tokyo, Japan). The leaves were then exposed to blue irradiation from blue-fluorescent tubes for 0.5 and 1 h (FL20S B-F; National Co., Osaka, Japan). In the case of Arabidopsis, 12 days-old Ler seedlings grown on agar medium were irradiated for 15 min at a distance of 15 cm from the GL. The seedlings were then exposed to blue irradiation from blue-fluorescent tubes for 10, 18, and 24 h or were immediately placed in a light-tight box for 24 h. The irradiation intensity was adjusted to approximately 60 µmol m−2 s−1. Then, the rice leaves and Arabidopsis seedlings were immediately harvested and frozen in liquid nitrogen, then stored at −80 °C until analysis. About 8 leaves for rice and 50 seedlings for Arabidopsis were used for each condition. Genomic DNA was isolated from the leaves and shoots of irradiated or nonirradiated samples, as described previously (Teranishi et al. 2004). Genomic DNA was digested using BamHI and XhoI for rice and BamHI for Arabidopsis. The digested DNA was divided into 2 equal aliquots; 1 aliquot was treated with T4 endonuclease V, and the other was left untreated. The samples were resolved by alkaline agarose gel electrophoresis, and the DNA was blotted onto a nylon membrane as described previously (Hidema et al. 2007).

To generate probes for rice, a nuclear-specific gene (phr: Os10g0167600), a mitochondrial-specific gene (cob: GenBank Accession DQ167400.1), and a chloroplast DNA containing chloroplast-specific gene (atpI: GenBank Accession ON101253.1) were amplified from rice genomic DNA using gene-specific primers (Supplementary Table S2). To generate probes for Arabidopsis, a nuclear-specific gene (NRPB1: AT4G35800), a mitochondrial DNA containing mitochondrial-specific gene (COX1: ATMG01360), and a chloroplast-specific gene (PSBA: ATCG00020) were amplified using gene-specific primers (Supplementary Table S2). The amplified fragments were subcloned into pGEM-T Easy Vector (Promega). The digested inserts were labeled with [α-32P] 2'-deoxycytidine 5'-triphosphate and hybridized individually onto nylon membranes (Hidema et al. 2007).

Statistical analysis

All data with error bars are presented as the standard error of the mean. Tukey's test was used to compare multiple samples, as indicated in the figure legends.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers in Supplementary Table S3.

Supplementary Material

kiae060_Supplementary_Data
kiae060_supplementary_data.zip (2.1MB, zip)

Acknowledgments

We thank Dr. Takayuki Kohchi (Kyoto University) for providing the pMpGWB106 vector, Dr. Yoshikatsu Sato (Nagoya University) for providing Kakshine PC3 reagent. We thank Drs. Seiichi Toki and Hiroaki Saika (National Institute of Agrobiological Sciences, Japan) for providing the vectors for constructing GT rice plants. We deeply thank Drs. Atsushi Higashitani and Shusei Sato (Tohoku University) for their valuable comments and discussions. We thank Ms. Chikako Mitsuoka, Technical Assistant, for her technical support in constructing GT rice plants. We thank Ms. Hiroko Yamaguchi, Technical Assistant, for her technical support in measuring the activity of the PHR.

Contributor Information

Momo Otake, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.

Mika Teranishi, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.

Chiharu Komatsu, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.

Mamoru Hara, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.

Kaoru Okamoto Yoshiyama, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.

Jun Hidema, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan; Division for the Establishment of Frontier Sciences of the Organization for Advanced Studies, Tohoku University, Sendai, Miyagi 980-8577, Japan.

Author contributions

M.O. and J.H. conceived and designed the experiments. M.O. performed most of the experiments. M.T. prepared the transgenic rice plants and measured CPD. C.K. and M.H. assisted in the subcellular localization analysis. K.Y. designed the constructs; M.O. M.T. and J.H. wrote the manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Effects of ultraviolet-B (UV-B) radiation on rice plants.

Supplementary Figure S2. Repair of CPD in rice (Oryza sativa, Os) genomic DNA.

Supplementary Figure S3. Confirmation that strong speckled citrine signals in chloroplasts of protoplasts transformed with full length-rice (Oryza sativa, Os) CPD photolyase (FL-OsPHR) were nucleoids.

Supplementary Figure S4. Effects of chloroplast translocation of RBCS by BFA treatment.

Supplementary Figure S5. Multiple sequence alignment of deduced amino acid sequences of CPD PHR in various plant species using the Clustal omega program.

Supplementary Figure S6. Overall view of each membrane in Fig. 5F. Repair of CPD in Arabidopsis genomic DNA.

Supplementary Figure S7. Cellular localization of CPD PHR in Poaceae species.

Supplementary Figure S8. Identification of important proline residues for chloroplast translocation in Poaceae-CPD PHR.

Supplementary Figure S9. Confocal microscopic images of Arabidopsis thaliana protoplasts expressing Citrine (as a control), Arabidopsis-CPD PHR-Citrine (AtPHR), or Oryza sativa PHR-Citrine (OsPHR).

Supplementary Figure S10. Construction of rice (Oryza sativa, Os) CPD PHR (OsPHR) GT rice plants.

Supplementary Table S1. TargetP performance values.

Supplementary Table S2. Primer sequences were used in this study.

Supplementary Table S3. Accession number in this study.

Funding

This work was supported by JST SPRING (Grant Number JPMJSP2114 to M.O.) and Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the promotion of Science (JSPS) KAKENHI (Grant Numbers JP21H05665 and JP20H04330 to J.H.).

Data availability

The data underlying this article are available in the article and in its online Supplemental Data.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

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