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Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 80 (2004) 65–74 www.elsevier.com/locate/ypest
Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance Y. Lee, S. Jung, K. Back* Department of Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute, Chonnam National University, Gwangju, 500-757, Republic of Korea Received 27 April 2004; accepted 30 June 2004 Available online 7 August 2004
Abstract A human protoporphyrinogen oxidase (Protox) coding sequence under the control of a ubiquitin promoter was introduced into rice to determine whether transgenic rice overexpressing the human Protox gene exhibits resistance against a peroxidizing herbicide. The transgenic rice lines (H3, H4, H5, H6, H9, and H10) transcribed the human Protox mRNA, whereas hybridizing RNA band was not detected in wild-type rice, indicating that the human Protox gene had been successfully transmitted into transgenic rice plants. The transgenic lines H9 and H10 showed growth retardation and light-dependent formation of necrotic lesions. Compared with wild-type rice plants, rice with a human Protox gene had increased Protox activity and content of the photosensitizer protoporphyrin IX, and reduced chlorophyll. The photosynthetic efficiency in lines H9 and H10, as indicated by Fv/Fm, was not different from that of wild type. The two transgenic lines had decreased levels of antheraxanthin, lutein, and b-carotene and similar levels of neoxanthin and violaxanthin as compared with wild-type plants. The staining activities of catalase, peroxidase, superoxide dismutase, and glutathione reductase were higher in transgenic lines than in wild type. Line H9 germinated in the presence of 20 lM oxyfluorfen, whereas 2 lM oxyfluorfen inhibited the germination of wild-type seeds. Thus, the transgenic rice plants exhibited enhanced resistance to oxyfluorfen. 2004 Elsevier Inc. All rights reserved.
1. Introduction Porphyrin compounds play an essential role in plant metabolism. Protoporphyrinogen oxidase
(Protox, EC 1.3.3.4),1 the last common enzyme in heme and chlorophyll (Chl) biosynthesis [1,2], catalyzes the exchange of six hydrogen atoms from 1
*
Corresponding author. Fax: +82-62-530-2169. E-mail address: [email protected] (K. Back).
Abbreviations used: Chl, chlorophyll; Pchlide, protochlorophyllide; Proto IX, protoporphyrin IX; Protox, protoporphyrinogen oxidase.
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.06.008
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protoporphyrinogen IX (Protogen IX) to O2, producing the aromatic heme and Chl precursor protoporphyrin IX (Proto IX) and H2O2. Proto IX, the substrate of iron or magnesium chelatases, is a photosensitizer and produces active oxygen species (AOS), such as 1O2, which is harmful to the cell and causes the peroxidation of membrane lipids. The biosynthesis of porphyrin is tightly regulated at several levels to coordinate apoprotein synthesis and to avoid the accumulation of Proto IX and protochlorophyllide (Pchlide) at the stage preceding Chl biosynthesis [3]. Plants suffer severe photodynamic damage if these control mechanisms are circumvented. AOS are eliminated efficiently by an integrated system of non-enzymatic and enzymatic antioxidants. The non-enzymatic reductants are ascorbate, glutathione, a-tocopherol, carotenoids, and phenolic compounds [4,5]. Xanthophyll cycle-dependent energy dissipation in the light-harvesting antennae is thought to play an important photoprotective role by mitigating oxidative stress [6]. The metabolism of AOS is also dependent on several functionally interrelated antioxidant enzymes. Superoxide dismutase (SOD, EC 1.15.1.1) is believed to play a crucial role in antioxidant defense, because it catalyzes the dismutation of O 2 into H2O2, whereas catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC 1.11.1.7) destroy H2O2 [7]. SOD, together with ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase (GR, EC 1.6.4.2), constitutes the major defense system against AOS in chloroplasts [8]. In plants, Protox is the target enzyme for the peroxidizing herbicides including oxyfluorfen. Phytotoxicity occurs when accumulated Protogen IX, resulting from the inhibition of Protox by oxyfluorfen, diffuses from chloroplast and is subjected to non-enzymatic oxidation to yield Proto IX, a powerful generator of 1O2 in light [9]. Typical symptoms of oxyfluorfen-treated plants include leaf desiccation, veinal necrosis, destruction of photosynthetic reactions, and leaf deformation [10–12]. To produce transgenic plants resistant to peroxidizing herbicides, plastidic expression of Protox genes from Bacillus subtilis and Arabidopsis thaliana has been used to transform tobacco
[13,14] and rice [15,16]. These transgenic plants were shown to be resistant to peroxidizing herbicides including oxyfluorfen and acifluorfen. Resistance to peroxidizing herbicides can also be achieved by overexpressing the mitochondrial forms of Protox. Oxyfluorfen-resistant soybean cells have a mitochondrial Protox activity that is 9-fold higher than the activity in non-resistant cells [17]. The herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox, and excess Protogen IX generated by inhibition of chloroplast Protox is rapidly utilized for heme synthesis in mitochondria by the abnormally high level of mitochondrial Protox [18]. In humans, a deficiency in Protox activity has been associated with variegated porphyria, in which skin lesions appear in areas exposed to light [19]. Human Protox consists of 477 amino acids with a molecular mass of 50.8 kDa [20]. The protein is located in the mitochondria, and the N-terminal portion contains three basic residues and no acidic residues, characteristic of a presequence. The mitochondrial Protox is closely involved in preventing the accumulation of the photosensitizer Proto IX. It is possible that overexpression of human Protox in mitochondria of plant cells enhances herbicide resistance of transgenic plants. To test this hypothesis, a human Protox coding sequence under the control of the ubiquitin promoter was introduced into rice, and the contribution of human Protox to the Protox activity of plants was assessed. The phenotypical characteristics as well as human Protox gene expression and Proto IX content were compared between transgenic rice and wild-type rice.
2. Materials and methods 2.1. Construction of binary vector Transgenic rice plants were generated using Agrobacterium-mediated transformation. Scutellumderived calli of rice (Oryza sativa cv. Dongjin) were co-cultured with Agrobacterium tumefaciens LBA4404 harboring the pGA1611:HP binary vector (Fig. 1). The human Protox gene was kindly provided by Professor S. Taketani (Kyoto Insti-
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2.3. Plant growth and oxyfluorfen treatment
Fig. 1. Schematic diagram of T-DNA of the binary vectors used for plant transformation. Ubi-P, maize ubiquitin promoter; HP, human protoporphyrinogen oxidase; Tnos, nopaline synthase terminator; CaMV 35S, cauliflower mosaic virus 35S promoter; HPT, hygromycin phosphotransferase; TiA6-7, TiA6-7 terminator.
tute of Technology, Kyoto, Japan). The complete open reading frame of human Protox was amplified using the primers 5 0 -CAAGCTTCCATGGGCCG GACCGTGGTC-3 0 (HindIII site underlined) and 5 0 -TTGGGTACCTCAGCTGTTAGGCTTTGT-3 0 (KpnI site underlined). The PCR product was digested with HindIII and KpnI, gel-purified, and ligated between the same restriction sites within pBluescript-SK (Stratagene, Cedar Creek, TX). After verifying the integrity of the sequence, the HindIII and KpnI fragments of Protox were fused between the same restriction enzyme sites of the pGA1611 binary vector between the maize ubiquitin promoter and the nos 3 0 terminator. The resulting pGA1611:HP vector was transformed into A. tumefaciens strain LBA4404 using the freeze– thaw method. 2.2. Plant transformation and regeneration Agrobacterium tumefaciens LBA4404 harboring pGA1611:HP was grown overnight at 28 C in YEP medium supplemented with 5 lg ml1 tetracycline and 40 lg ml1 hygromycin. The cultures were centrifuged and the pellets were re-suspended in an equal volume of AA medium containing 100 lM acetosyringone. Calli were induced from the scutellum of rice seeds on N6 medium as previously described [21]. The calli were transferred to a co-culture medium and cultured for 2–3 days in darkness at 25 C. Following 3–4 weeks of hygromycin selection, the calli were transferred to regeneration medium for shoot and root development. After roots had sufficiently developed, the transgenic plants were transferred to a greenhouse and grown to maturity.
The T0 generation of homozygous transgenic rice lines (H1, H3, H4, H5, H6, H9, and H10) expressing human Protox was used for Northern analysis, and the T1 generation of transgenic lines H9 and H10 was used for physiological experiments. Seeds of untransformed and transgenic lines were grown on half-strength Murashige and Skoog (MS) medium containing hygromycin (50 lg ml1) in the plant growth room at 28 C and 70% humidity in 12 h light (photon flux density of 200 lmol m2 s1)/12 h dark cycle. When mature rice plants were required, the seedlings were potted in paddy soil and grown in a greenhouse at 30 C. Leaf sections were taken from the 4week-old plants for determining hygromycin resistance, Protox activity, and Proto IX content. In chlorophyll a fluorescence measurement, pigment analysis, and antioxidant enzyme assays, leaves of the 6-week-old plants were used. For the germination assay, rice seeds were husked, surface-sterilized with 2% NaOCl, planted in a bottle containing MS medium supplemented with various concentrations of oxyfluorfen, and maintained under continuous light of 200 lmol m2 s1 following a 12-h dark incubation. Seedlings were grown in a plant growth room. Technical-grade oxyfluorfen was generously provided by Kyungnong (Kyungju, Korea). The physiological experiments were repeated twice each with three determinations. Data were analyzed by DuncanÕs Multiple Range Test at P < 0.05. 2.4. Isolation and analysis of nucleic acids Total RNA (10 lg) was isolated from the leaves of transgenic or wild-type rice plants using TRI reagent (Sigma Chemical, St. Louis, MO, USA) and fractionated on a 1% agarose gel containing formaldehyde, using 20 mM 3-(N-morpholino)propanesulfuric acid as a running buffer. After the RNA was stained with ethidium bromide, the gel was blotted onto a nylon membrane, and the RNA blots were hybridized with a human Protox cDNA sequence that had been radiolabeled using the Prime-It Kit (Stratagene, La Jolla, CA, USA). Hybridizations were performed at 60 C in 0.25 M
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sodium phosphate buffer (pH 7.5) containing 7% SDS, 1% bovine serum albumin (BSA), and 1 mM EDTA. After hybridization, the RNA blot was washed twice with 2· SSC (0.15 M NaCl, 1.5 mM sodium citrate, pH 7.0)/0.1% SDS and twice with 0.2· SSC/0.1% SDS at 55 C. 2.5. Protoporphyrinogen oxidase activity and porphyrin content
rescence yield, Fo, was obtained upon excitation with a weak measuring beam from a pulse lightemitting diode. The maximal fluorescence yield, Fm, was determined after exposure to a saturating pulse of white light to reduce all reaction centers. The ratio of Fv to Fm, representing the activity of photosystem (PS) II, was used to assess the functional damage to plants. 2.7. Pigment extraction and analysis
Rice seedlings were homogenized with buffer solution (400 mM sucrose, 20 mM Tris–HCl (pH 7.8), 1 mM MgCl2, 1 mM EDTA, and 0.1% BSA) and filtered through Miracloth. The mixture was centrifuged at 200g for 5 min at 4 C. The resulting supernatant was centrifuged at 3000g for 5 min at 4 C. The pellet was re-suspended in suspension buffer (330 mM sucrose and 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.8), and the retained supernatant was centrifuged at 10,000g for 20 min. The combined fractions were assayed for total extractable Protox activity. The substrate, Protogen IX, was prepared by chemical reduction of Proto IX with sodium mercury amalgam (Sigma–Aldrich, St. Louis, MO, USA). The enzyme reaction was incubated at 30 C for 10 min and stopped by adding methanol:DMSO (4:1, v/v) according to Lermontova and Grimm [14]. To measure the Proto IX content, plant tissue (0.1 g) was ground in 2 ml methanol:acetone:0.1 N NaOH (9:10:1, v/v/v), and the homogenate was centrifuged at 10,000g for 10 min to remove cell debris and proteins [14]. Porphyrins were separated by HPLC using a Novapak C18 column (4-lM particle size, 4.6 · 250 mm, Waters, Milford, MA, USA) at a flow rate of 1 ml min1. Porphyrins were eluted with a solvent system of 0.1 M ammonium phosphate (pH 5.8) and methanol. The column eluate was monitored with a fluorescence detector (474, Waters) at excitation and emission wavelengths of 400 and 630 nm, respectively. 2.6. Chlorophyll fluorescence Chl a fluorescence was measured in vivo using a pulse amplitude modulation fluorometer (Handy PEA, Hansatech Instruments, Norfolk, England) after 10 min dark incubation [22]. The initial fluo-
Chlorophyll content was determined spectrophotometrically according to the method of Lichtenthaler [23]. For carotenoid analysis, leaf tissues (0.1 g) were ground in 1 ml of 100% acetone containing 10 mg CaCO3. The extracts were centrifuged at 16,000g for 10 min and supernatants were collected. The pigments were separated by HPLC as previously described by Gilmore and Yamamoto [24] using a Waters 2690 System (Millipore, Milford, MA, USA) equipped with a Waters 2487 Absorbance Detector (Millipore, Milford, MA, USA). A Spherisorb ODS-1 column (5-lm particle size, 250 · 4.6 mm id) was obtained from Alltech (Deerfield, IL, USA). Solvent A (acetonitrile:methanol:0.1 M Tris–HCl buffer pH 8.0, 72:8:3, v/v/v) was run isocratically from 0 to 4 min followed by a 2.5 min linear gradient to 100% solvent B (methanol:hexane, 4:1, v/v) at a flow rate of 2 ml min1. The detector was set at 440 nm for integration of the peak areas. 2.8. Antioxidant enzymes Leaves (0.25 g) were macerated to a fine powder in a mortar under liquid N2. Soluble proteins were extracted by homogenizing the powder in 2 ml of 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM EDTA, 1% PVP-40, and 1 mM phenylmethanesulfonyl fluoride. Insoluble material was removed by centrifugation at 15,000g for 20 min at 4 C. Since maintenance of consistent CAT electrophoretic mobility and GR activity required the presence of DTT, an aliquot of each sample was mixed with DTT to 10 mM for CAT and GR zymograms. Equal amounts of protein were electrophoresed on 10% non-denaturing polyacrylamide gels at 4 C for 1.5 h at a constant current of
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30 mA. CAT activity was detected by incubating the gels in 3.27 mM H2O2 for 25 min, rinsing them in water, and staining them in a solution of 1% potassium ferricyanide and 1% ferric chloride for 4 min [25]. Staining of POD isozymes was achieved by incubating the gels in sodium citrate buffer (pH 5.0) containing 9.25 mM p-phenylenediamine and 3.92 mM H2O2 for 15 min [26]. To stain for SOD isoforms, gels were soaked in darkness for 25 min in 50 mM potassium phosphate (pH 7.8) containing 2.5 mM nitroblue tetrazolium (NBT), followed by a soaking in 50 mM potassium phosphate (pH 7.8) containing 28 mM NBT and 28 lM riboflavin in darkness for 30 min [27]. The gels were then exposed to light for approximately 30 min. To detect GR activity, the gels were incubated in darkness for 1 h in a solution of Tris–HCl (pH 7.5) containing 10 mg of 3-(4,5-dimethylthiazol-2-4)2,5-diphenyl tetrazolium bromide, 10 mg of 2,6-dichlorophenolindophenol, 3.4 mM GSSG, and 0.5 mM NADPH [27].
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express human Protox, were selected by screening for hygromycin resistance. The two lines exhibited a 3:1 Mendelian segregation ratio in the T1 generation and were used for further analyses, since they produced more seeds than the other transgenic lines (Table 1). 3.2. Effect of human Protox on Protox activity and Proto IX content The total Protox activities were measured and compared in transgenic and wild-type plants. The transgenic lines H9 and H10 showed 5.5- and 6.9-fold increases in Protox activity, respectively, compared with a wild-type line (Fig. 3). The intro-
Table 1 Segregation ratio of the introduced genetic material in human Protox-expressing transgenic rice plants Transformant
Hygromycin resistance in the T1 generation No. Resistant
No. Sensitive
v2
29 28
11 12
0.18 0.53
3. Results
H9 H10
3.1. Expression of human Protox in transgenic rice plants
Segregation ratios of the introduced genetic material were determined by the ratios of plants resistant to 50 mg l1 hygromycin. v2 was calculated on the basis of a 3:1 segregation ratio.
All transgenic rice lines transcribed human Protox mRNA, whereas no hybridizing band was detected in wild-type rice (Fig. 2). This result shows that the human Protox gene was incorporated into the rice genome and that the expression of human Protox in rice was successful. The transgenic lines H5 and H6 expressed human Protox at higher levels than the other transgenic lines (Fig. 2). Two independent T1 transgenic lines, H9 and H10, which
Fig. 2. Northern blot of human Protox in wild-type and transgenic rice plants expressing a human Protox gene. Total RNA (10 lg) was blotted onto a nylon membrane as described in Materials and methods. WT, wild type; H1, H3, H4, H5, H6, H9, and H10, transgenic lines.
Fig. 3. Protox activity of wild-type and transgenic rice plants expressing a human Protox gene. Plants were grown at 30 C in a greenhouse for 4 weeks and isolated chloroplasts and mitochondria were used for the experiments. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of four samples from two independent experiments.
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Fig. 4. Proto IX content of wild-type and transgenic rice plants expressing a human Protox gene. Plants were grown at 30 C in a greenhouse for 4 weeks and leaf sections were used for the experiments. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of six samples from two independent experiments.
duction of the human Protox gene into rice led to increased synthesis of an intermediate in the Chl biosynthetic pathway, Proto IX, which acts as a photosensitizer (Fig. 4). Proto IX accumulated significantly in the transgenic lines H9 and H10, with a greater increase in H10. 3.3. Photodynamic damage and antioxidant responses The transgenic lines H9 and H10 showed a characteristic phenotype of necrosis (Fig. 5). Growth was significantly retarded in H9 and H10, as compared with wild-type plants (Fig. 5). To confirm the involvement of PSII, chlorophyll fluorescence was determined. The photosynthetic efficiency in H9 and H10, as indicated by Fv/Fm, did not differ from wild type, but the initial fluorescence level, Fo, was lower in transgenic lines than in wild type (Table 2). The transgenic lines H9 and H10 had 16 and 35% lower levels of Chls, respectively, compared with the wild-type level (Table 3). The Chl a/b ratio was higher in transgenic lines H9 and H10 than in the wild type (Table 3). The content of the possible photoprotectant antheraxanthin was lower in transgenic lines H9 and H10 than in wild type (Table 3). Lutein and b-car-
Fig. 5. Phenotype of wild-type and transgenic rice plants expressing a human Protox gene. The plants were grown at 30 C in a greenhouse and photographed 6 weeks after seeding. WT, wild type; H9 and H10, transgenic lines. Table 2 Initial fluorescence (Fo) and photosystem II efficiency (Fv/Fm) in leaves of wild-type and transgenic rice plants expressing a human Protox gene Parameters
WT
H9
H10
Fo Fv/Fm
525 ± 9 0.80 ± 0.00
449 ± 15 0.79 ± 0.01
436 ± 7 8.00 ± 0.01
Plants were grown at 30 C in a greenhouse for 6 weeks. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of six plants from two independent experiments.
Table 3 Xanthophyll (mmol mol1 Chl a) and chlorophyll (lg g1 FW) contents in wild-type plants and transgenic rice plants expressing a human Protox gene Pigments
WT
H9
H10
Neoxanthin 34.1 ± 5.3 31.4 ± 1.5 30.2 ± 3.2 Violaxanthin 47.6 ± 2.4 53.1 ± 2.3 49.9 ± 5.1 Antheraxanthin 2.2 ± 0.7 1.9 ± 0.5 1.6 ± 0.2 Lutein 109.7 ± 6.4 100.9 ± 8.9 92.7 ± 2.8 b-Carotene 92.4 ± 3.6 78.3 ± 8.0 70.3 ± 5.4 Chlorophyll a + b 1987 ± 28.1 1669 ± 16.1 1299 ± 18.5 Chlorophyll a/b 1.34 ± 0.2 1.51 ± 0.1 1.49 ± 0.1 Plants were grown at 30 C in a greenhouse for 6 weeks. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of six samples from two independent experiments.
otene were also lower in the transgenic lines than in the wild type. The contents of other carotenoids, including neoxanthin and violaxanthin, were similar in transgenic and wild-type lines (Table 3).
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Fig. 6. Enzymatic antioxidants of wild-type and transgenic rice plants expressing a human Protox gene. (A) Catalase. (B) Peroxidase. (C) Superoxide dismutase. (D) Glutathione reductase. The plants were grown at 30 C in a greenhouse for 6 weeks. Non-denaturing activity gels were prepared and run as described in Materials and methods. WT, wild type; H9 and H10, transgenic lines.
CAT staining activities were higher in transgenic lines H9 and H10 than in wild type (Fig. 6A). The activities of all four POD isozymes were greater in lines H9 and H10 than in wild type, with the largest increases in POD isozymes 3 and 4 (Fig. 6B). Staining activities of chloroplastic Cu/ZnSOD (band 1), cytosolic Cu/Zn-SOD (bands 2 and 4), and mitochondrial Mn-SOD (band 3) were higher in lines H9 and H10 (Fig. 6C). Finally, the staining activities of glutathione-specific GR isozymes 1 and 2 were stronger in lines H9 and H10 than in wild type (Fig. 6D). 3.4. Oxyfluorfen resistance Herbicide resistance was compared in transgenic line H9 and a wild-type line using various concentrations of oxyfluorfen. Untreated plants of transgenic line H9 exhibited reduced growth compared with the wild-type line (Fig. 7). Although 2 lM oxyfluorfen inhibited the germination of the wild-type seeds, the seeds of transgenic line H9 germinated in the presence of 20 lM oxyfluorfen (Fig. 7). The transgenic line H9 grew quite well in the presence of 2 and 5 lM oxyfluorfen.
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Fig. 7. Wild-type (WT) and transgenic rice seeds (H9) germination as affected by oxyfluorfen. Seeds were sterilized and sown on half-strength MS medium containing various concentrations of technical-grade oxyfluorfen. Tissues were exposed to continuous light of 200 lmol m2 s1 following a 12-h dark incubation. Photographs were taken 10 days after seeding.
4. Discussion Although the primary target of peroxidizing herbicides is chloroplastic Protox, mitochondrial Protox is also important in preventing the accumulation of the photosensitizer Proto IX and in maintaining the flow of tetrapyrrole precursors for heme and chlorophyll biosynthesis [28]. Mammalian Protox interacts directly with ferrochelatase to facilitate the supply of Proto IX [29]. To increase the mitochondrial Protox activity, the human Protox gene was introduced into rice plants (Figs. 1 and 2). The Protox activity was higher in transgenic lines H9 and H10 than in the wild type, which was consistent with the increased synthesis of Proto IX (Figs. 3 and 4). This demonstrates that the human Protox gene produces a functionally active enzyme in transgenic rice plants and that the human Protox enzyme provides additional Protox for plant porphyrin synthesis. In an attempt to address the localization of human Protox, we did Western blot analysis using antibodies raised against the synthetic peptides of human Protox, but, we failed to detect the human Protox possibly due to low affinity of antibodies. We are now preparing other antibodies raised against the synthetic peptides, which correspond to various 14–18 amino acids of human Protox.
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The accumulated Proto IX in transgenic lines H9 and H10 may act as a photosensitizer for the formation of 1O2, which triggers photodynamic damage in transgenic rice plants expressing human Protox (Fig. 4). The increased Proto IX content led to a decline in Chls in the transgenic lines (Table 3). Elevated Chl a/b ratio in transgenic lines H9 and H10 (Table 3) could reflect either a reduced light-harvesting antenna size of PSII or increased PSI/PSII ratios. Many intermediates in the porphyrin biosynthetic pathway, such as Proto IX and its various Mg2+ derivatives including Pchlide, interact with O2 by triplet–triplet interchange to produce 1O2 after light absorption [30]. This compound is potentially harmful to the plant because it causes membrane peroxidation and pigment bleaching, effects that lead to photooxidative damage of the chloroplast, the site of porphyrin synthesis, and ultimately to cell death. Necrotic patches and greatly reduced shoot growth were observed in the leaves of transgenic lines H9 and H10, which may result from toxic level of Proto IX, whereas leaves of the wild-type line remained green (Figs. 4 and 5). A drop in the Fv/Fm ratio was not observed in transgenic lines, indicating no alteration of reaction centers to quenchers by photodynamic stress caused by increased Proto IX (Table 2). The Fo is known to be affected by environmental stresses that cause structural alterations in the PSII complex [22,31]. The decrease in Fo in lines H9 and H10 might arise from a markedly low level of the de-excitation of Chl molecules excited by fluorescence (Table 2). The introduction of human Protox did not produce an accumulation of photoprotective xanthophyll pigments, but a reduction in antheraxanthin, lutein, and b-carotene, which resulted from photodynamic damage of chloroplasts in transgenic lines (Table 3). Energy dissipation through the xanthophyll cycle does not explain the photoprotective process of the transgenic plants to the necrotic response caused by photodynamic stress. Thus, the plants required other components for the dissipative process. Enzymatic antioxidant systems provide protection against the toxic effects of AOS. CAT has a protective role against photodynamic stress in transgenic lines H9
and H10 (Fig. 6A). The transgenic lines H9 and H10 were capable of increasing POD isozymes, which may be a response to damage caused by photodynamic stress (Fig. 6B). Increased SOD activity is due to increased expression of chloroplastic Cu/Zn-SOD (band 1), cytosolic Cu/ Zn-SOD (bands 2 and 4), and mitochondrial Mn-SOD (band 3) (Fig. 6C). These enzymes may confer protection against photodynamic stress. The ascorbate–glutathione cycle has been known to be activated under oxidative stress conditions [32]. GR, a rate-limiting enzyme in the H2O2 scavenging cycle [33], increased in transgenic lines H9 and H10 (Fig. 6D), suggesting that the functioning of the cycle was efficiently activated in the transgenic plants. Antioxidant enzymes probably play a considerable part in the defense mechanism against photodynamic reaction in the transgenic lines. Several strategies have been used to produce peroxidizing herbicide-resistant transgenic plants by the heterologous overexpression of Protox genes from B. subtilis and A. thaliana [13–16,34]. Despite photodynamic damage in transgenic line H9, the transgenic plants were able to germinate in the presence of 20 lM oxyfluorfen, whereas 2 lM oxyfluorfen inhibited germination in the wild type. This shows that oxyfluorfen resistance is enhanced in transgenic rice plants that express human Protox (Fig. 7). Phytotoxicity in peroxidizing herbicide-treated plants is derived from a non-enzymatic accumulation of Proto IX in the cytosol [9]. In oxyfluorfen-treated transgenic plants expressing human Protox, the herbicide resistance may be attributed to the decreased accumulation of photodynamic Proto IX in the cytosol due to the high Protox activity, which metabolizes Protogen IX to Chl and heme. The elevated level of Proto IX that results from the expression of human Protox in rice was toxic even under optimal irradiance condition, developing a differential photosensitivity, as indicated by the reduced growth and appearance of necrotic lesions in transgenic plants. The transgenic plants appeared to develop an efficient defense mechanism upon photodynamic stress mainly through enzymatic antioxidants. Interestingly, the transgenic plants that had symptoms typical of photody-
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namic reactions exhibited increased resistance to oxyfluorfen. Further studies will be required to solve this paradox between photodynamic damage and herbicide resistance in the transgenic plants using in vivo and in vitro translocation experiments.
Acknowledgment This work was supported by grants from the Korea Science and Engineering Foundation (KOSEF) through the Agricultural Plant Stress Research Center (R11-2001-09203001-0).
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[25] W. Woodbury, A.K. Spencer, M.A. Stahman, An improved procedure for using ferricyanide for detecting catalase isozymes, Anal. Biochem. 44 (1971) 301– 305. [26] P.D. Olson, J.E. Varner, Hydrogen peroxide and lignification, Plant J. 4 (1993) 887–892. [27] M.V. Rao, G. Paliyath, D.P. Ormrod, Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana, Plant Physiol. 110 (1996) 125–136. [28] N. Watanabe, F.-S. Che, M. Iwano, S. Takayama, T. Nakano, S. Yoshida, A. Isogai, Molecular characterization of photomixotrophic tobacco cells resistant to protoporphyrinogen oxidase-inhibiting herbicides, Plant Physiol. 118 (1998) 751–758. [29] G.C. Ferreira, T.L. Andrew, S.W. Kair, H.A. Dailey, Organization of the terminal two enzymes of the heme biosynthetic pathway. Orientation of protoporphyrinogen oxidase and evidence for a membrane complex, J. Biol. Chem. 263 (1988) 3835–3839.
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Pesticide Biochemistry and Physiology 80 (2004) 65–74 www.elsevier.com/locate/ypest
Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance Y. Lee, S. Jung, K. Back* Department of Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute, Chonnam National University, Gwangju, 500-757, Republic of Korea Received 27 April 2004; accepted 30 June 2004 Available online 7 August 2004
Abstract A human protoporphyrinogen oxidase (Protox) coding sequence under the control of a ubiquitin promoter was introduced into rice to determine whether transgenic rice overexpressing the human Protox gene exhibits resistance against a peroxidizing herbicide. The transgenic rice lines (H3, H4, H5, H6, H9, and H10) transcribed the human Protox mRNA, whereas hybridizing RNA band was not detected in wild-type rice, indicating that the human Protox gene had been successfully transmitted into transgenic rice plants. The transgenic lines H9 and H10 showed growth retardation and light-dependent formation of necrotic lesions. Compared with wild-type rice plants, rice with a human Protox gene had increased Protox activity and content of the photosensitizer protoporphyrin IX, and reduced chlorophyll. The photosynthetic efficiency in lines H9 and H10, as indicated by Fv/Fm, was not different from that of wild type. The two transgenic lines had decreased levels of antheraxanthin, lutein, and b-carotene and similar levels of neoxanthin and violaxanthin as compared with wild-type plants. The staining activities of catalase, peroxidase, superoxide dismutase, and glutathione reductase were higher in transgenic lines than in wild type. Line H9 germinated in the presence of 20 lM oxyfluorfen, whereas 2 lM oxyfluorfen inhibited the germination of wild-type seeds. Thus, the transgenic rice plants exhibited enhanced resistance to oxyfluorfen. 2004 Elsevier Inc. All rights reserved.
1. Introduction Porphyrin compounds play an essential role in plant metabolism. Protoporphyrinogen oxidase
(Protox, EC 1.3.3.4),1 the last common enzyme in heme and chlorophyll (Chl) biosynthesis [1,2], catalyzes the exchange of six hydrogen atoms from 1
*
Corresponding author. Fax: +82-62-530-2169. E-mail address: [email protected] (K. Back).
Abbreviations used: Chl, chlorophyll; Pchlide, protochlorophyllide; Proto IX, protoporphyrin IX; Protox, protoporphyrinogen oxidase.
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.06.008
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protoporphyrinogen IX (Protogen IX) to O2, producing the aromatic heme and Chl precursor protoporphyrin IX (Proto IX) and H2O2. Proto IX, the substrate of iron or magnesium chelatases, is a photosensitizer and produces active oxygen species (AOS), such as 1O2, which is harmful to the cell and causes the peroxidation of membrane lipids. The biosynthesis of porphyrin is tightly regulated at several levels to coordinate apoprotein synthesis and to avoid the accumulation of Proto IX and protochlorophyllide (Pchlide) at the stage preceding Chl biosynthesis [3]. Plants suffer severe photodynamic damage if these control mechanisms are circumvented. AOS are eliminated efficiently by an integrated system of non-enzymatic and enzymatic antioxidants. The non-enzymatic reductants are ascorbate, glutathione, a-tocopherol, carotenoids, and phenolic compounds [4,5]. Xanthophyll cycle-dependent energy dissipation in the light-harvesting antennae is thought to play an important photoprotective role by mitigating oxidative stress [6]. The metabolism of AOS is also dependent on several functionally interrelated antioxidant enzymes. Superoxide dismutase (SOD, EC 1.15.1.1) is believed to play a crucial role in antioxidant defense, because it catalyzes the dismutation of O 2 into H2O2, whereas catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC 1.11.1.7) destroy H2O2 [7]. SOD, together with ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase (GR, EC 1.6.4.2), constitutes the major defense system against AOS in chloroplasts [8]. In plants, Protox is the target enzyme for the peroxidizing herbicides including oxyfluorfen. Phytotoxicity occurs when accumulated Protogen IX, resulting from the inhibition of Protox by oxyfluorfen, diffuses from chloroplast and is subjected to non-enzymatic oxidation to yield Proto IX, a powerful generator of 1O2 in light [9]. Typical symptoms of oxyfluorfen-treated plants include leaf desiccation, veinal necrosis, destruction of photosynthetic reactions, and leaf deformation [10–12]. To produce transgenic plants resistant to peroxidizing herbicides, plastidic expression of Protox genes from Bacillus subtilis and Arabidopsis thaliana has been used to transform tobacco
[13,14] and rice [15,16]. These transgenic plants were shown to be resistant to peroxidizing herbicides including oxyfluorfen and acifluorfen. Resistance to peroxidizing herbicides can also be achieved by overexpressing the mitochondrial forms of Protox. Oxyfluorfen-resistant soybean cells have a mitochondrial Protox activity that is 9-fold higher than the activity in non-resistant cells [17]. The herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox, and excess Protogen IX generated by inhibition of chloroplast Protox is rapidly utilized for heme synthesis in mitochondria by the abnormally high level of mitochondrial Protox [18]. In humans, a deficiency in Protox activity has been associated with variegated porphyria, in which skin lesions appear in areas exposed to light [19]. Human Protox consists of 477 amino acids with a molecular mass of 50.8 kDa [20]. The protein is located in the mitochondria, and the N-terminal portion contains three basic residues and no acidic residues, characteristic of a presequence. The mitochondrial Protox is closely involved in preventing the accumulation of the photosensitizer Proto IX. It is possible that overexpression of human Protox in mitochondria of plant cells enhances herbicide resistance of transgenic plants. To test this hypothesis, a human Protox coding sequence under the control of the ubiquitin promoter was introduced into rice, and the contribution of human Protox to the Protox activity of plants was assessed. The phenotypical characteristics as well as human Protox gene expression and Proto IX content were compared between transgenic rice and wild-type rice.
2. Materials and methods 2.1. Construction of binary vector Transgenic rice plants were generated using Agrobacterium-mediated transformation. Scutellumderived calli of rice (Oryza sativa cv. Dongjin) were co-cultured with Agrobacterium tumefaciens LBA4404 harboring the pGA1611:HP binary vector (Fig. 1). The human Protox gene was kindly provided by Professor S. Taketani (Kyoto Insti-
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2.3. Plant growth and oxyfluorfen treatment
Fig. 1. Schematic diagram of T-DNA of the binary vectors used for plant transformation. Ubi-P, maize ubiquitin promoter; HP, human protoporphyrinogen oxidase; Tnos, nopaline synthase terminator; CaMV 35S, cauliflower mosaic virus 35S promoter; HPT, hygromycin phosphotransferase; TiA6-7, TiA6-7 terminator.
tute of Technology, Kyoto, Japan). The complete open reading frame of human Protox was amplified using the primers 5 0 -CAAGCTTCCATGGGCCG GACCGTGGTC-3 0 (HindIII site underlined) and 5 0 -TTGGGTACCTCAGCTGTTAGGCTTTGT-3 0 (KpnI site underlined). The PCR product was digested with HindIII and KpnI, gel-purified, and ligated between the same restriction sites within pBluescript-SK (Stratagene, Cedar Creek, TX). After verifying the integrity of the sequence, the HindIII and KpnI fragments of Protox were fused between the same restriction enzyme sites of the pGA1611 binary vector between the maize ubiquitin promoter and the nos 3 0 terminator. The resulting pGA1611:HP vector was transformed into A. tumefaciens strain LBA4404 using the freeze– thaw method. 2.2. Plant transformation and regeneration Agrobacterium tumefaciens LBA4404 harboring pGA1611:HP was grown overnight at 28 C in YEP medium supplemented with 5 lg ml1 tetracycline and 40 lg ml1 hygromycin. The cultures were centrifuged and the pellets were re-suspended in an equal volume of AA medium containing 100 lM acetosyringone. Calli were induced from the scutellum of rice seeds on N6 medium as previously described [21]. The calli were transferred to a co-culture medium and cultured for 2–3 days in darkness at 25 C. Following 3–4 weeks of hygromycin selection, the calli were transferred to regeneration medium for shoot and root development. After roots had sufficiently developed, the transgenic plants were transferred to a greenhouse and grown to maturity.
The T0 generation of homozygous transgenic rice lines (H1, H3, H4, H5, H6, H9, and H10) expressing human Protox was used for Northern analysis, and the T1 generation of transgenic lines H9 and H10 was used for physiological experiments. Seeds of untransformed and transgenic lines were grown on half-strength Murashige and Skoog (MS) medium containing hygromycin (50 lg ml1) in the plant growth room at 28 C and 70% humidity in 12 h light (photon flux density of 200 lmol m2 s1)/12 h dark cycle. When mature rice plants were required, the seedlings were potted in paddy soil and grown in a greenhouse at 30 C. Leaf sections were taken from the 4week-old plants for determining hygromycin resistance, Protox activity, and Proto IX content. In chlorophyll a fluorescence measurement, pigment analysis, and antioxidant enzyme assays, leaves of the 6-week-old plants were used. For the germination assay, rice seeds were husked, surface-sterilized with 2% NaOCl, planted in a bottle containing MS medium supplemented with various concentrations of oxyfluorfen, and maintained under continuous light of 200 lmol m2 s1 following a 12-h dark incubation. Seedlings were grown in a plant growth room. Technical-grade oxyfluorfen was generously provided by Kyungnong (Kyungju, Korea). The physiological experiments were repeated twice each with three determinations. Data were analyzed by DuncanÕs Multiple Range Test at P < 0.05. 2.4. Isolation and analysis of nucleic acids Total RNA (10 lg) was isolated from the leaves of transgenic or wild-type rice plants using TRI reagent (Sigma Chemical, St. Louis, MO, USA) and fractionated on a 1% agarose gel containing formaldehyde, using 20 mM 3-(N-morpholino)propanesulfuric acid as a running buffer. After the RNA was stained with ethidium bromide, the gel was blotted onto a nylon membrane, and the RNA blots were hybridized with a human Protox cDNA sequence that had been radiolabeled using the Prime-It Kit (Stratagene, La Jolla, CA, USA). Hybridizations were performed at 60 C in 0.25 M
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sodium phosphate buffer (pH 7.5) containing 7% SDS, 1% bovine serum albumin (BSA), and 1 mM EDTA. After hybridization, the RNA blot was washed twice with 2· SSC (0.15 M NaCl, 1.5 mM sodium citrate, pH 7.0)/0.1% SDS and twice with 0.2· SSC/0.1% SDS at 55 C. 2.5. Protoporphyrinogen oxidase activity and porphyrin content
rescence yield, Fo, was obtained upon excitation with a weak measuring beam from a pulse lightemitting diode. The maximal fluorescence yield, Fm, was determined after exposure to a saturating pulse of white light to reduce all reaction centers. The ratio of Fv to Fm, representing the activity of photosystem (PS) II, was used to assess the functional damage to plants. 2.7. Pigment extraction and analysis
Rice seedlings were homogenized with buffer solution (400 mM sucrose, 20 mM Tris–HCl (pH 7.8), 1 mM MgCl2, 1 mM EDTA, and 0.1% BSA) and filtered through Miracloth. The mixture was centrifuged at 200g for 5 min at 4 C. The resulting supernatant was centrifuged at 3000g for 5 min at 4 C. The pellet was re-suspended in suspension buffer (330 mM sucrose and 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.8), and the retained supernatant was centrifuged at 10,000g for 20 min. The combined fractions were assayed for total extractable Protox activity. The substrate, Protogen IX, was prepared by chemical reduction of Proto IX with sodium mercury amalgam (Sigma–Aldrich, St. Louis, MO, USA). The enzyme reaction was incubated at 30 C for 10 min and stopped by adding methanol:DMSO (4:1, v/v) according to Lermontova and Grimm [14]. To measure the Proto IX content, plant tissue (0.1 g) was ground in 2 ml methanol:acetone:0.1 N NaOH (9:10:1, v/v/v), and the homogenate was centrifuged at 10,000g for 10 min to remove cell debris and proteins [14]. Porphyrins were separated by HPLC using a Novapak C18 column (4-lM particle size, 4.6 · 250 mm, Waters, Milford, MA, USA) at a flow rate of 1 ml min1. Porphyrins were eluted with a solvent system of 0.1 M ammonium phosphate (pH 5.8) and methanol. The column eluate was monitored with a fluorescence detector (474, Waters) at excitation and emission wavelengths of 400 and 630 nm, respectively. 2.6. Chlorophyll fluorescence Chl a fluorescence was measured in vivo using a pulse amplitude modulation fluorometer (Handy PEA, Hansatech Instruments, Norfolk, England) after 10 min dark incubation [22]. The initial fluo-
Chlorophyll content was determined spectrophotometrically according to the method of Lichtenthaler [23]. For carotenoid analysis, leaf tissues (0.1 g) were ground in 1 ml of 100% acetone containing 10 mg CaCO3. The extracts were centrifuged at 16,000g for 10 min and supernatants were collected. The pigments were separated by HPLC as previously described by Gilmore and Yamamoto [24] using a Waters 2690 System (Millipore, Milford, MA, USA) equipped with a Waters 2487 Absorbance Detector (Millipore, Milford, MA, USA). A Spherisorb ODS-1 column (5-lm particle size, 250 · 4.6 mm id) was obtained from Alltech (Deerfield, IL, USA). Solvent A (acetonitrile:methanol:0.1 M Tris–HCl buffer pH 8.0, 72:8:3, v/v/v) was run isocratically from 0 to 4 min followed by a 2.5 min linear gradient to 100% solvent B (methanol:hexane, 4:1, v/v) at a flow rate of 2 ml min1. The detector was set at 440 nm for integration of the peak areas. 2.8. Antioxidant enzymes Leaves (0.25 g) were macerated to a fine powder in a mortar under liquid N2. Soluble proteins were extracted by homogenizing the powder in 2 ml of 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM EDTA, 1% PVP-40, and 1 mM phenylmethanesulfonyl fluoride. Insoluble material was removed by centrifugation at 15,000g for 20 min at 4 C. Since maintenance of consistent CAT electrophoretic mobility and GR activity required the presence of DTT, an aliquot of each sample was mixed with DTT to 10 mM for CAT and GR zymograms. Equal amounts of protein were electrophoresed on 10% non-denaturing polyacrylamide gels at 4 C for 1.5 h at a constant current of
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30 mA. CAT activity was detected by incubating the gels in 3.27 mM H2O2 for 25 min, rinsing them in water, and staining them in a solution of 1% potassium ferricyanide and 1% ferric chloride for 4 min [25]. Staining of POD isozymes was achieved by incubating the gels in sodium citrate buffer (pH 5.0) containing 9.25 mM p-phenylenediamine and 3.92 mM H2O2 for 15 min [26]. To stain for SOD isoforms, gels were soaked in darkness for 25 min in 50 mM potassium phosphate (pH 7.8) containing 2.5 mM nitroblue tetrazolium (NBT), followed by a soaking in 50 mM potassium phosphate (pH 7.8) containing 28 mM NBT and 28 lM riboflavin in darkness for 30 min [27]. The gels were then exposed to light for approximately 30 min. To detect GR activity, the gels were incubated in darkness for 1 h in a solution of Tris–HCl (pH 7.5) containing 10 mg of 3-(4,5-dimethylthiazol-2-4)2,5-diphenyl tetrazolium bromide, 10 mg of 2,6-dichlorophenolindophenol, 3.4 mM GSSG, and 0.5 mM NADPH [27].
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express human Protox, were selected by screening for hygromycin resistance. The two lines exhibited a 3:1 Mendelian segregation ratio in the T1 generation and were used for further analyses, since they produced more seeds than the other transgenic lines (Table 1). 3.2. Effect of human Protox on Protox activity and Proto IX content The total Protox activities were measured and compared in transgenic and wild-type plants. The transgenic lines H9 and H10 showed 5.5- and 6.9-fold increases in Protox activity, respectively, compared with a wild-type line (Fig. 3). The intro-
Table 1 Segregation ratio of the introduced genetic material in human Protox-expressing transgenic rice plants Transformant
Hygromycin resistance in the T1 generation No. Resistant
No. Sensitive
v2
29 28
11 12
0.18 0.53
3. Results
H9 H10
3.1. Expression of human Protox in transgenic rice plants
Segregation ratios of the introduced genetic material were determined by the ratios of plants resistant to 50 mg l1 hygromycin. v2 was calculated on the basis of a 3:1 segregation ratio.
All transgenic rice lines transcribed human Protox mRNA, whereas no hybridizing band was detected in wild-type rice (Fig. 2). This result shows that the human Protox gene was incorporated into the rice genome and that the expression of human Protox in rice was successful. The transgenic lines H5 and H6 expressed human Protox at higher levels than the other transgenic lines (Fig. 2). Two independent T1 transgenic lines, H9 and H10, which
Fig. 2. Northern blot of human Protox in wild-type and transgenic rice plants expressing a human Protox gene. Total RNA (10 lg) was blotted onto a nylon membrane as described in Materials and methods. WT, wild type; H1, H3, H4, H5, H6, H9, and H10, transgenic lines.
Fig. 3. Protox activity of wild-type and transgenic rice plants expressing a human Protox gene. Plants were grown at 30 C in a greenhouse for 4 weeks and isolated chloroplasts and mitochondria were used for the experiments. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of four samples from two independent experiments.
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Fig. 4. Proto IX content of wild-type and transgenic rice plants expressing a human Protox gene. Plants were grown at 30 C in a greenhouse for 4 weeks and leaf sections were used for the experiments. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of six samples from two independent experiments.
duction of the human Protox gene into rice led to increased synthesis of an intermediate in the Chl biosynthetic pathway, Proto IX, which acts as a photosensitizer (Fig. 4). Proto IX accumulated significantly in the transgenic lines H9 and H10, with a greater increase in H10. 3.3. Photodynamic damage and antioxidant responses The transgenic lines H9 and H10 showed a characteristic phenotype of necrosis (Fig. 5). Growth was significantly retarded in H9 and H10, as compared with wild-type plants (Fig. 5). To confirm the involvement of PSII, chlorophyll fluorescence was determined. The photosynthetic efficiency in H9 and H10, as indicated by Fv/Fm, did not differ from wild type, but the initial fluorescence level, Fo, was lower in transgenic lines than in wild type (Table 2). The transgenic lines H9 and H10 had 16 and 35% lower levels of Chls, respectively, compared with the wild-type level (Table 3). The Chl a/b ratio was higher in transgenic lines H9 and H10 than in the wild type (Table 3). The content of the possible photoprotectant antheraxanthin was lower in transgenic lines H9 and H10 than in wild type (Table 3). Lutein and b-car-
Fig. 5. Phenotype of wild-type and transgenic rice plants expressing a human Protox gene. The plants were grown at 30 C in a greenhouse and photographed 6 weeks after seeding. WT, wild type; H9 and H10, transgenic lines. Table 2 Initial fluorescence (Fo) and photosystem II efficiency (Fv/Fm) in leaves of wild-type and transgenic rice plants expressing a human Protox gene Parameters
WT
H9
H10
Fo Fv/Fm
525 ± 9 0.80 ± 0.00
449 ± 15 0.79 ± 0.01
436 ± 7 8.00 ± 0.01
Plants were grown at 30 C in a greenhouse for 6 weeks. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of six plants from two independent experiments.
Table 3 Xanthophyll (mmol mol1 Chl a) and chlorophyll (lg g1 FW) contents in wild-type plants and transgenic rice plants expressing a human Protox gene Pigments
WT
H9
H10
Neoxanthin 34.1 ± 5.3 31.4 ± 1.5 30.2 ± 3.2 Violaxanthin 47.6 ± 2.4 53.1 ± 2.3 49.9 ± 5.1 Antheraxanthin 2.2 ± 0.7 1.9 ± 0.5 1.6 ± 0.2 Lutein 109.7 ± 6.4 100.9 ± 8.9 92.7 ± 2.8 b-Carotene 92.4 ± 3.6 78.3 ± 8.0 70.3 ± 5.4 Chlorophyll a + b 1987 ± 28.1 1669 ± 16.1 1299 ± 18.5 Chlorophyll a/b 1.34 ± 0.2 1.51 ± 0.1 1.49 ± 0.1 Plants were grown at 30 C in a greenhouse for 6 weeks. WT, wild type; H9 and H10, transgenic lines. The data represent means ± SE of six samples from two independent experiments.
otene were also lower in the transgenic lines than in the wild type. The contents of other carotenoids, including neoxanthin and violaxanthin, were similar in transgenic and wild-type lines (Table 3).
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Fig. 6. Enzymatic antioxidants of wild-type and transgenic rice plants expressing a human Protox gene. (A) Catalase. (B) Peroxidase. (C) Superoxide dismutase. (D) Glutathione reductase. The plants were grown at 30 C in a greenhouse for 6 weeks. Non-denaturing activity gels were prepared and run as described in Materials and methods. WT, wild type; H9 and H10, transgenic lines.
CAT staining activities were higher in transgenic lines H9 and H10 than in wild type (Fig. 6A). The activities of all four POD isozymes were greater in lines H9 and H10 than in wild type, with the largest increases in POD isozymes 3 and 4 (Fig. 6B). Staining activities of chloroplastic Cu/ZnSOD (band 1), cytosolic Cu/Zn-SOD (bands 2 and 4), and mitochondrial Mn-SOD (band 3) were higher in lines H9 and H10 (Fig. 6C). Finally, the staining activities of glutathione-specific GR isozymes 1 and 2 were stronger in lines H9 and H10 than in wild type (Fig. 6D). 3.4. Oxyfluorfen resistance Herbicide resistance was compared in transgenic line H9 and a wild-type line using various concentrations of oxyfluorfen. Untreated plants of transgenic line H9 exhibited reduced growth compared with the wild-type line (Fig. 7). Although 2 lM oxyfluorfen inhibited the germination of the wild-type seeds, the seeds of transgenic line H9 germinated in the presence of 20 lM oxyfluorfen (Fig. 7). The transgenic line H9 grew quite well in the presence of 2 and 5 lM oxyfluorfen.
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Fig. 7. Wild-type (WT) and transgenic rice seeds (H9) germination as affected by oxyfluorfen. Seeds were sterilized and sown on half-strength MS medium containing various concentrations of technical-grade oxyfluorfen. Tissues were exposed to continuous light of 200 lmol m2 s1 following a 12-h dark incubation. Photographs were taken 10 days after seeding.
4. Discussion Although the primary target of peroxidizing herbicides is chloroplastic Protox, mitochondrial Protox is also important in preventing the accumulation of the photosensitizer Proto IX and in maintaining the flow of tetrapyrrole precursors for heme and chlorophyll biosynthesis [28]. Mammalian Protox interacts directly with ferrochelatase to facilitate the supply of Proto IX [29]. To increase the mitochondrial Protox activity, the human Protox gene was introduced into rice plants (Figs. 1 and 2). The Protox activity was higher in transgenic lines H9 and H10 than in the wild type, which was consistent with the increased synthesis of Proto IX (Figs. 3 and 4). This demonstrates that the human Protox gene produces a functionally active enzyme in transgenic rice plants and that the human Protox enzyme provides additional Protox for plant porphyrin synthesis. In an attempt to address the localization of human Protox, we did Western blot analysis using antibodies raised against the synthetic peptides of human Protox, but, we failed to detect the human Protox possibly due to low affinity of antibodies. We are now preparing other antibodies raised against the synthetic peptides, which correspond to various 14–18 amino acids of human Protox.
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The accumulated Proto IX in transgenic lines H9 and H10 may act as a photosensitizer for the formation of 1O2, which triggers photodynamic damage in transgenic rice plants expressing human Protox (Fig. 4). The increased Proto IX content led to a decline in Chls in the transgenic lines (Table 3). Elevated Chl a/b ratio in transgenic lines H9 and H10 (Table 3) could reflect either a reduced light-harvesting antenna size of PSII or increased PSI/PSII ratios. Many intermediates in the porphyrin biosynthetic pathway, such as Proto IX and its various Mg2+ derivatives including Pchlide, interact with O2 by triplet–triplet interchange to produce 1O2 after light absorption [30]. This compound is potentially harmful to the plant because it causes membrane peroxidation and pigment bleaching, effects that lead to photooxidative damage of the chloroplast, the site of porphyrin synthesis, and ultimately to cell death. Necrotic patches and greatly reduced shoot growth were observed in the leaves of transgenic lines H9 and H10, which may result from toxic level of Proto IX, whereas leaves of the wild-type line remained green (Figs. 4 and 5). A drop in the Fv/Fm ratio was not observed in transgenic lines, indicating no alteration of reaction centers to quenchers by photodynamic stress caused by increased Proto IX (Table 2). The Fo is known to be affected by environmental stresses that cause structural alterations in the PSII complex [22,31]. The decrease in Fo in lines H9 and H10 might arise from a markedly low level of the de-excitation of Chl molecules excited by fluorescence (Table 2). The introduction of human Protox did not produce an accumulation of photoprotective xanthophyll pigments, but a reduction in antheraxanthin, lutein, and b-carotene, which resulted from photodynamic damage of chloroplasts in transgenic lines (Table 3). Energy dissipation through the xanthophyll cycle does not explain the photoprotective process of the transgenic plants to the necrotic response caused by photodynamic stress. Thus, the plants required other components for the dissipative process. Enzymatic antioxidant systems provide protection against the toxic effects of AOS. CAT has a protective role against photodynamic stress in transgenic lines H9
and H10 (Fig. 6A). The transgenic lines H9 and H10 were capable of increasing POD isozymes, which may be a response to damage caused by photodynamic stress (Fig. 6B). Increased SOD activity is due to increased expression of chloroplastic Cu/Zn-SOD (band 1), cytosolic Cu/ Zn-SOD (bands 2 and 4), and mitochondrial Mn-SOD (band 3) (Fig. 6C). These enzymes may confer protection against photodynamic stress. The ascorbate–glutathione cycle has been known to be activated under oxidative stress conditions [32]. GR, a rate-limiting enzyme in the H2O2 scavenging cycle [33], increased in transgenic lines H9 and H10 (Fig. 6D), suggesting that the functioning of the cycle was efficiently activated in the transgenic plants. Antioxidant enzymes probably play a considerable part in the defense mechanism against photodynamic reaction in the transgenic lines. Several strategies have been used to produce peroxidizing herbicide-resistant transgenic plants by the heterologous overexpression of Protox genes from B. subtilis and A. thaliana [13–16,34]. Despite photodynamic damage in transgenic line H9, the transgenic plants were able to germinate in the presence of 20 lM oxyfluorfen, whereas 2 lM oxyfluorfen inhibited germination in the wild type. This shows that oxyfluorfen resistance is enhanced in transgenic rice plants that express human Protox (Fig. 7). Phytotoxicity in peroxidizing herbicide-treated plants is derived from a non-enzymatic accumulation of Proto IX in the cytosol [9]. In oxyfluorfen-treated transgenic plants expressing human Protox, the herbicide resistance may be attributed to the decreased accumulation of photodynamic Proto IX in the cytosol due to the high Protox activity, which metabolizes Protogen IX to Chl and heme. The elevated level of Proto IX that results from the expression of human Protox in rice was toxic even under optimal irradiance condition, developing a differential photosensitivity, as indicated by the reduced growth and appearance of necrotic lesions in transgenic plants. The transgenic plants appeared to develop an efficient defense mechanism upon photodynamic stress mainly through enzymatic antioxidants. Interestingly, the transgenic plants that had symptoms typical of photody-
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namic reactions exhibited increased resistance to oxyfluorfen. Further studies will be required to solve this paradox between photodynamic damage and herbicide resistance in the transgenic plants using in vivo and in vitro translocation experiments.
Acknowledgment This work was supported by grants from the Korea Science and Engineering Foundation (KOSEF) through the Agricultural Plant Stress Research Center (R11-2001-09203001-0).
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