JXB Advance Access originally published online on August 16, 2005
Journal of Experimental Botany 2005 56(420):2673-2682; doi:10.1093/jxb/eri260
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RESEARCH PAPER |
Overexpression in transgenic tobacco reveals different roles for the rice homeodomain gene OsBIHD1 in biotic and abiotic stress responses
1Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310029, PR China
2Department of Plant Protection, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310029, PR China
3State Key Laboratory for Rice Biology, Zhejiang University, Hangzhou, Zhejiang 310029, PR China
To whom correspondence should be addressed. Fax: +086 0571 8604 9815. E-mail: fmsong{at}zju.edu.cn
Received 2 November 2004; Accepted 4 July 2005
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Abstract |
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The rice OsBIHD1 gene encodes a transcriptional factor belonging to the homeodomain class. It had previously been shown to be activated by treatment with benzothiadiazole, a chemical inducer of disease resistance, and in an incompatible interaction between rice and the blast fungus. To allow a better understanding of the function of OsBIHD1 in plant disease resistance response, the OsBIHD1 gene in tobacco was overexpressed by Agrobacterium-mediated leaf disc transformation with a construct containing the OsBIHD1 ORF under control of the 35S promoter. Overexpression of the rice OsBIHD1 gene in some of the transgenic tobacco lines led to some morphological abnormalities in the top buds and roots. The transgenic tobacco plants showed an elevated level of defence-related PR-1 gene expression and enhanced disease resistance against infection by tomato mosaic virus, tobacco mosaic virus, and Phytophthora parasitica var. nicotianae. However, the transgenic tobacco plants overexpressing OsBIHD1 showed enhanced sensitivity to salt and oxidative stress as compared with the wild-type plants. The results suggested that the OsBIHD1 protein may be positively involved in activating expression of the defence-related genes in disease resistance responses, and is also important in rice development and abiotic stress tolerance.
Key words: Disease resistance response, homeodomain, OsBIHD1, oxidative stress tolerance, salt tolerance, transgenic tobacco plant
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Introduction |
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Homeodomain proteins are present extensively in the genomes of animals, fungi, and plants and function as transcription factors in regulating expression of batteries of target genes (Chan et al., 1998
). According to sequence conservation within the homeodomain and the presence of additional sequences, the plant homeodomain proteins can be subdivided into different families, including Knotted 1, Hd-Zip, Glabra2, PHD finger, and Bell1 (Chan et al., 1998). The functions of the homeodomain proteins in plants have been demonstrated to be involved in various developmental processes and responses to hormones (Otting et al., 1990; Chang et al., 1997; Kerstetter et al., 1997; Hung et al., 1998; Mussig et al., 2000; Sentoku et al., 2000; Abe et al., 2001; Johannesson et al., 2001; Tang et al., 2001; Himmelbach et al., 2002; Ohashi et al., 2003). On the other hand, the homeodomain proteins have also been implicated in adaptation of plants to the environment and to biotic stress (Masucci and Schiefelbein, 1996; Soderman et al., 1996, 1999; Tamaoki et al., 1997; Frank et al., 1998; Kusaba et al., 1998; Lee and Chun, 1998; Sakamoto et al., 2001; Deng et al., 2002; Gago et al., 2002; Himmelbach et al., 2002; Sawa et al., 2002; Johannesson et al., 2003). Moreover, several lines of evidence have shown that some homeodomain proteins might play roles in transcriptional regulation of defence-related gene expression during disease resistance responses (Korfhage et al., 1994) and programmed cell death (Mayda et al., 1999).
Recently, a number of genes encoding the BELL type of homeodomain proteins have been identified from different plant species including Arabidopsis thaliana (Byrne et al., 2003; Smith and Hake, 2003), barley (Muller et al., 2001), potato (Chen et al., 2003), and apple (Dong et al., 2000). There is evidence to suggest that BELL-type homeodomain proteins have a role in the development of flowers, fruits, and tubers (Reiser et al., 1995; Dong et al., 2000; Chen et al., 2003). For example, the Arabidopsis BELL1 was found to be involved in the regulation of ovule development (Reiser et al., 1995) and might act, at least in part, to repress the function of the organ identity gene AGAMOUS during ovule development (Western and Haughn, 1999). Mutation in one of the Arabidopsis BELL genes, PENNYWISE (PNY), led to shorter internodes and a slight increase in the number of axillary branches (Smith and Hake, 2003). The Arabidopsis BELL1-related homeodomain protein BELLRINGER was implicated in maintenance of the spiral phyllotactic pattern and was also shown to be required for maintenance of stem cell fate in the absence of the regulatory genes SHOOT MERISTEMLESS and ASYMMETRIC LEAVES1 (Byrne et al., 2003). Overexpression of JuBel1, a barley gene encoding a BELL1-type transcription factor, in transgenic tobacco led to male sterility; however, the transgenic tobacco plants overexpressing a mutant version of JuBel2, which encoded a truncated N-terminal but containing all conserved domains, showed abnormal phenotypes; for example, the bushy growth habit, the divergence of leaf veins at the leaf base, and the appearance of ectopic outgrowths on the abaxial sides of both the petals and the flower base (Muller et al., 2001). The apple MDH1 and the potato StBEL5 have also been shown to play an important role in fruit and tuber development (Dong et al., 2000; Chen et al., 2003).
Previously, a rice gene, OsBIHD1, which encodes a protein belonging to the BELL class of the plant homeodomain transcriptional factor family, had been identified. The recombinant OsBIHD1 protein was shown to have DNA-binding activity in vitro and was located in the nucleus of the plant cells. The OsBIHD1 gene was activated during disease resistance responses, indicating that OsBIHD1 is associated with resistance response in rice (Luo et al., 2005). To understand better the function of OsBIHD1 in response to disease resistance, a functional analysis of OsBIHD1 in transgenic tobacco plants was performed. Overexpression of the rice OsBIHD1 gene in the transgenic tobacco plants led to some morphological abnormalities and constitutive expression of the defence-related PR-1 gene, and enhanced disease resistance against viral and oomycete pathogens. However, the overexpressing OsBIHD1 gene of transgenic tobacco plants showed reduced tolerance to salt and oxidative stress.
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Materials and methods |
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Construction of binary vector and transformation of tobacco
Five micrograms of plasmid pUCm-OsBIHD1-1, which contained the full-length cDNA of the OsBIHD1 gene (GenBank AY524972; Luo et al., 2005
), was digested with BamHI for 2 h at 37 °C, and the target fragment containing the full open reading frame (ORF) sequence was gel purified. The purified ORF fragment of the OsBIHD1 gene was ligated into plant binary vector CHF3 pp2p212, which was digested with BamHI and dephosphorylated with alkaline phosphatase, and placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in the sense orientation. The sequence correctness and orientation of the ORF fragment in the recombinant plasmid was confirmed by sequencing and restriction enzyme digestion. The resulting correct recombinant plasmid, designated as CHF3-OsBIHD1-1, was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation using a GENE PULSER II Electroporation System (Bio-Rad Laboratories, Hercules, CA, USA). The electroporation was carried out using 2500 V, 25 µF, 200 
, 5 m s–1 impulse conditions. Agrobacteria containing the plasmid CHF3-OsBIHD1-1 were grown at 28 °C with shaking (200 rpm) in YEP broth (peptone, 10 g l–1; yeast extract, 10 g l–1; NaCl, 5 g l–1; pH 7.2) supplemented with 100 µg ml–1 streptomycin and 100 µg ml–1 spectinomycin and collected by centrifugation, followed by resuspending in MS medium to a final OD600 of approximately 0.80.
Transformation of tobacco was performed using Agrobacterium-mediated leaf disc transformation as described previously (Horsch et al., 1985). Briefly, fully expanded leaves from 4-week-old tobacco plants were surface-sterilized with 70% ethanol and 0.5–2% NaClO3, respectively. Leaf discs (0.5 cm in diameter) were cut and pre-cultivated on MS medium for 3 d. The pre-cultivated leaf discs were infected by soaking them in Agrobacterium suspension for 5–10 min with occasionally shaking. Agrobacterium-infected leaf discs were cultivated on MS medium at 25 °C for 2 d, and then transferred to MS medium supplemented with 200 µg ml–1 kanamycin and 500 µg ml–1 carbenicillin. Shoots were rooted on MS medium containing 200 µg ml–1 kanamycin and 250 µg ml–1 carbenicillin, and transferred to soil and grown in a greenhouse.
Growth of tobacco plants
All transgenic and wild-type tobacco plants were cultivated in a growth room under a 16 h day/8 h night regime at a temperature of 20–25 °C. The T1 seeds of the transgenic tobacco lines were germinated on half-strength MS medium containing 200 µg ml–1 kanamycin in a growth chamber under 16 h day/8 h night regime at a temperature of 22–25 °C for 3–4 weeks. The surviving plants were transferred into natural soil and grown in the growth room under the above-mentioned conditions for another 3–4 weeks. Illumination was provided by cool-white fluorescent lamps at a light intensity of 150–190 µE cm–2 s–1. Fully expanded leaves from 6–8-week-old plants were collected and immediately frozen in liquid nitrogen until use. All experiments were repeated independently at least three times.
Genomic DNA extraction and PCR detection
Frozen leaves of 2-month-old transgenic tobacco plants were ground to a fine powder in a mortar and pestle with liquid nitrogen. Genomic DNA was extracted with 2 volumes 2x CTAB extraction buffer (2% CTAB, 10 mM TRIS-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 2% 2-ME). The mixture was incubated at 65 °C for 90 min with gentle shaking, and extracted with 1 volume of chloroform/isoamyl alcohol (24:1 v:v), followed by centrifugation at 7500 rpm for 10 min at 4 °C. The supernatant was extracted with 1/10 volume 10x CTAB buffer (10% CTAB, 0.7 M NaCl) and 1 volume of chloroform/isoamyl alcohol (24:1 v:v). The DNA in the supernatant was precipitated with 0.7 volume isopropanol and centrifuged, followed by washing with 70% ethanol. About 100 ng of genomic DNA was used to amplify the transgene as a template by PCR, using HIHN-W5-2F (5'-GGA TCC ATG GCT ACT TAC TAC T-3') and HIHN-W5-2R (5'-AAG CTT TCA GGC CAC AAA ATC ATG CA-3') as primers.
Southern blot hybridization
Fifteen micrograms of the transgenic tobacco plant genomic DNA were digested completely with HindIII, separated by electrophoresis on a 0.8% agarose gel, and transferred by capillary action overnight onto a Hybond-N+ nylon membrane (Amersham Biosciences, Little Chalfont, UK) using 0.4 M NaOH/1.0 M NaCl. An 897 bp fragment was prepared by digestion of the OsBIHD1 ORF sequence with PstI and labelled with [
-32P]dCTP (3000 Ci mM–1) by the random priming method using a Random Primed DNA Labeling Kit (Takara, Dalian, China). Prehybridization was performed at 42 °C for 30 min in ULTRAhyb hybridization buffer (Ambion, Austin, TX, USA) and hybridization was carried out overnight at 42 °C in the same hybridization buffer with the [-32P]-labelled probe. After hybridization, the blots were washed twice with 2x SSC, 0.1% SDS and 1x SSC, 0.1% SDS for 10 min each at 42 °C. The membrane was blotted between waxfilm (Whatman International, Maidstone, UK) and autoradiographed by exposure to X-ray film (Lucky Film Corporation, Baoding, China) for 2 d at –80 °C.
RNA extraction and northern blot hybridization
Total RNA was extracted using an acid phenol–guanidine isothiocyanate–chloroform one-step method (Sambrook and Russell, 2001). Leaf tissues were homogenized and extracted in 1.0 volume of denatured buffer [4 M guanidine isothiocyanate, 25 mM sodium citrate tribasic (pH 7.0), 0.5% (w/v) sodium lauroyl sarcosine, 0.1 M 2-mercaptoethanol]. The samples were vortexed after the addition of 0.1 volume of sodium acetate (pH 4.0), 1.0 volume acid phenol, and 0.2 volume of chloroform/isoamyl alcohol (49:1) and placed on ice for 15 min. After centrifugation, RNA in the supernatants was precipitated with an equal volume of isopropanol for 1 h at –20 °C. RNA pellets were dissolved in denatured buffer, and precipitated by adding an equal volume of isopropanol for 1 h at –20 °C, followed by washing with 70% ethanol. Twenty micrograms of total RNA were fractionated on a 1.0% agarose-formaldehyde gel and transferred by capillary action overnight to a Hybond-N+ nylon membrane (Amersham Biosciences) using 20x SSC. The RNA on the membrane was fixed by baking at 80 °C for 2 h. Probe labelling, hybridization, and detection were the same as in the procedure described for Southern blot hybridization.
Expression of the PR-1 gene in transgenic tobacco plants
According to the cDNA sequence of the tobacco PR-1a gene (GenBank accession no. X76982), primers NtPR1-1F (5'-ATG GGA TTT GTT CTC TTT TCA CA-3') and NtPR1-1R (5'-TTA GTA TGG ACT TTC GCC TCT-3') were used to amplify the ORF of PR-1a using tobacco genomic DNA as the template. The PCR product was cloned and confirmed by sequencing. Probe labelling, total RNA membrane transferring, hybridization, and detection were the same as in the procedure described for northern blot hybridization.
Disease resistance assay
Inoculation of the tobacco plants with tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) was performed using a standard mechanical rubbing method. To prepare the inocula, ToMV- or TMV-infected tobacco leaves were ground to a fine powder in 5 ml of potassium phosphate buffer (50 mM, pH 7.0). Fully expanded leaves were dusted with dry carborundum and inoculated by gently rubbing the upper leaf surface with 100 µl of the viral suspension inoculum, followed immediately by rinsing with deionized water. Control plants were also dusted with carborundum and mock inoculated with a sample volume of potassium phosphate buffer. Following inoculation, plants were maintained at 23–26 °C under continuous illumination provided by cool-white fluorescent lamps. The lesion numbers on the inoculated leaves were counted 5 d after inoculation.
Phytophthora parasitica var. nicotianae (Ppn) was cultivated on PDA medium (potato, 200 g l–1; sucrose 20 g l–1; agar 15 g l–1; pH 6.5) at 25 °C for 4–6 d. In the detached-leaf inoculation assay, fully expanded leaves from 2-month-old tobacco plants were inoculated with mycelium discs (0.5 cm in diameter) of the fungus and placed in Petri dishes containing filter paper saturated with sterilized distilled water and kept under a 16 h day/8 h night regime at 20–25 °C. Disease severity was evaluated 3 d after inoculation on a four-scale standard in which 0 = no symptoms; 1 = lesion diameter <0.75 cm; 2 = lesion diameter between 0.75 and 1.5 cm; 3 = lesion diameter >1.5 cm and lesions merged.
In the whole plant drenching inoculation assay, Ppn was cultivated in PDA broth with shaking (180 rpm) at 25 °C for 3 d. The mycelia of Ppn were collected and resuspended in water to OD600
0.5 for inoculation. Two-month-old tobacco plants were inoculated by pouring mycelium suspension on to the base of the stem (20 ml per plant) and then they were incubated in a growth room under the conditions described above. Disease severity was evaluated 30 d after inoculation on a four-scale standard as following: 0 = no symptoms; 1 = lesions on the stem base not encircled the stem and no leaves wilted; 2 = lesions on the stem base encircled the stem and two or three lower leaves turned yellowish and/or wilted; 3 = lesions on the stem base encircled the stem and the plants wilted.
Abiotic stress tolerance assay
Fully expanded leaves from 2-month-old plants of wild-type and transgenic lines were briefly washed in deionized water, and leaf discs (1 cm in diameter) were punched out using a cork borer. The leaf discs were floated in 10 ml of NaCl solution at different concentrations (0, 0.4, and 0.8 M,) for 3 d, and the chlorophyll contents measured as described previously by Ridderstrom and Mannervik (1997).
Assays for tolerance of the transgenic lines to oxidative stress were performed as described by Yoshimura et al. (2004). Leaf discs, 1 cm in diameter, from fully expanded leaves of 2-month-old plants were punched out and floated on a solution containing 50 µM methyl violet (MV) and 0.1% Tween-20 in the dark for 1 h, followed by illumination at moderate light intensity (200 µE cm–2 s–1) for 9 h at 25 °C. Electrolyte leakage was determined by using a DDS-IIAT-type conductivity meter at different time points during incubation. The percentage of electrolyte leakage attributable to the MV treatment was calculated as 100%x (conductivity of the test samples)/(conductivity after autoclaving).
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Results |
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Generation of transgenic tobacco lines overexpressing the rice OsBIHD1 gene
The rice gene, OsBIHD1, encoding a protein belonging to the BELL class of plant homeodomain transcriptional factors, had previously been shown to be activated in disease resistance responses against infection by the blast fungus (Magnaporthe grisea), indicating that OsBIHD1 is associated with the resistance response in rice (Luo et al., 2005
). In this study, a functional analysis in transgenic tobacco plants was performed by overexpressing the OsBIHD1 gene under the control of the CaMV 35S promoter using the Agrobacterium-mediated leaf disc transformation approach. A total of 21 independent transgenic lines was obtained by screening kanamycin-resistant regenerated plants. All of them contained the OsBIHD1 gene in their genomes as confirmed by PCR detection of the OsBIHD1 ORF fragment with genomic DNA as the template. Southern blot analysis revealed that most of the transgenic lines had a single copy of the OsBIHD1 gene in their genomes and some had two or more copies (Fig. 1A). The amounts of mRNA of the transgene OsBIHD1 in the transgenic lines were analysed by northern blotting. The expression level of the OsBIHD1 gene in the transgenic lines varied to some extent; for examples, lines 7 and 8 showed a relatively high level of expression of the OsBIHD1 gene (Fig. 1B).
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In the present study, it was found that some of the transgenic lines showed abnormal morphological phenotypes; for example, geminated top buds (Fig. 2E), abnormal roots (Fig. 2B and C), reduced fertility or infertility (Fig. 2G), and delayed germination of T1 seeds in some transgenic lines. Among the 21 transgenic lines obtained, 14 lines showed geminated top buds (66.7% of the total), 10 lines had an abnormal root system (47.6%), five lines showed geminated top buds but had an abnormal root system (23.8%), two lines showed reduced fertility or infertility (9.5%), and only six lines showed normal phenotype without any morphological abnormality. These results suggested a role for the OsBIHD1 gene in regulating various development processes.
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For downstream studies, eight transgenic lines were selected. The information on morphological phenotype, copy number, and expression level of the transgene is summarized in Table 1.
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Elevated level of defence-related gene expression in the OsBIHD1-overexpressing tobacco plants
Expression of genes encoding the pathogenesis-related (PR) protein is tightly correlated with the onset of systemic-acquired resistance, a defence response effective against a variety of fungal, viral, and bacterial pathogens. Constitutive high-level expression of PR-1a in transgenic tobacco conferred tolerance to infection by two oomycete pathogens, Peronospora tabacina and Phytophthora parasitica var. nicotianae (Alexander et al., 1993
). To confirm whether expression of defence-related genes in the OsBIHD1-overexpressing tobacco plants was activated due to overexpression of OsBIHD1, the expression of PR-1a in transgenic tobacco plants was examined. In the northern blot hybridizations with total RNA samples from two independent experiments, a relatively low level of PR-1a expression was detected in the wild-type plants and in transgenic lines 1 and 6, which showed no transcriptional expression of the transgene OsBIHD1 (Fig. 1B). However, the expression levels of PR-1a in the other six transgenic lines tested were higher than the level in the control (Fig. 1B). This result suggests that overexpression of the rice OsBIHD1 gene in transgenic tobacco plants leads to activation of defence-related gene expression. Based on this result, together with the phenotype of the transgenic lines and the copy number and expression level of the transgene OsBIHD1, six lines (lines 2, 3, 4, 5, 7, and 8) were selected to perform disease resistance and stress tolerance assays.
Enhanced disease resistance against viruses in the OsBIHD-overexpressing transgenic tobacco plants
First, the disease resistance of the transgenic lines was evaluated against ToMV and TMV. Necrotic lesions were seen on the leaves of the wild-type and the OsBIHD1-overexpressing transgenic plants 3–5 d after inoculation with ToMV; however, disease severity based on the lesion numbers and symptoms in the leaves of the OsBIHD1-overexpressing transgenic plants was less severe than those of the wild-type tobacco plants. The ToMV lesion numbers in the leaves of all transgenic lines were significantly reduced as compared with those in the wild-type plants (Fig. 3A). As with ToMV, necrotic lesions caused by TMV were observed on the leaves of the wild-type and the OsBIHD1-overexpressing transgenic plants 3 d after inoculation, and disease severity based on the lesion numbers and symptoms in the leaves of the OsBIHD1-overexpressing transgenic plants was less severe than those of the wild-type tobacco plants. The TMV lesion numbers in the leaves of the transgenic lines 2, 3, 4, 7, and 8 were significantly reduced as compared with those of the wild-type plants (Fig. 3B); however, the lesion numbers on the leaves of transgenic line 5 were not significantly different from those of the control. These results indicated that overexpression of the rice OsBIHD1 gene in most of the transgenic tobacco plants led to an enhanced disease resistance against both ToMV and TMV.
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0.05.Enhanced disease resistance against Ppn in the OsBIHD-overexpressing transgenic tobacco plants
Two different methods, detached-leaf inoculation and whole-plant drenching inoculation assays, were used to evaluate the disease resistance of the transgenic lines against Ppn. In the detached-leaf inoculation assay, water-soaked lesions were seen 36 h after inoculation in all leaves, both from the transgenic lines and wild-type plants. Lesions in leaves from the wild-type plants merged with each other 3 d after inoculation; lesions in leaves from the transgenic lines were slightly but not significantly smaller than those in the control leaves, although they varied among different transgenic lines (Fig. 4A). In the whole-plant drenching inoculation assays, disease symptoms were observed 10 d after inoculation. Some lower leaves wilted, and the stem base turned black and rotted. The disease severity 30 d after inoculation in most of the transgenic lines was significantly reduced as compared with those in wild-type plants (Fig. 4B). Plants of transgenic lines 2 and 3 showed no disease symptoms in any of the three independent experiments; disease severity in transgenic lines 4, 7, and 8 was markedly reduced by 50–87.5% as compared with the controls. However, the disease severity in transgenic line 5 was only slightly reduced by 25% as compared with the controls. Although the disease resistance against Ppn in the the OsBIHD1-overexpressing transgenic tobacco lines varied to some extent, the present results suggest that overexpression of the rice OsBIHD1 gene in transgenic tobacco plants confers an enhanced disease resistance against Ppn.
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Reduced tolerance of the OsBIHD1-overexpressing tobacco plants to salt and oxidative stress
The detached-leaf disc assay was used to assess the tolerance of the OsBIHD1-overexpressing transgenic tobacco plants against salinity stress. During the 3-d period of incubation in NaCl solutions at different concentrations, leaf discs treated with distilled water without NaCl maintained a normal shade of green. Leaf discs from the wild-type plants appeared slightly bleached when treated in NaCl solutions at concentrations of 0.4 M and 0.8 M. However, it was observed that leaf discs from most of the OsBIHD1-overexpressing transgenic lines turned chlorotic to some extent 30 h after treatment in NaCl solution and became bleached 3 d after treatment. Bleaching of the leaf discs varied among different transgenic lines; for example, transgenic lines 2 and 3 showed the highest level of bleaching, while transgenic line 5 showed slight bleaching as in the controls. To verify that the bleaching of the leaf discs from the transgenic lines was due to cellular damage, the chlorophyll content of the leaf discs was measured 3 d after treatment. In the water treatment control, only a slight reduction in chlorophyll content was detected and no significant difference in chlorophyll content was observed among the leaf discs from the wild-type and the transgenic lines (Fig. 5A). However, reduction in chlorophyll content showed a significant difference among the leaf discs from the wild-type and the transgenic lines when treated in NaCl solutions at concentrations of 0.4 M and 0.8 M. For example, a reduction in chlorophyll content in the leaf discs from the transgenic lines 2, 3, 4, 7, and 8 was significantly higher than those in the wild-type plants (Fig. 5A). Reduction in chlorophyll content in transgenic line 5 was higher than in the control when treated with the 0.4 M NaCl solution; however, the reduction was slightly lower than those in the control when treated with the 0.8 M NaCl solution. These results suggest that overexpression of the rice OsBIHD1 gene may lead to enhanced sensitivity to salt stress and thus result in a high level of cellular damage when plants are exposed to salt.
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Experiments were also performed to assess tolerance of the OsBIHD1-overexpressing transgenic tobacco lines to oxidative stress. Leaf discs from wild-type and the OsBIHD1-overexpressing transgenic tobacco plants were incubated in 50 µM MV solution under model light intensity. The rates of electrolyte leakage from the leaf discs were measured at different time points after treatment. There was no obvious change in electrolyte leakage among leaf discs from wild-type and the OsBIHD1-overexpressing transgenic tobacco plants for 1–3 h after MV application. However, the rates of electrolyte leakage in the leaf discs from transgenic lines 2, 3, 4, 7, and 8 were significantly higher than those in the wild-type plants 6 h after MV application, and the rates of electrolyte leakage in leaf discs from these transgenic lines increased markedly at 9 h after exposure to the oxidative stress condition (Fig. 5B). Rates of electrolyte leakage in leaf discs from transgenic line 5 were similar to those in the control. These results suggest that overexpression of the rice OsBIHD1 gene may also lead to enhanced sensitivity to oxidative stress and thus result in a high level of cell membrane damage when plants are exposed to oxidative stress.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The BELL-type homeodomain proteins have been extensively demonstrated to be involved in the development of flowers, fruits, and tubers (Reiser et al., 1995
; Dong et al., 2000; Chen et al., 2003), as well as in the regulation of inflorescence stem growth (Bhatt et al., 2004). Overexpression of genes encoding BELL-type homeodomain transcriptional factors has been found to lead to uncertain morphological phenotypes including dwarfing and reduced fertility (Dong et al., 2000; Chen et al., 2003). In this study, some of the transgenic lines that overexpressed the rice OsBIHD1 gene were found to show morphological abnormalities; for example, geminated top buds, abnormal roots, and reduced fertility. Together with the previous observation that the OsBIHD1 gene has a relatively high level of basal expression in rice (Luo et al., 2005), these results suggested a role for OsBIHD1 in rice development, as has been found for other BELL genes identified so far.
Several lines of evidence have also shown that homeodomain proteins play roles in regulating plant responses to hormones (Soderman et al., 1996; Tamaoki et al., 1997; Kusaba et al., 1998; Sakamoto et al., 2001; Hamant et al., 2002; Himmelbach et al., 2002; Sawa et al., 2002; Johannesson et al., 2003; Rosin et al., 2003; for review, see Hay et al., 2004), environmental stress (Soderman et al., 1996, 1999; Frank et al., 1998; Lee and Chun, 1998, Deng et al., 2002; Gago et al., 2002; Wang et al., 2003), and biotic pathogens (Korfhage et al., 1994; Mayda et al., 1999; Desvoyes et al., 2002). However, the function of the BELL-type proteins in plant responses to environmental stress remains unknown. In this study, it was found that overexpression of the rice OsBIHD1 gene in transgenic tobacco plants led to enhanced disease resistance against both viral and oomycete diseases and an elevated level of sensitivity to salt and oxidative stress. These findings provide new support for homeodomain proteins having a role in plant disease resistance and abiotic stress tolerance.
Some homeodomain proteins have been demonstrated to play important roles in the transcriptional regulation of defence-related gene expression (Korfhage et al., 1994) and programmed cell death (Mayda et al., 1999). In another study, it was found that expression of the OsBIHD1 gene was up-regulated in disease resistance responses induced by benzothiadiazole, a chemical inducer of system-acquired resistance, and in the incompatible interaction between rice and the blast fungus, suggesting the involvement of OsBIHD1 in rice disease resistance responses (Luo et al., 2005). In the present study, it was further observed that overexpression of the OsBIHD1 gene in transgenic tobacco plants activated an elevated level of defence-related PR-1a gene expression and conferred an enhanced disease resistance against virus and fungus. This finding is similar to the previous observation that antisense inhibition of H52 gene expression in transgenic tomato plants resulted in a mis-regulation of programmed cell death, activation of defence genes, and enhanced disease resistance against virulent pathogens (Mayda et al., 1999). However, further studies are still needed to elucidate the possible mechanisms by which OsBIHD1 protein regulates the transcription of PR genes.
The expression of several homeodomain genes is regulated by several environmental stresses, for example, water deficit conditions (Soderman et al., 1996, 1999; Frank et al., 1998; Lee and Chun, 1998, Deng et al., 2002; Gago et al., 2002) and oxidative stress (Mayda et al., 1999). Cellular redox status may also be involved in regulating activity of homeodomain transcriptional factors (Tron et al., 2002). In the present study, overexpression of the rice OsBIHD1 gene led to reduced tolerance to salt and oxidative stress in the transgenic tobacco plants. One possibility is that overproduction of the OsBIHD1 protein in transgenic lines suppresses abiotic stress response signalling pathways and acts as a negative regulator of stress tolerance. Another possibility is that activity of the OsBIHD1 protein as a transcriptional factor is suppressed by salt and oxidative stress. It would be worthwhile performing further experiments to clarify which of these two possibilities is the more likely.
It was not possible to detect any expression of the OsBIHD1 gene in some of the transgenic lines, for example, lines 1 and 6 in Fig. 1B, although these transgenic lines were verified as positive for the OsBIHD1 gene in their genomes by PCR detection. This is most likely to be a result of gene silencing due to multiple copies of the transgene. It was also noted that phenotypes of disease resistance and abiotic stress tolerance in transgenic line 5 were different from those of the five other transgenic lines tested. Levels of disease resistance and tolerance to salt and oxidative stresses were almost unchanged in transgenic line 5 as compared with those of the wild-type control. The expression level of the OsBIHD1 gene in transgenic line 5 was similar to those of other transgenic lines tested; however, this line contained two copies of the transgene in its genome.
In conclusion, the results suggest that OsBIHD1 protein may be involved in different signal transduction pathways to regulate developmental processes and responses to biotic and abiotic stress. Regulatory functions of the OsBIHD1 protein in response to biotic and abiotic stress are also different. Upon pathogen infection, the OsBIHD1 protein activates expression of defence-related genes and thus positively regulates disease-resistance responses. Further, the OsBIHD1 protein is also important for tolerance of stress; for example, it acts as a negative regulator of abiotic stress tolerance. Further studies with knockout mutant or transgenic rice plants, in which the endogenous OsBIHD1 gene is suppressed by an RNA interference approach, will provide new insights into the precise functions of the OsBIHD1 gene in regulating responses to biotic and abiotic stress as well as in development.
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Acknowledgements |
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This study was supported by the National Natural Science Foundation of China (grant no. 30170494), the Fund for the New Century Talent Program from the Education Ministry of China, and by the Rice Science Development Foundation of China to FMS. The authors thank Dr Xueping Zhou (Institute of Biotechnology, Zhejiang University) for providing tobacco mosaic virus and tomato mosaic virus.
While the manuscript has been in the process of preparation for publication, a highly related paper describing the involvement of an Arabidopsis homeodomain transcription factor in disease resistance against necrotrophic fungal pathogens was published in The Plant Cell. For details, see the following reference. Coego A, Ramirez V, Gil MJ, Flors V, Mauch-Mani B, Vera P. 2005. An Arabidopsis homeodomain transcription factor, OVEREXPRESSOR OF CATIONIC PEROXIDASE 3, mediates resistance to infection by necrotrophic pathogens. The Plant Cell 17, 2123–2137.
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Footnotes |
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* Present address: Institute of Biotechnology, Hainan University, Haikou, Hainan 570000, PR China.
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