The photosensitive phs1 mutant is impaired in the riboflavin biogenesis pathway

Journal of Plant Physiology 167 (2010) 1466–1476

Contents lists available at ScienceDirect

Journal of Plant Physiology journal homepage: www.elsevier.de/jplph

The photosensitive phs1 mutant is impaired in the riboflavin biogenesis pathway Min Ouyang, Jinfang Ma, Meijuan Zou, Jinkui Guo, Liyuan Wang, Congming Lu, Lixin Zhang ∗ Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

a r t i c l e

i n f o

Article history: Received 17 March 2010 Received in revised form 17 May 2010 Accepted 19 May 2010 Keywords: Arabidopsis Photooxidative damage Riboflavin pathway

a b s t r a c t A photosensitive (phs1) mutant of Arabidopsis thaliana was isolated and characterized. The PHS1 gene was cloned using a map-based approach. The gene was found to encode a protein containing a deaminase–reductase domain that is involved in the riboflavin pathway. The phenotype and growth of the phs1 mutant were comparable to that of the wild-type when the plants were grown under low light conditions. When the light intensity was increased, the mutant was characterized by stunted growth and bleached leaves as well as a decrease in FNR activity. The NADPH levels declined, whereas the NADP+ levels increased, leading to a decrease in the NADPH/NADP+ ratio. The mutant suffered from severe photooxidative damage with an increase in antioxidant enzyme activity and a drastic reduction in the levels of chlorophyll and photosynthetic proteins. Supplementing the mutant with exogenous FAD rescued the photosensitive phenotype, even under increasing light intensity. The riboflavin pathway therefore plays an important role in protecting plants from photooxidative damage. © 2010 Elsevier GmbH. All rights reserved.

Introduction Riboflavin is the precursor of the coenzymes riboflavin monophosphate and flavin adenine dinucleotide, which serve as indispensable redox cofactors in all organisms. In plants, these cofactors are required for numerous critical cellular processes as diverse as the citric acid cycle, fatty acid oxidation, mitochondrial electron transport and de novo pyrimidine biosynthesis. They also participate in a variety of other vital processes, such as DNA photorepair (Sancar, 1994; Hitomi et al., 2009), light sensing (Salomon et al., 2001; Briggs and Huala, 1999), bioluminescence (Lee et al., 1992; Meighen, 1993), jasmonate (JA) signal transduction (Xiao et al., 2004) and the functioning of circadian and seasonal clocks (Imaizumi et al., 2003; Hitomi et al., 2009). The biosynthesis of riboflavin has been studied in some detail in eubacteria and fungi (Richter et al., 1993, 1997; Ritz et al., 2001; Kaiser et al., 2002). It consists of seven distinct enzyme-catalyzed reactions and requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates, which are catalyzed in turn by GTP cyclohydrolase II, dihydroxybutanone phosphate synthase, pyrimidine deaminases, pyrimidine reductases, hypothetical phos-

Abbreviations: FNR, ferredoxin-NADP+ oxidoreductase; NBT, nitroblue tetrazolium; SOD, superoxide dismutase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; PET, photosynthetic electron transport; ROS, reactive oxygen species; SSLP, simple sequence length polymorphism. ∗ Corresponding author. Tel.: +86 10 62836256; fax: +86 10 82599384. E-mail address: [email protected] (L. Zhang). 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.05.005

phatase, lumazine synthase and riboflavin synthase (Fischer and Bacher, 2008). Recently, several homologous genes involved in the riboflavin pathway were cloned in plants. The ribA gene, which encodes a bifunctional GTP cyclohydrolase II/dihydroxybutanone phosphate synthase, was cloned from tomato and Arabidopsis thaliana (Herz et al., 2000). The lumazine synthase gene was cloned from spinach, tobacco and A. thaliana (Jordan et al., 1999); this gene was shown to be involved in JA signaling transduction in A. thaliana (Xiao et al., 2004). The riboflavin synthase gene and the pyrimidine deaminases gene were both cloned from A. thaliana (Bacher and Eberhardt, 2001; Fischer et al., 2004), but little is known about the enzyme catalyzing the reduction of the ribosyl side chain in plants. In A. thaliana, there are two genes (At4g20960, At3g47390) that contain riboflavin deaminase–reductase domains; the At4g20960 protein was shown to demonstrate deamination activity (Fischer et al., 2004), and it is likely that the At3g47390 protein shows pyrimidine reductases activity. In plant thylakoid membranes, light drives the flow of electrons from water via photosystem II (PSII), cytochrome b6 f (cytb6 f) and PSI to ferredoxin. Light energy is important for maintaining plant growth and metabolism, but when the amount of energy absorbed exceeds the amount needed to drive metabolism, plants will likely suffer from photoinhibition and oxidative stress (Krieger-Liszkay et al., 2008). Several biosynthetic processes and metabolites might protect plants from excessive light and photooxidative stress, including the ascorbate–glutathione cycle (May et al., 1998; Noctor and Foyer, 1998; Ding et al., 2009), the xanthophyll cycle (Arnoux et al., 2009; Li et al., 2009), carotenoid (Davison et al., 2002), vitamin B6 (Havaux et al., 2009), vitamin E (Havaux et al., 2005) and others.

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

In this study, we isolated and characterized a photosensitive mutant (phs1). The PHS1 gene was cloned using a map-based approach. It was found to encode a protein containing a deaminase–reductase domain, which is a key component in the riboflavin pathway. We show that the riboflavin pathway is important for protecting plants from photooxidative stress. Materials and methods 1.1. Plant material and growth conditions The phs1 mutant was screened from a collection of pSKI015 TDNA mutagenized Arabidopsis (ecotype Columbia) lines from the ABRC. The mutants were selected based on the presence of bleaching when the plants were grown under 140 ␮mol m−2 s−1 of light. Wild-type and homozygous mutant plants were grown on either Murashige or Skoog (MS) medium containing 3% sucrose or on soil. The plants were grown at 22 ◦ C under short-day conditions (10 h of light/14 h of dark) with a photon flux density of 30 ␮mol m−2 s−1 , 80 ␮mol m−2 s−1 or 140 ␮mol m−2 s−1 . To determine the effects of chemical treatments on plant growth and phenotypes, the mutant and wild-type plants were grown on MS medium supplemented with 10 mg/L riboflavin, FMN or FAD. 1.2. Map-based cloning and complementation A mapping population was obtained by crossing wild-type plants of the ecotype Landsberg erecta (Ler) with ecotype Columbia (Col) heterozygous phs1 plants. Homozygous F2 mutant plants were screened based on the photosensitive phenotype described above. Genomic DNA was extracted from 1024 mutant plants and subjected to PCR analysis using specific molecular markers. The mutation was mapped to a 120-kb region between two bacterial artificial chromosomes (F1P2 and T21L8) on chromosome 3. Candidate genes with predicted chloroplast transit peptides were sequenced and analyzed with cDNA from phs1 and wild-type plants (Col). To confirm the mutation, a complementation experiment was performed as follows. A fragment containing the full-length PHS1 coding sequence was amplified with primers 5 -CTGGATCCATGGCTCTTTCATTTCG-3 and 5 -TTTGAATTCTCAGGCCGAAGAAGTCT-3 and subcloned into the pBI121 vector under the control of the cauliflower mosaic virus 35S promoter. The constructs were then transformed into Agrobacterium tumefaciens strain EHA105 and introduced into the phs1 mutant plants using the floral dip method (Clough and Bent, 1998). Transgenic plants were planted on MS medium containing 40 ␮g ml−1 kanamycin monosulfate. Resistant plants were transferred to soil and grown in a growth chamber to produce seeds. 1.3. Nucleic acid preparation and analysis Total RNA was isolated from 100-mg samples of fresh plant leaves using the TRIZOL reagent, and DNA was isolated as described by Liu et al. (1995). The expression of PHS1 was determined using RT-PCR analysis. Equal amounts of RNA were loaded for each sample, and RT-PCR analysis of actin cDNA was performed using the following primers: sense primer (5 -AACTGGGATGATATGGAGAA3 ) and antisense primer (5 -CCTCCAATCCAGACACTGTA-3 ). 1.4. Chlorophyll fluorescence analysis and oxido-reduction of P700 Chlorophyll fluorescence images and data were obtained as described by Chi et al. (2008). The image acquired in each experiment was normalized to a false color scale; the lightest areas were arbitrarily assigned a value of 0, whereas the darkest areas were

1467

assigned a value of 0.85. As a result, the highest and lowest Fv/Fm values were represented by the red and blue extremes of the color scale, respectively (CF Imager; Technologica). The maximum quantum efficiency of PSII photochemistry was expresses as the ratio of variable to maximal fluorescence, Fv/Fm; this was measured in intact leaves using a PAM 2000 portable chlorophyll fluorometer (Walz). For the measurement of light-induced P700 absorbance changes at 820 nm, the PAM chlorophyll fluorometer was equipped with an ED 800T (Walz) emitter–detector unit, and the measurements were performed according to the methods of Peng et al. (2006). 1.5. Transmission electron microscopy Samples of wild-type and mutant leaves were prepared for transmission electron microscopy as described by Peng et al. (2006). The samples were then viewed with a transmission electron microscope (JEM-1230; JEOL). 1.6. Total protein preparation, chlorophyll content determination and protein-blot analysis Total proteins were extracted from the samples as described by Martinez-Garcia et al. (1999). The protein concentrations were determined using the BioRad DC protein assay (Bio-Rad, Hercules, CA, USA). The quantitative determination of chlorophyll content was carried out as described by Lichtenthaler (1987). For protein-blot analysis, total proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with specific primary antibodies, and the signals from secondary conjugated antibodies were detected using the enhanced chemiluminescence method. 1.7. Antiserum production The nucleotide sequences encoding the N-terminus of PHS1 (amino acids 196–314 corresponding to nucleotide positions 586–942 of the PHS1 gene) were amplified by PCR using the primers 5 -CACGCTAGCCTTGATGGTAAGATTGC-3 and 5 CCCAAGCTTTTCCATAACTTCTCTTG-3 . The resulting DNA fragment was cleaved with Nhe I and Hind III and fused in frame with the N-terminal His affinity tag of pET28a. The fusion protein expression, purification and antiserum production were carried out as described by Ma et al. (2007). 1.8. Isolation of chloroplasts and measurement of ferredoxin-NADP+ oxidoreductase (FNR)-dependent cytochrome c reductase activity The chloroplasts were isolated as described in Zhang et al. (1999). Briefly, the leaves were homogenized in 330 mM sorbitol, 5 mM ascorbate, 0.05% bovine serum albumin, 2 mM EDTA, 1 mM MgCl2 and 50 mM Hepes-KOH, pH 7.6, filtered through Miracloth and centrifuged for 1 min at 1000 × g. The pellets were resuspended in Medium A (330 mM sorbitol, 2 mM dithiothreitol, 50 mM HepesKOH, pH 8.0) and loaded onto Percoll step gradients (40% and 70% in Medium A) and spun for 5 min at 4500 × g at 4 ◦ C. The chloroplasts in 70% Percoll were diluted with Medium A, spun for 2 min at 1300 × g, and washed once with Medium A. All subsequent manipulations were carried out on ice or at 4 ◦ C. The chloroplasts were osmotically shocked on ice for 10 min in a hypotonic buffer containing 50 mM Hepes-KOH, pH 8.0, and 5 mM MgCl2 . FNRdependent cytochrome c reductase activity was determined using the methods of Orellano et al. (1993). The reaction medium (1 ml) contained 50 mM Tris–HCl (pH 8.0), 0.3 mM NADP+ , 3 mM glucose6-phosphate, 1 U of glucose-6-phosphate dehydrogenase, 50 ␮M

1468

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

cytochrome c, 5 ␮M Fd, and 5 ␮g of the protein sample. The reaction was monitored spectrophotometrically by following cytochrome c reduction at 550 nm (ε550 = 19 mM−1 cm−1 ).

was determined by following the rate of GSH formation at 412 nm (González et al., 1998). Results

1.9. Measurement of NADP+ and NADPH contents 1.13. Isolation of a photosensitive mutant Phosphorylated pyridine nucleotides (NADP+ , NADPH) were extracted from fresh leaf tissue and assayed as described by Foyer et al. (1991). Leaves were ground into a fine powder in liquid nitrogen. The resulting powder was divided into two approximately equal samples. For NADP+ determination, the powder was mixed with 10 ml of 0.1 M HClO4 . The suspension was boiled for 2 min and centrifuged at 10,000 × g for 5 min. One ml of the supernatant was neutralized with 0.2 ml of 1 M Tris–HCl, pH 7.4 and 1 ml of 0.1 M KOH and incubated on ice for 15 min. For NADPH determination, the powder was mixed with 10 ml of 0.1 M KOH. The suspension was treated as above, except that it was neutralized with 0.2 ml of 1 M Tris–HCl (pH 7.4) and 1 ml of 0.1 M HClO4 . The neutralized supernatants were assayed for either NADP+ or NADPH using the enzyme cycling method (Slater and Sawyer, 1962). 1.10. Determination of FAD concentration FAD was extracted as described by Bafunno et al. (2004). Onehalf gram of leaves was ground into a fine powder in liquid nitrogen, and the powder was treated with detergent extraction buffer (50 mM Tris–HCl pH 7.0, 1% [w/v] digitonin). The supernatant was collected from the digitonin-treated sample after centrifugation at 15,000 × g for 5 min and used for the determination of FAD content. The protein concentration was determined using the BioRad DC protein assay (Bio-Rad, Hercules, CA, USA). The concentration of FAD was determined from the rate of oxidation of NADPH by PHBH during the catalysis of the conversion of POHB, as described by Müller and van Berkel (1982). After 2.5 min of incubation of apoPHBH with 0.2 mM POHB and 0.2 mM NADPH in 0.1 mM Tris–HCl, pH 8.0, at 25 ◦ C, the reaction was initiated by the addition of either FAD or the protein sample. The oxidation of NADPH was monitored at 340 nm. The FAD concentration of the sample was determined using a calibration curve (rate of NADPH oxidation vs FAD concentration) that was obtained by the addition of aliquots of a standard FAD solution to the reaction mixture. 1.11. In situ detection of O2 − The in situ detection of O2 − was performed using nitroblue tetrazolium (NBT) staining (Rao and Davis, 1999). The detached leaves were submerged in an NBT (1 mg ml−1 ) solution containing 10 mM NaN3 (10 mM) in 10 mM potassium phosphate buffer (pH 7.8). After a brief vacuum infiltration, the leaves were stained in NBT for 30 min at room temperature. The stained leaves were cleared by boiling them in an acetic acid:glycerol:ethanol (1:1:3 [v/v/v]) solution prior to photography. 1.12. Measurement of antioxidant enzyme activities One gram of fresh leaves was ground into a fine powder in liquid nitrogen and homogenized in 2 ml of extraction buffer (0.1 M Tris–HCl, pH 7.8, 10 mM MgCl2 , 1.0 mM EDTA, and 2% PVP). After centrifugation at 13,000 × g for 10 min, the supernatant was collected and assayed for enzyme activity. Superoxide dismutase (SOD, EC1.15.1.1) activity was estimated by employing its ability to inhibit the photoreduction of nitroblue tetrazolium (Beyer and Fridovich, 1987). Dehydroascorbate reductase (DHAR, EC1.8.5.1) activity was determined by measuring the increase in absorbance at 265 nm caused by DHA reduction (Knörzer et al., 1996). GR activity

The photosensitive mutant was isolated by screening for the photobleaching phenotype at 140 ␮mol m−2 s−1 from the Scheible and Somerville T-DNA Arabidopsis lines (Weigel et al., 2000). To study the effects of light intensity on the growth and phenotype of the mutants, the wild-type and mutant plants were grown under low light (30 ␮mol m−2 s−1 ) for four weeks followed by growth under low light (30 ␮mol m−2 s−1 ), normal light (80 ␮mol m−2 s−1 ) or high light (140 ␮mol m−2 s−1 ) for two weeks. Under low light conditions, the mutant displayed a phenotype similar to that of the wild-type; the Fv/Fm ratios (indicating the maximum potential capacity of the photochemical reactions of PSII) and chlorophyll contents of the phs1 mutant were similar to those of wild-type plants. Under normal light conditions, the mutant plants showed reduced growth and increasingly chlorotic leaves. The phs1 mutant contained about 30% as much chlorophyll as the wild-type. Under high light conditions, some of the mutant leaves were severely bleached, and the spreading chlorosis covered nearly all of the leaves. The levels of chlorophyll in the mutant were reduced to only about 15% of the wild-type levels (Fig. 1). Meanwhile, PSII activity in the phs1 mutant plants also gradually decreased with increasing light intensity relative to wild-type levels (0.85). The Fv/Fm values of the bleached leaves of the phs1 mutant were only about 0.5 in normal light and 0.3 in high light, compared to 0.85 for wild-type plants (Fig. 1). 1.14. Ultrastructure of the wild-type and mutant chloroplasts We next investigated the ultrastructure of chloroplasts from wild-type and mutant plants grown under different light conditions by examining electron micrographs of ultrathin sections obtained from leaf samples (Fig. 2). The ultrastructures of wildtype chloroplasts were similar in plants grown under different light conditions. The chloroplasts displayed well-developed membrane systems composed of grana connected by stroma lamellae. Under low light, the ultrastructure of mutant chloroplasts was similar to that of wild-type chloroplasts. However, under normal light conditions, the thylakoid membrane systems of mutant chloroplasts were disturbed and the membrane spacing was not as clear as that of wild-type chloroplasts. Under high light conditions, the mutant chloroplasts began to enlarge, and their thylakoid membranes were distorted. 1.15. Map-based cloning of the mutated gene Genetic analysis showed that the mutation was recessive and that the mutant phenotype did not cosegregate with the phosphinothricin resistance marker, indicating that the mutated gene was not tagged by the T-DNA (data not shown). Map-based cloning of the mutated gene was performed using simple sequence length polymorphism (SSLP) molecular markers. Using several molecular markers (Lukowitz et al., 2000), the gene was mapped to chromosome 3. Further mapping using two newly-developed SSLP markers (A18000 and A19000) placed the gene between BAC clones T21L8 and F1P2, which contains a 120-kb Arabidopsis genomic insert and 21 annotated ORFs. The cDNAs from each ORF were amplified from total RNA isolated from the mutant using a series of overlapping, gene-specific primers to each ORF, and the RT-PCR products were sequenced. One ORF (designated At3g47390) was found to have a PCR product with a 14-nucleotide deletion from base 1247 to 1260

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

1469

Fig. 1. Characterization of wild-type and phs1 plants grown under low light (30 ␮mol m−2 s−1 ) for four weeks, followed by growth under low light (LL, 30 ␮mol m−2 s−1 ), normal light (NL, 80 ␮mol m−2 s−1 ) or high light (HL, 140 ␮mol m−2 s−1 ) for two weeks. Leaves indicated with a red arrow were used for the chlorophyll fluorescence measurement. (a) Photographs and chlorophyll fluorescence images of wild-type and phs1 plants. Fluorescence was measured by the FluorCam700MF and visualized using a pseudocolor index as indicated at the bottom. The highest (0.85) and lowest (0) Fv/Fm values were represented by the red and blue extremes of the color scale, respectively. (b) Chlorophyll (Chl) contents per leaf area from wild-type and phs1 plants. All of the leaves of wild-type and phs1 plants were prepared for chlorophyll content analysis. The values are mean ± SD of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

in the coding region of the gene At3g47390; this resulted in the truncation of the protein due to the presence of the premature stop codon TGA (Fig. 3). To confirm that the phenotype of the phs1 mutant was due to the mutation in At3g47390, a complementation experiment was carried out using full-length At3g47390 cDNA. Twenty-one successfully complemented transgenic plants had growth rates and

phenotypes that were similar to those of wild-type plants. Thus, the inactivation of the At3g47390 gene was responsible for the phs1 mutant phenotype. BLAST searches revealed that the PHS1 gene encodes a putative protein with a riboflavin deaminase–reductase domain (Fig. 4). Protein alignments showed that it shares significant identity at the amino acid level with proteins from Oryza sativa (69%), Sorghum

1470

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

Fig. 2. Transmission electron microscopic images of chloroplasts of wild-type and phs1 plants grown under different light conditions as described in Fig. 1. Leaves indicated with a red arrow in Fig. 1 were used for transmission electron microscopy analysis. Bars = 1 ␮m.

bicolor (62%), Chlamydomonas reinhardtii (50%) and another A. thaliana protein, At4g20960 (25%).

only FAD could rescue the photosensitive phenotype under normal light and that the photosensitive phenotype could be partially reversed under high light (Fig. 6).

1.16. RT-PCR and immunoblot analysis To determine whether the 14-bp deletion in the mutant affects PHS1 expression, RT-PCR and immunoblot analysis was performed. RT-PCR analysis showed that the expression of the At3g47390 gene was similar in wild-type and phs1 plants after 30 cycles, which suggested that the 14-bp deletion does not affect the transcript accumulation of PHS1. The 40-kD PHS1 truncated protein fragments were detected in phs1 plants, but not in wild-type plants, using a specific PHS1 antibody (Fig. 5).Exogenous FAD can rescue the mutant phenotype Because the PHS1 gene encodes a protein that is involved in the riboflavin biosynthetic process, and because riboflavin, FMN and FAD are the final products of the riboflavin pathway, we examined whether exogenous riboflavin, FMN or FAD could rescue the phenotype of the mutant grown on MS medium. Our results showed that

Fig. 3. Map-based cloning of the PHS1 gene. (a) SSLP markers were used to map the PHS1 gene to the ciw4 region of chromosome III. A mapping population was obtained by crossing wild-type plants of the ecotype Landsberg erecta (Ler) with ecotype Columbia (Col) heterozygous phs1 plants. Genomic DNA was extracted from 1024 homozygous F2 mutant plants and subjected to PCR analysis using specific molecular markers. The gene was fine-mapped to a 120-kbp interval between markers A18000 and A19000 (supporting information). This region is covered by two bacterial artificial chromosome clones, T21L8 and F1P2. (b) The phs1 mutant has a 14-nucleotide deletion from base 1247 to 1260 (red font) in the coding regions of the At3g47390 gene, resulting in the truncation of the protein by a following stop codon TGA (purple font). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

1.18. Changes of the FAD content, FNR activity and the NADPH/NADP+ ratio Because FAD could rescue the mutant phenotype, we determined the FAD content of wild-type and mutant leaves under various light conditions. The FAD contents were similar in wildtype leaves grown under different light conditions. Under low light conditions, the FAD content was lower in mutant plants than in wild-type plants. In addition, the levels of FAD in the mutant under both normal and high light conditions were comparable to that under low light conditions. In addition, because of the variable phenotype seen in mutant plants grown under normal and high light conditions, we determined the FAD content in bleached and green mutant leaf tissue separately. Interestingly, the bleached and green leaf tissue had similar levels of FAD under the same light conditions. Because FAD acts as the cofactor of ferredoxin-NADP+ oxidoreductase (FNR), we further investigated the FNR protein content and FNR-specific cytchrome c reductase activity in total leaf extracts from the mutant and wild-type plants. No significant difference in the levels of FNR protein was observed between wild-type and mutant plants grown under various light conditions (Fig. 7). The FNR activities were similar in wild-type and mutant plants grown under low light conditions. However, the FNR activities in the mutant were reduced to ∼38% and 20% of wild-type levels under normal and high light conditions, respectively (Fig. 7). Using FAD as a cofactor, FNR mediates the last step of photosynthetic electron flow by transferring electrons from ferredoxin to NADP+ , which produces the reducing power (NADPH) required for CO2 fixation and other biosynthetic pathways. Previous reports showed that FNR deficiency could lead to the oxidation of the NADPH pool (Hajirezaei et al., 2002; Palatnik et al., 2003). In this study, the ratios of NADPH to NADP+ in the mutant were reduced to ∼36% and 24% of wild-type levels under normal light and high light conditions, respectively (Fig. 7). 1.19. The kinetics of P700 oxidation rates To understand whether there is a limitation in electron flow at PSI in mutant plants grown under normal or high light condi-

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

1471

Fig. 4. Amino acid sequence alignment of PHS1 deaminase–reductase domains. Comparison of the amino acid sequence of the PHS1 deaminase–reductase domains with the proteins from Oryza sativa, Sorghum bicolor, Chlamydomonas reinhardtii and Arabidopsis thaliana. Black boxes indicate strictly-conserved amino acids among these proteins.

tions, the kinetics of the P700 oxidation and re-reduction rates of wild-type and phs1 plants grown under various light levels were measured. The P700 oxidation and re-reduction rates of wild-type plants under all of the light conditions were similar to that of the phs1 mutant plants grown under low light condition. However, the phs1 plants grown in normal or high light showed significantly slower oxidation and re-reduction of P700+ than did the wild-type plants as reflected by the increased tox1/2 (the time of reaching the half of steady-state level of P700+ ) and tre1/2 (the time of the half re-reduction of P700 oxidized) of mutants grown under normal or high light conditions (Table 1). 1.20. Oxidative stress responses of phs1 mutant The decreased FNR activity and NADPH/NADP+ ratios, slower P700 oxidation and re-reduction rates observed in the mutants

might lead to the channeling of electrons from PSI to molecular oxygen via the Mehler reaction (Mehler, 1951), which results in the formation of superoxide radicals (O2 − ). To examine the enhanced oxidative stress and increased ROS levels occurring in the mutant, we examined the accumulation of O2 − in the mutant and in the wild-type under various light conditions. Under normal and high light conditions, the contents of O2 − were significantly higher in the mutant than in the wild-type (Fig. 8). We further measured the activities of various antioxidant enzymes of ascorbate–glutathione cycle in total leaf extracts from mutant and wild-type plants. The activities of total SOD and DHAR in the mutant were increased by about 20% and 30% under normal and high light conditions, respectively, compared to that of the wild-type (Fig. 8). Interestingly, there were significant differences in the GR activity between the mutant and wild-type under both normal and high light conditions. The GR activity in the mutant showed a strong increase compared to

1472

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476 Table 1 P700 oxidation and re-reduction rates of wild-type and phs1 plants. tox 1/2 (s) WTLL WTNL WTHL phs1LL phs1NL phs1HL

1.04 1.10 1.06 1.15 1.53 1.65

± ± ± ± ± ±

0.05 0.07 0.1 0.04 0.10 0.08

(%) (100) (105) (102) (109) (147) (161)

tre1/2 (s) 1.10 1.20 1.15 1.14 1.47 1.75

± ± ± ± ± ±

0.10 0.06 0.11 0.09 0.12 0.15

(%) (100) (109) (104) (103) (134) (164)

The wild-type and phs1 plants were grown under low light (30 ␮mol m−2 s−1 ) for four weeks, followed by growth under low light (LL, 30 ␮mol m−2 s−1 ), normal light (NL, 80 ␮mol m−2 s−1 ) or high light (HL, 140 ␮mol m−2 s−1 ) for two weeks. The redox kinetics of wild-type and phs1 mutant plants were investigated by measuring absorbance changes of P700 at 820 nm induced by far-red light (FR; 720 nm). Both the tox1/2 (the time of reaching the half of steady-state level of P700+ ) and the tre1/2 (the time of the half of re-reduction of P700 oxidized) of mutant under NL or HL conditions are more than those of wild-type plants. Taking the t1/2 value of wildtype under low light conditions as control (100), the relative t1/2 values were also calculated.

the wild-type, with a 280% and 360% increase under normal and high light conditions, respectively. Under low light conditions, not only the contents of O2 − , but also the antioxidant activities of the mutant were similar to that of the wild-type. Fig. 5. Expression of the PHS1 gene. (a) RT-PCR analysis. Total RNA was isolated from 100-mg samples of fresh plant leaves using the TRIZOL reagent. The expression of PHS1 was determined using RT-PCR analysis with specific primers for At3g47390 and actin-specific primers. (b) Leaves of wild-type and phs1 plants under LL conditions for six weeks were prepared for truncated protein detection. The total proteins were extracted, separated by SDS-PAGE and immunodetected with antibodies raised against the N-terminal residues (196–314) of the PHS1 protein. The mutant truncated protein fragment is indicated with an arrow.

1.21. Changes in photosynthetic protein content To assess whether the photooxidative damage might be reflected at the level of photosynthetic proteins, immunoblot analysis was performed using specific antibodies against the subunits of photosynthetic protein complexes using total protein extracts prepared from the leaves of mutant and wild-type plants (Fig. 9).

Fig. 6. Effects of riboflavin, FMN and FAD on the phenotype. The wild-type and phs1 mutant plants were grown on MS medium supplemented with riboflavin, FMN or FAD under different light conditions (30 ␮mol m−2 s−1 , 80 ␮mol m−2 s−1 or 140 ␮mol m−2 s−1 ) for three weeks.

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

1473

Fig. 7. The FAD content, FNR activity and NADPH/NADP+ ratios in wild-type and phs1 plants under LL, NL or HL growth conditions as described in Fig. 1. The values are mean ± SD of three independent experiments. (a) FAD content. The green and bleached sections of wild-type and mutant leaves were separately harvested for FAD content determination. (b) The activity of FNR-specific cytchrome c reductase. Intact chloroplasts isolated from the leaves of wild-type and phs1 plants were osmotically shocked and FNR-specific cytchrome c reductase activity was measured. (c) FNR protein levels. Total proteins extracted, separated by SDS-PAGE followed by immunodetection with the specific antibody against FNR. (d) NADPH/NADP+ ratios in wild-type and phs1 plants. All of the leaves of wild-type and phs1 plants were used for the measurement of NADP+ and NADPH contents.

Under low light conditions, the photosynthetic protein levels of the mutant were unchanged compared to the wild-type; however, the photosynthetic protein levels of the phs1 mutant were reduced with increasing light intensity. The levels of photosystem II proteins were more reduced than that the levels of photosystem I proteins. The photosystem I proteins (PsaA/B, PsaN) were reduced to 50% and 30% of wild-type levels under normal and high light conditions, respectively, whereas the levels of photosystem II proteins (D1, D2, CP43, CP47) were reduced to about 30% and 10% of wildtype levels under normal and high light condition, respectively. Conversely, the levels of CF1␤-subunit protein from ATP synthase were relatively unchanged in the phs1 mutant. Discussion The riboflavin pathway functions as an essential cofactor in a wide range of biochemical reactions (Sancar, 1994; Hitomi et al., 2009; Imaizumi et al., 2003). A complete abolishment of the riboflavin pathway would produce a range of phenotypes and would probably affect the survival of the plant (Xiao et al., 2004). For many years, the biochemical functions of the enzymes involved

in the riboflavin pathway have been studied in some detail (Herz et al., 2000; Bacher and Eberhardt, 2001; Fischer et al., 2004; Sandoval et al., 2008). Although seven distinct enzymes are known to be involved in the riboflavin pathway in plants, only the A. thaliana cos1 mutant lacking lumazine synthase has been characterized (Xiao et al., 2004). In this study, we report the characterization of the phs1 mutant. The PHS1 gene was cloned via a map-based approach and found to encode a protein that possesses a riboflavin deaminase–reductase domain. The phs1 mutant is characterized by its photosensitive phenotype that could be rescued by FAD supplementation. However, supplementation with the same concentration of riboflavin or FMN failed to rescue this phenotype (Fig. 6). Several riboflavin transporters have been characterized in Bacillus subtilis, Saccharomyces cerevisiae and Mammals, such as YpaA, Mch5p, hRFT1 and rRFT1. No sequence homologs have been identified in either A. thaliana or O. sativa (Kreneva et al., 2000; Reihl and Stolz, 2005; Yonezawa et al., 2008). In contrast, the FAD transporter (Flx1p) has been characterized in S. cerevisia. This protein has more than 30 sequence homologs in A. thaliana and O. sativa, as identified using FASTA searches (http://www.ebi.ac.uk/Tools/fasta) (Giancaspero et

1474

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

Fig. 8. Oxidative state of wild-type and phs1 plants grown under LL, NL or HL growth conditions as described in Fig. 1. The leaves of the wild-type and phs1 plants were used for O2 − detection and measurement of antioxidant enzyme activities. The values are mean ± SD of three independent experiments. (a) In situ detection of O2 − . The detached leaves were stained in NBT for 30 min at room temperature after a brief vacuum infiltration. The stained leaves were cleared by boiling them in an acetic acid:glycerol:ethanol (1:1:3 [v/v/v]) solution prior to photography. The blue color indicates O2 − accumulation. (b) Superoxide dismutase (SOD) activity in wild-type and phs1 plants. The fresh leaves were ground and homogenized, and the supernatant was collected for the assay of SOD activity after centrifugation. (c) Dehydroascorbate reductase (DHAR) activity in wild-type and phs1 plants. The fresh leaves were ground and homogenized, and the supernatant was collected for the assay of DHAR activity after centrifugation. (d) Glutathione reductase (GR) activity in wild-type and phs1 plants. The fresh leaves were ground and homogenized, and the supernatant was collected for the assay of GR activity after centrifugation.

al., 2009). It is likely that FAD can be transported across the plasma membrane or across the chloroplast envelope using FAD transporters, but riboflavin or FMN cannot. Meanwhile, the severely photosensitive phenotype might be caused by a FAD deficiency that increases with increasing light intensity. In plants, FAD is an essential cofactor for a variety of enzymes that participate in many metabolic processes, including DNA repair and the circadian clock (Hitomi et al., 2009), glycerol catabolism (Quettier et al., 2008), vitamin C biosynthesis (Leferink et al., 2008), and others. FNR also uses FAD as a cofactor to catalyze the final step of photosynthetic electron transport in chloroplasts. FAD-depleted FNR assumes a partially-folded structure with a residual NADP+ binding domain but a disordered FAD binding domain (Maeda et al., 2002), which results in reduced FNR activity and an impaired electron transport chain. The ascorbate (ASC)–glutathione (GSH) cycle plays a pivotal role in protecting plant from photodamage, and several antioxidant enzymes involved in ASC–GSH cycle are flavoenzyme. FAD is required by GR as a co-enzyme to regenerate GSH, an important cellular antioxidant as well as a substrate for the enzyme GPx (Beutler, 1969; Ermler et al., 1991; May et

al., 1998; Noctor and Foyer, 1998; Ding et al., 2009). Increased GR activity with increasing light intensity scavenges reactive oxygen species more effectively, but at the same time it also leads to increased consumption of FAD. In addition, monodehydroascorbate reductase (MDHAR), another antioxidant enzyme of the ascorbate–glutathione cycle, also uses FAD to regenerate ascorbate so as to maintain reduced pools of ascorbate (Murthy and Zilinskas, 1994). These antioxidant enzymes showed increased activity in response to high light (Bowler et al., 1994; Grace and Logan, 1996). It is reasonable to speculate that the increased activities of antioxidant enzymes or other enzymes using FAD as co-enzyme will also lead to increased consumption of FAD. Accordingly, the activity of FNR will be reduced due to decreased availability of FAD with increasing light intensity, which in turn affects the electron transfer at PSI in the phs1 mutant. Indeed, measurement of P700 oxidation and re-reduction rates showed that the PSI electron transfer was impaired in the phs1 mutant under normal or high light conditions. It is likely that the flow of electrons through the electron transport chain exceeds the capacity of metabolism to consume the reductant produced, then the electron flow to oxygen, the Mehler

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

1475

3H)-pyrimidinedione by sequential deamination, side chain reduction and dephosphorylation (Fischer and Bacher, 2008). Although bifunctional proteins with both deaminase and reductase activity were found in the eubacteria Escherichia coli and B. subtilis, reductase and deaminase are present as separate enzymes in S. cerevisiae, fungi and higher plants (Richter et al., 1997; Fischer et al., 2004). There are two genes (At4g20960, At3g47390) that contain riboflavin deaminase–reductase domains in A. thaliana. At4g20960 was shown to only have deamination activity in A. thaliana (Fischer et al., 2004). Our efforts to isolate a null mutant of At4g20960 failed. Considering that the null mutations in the At4g20960 gene will not cause lethality if the PHS1 protein has both deaminase activity and reductase activity, it is likely the PHS1 protein has pyrimidine reductase activity. Acknowledgement This study is supported by the State Key Basic Research and Development Plan of China (2009CB118500). References

Fig. 9. Levels of chloroplast proteins of wild-type and phs1 mutant plants under LL, NL or HL growth conditions as described in Fig. 1. The total protein extracts (10 ␮g of proteins) from all of the wild-type and phs1 leaves were separated by 15% SDS-ureaPAGE and blotted, and the blots were probed with specific antibodies, including: anti-D1, anti-D2, anti-CP43, anti-CP47, anti-PsaA/B, anti-PsaN, and anti-CF1␤.

reaction (Mehler, 1951), which results in the production of superoxide at the acceptor side of PSI (Allen, 2003; Asada, 2006). In addition, over-reduction of PSII leads to charge recombination reactions, producing singlet excited oxygen (Keren et al., 1997; Telfer, 2002; Fufezan et al., 2002). Thus, the phs1 mutant plants are likely to suffer from oxidative stress. Indeed, a drastic reduction in the contents of chlorophyll and photosynthetic proteins was observed with increasing light intensities. The phs1 mutant displayed a variable yellow–green phenotype under normal or high light conditions. It is unlikely that the variable phenotype is caused by the inactivation of ferredoxin-NADP(H) reductase (FNR) due to decreased FAD levels in the bleached leaves or decreased FAD with increasing light intensity because the FAD content is similar in bleached and green leaf sections in the phs1 mutant. Several mutants exhibiting variable phenotypes have previously been characterized, such as the A. thaliana variegated mutants immutans, spotty, var1, and var2 (Rosso et al., 2009). Tobacco plants deficient in ferredoxinNADP(H) reductase also exhibit a yellow–green phenotype due to the over-reduction of the intersystem PET chain (Palatnik et al., 2003). A common feature of these mutants is that the mutations affect components of the photosynthetic electron transport (PET) chain (Giacomelli et al., 2007), which results in the impaired PET chain and photosynthetic redox imbalance. Thus, the availability of FAD for FNR function may be insufficient for optimal photosynthetic electron transfer in the mutant with increasing light intensity, which may account for the variable mutant phenotype. In phs1 mutant plants, a 14-nucleotide deletion from base 1247 to 1260 in the coding regions of the PHS1 gene results in the truncation of the protein due to the presence of the premature stop codon TGA (Fig. 3). Because the riboflavin pathway is essential for plant growth, the complete abolishment of the riboflavin pathway would produce pleiotrophic phenotypes (Xiao et al., 2004). It is possible that the truncated protein still retains partial function, and thus FAD can still be synthesized in the phs1 mutant to maintain plant growth under very low light. In the earlier steps of the riboflavin pathway, GTP is converted into 5-amino-6-ribitylamino-2,4(1H,

Allen JF. Superoxide as an obligatory, catalytic intermediate in photosynthetic reduction of oxygen by adrenaline and dopamine. Antioxid Redox Signal 2003;5:7–14. Arnoux P, Morosinotto T, Saga G, Bassi R, Pignol D. A structural basis for the pH-dependent xanthophyll cycle in Arabidopsis thaliana. Plant Cell 2009;21:2036–44. Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 2006;141:391–6. Bacher A, Eberhardt S. German Patent WO 2001; 2000-EP11145. Bafunno V, Giancaspero TA, Brizio C. Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier in FAD export. J Biol Chem 2004;279:95–102. Beutler E. Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies. J Clin Invest 1969;48:1957–66. Beyer WF, Fridovich I. Assaying for superoxide dismutase activity: some large consequences of minor changes in condition. Anal Biochem 1987;72:248–54. Briggs WR, Huala E. Blue-light photoreceptors in higher plants. Annu Rev Cell Dev Biol 1999;15:33–62. Bowler C, Van Camp W, Van Montagu M, Inzé D. Superoxide dismutase in plants. Crit Rev Plant Sci 1994;13:199–218. Chi W, Ma JF, Zhang DY, Guo JK, Chen F, Lu CM, et al. The pentratricopeptide repeat protein delayed green1 is involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis. Plant Physiol 2008;147:573–84. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 1998;16:735–43. Davison PA, Hunter CN, Horton P. Overexpression of beta-carotene hydroxylase enhances stress tolerance in Arabidopsis. Nature 2002;418:203–6. Ding SH, Lu QT, Zhang Y, Yang ZP, Wen XG, Zhang LX, et al. Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state. Plant Mol Biol 2009;69:577–92. Ermler U, Ghisla S, Massey V, Schulz GE. Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues. Eur J Biochem 1991;199:133–8. Fischer M, Römisch W, Saller S, Illarionov B, Richter G, Rohdich F, et al. Evolution of vitamin B2 biosynthesis structural and functional similarity between pyrimidine deaminases of eubacterial and plant origin. J Biol Chem 2004;279:36299–308. Fischer M, Bacher A. Biosynthesis of vitamin B2: structure and mechanism of riboflavin synthase. Arch Biochem Biophys 2008;474:252–65. Foyer C, Lelandais M, Galap C, Kunert KJ. Effects of elevated cytosolic glutathione reductase activity on the cellular glutathione pool and photosynthesis in leaves under normal and stress conditions. Plant Physiol 1991;97:863–72. Fufezan C, Rutherford AW, Krieger-Liszkay A. Singlet oxygen production in herbicide-treated photosystem II. FEBS Lett 2002;532:407–10. Giancaspero TA, Locato V, de Pinto MC, De Gara L, Barile M. The occurrence of riboflavin kinase and FAD synthetase ensures FAD synthesis in tobacco mitochondria and maintenance of cellular redox status. FEBS J 2009;276:219–31. Giacomelli L, Masi A, Ripoll DR, Lee MJ, van Wijk KJ. Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low. Plant Mol Biol 2007;65:627–44. González A, Steffen KL, Lynch JP. Light and excess manganese. Implications for oxidative stress in common bean. Plant Physiol 1998;118:493–504. Grace SC, Logan BA. Acclimation of foliar antioxidant systems to growth irradiance in three broad-leaved evergreen species. Plant Physiol 1996;112:1631–40. Hajirezaei MR, Peisker M, Tschiersch H, Palatnik JF, Valle EM, Carrillo N, et al. Small changes in the activity of chloroplast NADP+ -dependent ferredoxin reductase lead to impaired plant growth and restrict photosynthetic capacity of transgenic tobacco plants. Plant J 2002;29:281–93.

1476

M. Ouyang et al. / Journal of Plant Physiology 167 (2010) 1466–1476

Havaux M, Eymery F, Porfirova S, Rey P, Dormann P. Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana. Plant Cell 2005;17:3451–69. Havaux M, Ksas B, Szewczyk A, Rumeau D, Franck F, Caffarri S, et al. Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress. BMC Plant Biol 2009;2229:109–30. Herz S, Eberhardt S, Bacher A. Biosynthesis of riboflavin in plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3, 4-dihydroxy-2-butanone 4-phosphate synthase. Phytochemistry 2000;53:723–31. Hitomi K, DiTacchio L, Arvai AS, Yamamoto J, Kim ST, Todo T, et al. Functional motifs in the (6–4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes. Proc Natl Acad Sci USA 2009;106:6962–7. Imaizumi T, Tran HG, Swartz TE, Briggs WR, Kay SA. FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis. Nature 2003;426:302–6. Jordan DB, Bacot KO, Carlson TJ, Kessel M, Viitanen PV. Plant riboflavin biosynthesis, cloning, chloroplast localization, expression, purification, and partial characterization of spinach lumazine synthase. J Biol Chem 1999;274:22114–21. Kaiser J, Schramek N, Eberhardt S, Puttmer S, Schuster M, Bacher A. Biosynthesis of vitamin B2. Eur J Biochem 2002;269:5264–70. Keren N, Berg A, van Kan PJ, Levanon H, Ohad I. Mechanism of photosystem II photoinactivation and D1 protein degradation at low light: the role of back electron flow. Proc Natl Acad Sci USA 1997;94:1579–84. Knörzer OC, Durner J, Böger P. Alterations in the antioxidative system of suspensioncultured soybean cells (Glycine max) induced by oxidative stress. Physiol Plant 1996;97:388–96. Kreneva RA, Gelfand MS, Mironov AA, Iomantas YA, Kozlov YI, Mironov AS, et al. Study of the phenotypic occurrence of ura gene inactivation in Bacillus subtilis. Genetika 2000;36:1166–8. Krieger-Liszkay A, Fufezan C, Trebst A. Singlet oxygen production in photosystem II and related protection mechanism. Photosynth Res 2008;98:551–64. , Lee J, Gibson BG, O Kane DJ, Kohnle A, Bacher A. Fluorescence study of the ligand stereospecificity for binding to lumazine protein. Eur J Biochem 1992;210:711–9. Leferink NG, van den Berg WA, van Berkel WJ. l-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis. FEBS J 2008;275:713–26. Li Z, Ahn TK, Avenson TJ, Ballottari M, Cruz JA, Kramer DM, et al. Lutein accumulation in the absence of zeaxanthin restores nonphotochemical quenching in the Arabidopsis thaliana npq1 mutant. Plant Cell 2009;21:1798–812. Lichtenthaler HK. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 1987;148:350–82. Liu YG, Mitsukawa N, Oosumi T, Whittie RF. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junction by thermal asymmetric interlaced PCR. Plant J 1995;8:457–63. Lukowitz W, Gillmor CS, Scheible WR. Positional cloning in Arabidopsis: why it feels good to have a genome initiative working for you. Plant Physiol 2000;123:795–806. Ma J, Peng L, Guo J, Lu Q, Lu C, Zhang L. LPA2 is required for efficient assembly of photosystem II in Arabidopsis thaliana. Plant Cell 2007;19:1980–93. Maeda M, Hamada D, Hoshino M, Onda Y, Hase T, Goto Y. Partially folded structure of flavin adenine dinucleotide-depleted ferredoxin-NADP+ reductase with residual NADP+ binding domain. J Biol Chem 2002;277:17101–7. May MJ, Vernoux T, Leaver C, Montagu MV, Inzé D. Glutathione homeostasis in plants: implications for environmental sensing and plant development. J Exp Bot 1998;49:649–67. Martinez-Garcia JF, Monte E, Quail PH. A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis. Plant J 1999;20:251–7. Mehler AH. Studies on reactions of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents. Arch Biochem Biophys 1951;33:65–77.

Meighen EA. Bacterial bioluminescence: organization, regulation and application of the lux genes. FASEB J 1993;7:1016–22. Müller F, van Berkel WJH. A study on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. A convenient method of preparation and some properties of the apoenzyme. Eur J Biochem 1982;128:21–7. Murthy SS, Zilinskas BA. Molecular cloning and characterization of a cDNA encoding pea monodehydroascorbate reductase. J Biol Chem 1994;269:31129–33. Noctor G, Foyer CH. Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 1998;49:249–79. Orellano EG, Calcaterra NB, Carrillo N, Ceccarelli EA. Probing the role of the carboxyl–terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. J Biol Chem 1993;268:19267–73. Palatnik JF, Tognetti VB, Poli HO, Rodriguez RE, Blanco N, Gattuso M, et al. Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photooxidative damage. Plant J 2003;35:332–41. Peng L, Ma J, Chi W, Guo J, Zhu S, Lu Q, Lu C, Zhang L. Low PSII accumulation1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana. Plant Cell 2006;18:955–69. Quettier AL, Shaw E, Eastmond PJ. Sugar-dependent 6 encodes a mitochondrial flavin adenine dinucleotide-dependent glycerol-3-p dehydrogenase, which is required for glycerol catabolism and post germinative seedling growth in Arabidopsis. Plant Physiol 2008;148:519–28. Rao MV, Davis RD. Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid. Plant J 1999;17:603–14. Reihl P, Stolz J. The monocarboxylate transporter homolog Mch5p catalyzes riboflavin (vitamin B2) uptake in Saccharomyces cerevisiae. J Biol Chem 2005;280:39809–17. Richter G, Richter H, Ritz G, Katzenmeier R, Volk A, Kohnle F, et al. Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli. J Bacteriol 1993;175:4045–51. Richter G, Fischer M, Krieger C, Eberhardt S, Lüttgen H, Gerstenschläger I, et al. Biosynthesis of riboflavin: characterization of the bifunctional deaminase–reductase of Escherichia coli and Bacillus subtilis. J Bacteriol 1997;179:2022–8. Ritz H, Schramek N, Bracher A, Herz S, Eisenreich W, Richter G, et al. Biosynthesis of riboflavin. Studies on the mechanism of GTP cyclohydrolase II. J Biol Chem 2001;276:22273–7. Rosso D, Bode R, Li W, Krol M, Saccon D, Wang S, et al. Photosynthetic redox imbalance governs leaf sectoring in the Arabidopsis thaliana variegation mutants immutans, spotty, var1, and var2. Plant Cell 2009;21:3473–92. Salomon M, Eisenreich W, Durr H, Schleicher E, Knieb E, Massey V, et al. An optomechanical transducer in the blue light receptor phototropin from Avena sativa. Proc Natl Acad Sci USA 2001;98:12357–61. Sancar A. Structure and function of DNA photolyase. Biochemistry 1994;33:2–9. Sandoval FJ, Zhang Y, Roje S. Flavin nucleotide metabolism in plants: monofunctional enzymes synthesize fad in plastids. J Biol Chem 2008;283:30890–900. Slater TF, Sawyer B. A colorimetric method for estimating the pyridine nucleotide content of small amounts of animal tissue. Nature 1962;193:454–6. Telfer A. What is beta-carotene doing in the photosystem II reaction centre? Philos Trans R Soc Lond B Biol Sci 2002;357:1431–9. Weigel D, Ahn JH, Blázquez MA, Borevitz JO, Christensen SK, Fankhauser C, et al. Activation tagging in Arabidopsis. Plant Physiol 2000;122:1003–14. Xiao S, Dai L, Liu F, Wang Z, Peng W, Xie D, et al. An Arabidopsis coronatine insensitive1 Suppressor essential for regulation of jasmonate-mediated plant defense and senescence. Plant Cell 2004;16:1132–42. Yonezawa A, Masuda S, Katsura T, Inui K. Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1. Am J Physiol Cell Physiol 2008;295:632–41. Zhang LX, Paakkarinen V, van Wijk KJ, Aro EM. Co-translational assembly of the D1 protein into photosystem II. J Biol Chem 1999;274:16062–7.