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Proc Natl Acad Sci U S A. May 10, 2005; 102(19): 7020–7025.
Published online Apr 29, 2005. doi:  10.1073/pnas.0502556102
PMCID: PMC1100798
Plant Biology

Enhanced dihydroflavonol-4-reductase activity and NAD homeostasis leading to cell death tolerance in transgenic rice

Abstract

The maize Hm1 gene encoding the NADPH-dependent HC-toxin reductase is capable of detoxifying HC-toxin of fungus Cochliobolus carbonum. Here, we conducted the metabolic and biochemical analysis in transgenic rice plants overexpressing an HC-toxin reductase-like gene in rice (YK1 gene). Methods employing NADPH oxidation and capillary electrophoresis mass spectrometry analysis confirmed that YK1 possessed dihydroflavonol-4-reductase activity in vitro and in vivo. The overexpression of YK1 in both suspension-cultured cells and rice plants increased NAD(H) and NADP(H) levels by causing an increase in NAD synthetase and NAD kinase activities. Activity changes in enzymes that require NAD(P) as coenzymes were also noted in rice cells ectopically expressing YK1, where the cell death caused by hydrogen peroxide and bacterial disease was down-regulated. Thus, a strategy was proposed that the combination of dihydroflavonol-4-reductase activity and the elevated level of NAD(P)H pool may confer the prevention of induced cell death in planta.

Keywords: NADPH-dependent HC-toxin reductase

In maize plants, the NADPH-dependent HC-toxin reductase (HCTR) detoxifies the HC-toxin produced by the fungus Cochliobolus carbonum (1). HCTR is encoded by the maize Hm1 gene (2). A similar gene is conserved in monocots such as rice (3) and barley (4). The molecular mechanism of the maize defense system against C. carbonum invasion remains largely unknown (5). The fungus C. carbonum is specialized for growth on maize, and HC-toxin is only biosynthesized in race 1. Therefore, it is uncertain that the role of Hm1 homologues in other monocots is detoxification (6). To determine the biological function of the Hm1 homologue in rice (the YK1 gene), we established transgenic rice plants with ectopic expression of YK1. The YK1-overexpressing rice plants showed enhanced resistance to rice blast disease and to abiotic stresses like UV radiation, increased salinity, and submergence (7). Here, we demonstrate that YK1 possesses dihydroflavonol-4-reductase (DFR) activity; to our knowledge, this is the first confirmation of such biochemical evidences for HCTR-like protein. Furthermore, the overexpression of YK1 in rice altered the activities of enzymes engaged in NAD biosynthesis, and of enzymes requiring NAD(P) as coenzymes. Moreover, overexpression of YK1 in rice conferred the prevention of cell death caused by the hydrogen peroxide as well as the bacterial disease.

Materials and Methods

Plant Materials. Transgenic rice plants (Oryza sativa L. cv. Nipponbare) possessing either vector alone (the control line) or the YK1 gene (L-1 and L-2) were grown in a growth-cabinet (7). L-1 and L-2 are independent lines overexpressing the YK1 gene under the maize ubiquitin promoter. Suspension-cultured cells of rice were transferred weekly into liquid MS medium supplemented with sucrose [3% (wt/wt)], KH2PO4 (340 mg/liter), thiamine (1 mg/liter), and 2,4-dichlorophenoxyacetic acid (0.2 mg/liter). Seedlings were maintained in the same medium devoid of 2,4-dichlorophenoxyacetic acid.

Measurements of DFR Activity. Plant samples were crushed under liquid N2, and total protein was extracted with PBS containing 137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4 (pH 7.4). After centrifugation (15,000 × g for 15 min at 4°C), the supernatant was used for further analysis. DFR activity was measured according to De-Yu et al. (8) with some modifications. The reaction mixture contained 25 mM Tris·HCl (pH 7.0), 4 mM NADPH, 100 μM taxifolin (dihydroquercetin), and protein samples. The reaction was initiated by addition of NADPH at 25°C, followed by measuring the rate of NADPH oxidation at 340 nm. The enzyme activity was calculated by using the extinction coefficient of NADPH, 6.22 mM–1·cm–1. One unit of enzyme activity was equivalent to the oxidation of 1 μmol of NADPH per min.

Characterization of GST-YK1 Fusion Protein. The coding region of YK1 cDNA (1.1 kbp) was ligated into the EcoRI site of the vector pGEX2T (Amersham Pharmacia) to produce GST-YK1 fusion protein in Escherichia coli. Purification of GST-YK1 protein was as reported by Kawai and Uchimiya (9), where protein purity was confirmed by gel electrophoresis and detection with an anti-YK1 antibody. Determination of the optimum pH for DFR activity was performed at 25°C for 30 min in a reaction mixture (0.5 ml) containing 40 mM citrate/phosphate buffer (pH 4.6–7.0) or 100 mM Tris·HCl (pH 6.8–8.8), 1 mM NADPH, 0.5 mM taxifolin, and protein sample. Determination of the optimum temperature was performed at 25–55°C for 30 min with a reaction mixture containing 100 mM Tris·HCl (pH 6.8–8.8), 1 mM NADPH, 0.5 mM taxifolin, and protein sample.

Detection of Reaction Products by Capillary Electrophoresis (CE)-MS. The reaction mixture, containing 25 mM Tris·HCl (pH 7.0), GST-YK1 (0.06, 0.3, and 1.2 μg) or GST (3 μg), 4 mM NADPH, and 100 μM taxifolin, was incubated at 25°C for 1 h. After addition of 10 μM methionine sulfone as an internal standard, centrifuged mixtures (10,000 × g for 10 min at 4°C) were filtered through 5-kDa cutoff filters (Millipore). The filtrates were analyzed by the CE-MS method of Soga and David (10). Methionine sulfone, taxifolin, and leucocyanidin were analyzed in cation mode at m/z 182, 305, and 307, respectively.

Measurement of Nicotinamide Coenzymes. Measurement of nicotinamide coenzymes was performed as described by Tezuka et al. (11). Plant samples homogenized with 0.1 M HCl (for NAD and NADP) or 0.1 M NaOH (for NADH and NADPH) at 95°C were cooled in an ice bath, then the pH was adjusted to 6.5 with NaOH (for NAD and NADP) or 7.5 with HCl (for NADH and NADPH). After the addition (0.5 ml) of 0.2 M glycylglycine (pH 6.5 or 7.5) to oxidized or reduced coenzyme fractions, respectively, the volume of each fraction was measured. Each fraction was centrifuged (10,000 × g for 20 min at 4°C), and the resulting supernatants were stored for further analyses. For NAD and NADH measurements, samples were added to the reaction mixture containing 50 mM glycylglycine (pH 7.4), 20 mM nicotinamide, 1 mM phenazine methosulfate (PMS), 1 mM thiazolyl blue (MTT), and alcohol dehydrogenase (final concentration, 40 μg/ml). After placing the cuvette containing the reaction mixture in a UV-visible spectrophotometer for measurement at 570 nm, 8% ethanol was added to start the reaction. For NADP and NADPH measurements, the samples were added to a reaction mixture containing 50 mM glycylglycine (pH 7.4), 20 mM nicotinamide, 1 mM PMS, 1 mM MTT, and 2 mM glucose-6-phosphate. After the cuvette was placed in a spectrophotometer for measurement at 570 nm, glucose-6-phosphate dehydrogenase (final concentration, 1 μg/ml) was added to start the reaction.

Analysis of Enzyme Activities in NAD(P) Synthesis Pathway. Analysis of enzyme activities was performed according to the modified method of Tezuka and Murayama (12). Plant samples crushed under liquid N2 were suspended in 50 mM potassium phosphate and centrifuged (10,000 × g for 10 min at 4°C). The supernatant was stored for further analysis. For the measurement of either NAD synthetase or ATP-NMN adenylyltransferase (NMNAT) activities, a reaction mixture containing 25 mM TES/KOH (pH 7.6), 5 mM ATP, 150 mM nicotinamide, 1 mM MgCl2, 1 mM deamido-NAD or 4 mM NMN, 20 mM l-glutamine (NAD synthetase only), and the enzyme fraction were incubated at 30°C for 30 min. Immediately after the incubation, 1 M HCl was added, and the mixture was heated at 95°C for 2 min. After cooling in an ice bath, 1 M NaOH and 250 mM TES/KOH (pH 7.6) were added to the mixture to achieve pH 7.0, followed by centrifugation (10,000 × g for 10 min at 4°C). For NAD kinase measurements, a reaction mixture containing 34 mM Tricine/KOH (pH 7.6), 5 mM ATP, 6 mM nicotinamide, 7 mM MgCl2, 5 mM NAD, and the protein sample was incubated at 30°C for 30 min. The reaction was quenched by adding 1 M HCl and then heating at 95°C for 2 min. The acidified sample was neutralized with 1 M NaOH and 250 mM Tris·HCl (pH 7.6), and cooled in an ice bath. After centrifugation (10,000 × g for 10 min at 4°C), the NAD(P) produced was measured as described by Tezuka et al. (11).

Enzyme Activities Associated with NAD(P) Reduction. NAD(P)H-generating enzyme activities were measured by a modified method of Muscolo et al. (13). For NAD(P)-dependent dehydrogenase assays, plant samples ground under liquid N2 were treated with 100 mM Hepes-NaOH (pH 7.5), 5 mM MgCl2, and 1 mM DTT. After centrifugation (15,000 × g for 10 min at 4°C), the supernatant was used for analysis of NAD(P) reduction at 340 nm. The reaction mixture contained 30 mM Hepes-NaOH (pH 7.6), 3 mM MgCl2, 0.5 mM NAD(P), and the protein extract. The reaction was initiated by the addition of respective substrates at 25°C. For isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), malic enzyme (ME), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH), we used 2.5 mM isocitrate, 10 mM malate, 0.8% ethanol, 0.05 mM glucose-6-phosphate, and 0.05 mM 6-phosphogluconate, respectively, as substrates. GAPDH activity was measured in a reaction mixture containing 50 mM sodium phosphate (pH 7.8), 1 mM EDTA, 1 mM glyceraldehyde-3-phosphate, 0.5 mM NAD, and the protein extract. Activity was monitored by following NAD(P) reduction at 340 nm.

Evans Blue Staining for H2O2-Induced Cell Death. Suspension-cultured cells of rice (7 days old) were treated with H2O2 (0–5 mM) for 2 h. Cell samples were then incubated with 0.05% Evans blue for 15 min, followed by extensive washing to remove unbound dye. Dye bound to dead cells was solubilized in 50% methanol with 1% SDS at 50°C for 30 min and quantified by absorbance at 600 nm (14).

Ion Leakage Measurement. Leaf segments (≈5 mm in size) obtained from 7-day-old rice seedlings were floated on distilled water with or without 30 mM H2O2. Electrolytes that leaked from leaf tissues into the water were measured by using an electrical conductivity meter (Horiba B-173) during 24 h (15).

Pathogen Inoculation and Disease Scoring. Xanthomonas oryzae pv. oryzae ATCC35933 and Acidovorax avenae PAVEN8101, which cause the bacterial blight and bacterial brown stripe, respectively, were used as compatible pathogens for inoculation. The bacterial inocula (5 × 108 cfu/ml) were prepared as described in ref. 16, and leaf blades of 1-month-old plants were inoculated by the leaf-clipping method (17). After inoculation, the plants were incubated under dark conditions at 25°C with 100% humidity for 24 h, and then kept at 28°C in daytime and 22°C at night with a 12-h photoperiod. The degree of disease symptom was scored by measuring the lesion length (in mm) 2 weeks after inoculation.

Results

YK1 Possesses DFR Activity. A blast search suggested the presence of six YK1 homologues in the rice genome, which include DFR-like genes. Thus, we measured DFR activity to investigate whether YK1-overexpressing cells show an increase in such activity. DFR activities in different protein samples were compared by measuring oxidized NADPH, using taxifolin as a substrate. Higher DFR activity was observed in 5-day-old transgenic YK1 calli, as well as 7-day-old seedlings (Table 1). Similar results were constant with 90-day-old plants (data not shown). By using purified GST-YK1 fusion protein, the effects of pH and temperature on DFR activity were evaluated. As shown in Fig. 1 A and B, a pH of ≈7.5 and a temperature of 30°C were found to be optimum. No DFR activity was detected in reaction mixtures containing GST alone.

Fig. 1.
Characterization of DFR activity of YK1 protein. (A and B) Evaluation of optimum pH (A) and temperature (B) for DFR activity of GST-YK1 fusion protein. (C) Analysis of DFR activity by CE-MS. Shown is quantitative analysis in cation mode at m/z 307 for ...
Table 1.
Comparison of DFR activities in callus and plant samples of rice

CE-MS Determination of Reaction Products. We measured DFR reaction products (leucocyanidin, derived from taxifolin) by CE-MS at pH 7.5 and 30°C. After incubation for 30 min, a reaction mixture containing protein samples and taxifolin as a substrate was analyzed by CE-MS. We analyzed three molecules by cation mode at m/z 182 (methionine sulfone), 305 (taxifolin), and 307 (leucocyanidin). Two peaks, corresponding to a mixture of methionine sulfone and leucocyanidin (migration time, 12 min) and taxifolin (migration time, 18 min), were detected (data not shown). The product (leucocyanidin) was detected at m/z 307, where no molecular ion peak was seen in GST alone (Fig. 1C). The amount of leucocyanidin increased according to the amount of GST-YK1 added in the reaction mixture (Fig. 1C), confirming that YK1 possesses the catalytic activity to convert taxifolin to leucocyanidin.

NAD(H) and NADP(H) Levels and Activities of NAD Synthetase, NMNAT, and NAD Kinase. DFR is an NADPH-dependent enzyme. Thus, we quantitated the nicotinamide dinucleotide contents and the activities of NAD(P) biosynthesis-related enzymes in YK1-overexpressing calli and plants. The total amounts of NAD(H) and NADP(H) in 5-day-old YK1 calli (L-1 and L-2) were higher than the control (Fig. 2A). In 7-day-old seedlings, the total amount of NAD(H) and NADP(H) was also higher in YK1 plants than the control (Fig. 2B). In the case of NAD synthetase and NAD kinase, both L-1 and L-2 callus lines showed higher activities than the control (Fig. 3 A and C). On the other hand, NMNAT activity was lower in YK1 lines than in the control suspension cells (Fig. 3B). In plants, NAD synthetase and NAD kinase activities showed the same trend (Fig. 3 D and F), whereas NMNAT activity was the same level in YK1 lines and the control (Fig. 3E).

Fig. 2.
Quantitative measurements of NAD(H) and NADP(H) levels in rice suspension-cultured cells and seedlings. (A) NAD(H) and NADP(H) level in 5-day-old callus. (B) NAD(H) and NADP(H) level in 7-day-old seedlings. C, control; L-1 and L-2, cell lines overexpressing ...
Fig. 3.
Comparison of enzyme activities associated with the NAD(P) biosynthetic pathways in rice suspension-cultured cells (AC) and seedlings (DF). (A and D) NAD synthetase activities. (B and E) NMNAT activities. (C and F) NAD kinase activities. ...

NAD(P)-Reducing Enzymes. As described earlier, overexpression of YK1 in rice was associated with up-regulation of enzyme activities in the NAD(P) pathway, namely, NAD synthetase and NAD kinase. Therefore, several enzymes responsible for NAD(P)H generation were investigated. ADH, GAPDH, and ME activity were enhanced in YK1-overexpressing cultured cells (L-1). On the other hand, activation of GAPDH, MDH, and G6PDH was observed in YK1-overexpressing plants (Table 2).

Table 2.
Analysis of several NAD(P)H-generating enzyme activities in rice callus and plant

Cell Death Analysis. Based on the observation that YK1 possessed DFR activity and altered NAD homeostasis, we were prompted to analyze the cellular responses of YK1 cells to ROS-induced cell death. Studies were conducted with 7-day-old rice suspension-cultured cells in a liquid medium with H2O2 (0–5 mM). After 2 h, callus was stained with Evans blue dye to evaluate ROS tolerance. L-1 and L-2 transgenic cells showed apparent H2O2 stress tolerance (Fig. 4A). Moreover, an ion leakage assay using excised leaves also demonstrated that both L-1 and L-2 lines leaked fewer ions than the control (Fig. 4B).

Fig. 4.
Analysis of cell death in calli and leaves. (A) Evans blue staining to monitor cell death, which was expressed as a cell death fold relative to 0 mM H2O2 (n = 3). (B) Ion leakage assays from leaf samples in the presence or absence of 30 mM H2O2 to monitor ...

Disease Response to Bacterial Infections. To examine the functional relevance of the YK1 gene, leaves of rice plants were tested for resistance to the bacterial blight and bacterial brown stripe. In the case of the former disease, the control and the YK1 plants showed similar levels of disease symptom (Fig. 5A), suggesting that the YK1 gene expression has less or no effects for tolerance to infection with the bacterial blight. In contrast, when the rice plants were challenged with the bacterial brown stripe pathogen, different degrees of disease symptom were observed (Fig. 5A). Both L-1 and L-2 lines exhibited relatively reduced disease severity compared with the control plants where actively spreading lesions were developed on leaves of the control plants, whereas unexpanded lesions were formed on the YK1 lines (Fig. 5B). These indicate that the overexpression of YK1 conferred resistance phenotype to the bacterial brown stripe.

Fig. 5.
Responses of transgenic rice plants expressing the YK1 to infection with bacterial pathogens. (A) Lesion phenotypes of the control and YK1 (L-1 and L-2) plants infected with X. oryzae pv. oryzae or A. avenae. Photographs were taken 14 days after inoculation. ...

Discussion

The HCTR encoded by the maize Hm1 gene has been a well characterized example for the molecular understanding of plant–pathogen interaction (2, 5, 18, 19). The ubiquitous presence of similar genes in other monocots has cast another potential nature of this gene product in metabolism. In this study, we used the methods integrating the enzymatic reaction and the mass spectrometry to identify the biochemical nature of the rice HCTR-like protein (YK1). Furthermore, by using the cell death assay, the physiological basis of ROS-stress tolerance in transgenic YK1 was evaluated at the cellular level.

YK1 Possesses DFR Activity. Nagabhushana and Arjula (20) reported that rice MYB up-regulated the DFR gene, leading to anthocyanin accumulation. A high flavonoid level may be related to dehydration and high salt stress tolerance through scavenging of stress-induced ROS (21). From blast searches, we identified six YK1 homologues to DFR-or HCTR-like gene in the rice genome. Using HC-toxin as a substrate, we confirmed that YK1 also possesses HCTR activity in vitro (data not shown). Moreover, our present study indicates that YK1 possesses DFR activity. Evidence in support of DFR activity of YK1 was obtained from an in vitro assay, using GST-YK1 fusion protein and taxifolin as a substrate. In addition, the optimum pH and temperature for GST-YK1 activity were similar to Medicago truncatula DFR (8). Using a CE-MS technique, we demonstrated that YK1 converted taxifolin to leucocyanidin in vitro. Furthermore, transgenic rice calli and plants had higher DFR activities than the control, confirming that YK1 is a protein possessing DFR activity. As a matter of fact, domains consisting of ≈50 amino acids important for the DFR catalysis are well conserved between YK1 and other DFRs. To our knowledge, this is the first report demonstrating that Hm1-like protein possesses DFR activities both in vitro and in vivo. Accordingly, we tentatively denoted the YK1 gene to OsDFR/HCTR.

Changes in NAD Homeostasis. Elevated levels of both NAD(H) and NADP(H) were detected in YK1-overexpressing transgenic rice. Moreover, the activities of NAD synthetase and NAD kinase were also increased. This study also indicated activity changes in NAD(P)H-generating enzymes. Nicotinamide dinucleotides play important roles in redox reactions and in the regulation of metabolic pathways. The pentose phosphate pathway and ME are the major sources of NADPH (22). NADPH is required for several biosynthetic processes, such as fatty acid synthesis, and is supplied by the glutathione redox cycle for protection against ROS (23). DFR requires NADPH as a coenzyme. Thus, its overexpression may facilitate oxidation of this coenzyme, leading to changes of enzyme activities associated with NAD homeostasis.

ROS-Induced Cell Death. We evaluated cell death caused by exogenous H2O2 with suspension-cultured cells and 7-day-old seedlings. The results showed that incubation of control cells with 2–5 mM (in calli) or 30 mM (in plants) H2O2 caused a marked increase in cell death as judged by Evans blue staining in calli and ion leakage in seedling. In contrast, cell death in YK1 cells was at a low level. Our unpublished results indicate that delphinidin, one of the anthocyanidins, was increased in YK1 overexpressing plants, suggesting a reason for ROS stress resistance. This result indeed reflected the fact that YK1 functions as a positive factor to suppress cell death under oxidative stress. In this connection, we found that H2O2 treatment of a recessive mutant (hm1) of maize resulted in accelerated death in excised leaves (24). Thus, it is possible that Hm1 and related genes may confer ROS-stress tolerance.

Response to Bacterial Disease. Previous study has shown that the HCTR functions in increased tolerance to infection with the rice blast fungus (7). In this study, we investigated roles of the enzyme in relation to bacterial diseases, and results showed different disease responses of the transgenic rice lines between bacterial blight and brown stripe. The rice bacterial blight pathogen, X. oryzae pv. oryzae, is a vascular pathogen, and entering and multiplying of the bacterial cells in xylem vessels are essential for symptom development (25). Because no obvious difference in disease response was obtained between the control and the YK1 plants, it is inferred that YK1 has no function in preventing the infection processes, or that it may not be expressed around the xylem tissues. By contrast, YK1-transformed plants showed increased resistance to the bacterial brown stripe. Although infection mechanisms of the disease are largely unknown, because the pathogen forms initially water-soaked lesions on infected leaves that soon elongate and coalesce into irregular yellowish or brownish stripes, the primary infection site is considered to be mesophyll parenchyma. As reported earlier, the maize ubiquitin (Ubi-1) promoter warrants the site-specific expression in plant tissue that has been exposed to external stresses like sheath blight pathogen (Rhizoctonia solani) (26). Thus, this may explain why infection by brown stripe pathogen was prevented by YK1 overexpression in rice leaves.

The present study also suggests that the overexpression of a single gene can cause metabolic alterations by changing the activities of other enzymes related to NAD homeostasis. It may be presumed that overexpressed YK1 protein may use more NAD(P)H than normal level, which in turn pumps up nicotinamide coenzymes by activating responsible enzymes. Such concomitant relation leading to cell-death tolerance is presented in Fig. 6. In addition, high-throughput analysis of metabolites by Fourier transform ion cyclotron resonance MS in transgenic YK1 plants suggested alteration in other metabolite levels (27).

Fig. 6.
Diagram illustrating the biochemical functions of YK1 protein in relation to flavonoid and NAD homeostasis in the metabolic pathways. Concordant interaction of metabolic pathways may contribute the basis of cell death tolerance in transgenic rice. Red ...

Acknowledgments

We thank M. Uchimiya for editing the manuscript and T. Fushimi for material gifts. This work was supported in part by Research for the Future from the Japan Society for the Promotion of Science and grants from the Ministry of Agriculture, Forestry, and Fisheries of Japan.

Notes

Author contributions: K.T., M.K.-Y., T.T., and H.U. designed research; M.H., H.T., K.T., J.H., and L.-H.Y. performed research; M.H., H.T., K.T., and H.U. analyzed data; and M.H., H.T., K.T., and H.U. wrote the paper.

Abbreviations: CE, capillary electrophoresis; DFR, dihydroflavonol-4-reductase; HCTR, NADPH-dependent HC-toxin reductase; NMNAT, ATP-NMN adenylyltransferase.

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