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Keywords:

  • salicylic acid;
  • salicylate hydroxylase;
  • Oryza sativa;
  • Magnaporthe grisea;
  • reactive oxygen species;
  • disease resistance

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Salicylic acid (SA) is a key endogenous signal that mediates defense gene expression and disease resistance in many dicotyledonous species. In contrast to tobacco and Arabidopsis, which contain low basal levels of SA, rice has two orders of magnitude higher levels of SA and appears to be insensitive to exogenous SA treatment. To determine the role of SA in rice plants, we have generated SA-deficient transgenic rice by expressing the bacterial salicylate hydroxylase that degrades SA. Depletion of high levels of endogenous SA in transgenic rice does not measurably affect defense gene expression, but reduces the plant's capacity to detoxify reactive oxygen intermediates (ROI). SA-deficient transgenic rice contains elevated levels of superoxide and H2O2, and exhibits spontaneous lesion formation in an age- and light-dependent manner. Exogenous application of SA analog benzothiadiazole complements SA deficiency and suppresses ROI levels and lesion formation. Although an increase of conjugated catechol was detected in SA-deficient rice, catechol does not appear to significantly affect ROI levels based on the endogenous catechol data and exogenous catechol treatment. When infected with the blast fungus (Magnaporthe grisea), SA-deficient rice exhibits increased susceptibility to oxidative bursts elicited by avirulent isolates. Furthermore, SA-deficient rice is hyperresponsive to oxidative damage caused by paraquat treatment. Taken together, our results strongly suggest that SA plays an important role to modulate redox balance and protect rice plants from oxidative stress.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Salicylic acid (SA) is a natural phenolic compound present in many plant species at various levels. While the therapeutic benefits of SA and its acetylated derivative, aspirin, have been widely known for a long time, our insights into the role of SA in plants have emerged only during the past decade. A large body of evidence has shown that SA is a key endogenous signal involved in plant defense responses as well as flowering and thermogenesis (Dempsey et al., 1999; Raskin, 1992). After pathogen infection, endogenous levels of SA and its conjugates increase significantly in tobacco, cucumber and Arabidopsis immediately preceding the induction of pathogenesis-related (PR) genes and the onset of disease resistance (Malamy et al., 1990; Métraux et al., 1990; Rasmussen et al., 1991). Exogenous application of SA or aspirin induces PR genes and activates local and systemic acquired resistance in a wide variety of plant species (Ryals et al., 1996). Overproduction of endogenous SA in transgenic tobacco and Arabidopsis also enhances disease resistance (Mauch et al., 2000; Verberne et al., 2000). Using transgenic tobacco and Arabidopsis expressing the Pseudomonas putida nahG gene that encodes the SA-degrading enzyme salicylate hydroxylase, SA was clearly demonstrated to play a central role in mediating local and systemic resistance against pathogen infection (Delaney et al., 1994; Gaffney et al., 1993).

In contrast to tobacco and Arabidopsis, which contain low basal levels of SA (less than 100 ng g−1 fresh weight), rice has basal SA levels (5000–30 000 ng g−1 fresh weight) far exceeding the elevated SA levels (500–2000 ng g−1 fresh weight) in infected tobacco or Arabidopsis tissues (Chen et al., 1997; Raskin et al., 1990; Silverman et al., 1995). Most endogenous SA in rice remains as the free acid rather than being rapidly glycosylated as is the case for de novo synthesized SA in pathogen-infected tobacco tissue. Little or no change in SA levels in rice shoots was found following infection by either bacterial or fungal pathogens. When exogenously applied to rice plants, SA is a poor activator of PR gene expression and induced resistance (M. Qi and Y. Yang, unpublished data). These observations suggest that endogenous SA may not act as a secondary signal molecule for induced resistance in rice. However, SA may serve as a preformed chemical barrier against pathogen infection and/or has other biological functions in rice plants.

In this study, we generated SA-deficient transgenic rice by overexpression of the bacterial nahG gene. The NahG rice has elevated levels of superoxide and H2O2 and exhibits spontaneous lesion formation in an age- and light-dependent manner. The NahG rice is also hyperresponsive to oxidative damage caused by pathogen infection or paraquat treatment. Using a combination of molecular, biochemical, genetic, and pathological analyses, we have shown that endogenous SA plays an important anti-oxidative role to protect rice plants from oxidative stress.

Generation of SA-deficient transgenic rice

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

To determine the role of high SA levels in rice plants, we have generated SA-deficient transgenic rice (O. sativa spp. japonica, cv. Nipponbare) by overexpression of the bacterial nahG gene via Agrobacterium-mediated transformation. Eleven T0 NahG transgenic lines were obtained, including eight lines with a single copy insertion of nahG transgene as identified by Southern hybridization and segregation analysis. As expected, these transgenic lines constitutively express the nahG transgene (Figure 1). High-performance liquid chromatography (HPLC) analysis reveals that free SA levels in NahG lines are greatly reduced to 100–400 ng g−1 fresh weight, which is about 20–80 times below the basal levels (8000–15 000 ng g−1 fresh weight) of SA in wild type or vector-transformed Nipponbare rice cultivar (Table 1; also see HPLC chromatograms in Figure 3d). Subsequently, molecular, biochemical, and pathological analyses were conducted on the first (T0), second (T1), and third (T2) generations of three NahG lines.

image

Figure 1. Constitutive expression of the bacterial nahG gene in transgenic rice. Total RNA was isolated from leaves of T0 control (CK, vector-transformed plants) and NahG lines, and analyzed by Northern hybridization with a 32P-labeled nahG DNA fragment.

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Table 1.  SA and catechol levels in control and NahG transgenic rice
Plant materialFree SA (ng g−1 FW)Catechol (ng g−1 FW)a
  1. NA, not analyzed; ND, not detectable.

  2. aLevels of conjugated catechol were determined in T2 generation. Free catechol was not detectable in both control and NahG rice plants.

  3. bThree-week-old seedling leaves of T1 generation were used for SA assay. Control lines (CK1-3) were transformed with the empty vector.

  4. cThree- and 9-week old plants of T2 generation were used for SA and catechol assays. Segregation lines that lost NahG transgene were used as control (CK).

  5. dStandard deviation of three independent samples for all SA and catechol assays.

T1 generationb
 CK112 917 ± 2781NA
 CK29868 ± 638NA
 CK311 281 ± 2111NA
 NahG1237 ± 161NA
 NahG2174 ± 65NA
 NahG3166 ± 57NA
T2 generationc
 CK (3 weeks old)14 213 ± 1503 102 ± 57
 CK (9 weeks old)6948 ± 1341ND
 NahG (3 weeks old)341 ± 2021580 ± 412
 NahG (9 weeks old)130 ± 48 848 ± 342d
image

Figure 3. Spontaneous lesion formation in NahG rice. (a) Lesion formation in leaves of 10-week-old T0 NahG plants; vector-transformed plants were used as control (CK). (b) Activation of PR-1 and PBZ1 genes during lesion formation in T0 NahG plants. −, non-lesioned leaf; +, lesioned leaf. (c) Restoration of normal phenotype in T2 segregation lines (CK) that lost nahG transgene. Extensive lesions were seen in 12-week-old plants of T2 homozygous NahG lines. (d) HPLC chromatographs showing a nearly undetectable level of SA in the T2 NahG line and a high SA peak in the segregation line that lost the nahG transgene.

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SA-deficient NahG rice contains elevated levels of superoxide and H2O2

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

SA-deficient NahG rice appears to have normal growth and reproduction. However, leaves of NahG rice seedlings (2–4 weeks old) contain slightly higher levels of superoxide and H2O2 than control plants (data not shown). As rice growth enters the reproductive stage (8 weeks old), leaves of NahG plants accumulate significantly higher levels of superoxide and H2O2 than control plants (Figure 2a,b). All control plants (non-transformed, vector-transformed, and segregated progeny that lost the nahG transgene) have similar levels of superoxide and H2O2 staining. Treatment of detached NahG leaves with superoxide dismutase (SOD) or catalase (CAT) greatly reduced superoxide and H2O2 levels, respectively, confirming that extracellular superoxide and H2O2 were being detected in the assays. In addition, diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase, significantly inhibited the production of superoxide and H2O2, suggesting that increased levels of superoxide and H2O2 in NahG plants are mainly dependent on NADPH oxidase.

image

Figure 2. Elevated levels of superoxide and H2O2 in SA-deficient NahG rice. Leaves of 8-week-old control (CK, vector-transformed lines) and NahG plants were stained with nitro blue tetrazolium (NBT, 1 mg ml−1) or 3,3-diaminobenzidine (DAB, 0.5 mg ml−1) to visually detect superoxide (a) and H2O2 (b), respectively. For enzyme or inhibitor treatments, detached rice leaves were placed in SOD (1500 U ml−1), CAT (120 U ml−1) or DPI (250 μm) solutions for 1 h before staining with NBT or DAB. Similar results were obtained in both T1 and T2 NahG lines.

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Development- and light-regulated lesion formation in NahG rice

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Following the rise of superoxide and H2O2 levels, older leaves of all NahG lines began to produce brownish lesions (Figure 3a). Lesion formation was accompanied by induction of rice PR genes such as PR-1 and PBZ1 (a member of the PR10 gene family) in an SA-independent manner (Figure 3b). As NahG plants aged, brownish lesions formed on most leaves except young shoots. We found that NahG plants exhibited earlier and more extensive lesion formation under high-intensity sunlight. In contrast, none of the vector-transformed plants grown under the same conditions exhibit lesion formation. This nahG-dependent lesion mimic phenotype was heritable and clearly seen in T1 and T2 homozygous and heterozygous progeny carrying nahG transgene (Figure 3c). However, segregation lines that lost the nahG transgene and restored normal levels of SA showed no lesions (Figure 3c,d), confirming that the lesion phenotype results from SA deficiency rather than other genetic defects. Developmentally regulated and light-dependent lesion formation was also observed in NahG transgenic tomato (Brading et al., 2000). In contrast, NahG tobacco does not have spontaneous lesion formation, but does exhibit larger hypersensitive lesions upon viral infection during the resistant interaction (Gaffney et al., 1993).

SA analog benzothiadiazole suppresses ROI levels and lesion formation

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

To examine whether exogenous application of SA or SA analogs such as benzothiadiazole (BTH) could complement endogenous SA deficiency, leaves of 8-week-old NahG rice were treated with SA (2 mm) and BTH (250 μm) before the onset of lesion formation. Exogenous application of SA, which could be rapidly degraded once it enters the NahG plants, was ineffective in rescuing the phenotypic defects caused by endogenous SA deficiency. However, BTH could functionally complement the endogenous SA deficiency by suppressing elevated ROI accumulation and lesion formation in NahG plants (Figure 4). SA analogs such as BTH and 2,6-dichloroionicotinic acid could induce protein levels of copper–zinc SOD (an antioxidant enzyme) (Kliebenstein et al., 1999). Therefore, BTH might have activated antioxidant defense response, or acted directly as an antioxidant, leading to the reduction in ROI levels and lesion formation.

image

Figure 4. Reduction in lesion formation as well as superoxide and H2O2 levels in NahG plants treated with BTH. Leaves of 8-week-old NahG plants were sprayed with BTH (250 μm) or H2O before the onset of lesion formation. Similar results were obtained with T1 and T2 NahG lines in three experiments.

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Catechol does not significantly affect ROI levels in rice

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Catechol might induce H2O2 generation and oxidative DNA damage in animal cells (Schweigert et al., 2001). To assess if catechol, the direct product of SA degradation by the bacterial salicylate hydroxylase, might have led to the increased ROI levels in transgenic NahG rice, free and conjugated catechol levels were determined by HPLC analysis using 3- and 9-week-old T2 plants (Table 1). Free catechol was undetectable in both control and NahG plants. However, higher levels of conjugated catechol were detected in 3-week-old NahG rice than in 9-week-old plants. A very low level of conjugated catechol was also found in 3-week-old control plants, but not in 9-week-old control plants. As described previously, a significant increase in superoxide and H2O2 was not observed in 2–4-week-old NahG plants that contained a high level of conjugated catechol, but rather in 8–10-week-old NahG plants that contained a relatively low level of conjugated catechol. Therefore, catechol is probably not the main cause that leads to the significant elevation of ROIs in NahG rice.

Besides the measurement of endogenous catechol levels, exogenous catechol has been tested for its effect on ROI levels in 3-week-old wild-type rice plants. Catechol solutions (0.1 and 0.5 mm) were sprayed onto intact plant or supplied to excised whole leaf through the cut end for 24 h. However, no significant increase in superoxide or H2O2 levels in rice leaf was observed in both treatments (data not shown).

NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Infection of plants by pathogens (particularly avirulent isolates) induces rapid production of superoxide and H2O2, an event known as oxidative burst. Significant accumulation of H2O2 was observed in rice leaves during the resistant interaction with the blast fungus (Ganesan and Thomas, 2001). To examine the potential susceptibility of NahG plants to pathogen infection, we inoculated 2-week-old seedlings with avirulent and virulent isolates of Magnaporthe grisea, respectively. After infection with an avirulent isolate (PO6-6) of M. grisea, the control plants exhibited an hypersensitive response as indicated by small, reddish lesions on the leaves (Figure 5a). In contrast, SA-deficient NahG plants showed spreading lesions with whitish brown color, which resembles the runaway cell death phenomenon in some lesion mimic mutants of Arabidopsis (Jabs et al., 1996). Furthermore, the fungal growth in planta increased nearly fivefold in the NahG plants compared with the control plants (Figure 5b). Nevertheless, the disease symptoms caused by virulent blast isolates (which elicit weak oxidative bursts) were no more severe in 2-week-old NahG seedlings than in control seedlings (data not shown). This is similar to NahG transgenic potato, in which a drastic reduction in endogenous SA levels resulted in no significant increase in disease severity when infected by virulent Phytophthora infestans (Yu et al., 1997).

image

Figure 5. Increased susceptibility of SA-deficient NahG rice to the blast fungus. (a) Blast lesions in 2-week-old seedlings of control (CK, vector-transformed lines) and NahG rice at 5 days after inoculation with an avirulent isolate (PO6-6 strain) of Magnaporthe grisea. (b) Growth of the PO6-6 strain in control and NahG plants as determined by RNA blot/phosphoimaging quantification of the fungal 28S rRNA. (c) SA-independent induction of rice PR-1 and PBZ1 genes by the infection of the PO6-6 strain. (d) Lesion formation in 10-week-old plants of control and NahG rice at 10 days after inoculation with a virulent isolate (IC17/18-1 strain) of M. grisea. Note very few and small pathogenic lesions in the control leaf, and a few pathogenic lesions and numerous spontaneous lesions in the NahG rice leaf.

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Adult rice plants (8–12 weeks old) are generally very resistant to the blast fungus. Under our experimental conditions, 10-week-old control plants (segregation lines that lost nahG transgene) are immune to the avirulent isolate, and only produce very few HR-like lesions when inoculated with the virulent isolate (IC17/18-1). In contrast, 10-week-old NahG plants were hyperresponsive and more susceptible to infection by the virulent isolate (Figure 5d). The virulent isolate causes more pathogenic lesions at the site of infection and stimulates spontaneous formation of numerous lesions throughout leaves of NahG rice.

Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

In tobacco, Arabidopsis and many other dicots, the level of endogenous SA significantly affects the expression of some defense genes such as PR-1 and PR-10. Interestingly, depletion of endogenous SA in rice does not measurably affect pathogen-induced PR-1 and PR-10 gene expression. As shown in Figure 5(c), the blast fungus induced similar levels of PR-1 and PBZ1 transcripts in NahG and control plants. The PR-1 and PBZ1 genes were also activated during lesion formation in NahG rice (Figure 3b). Therefore, SA is not essential to the induction of PR-1 and PR-10 genes in rice plants, despite its importance in signaling defense gene expression in many dicotyledonous plants such as tobacco and Arabidopsis.

NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

In addition to biotic stresses, NahG plants are more susceptible to oxidative damage caused by abiotic factors such as herbicide treatment. Paraquat, also known as methyl viologen, inhibits ferredoxin reduction in photosystem I by autooxidizing to a radical, resulting in the production of superoxide and H2O2 (Babbs et al., 1989). Application of paraquat induced localized cell death in the leaves of control plants, but much more extensive cell death/tissue damage in NahG plants (Figure 6). These results again demonstrate that SA functions as a preformed antioxidant to protect rice plants from oxidative damage.

image

Figure 6. Hyperresponsiveness of NahG rice to oxidative damage caused by paraquat treatment. Leaves of 8-week-old T1 control (CK, vector transformed) and NahG plants were treated with different concentrations of paraquat solutions. The picture was taken 24 h after treatment.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Salicylic acid is a key secondary signal molecule that mediates defense gene expression and disease resistance in many plant species such as tobacco, Arabidopsis and cucumber. In rice plants that contain very high basal levels of SA, however, SA does not appear to act as an effective signal molecule to activate many defense genes and induce disease resistance. In this study, SA-deficient NahG rice had elevated ROI levels and reduced antioxidant capacity as shown by increased susceptibility to oxidative damage caused by aging as well as biotic and abiotic stresses. Our data strongly suggest that endogenous SA plays an important anti-oxidative role in protecting rice plants from oxidative stress. Although the result is somewhat surprising, it is not totally unexpected. In animal systems, SA and aspirin (which is rapidly converted to SA in vivo) directly function as antioxidants and/or indirectly activate antioxidant pathways to protect cells from oxidative damage caused by ROI (Castagne et al., 1999; Oberle et al., 1998; Podhaisky et al., 1997). SA is a direct scavenger of hydroxyl radical and an iron-chelating compound, thereby inhibiting the direct impact of hydroxyl radicals as well as their generation via the Fenton reaction (Dinis et al., 1994; Halliwell et al., 1988, 1995). The salicylate–iron complex was shown to have SOD activity to catalyze the dismutation of superoxide radicals (Jay et al., 1999). SA can also protect plant and mammalian catalases against inactivation by H2O2in vitro and potentially reduce H2O2 accumulation in vivo (Durner and Klessig, 1996). Therefore, high levels of SA in rice plants may act directly as a preformed antioxidant to scavenge ROI and/or indirectly modulate redox balance through activation of antioxidant responses.

Catechol production in NahG Arabidopsis may lead to inappropriate generation of H2O2 and increased disease susceptibility (van Wees and Glazebrook, 2003). Previous studies either did not assay or failed to detect free or conjugated catechol in NahG Arabidopsis or tobacco (Bi et al., 1995; Mur et al., 1997; van Wees and Glazebrook, 2003). Conjugated catechol (2000–9000 ng g−1 FW) was detected in NahG tomato (Brading et al., 2000), but the recovery rate was very low (3%). As a result, the tomato catechol data (after correction for recovery) may not be very accurate. In our study, a much higher recovery rate (51–64%) was achieved based on spiked catechol control. As is the case of NahG tomato, free catechol was undetectable in control and NahG rice. But conjugated catechol was detected in NahG rice at a relatively higher level in 3-week-old plants (1580 ± 412 ng g−1 FW) than in 9-week-old plants (848 ± 342 ng g−1 FW). As a significant increase in ROI was not observed in 3-week-old NahG rice that contains a higher level of conjugated catechol and because exogenous application of catechol did not significantly increase ROI levels in rice plants, we conclude that SA deficiency (and reduced antioxidant capacity) rather than catechol production is mainly responsible for increased susceptibility of NahG rice to oxidative damage caused by aging as well as biotic and abiotic stress. However, it is possible that catechol may partially contribute to the elevated oxidative stress in NahG rice.

Reactive oxygen intermediates play a key role in the initiation and propagation of programmed cell death in plants (Grant and Loak, 2000). H2O2 acts as a secondary messenger for activation of defense genes and programmed cell death (Alvarez et al., 1998; Levine et al., 1994; Orozco-Cárdenas et al., 2001; Pellinen et al., 2002). In some lesion mimic mutants (e.g. lsd1) of Arabidopsis, elevated levels of extracellular superoxide were responsible for programmed cell death and lesion formation (Jabs et al., 1996). In addition, constitutive expression of OsRac1, a rice homolog of mammalian Rac GTPase (a component of NADPH oxidase), induces ROI production and cell death in transgenic suspension cultures and plants (Kawasaki et al., 1999). As high levels of superoxide and H2O2 accumulate before the onset of lesion formation and BTH suppresses cell death as well as superoxide and H2O2 levels, we believe that lesion formation in NahG rice results from its reduced capability to detoxify ROI which are generated during aging and under high-intensity light. Transgenic tobacco plants with reduced antioxidant capacity (due to antisense suppression of CAT or ascorbate peroxidase) contain increased levels of H2O2 and often exhibit lesion formation in older leaves, particularly under high-intensity light that can lead to the generation of ROI through the Mehler reaction (Chamnongpol et al., 1998; Mittler et al., 1999; Takahashi et al., 1997).

In addition to aging-induced lesion formation, SA-deficient rice is hyperresponsive (as shown by spreading lesions and tissue damage in Figure 5a) to an avirulent blast isolate that elicits strong oxidative bursts, but does not exhibit significantly increased susceptibility to a virulent isolate that elicits weak oxidative bursts. Generally, adult rice plants (8 weeks old) are very resistant to blast infection. The virulent isolate of M. grisea only elicited small, HR-like lesions (and presumably strong oxidative bursts) in adult control plants, but caused larger lesions in adult NahG plants and stimulated spontaneous lesion formation throughout leaves. In addition, NahG rice is also hyperresponsive to oxidative damage caused by paraquat that generates ROI in plants. These data suggest that the reduced resistance of NahG rice to the avirulent pathogen likely results from its decreased antioxidant capacity and increased susceptibility to oxidative damage.

Transgenic plants and lesion mimic mutants with increased levels of superoxide and/or H2O2 often exhibit enhanced disease resistance. For example, a number of lesion mimic mutants of Arabidopsis (e.g. lsd1) and rice (e.g. cdr) have increased levels of superoxide and H2O2, and exhibit enhanced resistance to pathogen infection (Jabs et al., 1996; Takahashi et al., 1999). Transgenic rice with overexpression of OsRac1 and elevated levels of ROI also exhibits cell death and enhanced disease resistance (Ono et al., 2001). In these cases, ROI likely act as secondary signals to induce defense genes and enhance disease resistance. By contrast, elevated levels of ROI promote oxidative damage and disease symptoms in NahG rice because of its reduced antioxidant capacity. Our results are consistent with many previous findings about ROI and stress tolerance in transgenic plants. Suppression of antioxidant enzymes reduced oxidative stress protection and enhanced pathogen-induced cell death (Dorey et al., 1998; Mittler et al., 1998). Transgenic tobacco (e.g. CAT or ascorbate peroxidase antisense plants) with reduced capability to detoxify ROI is hyperresponsive to pathogen infection (Mittler et al., 1999). In contrast, overexpression of ROI-scavenging enzymes such as SOD generally increases stress tolerance (Allen, 1995).

Depletion of endogenous SA in tobacco and Arabidopsis typically abolishes or greatly reduces pathogen-induced expression of defense genes, leading to reduced disease resistance. In contrast, SA deficiency in rice does not measurably affect the induction of PR genes such as PR-1 and PBZ1 and other defense genes such as JAmyb (Lee et al., 2001), suggesting that induction of many defense genes is independent of SA in rice. Further expression profiling with microarray should allow us to determine what defense genes, if any, are dependent on SA in rice. Despite reduced resistance, PR-1 and PBZ1 genes were readily inducible in NahG rice by M. grisea infection. The lack of correspondence between PR-1 and PBZ1 expression and blast resistance in NahG rice is not totally surprising because there is relatively small difference in rice PR-1 and PBZ1 expression during resistant and susceptible interactions. Increased susceptibility of NahG rice is mainly due to decreased antioxidant capacity to detoxify ROI, whereas increased susceptibility of NahG tobacco and Arabidopsis at least partly results from reduction in PR gene expression.

Based on our data and previous studies in rice, tobacco, Arabidopsis, and other plants, we propose a model to explain different roles of SA in mediating defense response and cell death in SA-sensitive and -insensitive plant species (Figure 7). In SA-insensitive plants such as rice, SA is not an effective secondary signal for activation of defense genes and induced resistance. Rather, a high level of endogenous SA may exert direct and/or indirect antioxidant effects to minimize oxidative damage caused by various biotic and abiotic factors. It is possible that a phenolic derivative structurally similar to SA may play an active role in rice defense signaling. This is consistent with the previous findings that BTH induces rice defense response and that NPR1/NIM1-mediated signaling pathway may exist in rice plants (Chern et al., 2001; Fitzgerald et al., 2004; Schweizer et al., 1999). In SA-sensitive plants such as tobacco and Arabidopsis, SA acts primarily as a secondary signal for activation of defense genes (antimicrobial and/or antioxidant) and induced resistance, but may also function as an antioxidant to limit oxidative damage at the site of infection (which has relatively high levels of SA). Rao and Davis (1999) proposed that SA influences ozone-induced cell death via two distinct mechanisms (Rao and Davis, 1999). In some cases, SA potentiates the activation of antioxidant defense responses to minimize ozone-induced oxidative stress, while in other cases, high levels of SA potentiate activation of oxidative bursts and cell death in Arabidopsis. In some lesion mimic mutants of Arabidopsis (e.g. lsd2 and lsd4), the lesion phenotype is more severe in the absence of endogenous SA (Hunt et al., 1997). Depletion of SA enhanced lesion phenotype in Arabidopsis mutants acd2 (defective in red chlorophyll catabolism, Mach et al., 2001) and lin2 (defective in coproporphyringogen III oxidase, Ishikawa et al., 2001). In other Arabidopsis mutants (e.g. lsd1, lsd6, lsd7), elevated levels of SA are required for lesion formation (Aviv et al., 2002; Dangl et al., 1996; Greenberg, 1997). SA was also required for necrosis during leaf senescence, salt and osmotic stress or susceptible responses to pathogens (Borsani et al., 2001; Morris et al., 2000; O'Donnell et al., 2001). Recently, induction of H2O2 was observed in rice leaf segments soaked in 2 or 5 mm SA solutions (Ganesan and Thomas, 2001). We found that detached NahG rice leaves soaked in 2 mm SA solution exhibited severe tissue damage and even developed lesion-like symptoms (Y. Yang and M. Qi, unpublished data), suggesting that SA-deficient rice is more susceptible to exogenous SA-induced oxidative damage. These seemly contradicting examples demonstrate that SA is an important component for modulating redox balance and may play pro- or anti-oxidative roles based on its endogenous levels and interaction with other cellular components (e.g. ROI, jasmonic acid, ethylene, etc.) in particular plant species under specific developmental or environmental conditions.

image

Figure 7. Role of SA in mediating defense response and cell death in SA-sensitive and -insensitive plants. (+), positive regulation; (−), negative regulation; (0), no significant effect.

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DNA and RNA blot analyses

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Genomic DNA was isolated from leaf tissues with the DNAzol reagent (Invitrogen, Carlsbad, CA, USA). To determine the copy number of nahG transgene, 5 μg DNA was digested with restriction enzymes, fractionated on a 0.8% agarose gel and blotted onto a nylon membrane. Total RNAs were isolated from leaf tissues with the TRIzol reagent (Invitrogen). Ten micrograms of total RNAs was fractionated on a 1.2% agarose gel containing formaldehyde and then transferred onto a nylon membrane. Both DNA and RNA blots were hybridized with the [α-32P] dCTP-labeled probes (nahG, PR1, PBZ1, or 25S rDNA) using PerfectHyb Plus hybridization buffer (Sigma, St Louis, MO, USA) according to the manufacturer's instruction.

Determination of SA and catechol levels in rice leaves

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Free SA was extracted from leaf tissues and quantified by HPLC according to the previously reported method (Bowling et al., 1994). Free and conjugated catechols were extracted using the same protocol as SA extraction but analyzed according to the HPLC method described by Alva and Peyton (2003). The running conditions for catechol were 1 ml min−1 of flow rate with solvents of 80% of acetic acid: H2O (2:98) and 20% of butanol:methanol:acetic acid:H2O (2.5:12.5:2:83) containing 18 mm ammonium acetate. Levels of catechol were quantified by a fluorescence detector (Waters Alliance HPLC, Waters Corporation USA, Milford, MA, USA) with excitation wavelength at 270 nm and emission wavelength at 330 nm. The authenticity of the SA and catechol from rice leaf extracts was verified based on the retention times and spectral properties that matched perfectly to that of commercial SA and catechol standards.

Detection of superoxide and H2O2 in rice leaves

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Superoxide and H2O2 levels were visually detected, respectively, with nitro blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) as described previously (Jabs et al., 1996; Orozco-Cárdenas and Ryan, 1999; Thordal-Christensen et al., 1997). Rice leaves were excised at the base with a razor blade and supplied through the cut ends with NBT (1 mg ml−1) or DAB (0.5 mg ml−1) solutions for 8 h. Leaves were then decolorized in boiling ethanol (95%) for 15 min. The superoxide and H2O2 staining were repeated four times with similar results in both T1 and T2 transgenic plants. At least five leaves were used for each treatment in these experiments.

Fungal infection and growth curve

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References

Both avirulent (PO6-6 strain) and virulent (IC17/18-1 strain) isolates of the blast fungus (M. grisea) were used in this study. Two-week old seedlings and 10-week-old mature plants of control and NahG lines were spray-inoculated with M. grisea at a concentration of 300 000 spores per ml. After incubation in a dew chamber (22°C) for 24 h, plants were moved to a Conviron growth chamber and maintained at 28°C under 16 h of light. Disease symptoms were visually examined and photographed. To quantify the fungal growth in planta, total RNAs were isolated from infected leaf tissues and hybridized with the 3′ region (about 250 bp) of M. grisea 28S rDNA. Relative fungal growth was calculated based on phosphoimaging quantification of 28S rRNA of M. grisea in rice leaves (Qi and Yang, 2002). The experiment was repeated three times in rice seedlings and two times in mature plants with similar results.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Generation of SA-deficient transgenic rice
  6. SA-deficient NahG rice contains elevated levels of superoxide and H2O2
  7. Development- and light-regulated lesion formation in NahG rice
  8. SA analog benzothiadiazole suppresses ROI levels and lesion formation
  9. Catechol does not significantly affect ROI levels in rice
  10. NahG rice exhibits increased susceptibility to oxidative damage caused by pathogen
  11. Pathogen-induced PR-1 and PR-10 expression in rice is independent of SA
  12. NahG rice is hyperresponsive to oxidative damage caused by paraquat treatment
  13. Discussion
  14. Experimental procedures
  15. Gene constructs and bacterial strains
  16. Rice transformation and plant growth conditions
  17. DNA and RNA blot analyses
  18. Determination of SA and catechol levels in rice leaves
  19. Detection of superoxide and H2O2 in rice leaves
  20. Fungal infection and growth curve
  21. Acknowledgements
  22. References