Introduction
Reactive oxygen species (ROS), which are formed from excitation or incomplete reduction of molecular oxygen, are unwelcome harmful by-products of normal cellular metabolism in aerobic organisms [13]. These partially reduced or activated derivatives of oxygen, such as singlet oxygen, superoxide anions, hydrogen peroxide and hydroxyl radical, are highly reactive and toxic not only to mammals, but also to the other life forms including plants, and can lead to the oxidative destruction of cells [2]. H2O2 is a major ROS which is generated in cells by the direct transfer of two electrons to the superoxide anion [8]. During the perception process of abiotic and biotic stresses, the rate of cellular H2O2 production is enhanced dramatically as a consequence of superoxide dismutase activation, which catalyze the conversion of O2- to H2O2, and also activates the NADPH-dependent oxidase system [5, 15].
In plant cells, ROS overproduction is induced by biotic and abiotic stresses, such as salt, drought, high light, pathogen, etc. To avoid the toxicity of ROS, aerobic cells are provided with a flexible set of enzymes and metabolites involved in ROS catabolism, which often acts at the site of ROS production [12]. These enzymes and metabolites maintain the homeostasis of ROS in plant cells. Major ROS-scavenging enzymes in plants include superoxide dismutase (SOD), ascorbate peroxidase (APx), catalase (CAT), glutathione peroxidase (GPx) and peroxiredoxin (PrxR). Together with the antixoidants ascorbic acid and glutathione, these enzymes provide cells with highly efficient machinery for detoxifying O2- and H2O2. The ROS network of these various scavenging enzymes can be found in almost every subcellular compartment. A particular ROS signal can be scavenged by more than one enzymatic scavenger in each of the different compartments [16].
Ascorbate peroxidase (APx) exists as isoenzymes and plays an important role in the metabolism of H2O2 in higher plants. APx utilizes ascorbate (ASC) as its specific electron donor to reduce H2O2 to water with the concomitant generation of monodyehydroascorbate (MDHAR), a univalent oxidant of ASC. In ascorbate-glutathione cycle, APx functions to prevent the accumulation of toxic levels of H2O2 in photosynthetic organisms [3]. Recent studies showed that APx isoenzymes subjected to several environmental stresses such as ozone, high light, and extremes of temperature, salt, and paraquat. In Arabidopsis and Tobacco, APx was reported plays a key role in elevating H2O2 and results in enhanced cell death in response to pathogen attack. The regulation of APx may play an essential part of regulation of terminal and beneficial roles of H2O2 in plant cell. Nitric oxygen is also a dose-dependent inhibitor of APx during the process of programmed cell death (PCD) which also can influence the H2O2 cellular levels [6].
In rice genome, eight different ascorbate peroxidase isoforms were already characterized by southern blotting analyses (OsAPx1 – OsAPx8) [23]. Two of them are cytosolic, two are putative peroxisomal, while four are putative chloroplastic. OsAPx1 and OsAPx2 are involved in pathogen response and growth, and plant hormones which trigger the defense/stress phenomenon [1]. Previously, based on the 2-DE-MS results, we identified 21 different oxygen detoxifying proteins in the roots of rice [18]. We demonstrated that OsAPx1 is involved in H2O2 scavenging system. In this study, the infection of Magnaporthe oryzae (M. oryzae) fungal elicitor, M. oryzae, and Xanthomonas oryzae pv. oryzae (Xoo) and phytohormone illuminated that OsAPx1 is associated with response to the pathogen defense via Jasmonic acid signaling pathway.
Materials and Methods
Plants material and growth conditions
Mature rice seeds (Oryza sativa L.) were obtained from the National Gyeongnam Agricultural Experimentation Station. The dehulled seeds were sterilized in 70% ethanol for 10 min and then in 3% sodium hypochlorite for 20 min. To induce the callus formation, sterilized seeds were placed in R2 medium and allowed to culture in the dark at 25℃. For raising the rice seedlings, the dehulled seeds were put onto the Kimwipe paper with water in a plant culture dish, and kept in the light chambers for 3 days for germination. Germinated rice seedlings were then shifted to the greenhouse for further growth. Fourth- and fifth-leaf stage rice seedlings grown under natural light conditions in a greenhouse (20-30℃) were inoculated with the rice blast fungus, M. oryzae.
Arabidopsis thaliana plants were grown in growth chambers under controlled conditions: 21-22℃, with 18 hr or constant light cycle, 100 μmol m-2 sec-1, and a relative humidity of 70%. Knockout Arabidopsis plants containing a T-DNA insert in Apx1 was obtained from the Arabidopsis knockout facility at the Arabidopsis Biological Resource Center (ABRC) and confirmed according to the knockout facility recommended protocols (http://www. arabidopsis.org/) using the following DNA Primers: LP 5’-ATTATAAGCCAGAGCCATCCG-3’ and RP 5’-AACTCTTGAGCGGAGAGAAGG-3’. The plants were selected to obtain a pure homozygote line.
Chemical treatment
The calli were treated with 50 μg/ml fungal elicitor, 250 μM Jasmonic acid (JA), 5 mM Salicylic acid (SA), or 200 μM abscisic acid (ABA), and harvested at 24 and 48 hr after treatment [20]. RNA was extracted from whole plant after 3 and 6 days of treatments.
Infection with rice blast fungus and rice blight bacteria
Two races of rice blast fungus, M. oryzae races KJ401, were used for infecting the rice plants. KJ401 is incompatible with the Jinheung cultivar. Conidia were spread on rice polish agar medium (25 g of rice polish, 20 g of agar/1 l H2O) and grown in the dark at 28℃ for 3 days. For conidia production, the plates were incubated at 28℃ for 3−4 more days under fluorescent light, after removing aerial mycelia with a sterile loop. The conidia were collected by agitating the cultures with distilled H2O containing 0.02% Tween 20, filtered through two layers of Kimwipe to remove aerial mycelia and cell debris and washed twice [20]. The rice calli (ca. 500 mg fresh weight) were suspended in 5 ml of R2 medium in 6-well plates, and inoculated with suspensions of conidia of each race to a final concentration of 1×105 conidia/ml. To prepare the fungal elicitor, mycelia of rice blast fungus (race KJ401), which is avirulent to the rice cultivar Jinheung, were used. The mycelia were chopped, homogenized in 50 mM sodium acetate buffer (pH 4.5), sonicated, and centrifuged. The pellet was homogenized again in 0.1 M borate buffer, pH 8.8, and autoclaved, and the supernatant was extensively dialyzed against distilled water at 4℃ and freeze-dried. Reducing sugar was determined by the dinitrosalicylic acid method using glucose as standard [9, 10].
Northern blot analysis
Total RNA was isolated from rice leaves or suspension cells. RNA was separated on formaldehyde -denaturing agarose gel, and blotted onto nylon membranes. Each 20 μg RNA sample blotted onto a nylon membrane was hybridized with [α-32P]dCTP labeled OsAPx1gene probe using the Prime-a-Gene labeling system (Promega, Madison, USA). Hybridization and washing conditions were as described for Southern blot analysis [21]. Equal loading of RNA were verified by intensity of rRNA staining by Ethidium bromide stain.
Semi-quantitative RT-PCR
RNA isolation and first-strand cDNA synthesis was carried out as mentioned above. A mixture comprising 500 ng mRNA, 500 ng Oligo dT18, and 500 μM dNTP was prepared at 65℃ and chilled on ice. To the mixture, the following reagents were added to the respective final concentrations: 1× First-strand buffer, 5 mM Dithiothreitol, 2 units RNaseOUT, and 10 units SuperScript II RT enzyme. The mixture was incubated at 50℃ for 60 min and inactivated at 70℃ for 15 min. PCR was performed on a PTC-0220 PCR machine (MJ Research, Waltham, MA, USA), using synthesized cDNA as template. Gene-specific primers were designed from the coding sequence of the desired gene (Forward primer, 5’-CTTGAGTGATCAGGACATTG-3’, and Reverse primer, 5’-AGCAGTAGTAGACTAGAAACCTCT-3’). An equal amount of cDNA was used for each PCR reaction by normalization to the actin gene (Forward primer, 5’-AGGAATGGAAGCTGCGGGTAT-3’ and Reverse primer, 5’-GCAGGAGGACGGCGATAACA-3’).
Protein extraction and Western blot
Control and treated samples were harvested and pulverized in liquid nitrogen. Samples were then homogenized with protein extraction buffer (0.5 M Tris-Cl (pH 8.3), 2% (v/v) NP-40, 20 mM MgCl2, 1 mM phenyl methyl sulfonyl fluoride, 2% (v/v) β-mercaptoethanol) and proteins were extracted using phenol-methanolic ammonium acetate precipitation as described previously [20].
For Western blotting, proteins (20 μg) were resolved on SDS-PAGE, and transferred to a Polyvinylidene fluoride (PVDF) membrane using a semidry electrophoretic apparatus (Hoefer, Holliston, MA). Protein detection was carried out using anti-Apx1, anti-CuZnSOD (1:1,000) and anti-Rubisco large subunit (1:5,000) as primary antibodies [20] and anti-rabbit (1:10,000) conjugated with horseradish peroxidase as secondary antibodies.
Detection of H2O2 and O2- with DAB and NBT stain
Leaves from both wild type and APx1 knockout mutant Arabidopsis were put into 10 mM H2O2 for 4 hr. Leaves were put into pH 3.8 ddH2O with 0.05% tween-20, and infiltrated for 5 min under vacuum. For 3,3'-Diaminobenzidine (DAB) staining, leaves were infiltrated with 1mg/ml DAB solution at 28℃ for 4 hr, and cleared by 10% lactic acid for 4 hr. To remove the chlorophylls, the stained samples were transferred to 70% ethanol, and incubated at 70℃ for 10 min. For nitro blue tetrazolium (NBT) staining, leaves were infiltrated with 0.1% NBT solution at room temperature for 2 hr, and washed with 70% ethanol for 4hr.
Results and Discussion
Expression pattern in rice tissues
For analyzing the tissue specific expression of OsAPx1 in rice, total RNA was extracted from different tissues of either rice seedlings or mature plants, including root (R), leaf (L), stem (S), flower (F), short apical meristem (SAM) and leaf sheath (SH). At young seedling stage, OsAPx1 mRNA was highly expressed in root, SAM and leaf sheath, but expressed low in leaf (Fig. 1A). In mature plants, a higher level expression of OsAPx1 was observed in root, stem and flower but weakly in rice leaf (Fig. 1B). Western blot analysis revealed that accumulation pattern of OsAPx1 was consistent with that of RNA level (Fig. 1C). Apx is an important ROS scavenger which maintains a particular level of ROS. ROS are continuously produced during different stages of plant growth and development. Here, we also observed the transcriptional and translational accumulation of OsAPx1 protein in different developmental organs of rice which may help in maintaining the ROS levels in the cells.
Fig. 1.Northern blot analysis of OsAPx1 expression in different rice tissues. Total RNA were extracted form root, shoot apical meristem and leaf sheath at two leaf stage. (B) Total RNA were extracted form root, leaf, stem and flower at mature stage. (Each RNA sample (20 μg) was blotted onto nylon membranes, hybridized with 32P-labeled probes, and washed at high stringency. Equal loading was verified by staining rRNA with ethidium bromide. Blots were exposed to X-ray film at -70℃ for 1 day. (C) Western blot analysis of OsAPx1 in the leaf, stem and root tissues of rice.
Interestingly, a previous report categorized many ROS-related proteins as belonging to the defense/stress group. It was also reported that ROS level was highly accumulated in the rice root and the growth was dramatically inhibited by ROS quenching chemicals which inhibited ROS generation in the rice root [18]. Thus, functions of APxs are to detoxify cytotoxic substrates and protect cells against oxidative damage in the rice root system.
Semi quantitative RT-PCR and Western blot based validation of OsAPx1 expression
Here we characterized several proteins involved in redox regulation including OsAPx1 and cupper/zinc- superoxide dismutase (Cu/Zn-SOD) were characterized in response to Xoo and M. oryzae infection. Transcription of OsApx1 and Cu/Zn-SOD were detected at 12 hpi, and increased further at 48 and 72 hpi (Fig. 2A and 2C), indicated that OsApx1 and Cu/Zn-SOD was induced in response to Xoo and M. oryzae infections. These results were further validated by Western blot analysis. The secretion of APx1 and Cu/Zn-SOD proteins were detected at 24 hpi, and were further increased at 48 hpi and 72 hpi (Fig. 2B and 2D). The intracellular protein RuBisCO was used for loading control.
Fig. 2.Valiation of expression patterns of OsAPX1 in leaves inoculated with Xoo and with M. oryzae. (A) Semiquantitative analysis of Apx1 and Cu/Zn-SOD transcriptional regulation in response to X. oryzae infection. (B) Western blot analysis of secreted Apx1 and Cu/Zn-SOD level in response to X. oryzae infection. (C) Semiquantitative analysis of Apx1 and Cu/Zn-SOD transcriptional regulation in response to M. oryzae infection. (D) Western blot analysis of secreted Apx1 and Cu/Zn-SOD level in response to M. oryzae infection.
In plant cells, ROS formation is induced by various factors including different biotic and abiotic stress [4, 17]. It was also reported that the ROS related-proteins including 7 glutathione S-transferases, 6 ascorbate peroxidases, 4 catalase isozymes, 3 superoxide dismutase, glutathione (GSH)-dependent dehydroascorbate reductase 1, and glutathione reductase comprised the most abundant group in root proteome [18]. These data suggest that ROS-scavenging activities may differentially regulate protein expression levels and patterns because GSH, ascorbate and diphenyleneiodonium (DPI), which were ROS quenching chemicals, have other cellular functions in addition to ROS scavenging. Thus, during plant-microbe interaction, OsAPx plays a critical role for homeostasis of ROS levels in leaves inoculated with pathogens.
Induction of OsAPx1 gene by JA
In plant cells, biotic stress and different stress signals such as JA can induce ROS overproduction. JA is a phytohormone which is produced in response to pathogen attack. Here, we observed that the OsAPx1 mRNA was strongly upregulated in response to JA treatment. When JA treatment was given along with either SA or ABA, a reduction in its m-RNA expression was observed. Moreover, when SA and ABA were applied together, OsAPx1 transcription was completely inhibited (Fig. 3).
Fig. 3.Northern blot analyses of OsAPx1 gene in treatment of plant hormones. Total RNA was extracted from rice callus after different plant hormone treatment which described in pictures. RNA was extracted. Each RNA sample (20 μg) was blotted onto nylon membranes, hybridized with 32P-labeled probes, and washed at high stringency. Equal loading was verified by staining rRNA with ethidium bromide. Blots were exposed to X-ray film at –70℃ for 1 day. Abbreviations: ABA, abscisic acid; JA, jasmonic acid; SA, salicylic acid.
SA and JA, produced during pathogen attack, act as signaling molecules which lead to the transcriptional induction of several PR genes [14, 24]. Detailed analysis of the SA and JA induced signaling has shown a crosstalk between these two signaling pathways each other and fungal elicitors in previous studies [7, 11]. Our results showed plant hormones also have great effect on OsAPX1 expression. Its mRNA was strongly upregulated by the presence of JA, but significantly reduced by SA and ABA. Both SA and ABA reduced the transcription level of OsAPx1, and also restricted the JA induced OsAPx1 gene expression. It has been reported that increased concentrations of H2O2 have beneficial effects on plant defense. Several reports have suggested that pathogen attack leads to the accumulation of H2O2 which results in cell death (hypersensitive response, HR). Therefore, SA and ABA based inhibition of OsAPx1 may be beneficial during pathogen attack as it would lead to the increased concentration of H2O2 by reducing the expression of redox detoxifying enzymes including APx1. However, which pathways and cross-talk regulate the OsAPx1 expression is still unknown.
Phylogenetic relationship analysis and characterization of AtAPx1 knockout plant
To understand the phylogenetic relationship among ascorbate peroxidases of Rice and Arabidopsis, APx protein sequences of 9 isoforms of rice and 8 isoforms of Arabidopsis were aligned by Bio-Edit software. The phylogenetic tree showed that OsAPx1 protein has the close relationship with AtAPx1 (Fig. 4A), and shares 79.3% homology with each other. To further understand the function of APx gene, we studied the function of ascorbate peroxidase gene in Arabidopsis along with the rice. Therefore, we identified an AtAPx1 knockout line (apx1) from the SALK collection, containing T-DNA insertions in the AtAPx1 gene. We named it apx1. The insertion is located in the 3’-UTR in apx1. We use PCR method which described in 2.1 to selected homology apx1 plant. Western blot analysis showed that the expression of OsAPx1 gene in these apx1 plants was eliminated (data not shown).
Fig. 4.Phylogenetic relationship of APx protein from Rice and Arabidopsis and Detection of O2- and H2O2 in Arabidopsis leaves (A) The dendrogram was constructed using BioEdit program. Bootstrap values are shown at the branch. Protein sequences were identified by the accession numbers in the NCBI database. (B) Wild type and apx1 line were infiltrated with 10mM H2O2. After 4 hr, plant leaves were stained with nitro blue tetrazolium (NBT) and 3,3’-diaminobenzidine (DAB) solution, respectively, for 2 hr. Leaves was washed with 98% ethanol at 70℃ for 10 min, and then washed with 10% Lactic Acid or 70% ethanol.
H2O2 treatment and Superoxide anion, hydrogen peroxide detection
Wild type and apx1 seeds were planted on the Phytagel solid MS medium contains 2.5 mM H2O2. H2O2 showed an inhibitory effect on the growth of both wild type and apx1 lines were. However, the degree of inhibition was higher in the apx1 lines as compared with the wild type (Fig. 4B). The effect of H2O2 was further tested on the mature plants. The wild type and apx1 root were dip into MS medium which contains 10 mM H2O2. The apx1 lines showed early senescence just after 3 days treatment with H2O2 (Fig. 4B). H2O2 not only acts as a signal for biosynthesis and development, but it also acts as a signal for PCD when its concentration is increased over a threshold value. Therefore, we tried to examine whether the growth inhibit and early senescence is caused by the oxidative damage to cell. To detect the accumulation of O2- and H2O2 levels in Arabidopsis leaves, we stained it with NBT and DAB. The Arabidopsis leaves were infiltrated with 10 mM H2O2 for 4 hr, then stained and washed as described in Materials and methods section. In apx1 lines, the accumulation of both O2- and H2O2 were higher than the wild type (Fig. 4B). These results indicated that the high concentration of exogenous H2O2 cause the intercellular superoxide anion and hydrogen peroxide accumulation in AtAPx1 knockout plant, and also lead to growth suppression and early senescence than wild type. Therefore, ascorbate peroxidase may have the function of scavenging H2O2 to reduce the oxidative damage of cell.
Conclusion
The fact that OsAPx1 expression is activated both by pathogens (M. oryzae and Xoo) and JA raised the possibility that signal transduction leading to OsAPx1 transcription occurs through a shared pathway. However, we first report that SA and ABA had antagonistic effects on pathogen responsive OsAPx1 expression. In a similar way, PR genes including OsGlu1, OsGlu2, OsTLP, OsRLK, and OsPR-10 transcription is induced by JA but inhibited by SA and ABA [19]. Taken together, these results suggest that the induction of OsAPx is differentially regulated by the signaling molecules, that is, positively by JA and negatively by SA and ABA.