JXB Advance Access originally published online on May 11, 2006
Journal of Experimental Botany 2006 57(9):1957-1969; doi:10.1093/jxb/erj143
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RESEARCH PAPER |
Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia
1International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
2Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi, Bangalore 560056, India
3Centro de Ciencias Genómicas, Programa de Genómica Funcional Eucariotes, Universidad Nacional Autónoma de México, Apdo. Postal 565-A, Cuernavaca, Morelos 62210, México
To whom correspondence should be addressed in Mexico. E-mail: pmreddy{at}ccg.unam.mx
Received 1 November 2005; Accepted 1 February 2006
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Abstract |
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Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.
Key words: Flavonoids, isoflavone synthase, nod gene induction, Oryza sativa, rhizobia
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Introduction |
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Flavonoids are secondary plant metabolites, derived from the phenylpropanoid pathway (Dixon and Steele, 1999; Winkely-Shirley, 2001). Isoflavonoids, which are limited primarily to leguminous plants, are produced by a branch of the phenylpropanoid pathway (Fig. 1). Many studies link the dietary consumption of the isoflavones genestein and daidzein to a range of health benefits (for references see Fader et al., 2000; Liu et al., 2002). In plants, these compounds are known to be involved in interactions with other organisms and participate in the defence responses of legumes against phytopathogenic micro-organisms (Fader et al., 2000). In addition, isoflavonoids also participate in promoting symbiotic relationship between legume plants and rhizobia leading to the formation of nitrogen-fixing root nodules (Broughton et al., 2000; Spaink, 2000). Isoflavones act as chemoattractants for the rhizobia and as inducers of nodulation (nod) gene expression, leading to the production of Nod factors, which in turn aid in the development of nodules in legume plants.
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The substrate for producing the isoflavone genistein is naringenin, an intermediate in the branch of the phenylpropanoid pathway that leads to synthesis of the flavonoids, which include flavanones, flavones, flavonols, proanthocyanidins, and anthocyanins. Naringenin is the product of chalcone synthase and chalcone isomerase enzymes that are common to most plants (Fig. 1). Legumes have a unique enzyme that carries out aryl migration of the B-ring to the 3-position of naringenin, paving the way for the production of the isoflavone (Kochs and Grisebach, 1986). This key enzyme that redirects phenylpropanoid pathway-flavonone intermediates to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In most plants other than legumes, isoflavones are not among the complex variety of secondary metabolites synthesized by the phenylpropanoid pathway. This difference is due to the absence of isoflavone synthase, which converts naringenin into an isoflavone. The identification of the gene encoding isoflavone synthase (Akashi et al., 1999; Steele et al., 1999; Jung et al., 2000) allowed the introduction of an isoflavone synthesizing capacity into Arabidopsis and tobacco, and maize BMS cell cultures that do not naturally produce isoflavones (Jung et al., 2000; Yu et al., 2000; Liu et al., 2002). These studies demonstrated that the isoflavone genistein could be synthesized in Arabidopsis and tobacco, thus proving that the naringenin substrate present in these anthocyanin-producing plants was available for isoflavone synthase to convert to genistein.
Rice is one of the most important cereal crops of the world, and recently much effort has been directed towards reducing the nitrogen input from chemical fertilizers and providing nitrogen to rice by biological nitrogen fixation (Ladha and Reddy, 2000, 2003). Towards this goal, studies have focused on how to encourage the interaction of rice with rhizobia in order to develop a stable symbiotic association (Reddy et al., 1997, 2000a, b; Sreevidya et al., 2005). Rice was shown to harbour at least partial genetic make up in its genome for interacting with rhizobia (Reddy et al., 2000b, 2002). However, rice varieties have a very low capacity to induce nod genes in rhizobia, presumably because of a lack of ability to synthesize nod gene-inducing flavonoids (Reddy et al., 2000b; Rolfe et al., 2000). Hence, isoflavone production may pave the way for rice plants to enter into a symbiotic relationship with rhizobia. Because secretion of flavonoids is a first step in the legume-Rhizobium interactions, and rice lacks the pathway to produce isoflavones from flavanones such as naringenin, in this study the aim was to transfer the key enzyme, isoflavone synthase for isoflavonoid production in rice, and to determine its ability to synthesize the isoflavonoid genistein, and to induce nod genes in rhizobia. In this study, a pigmented rice variety (Oryza sativa L. cv. Murasaki R86) was used which produces anthocyanins in leaves, culm, and seed coat (Nakai et al., 1998). Any plant that can produce anthocynins must have the ability to generate naringenin, a substrate for isoflavone synthase for the production of genistein (Fig. 1).
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Rhizobial strains
Rhizobial strains, Rhizobium NGR234, A. caulinodans ORS571, R. meliloti 1021, and B. japonicum USDA110 harbouring nod::lacZ fusions, used in the present study are listed in Table 1, and will be referred to in the text as NGR234, ORS571, Rm1021, and USDA110, respectively. The Rhizobium and Bradyrhizobium strains were routinely grown under aerobic conditions in the dark at 30 °C in yeast mannitol (YM) broth or agar plates (Vincent, 1970), supplemented with the appropriate antibiotics, if needed.
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Construction of plant transformation vector
The binary transformation vector pMSH2-cyp93C1 (Fig. 2A) containing the soybean isoflavone synthase gene (IFS; cyp93C1v1) under the control of CaMV 35S promoter and the nopaline synthase gene (nos) terminator was constructed by ligating the 1.8 kb SpeI/XbaI fragment of the cyp93C1v1 cDNA, derived from pCR2.1/cyp93C1v1 (Siminszky et al., 1999), downstream of the CaMV 35S promoter in the SpeI/XbaI restricted binary vector pMSH2 (Kawasaki et al., 1999), in sense orientation.
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Rice transformation and growth conditions
Binary vectors used for rice transformation, pVM200 (a derivative of the pMSH lacking the CaMV 35S promoter at the multiple cloning site; control vector) or pMSH2-cyp93C1 (35S-IFS), were introduced into Agrobacterium tumefaciens EHA105 by electroporation (Nagel et al., 1990). Agrobacterium-mediated transformation of rice (Oryza sativa L. cv. Murasaki R86) calli was performed according to Sreevidya et al. (2005). Plants were regenerated from transformed calli selected by hygromycin resistance. Fully regenerated plantlets were acclimatized hydroponically in Yoshida nutrient solution (Yoshida et al., 1976) for 15 d and then the putative transgenic plants (T0 generation) were transferred to soil in pots and grown in a transgenic containment greenhouse.
Polymerase chain reaction (PCR), and Southern and northern blot analyses
Genomic DNA was prepared from young rice leaves using the procedure developed by Dellaporta et al. (1983) for Southern analysis, and by the CTAB method (Taylor et al., 1993) for PCR analysis.
For PCR analysis 
5 ng of genomic DNA was used as a template. The primers, designed to generate the entire coding sequences of IFS, were used at 10 µM concentration in a 20 µl reaction mixture containing 0.2 mM of each dNTPs, 2 mM MgCl2, 2 U Taq DNA polymerase in PCR buffer, and PCR was performed on Biometra Uno-Thermocycler (Biometra, Gottingen, Germany). The forward and reverse primers were 5'-ATG TTG CTT GAA CTT GCA CT-3' and 5'-TTA AGA AAG GAG TTT AGA TG-3', respectively, and the amplification was carried out with an initial cycle of 3 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 45 s at 53 °C, and 1.5 min at 72 °C, and a single final cycle of 10 min at 72 °C. After the completion of PCR, samples were separated on 0.8% (wt/vol) agarose gels, stained with ethidium bromide and visualized on an UV transilluminator.
For Southern analysis aliquots of 10 µg of purified genomic DNA were digested with XbaI, size-fractionated on a 0.8% (w/v) agarose gel, alkali transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences, Buckinghamshire, UK), and hybridized overnight at 42 °C to digoxigenin-11-dUTP labelled PCR fragments of the coding sequence of IFS. DNA probe preparation using random primers, hybridization and stringency washing of the membranes, and chemiluminescent detection of the bands with anti-DIG-AP and CSPD were done using the DIG-High Prime DNA Labeling and Detection Kit II as per the manufacturer's instructions (Roche Applied Science, Mannheim, Germany). The blots were exposed to X-ray film (Kodak, Rochester, NY, USA) for 1–2 h.
For northern analysis, total RNA was isolated from young rice leaves by guanidine isothiocyanate extraction as per the manufacturer's instructions (TRIzol reagent; GIBCO-BRL, Grand Island, NY). Total RNA (20 µg) was subjected to denaturing agarose gel electrophoresis and blotted onto the Hybond-N+ membrane (Amersham) using standard methods (Sambrook et al., 1989). The membrane was hybridized with a DIG-labelled IFS probe and processed as described above.
Identification of free isoflavones in 35S-IFS transgenic plants
Plant tissues were ground in liquid nitrogen, extracted with methanol at 100 mg ml–1, and filtered through 0.2 µm Acrodisc syringe filters (Gelman Sciences, Ann Arbor, MI, USA). To hydrolyse any possible isoflavone conjugates, 3 ml of 1 N HCl were added to 1 ml of the extract and the sample was incubated at 95 °C for 2 h, followed by extraction using 1 ml ethyl acetate. Both unhydrolysed and hydrolysed methanol extracts were separated by HPLC system (Shimadzu SCL10A, Japan) using Luna C18 column (3 µm, 150 mmx4.6 mm; Phenomenex, USA), by isocratic separation with 65% methanol in water at a flow rate of 0.5 ml min–1. The peaks were monitored at 260 nm and 280 nm, and absorbance of the eluents was recorded on UV detector (SPD10A, Shimadzu, Japan). Absorption spectra of the eluents were obtained using a spectrophotometer (DU800, Beckman, USA). The eluent fractions collected at 1 min intervals were evaporated in Speedvac (SC110A Speedvac Plus, Savant, Germany), dissolved in methanol at a concentration of 10 mg ml–1 and used for nod gene induction studies. Authentic naringenin, genistein (Sigma, St Louis, USA), liquiritigenin (Indofine Chemical Company, Somerville, NJ, USA), and daidzein (Research Biochemicals International, Natick, MA, USA) were dissolved at a concentration of 10 µM in methanol and used as standards in HPLC and spectral analyses.
Isoflavone synthase activity assay
For Isoflavone synthase assays, soluble and membrane protein fractions from control and 35S-IFS transgenic rice plants were prepared as follows. Root/leaf tissues (1 g fresh weight) of 2 month-old rice plants were frozen with liquid nitrogen, ground to a fine powder, and homogenized in 10 ml of extraction buffer (50 mM TRIS-HCl, pH 8.2, 0.4 M sucrose, 2 mM EDTA, 0.5 mM DTT, 0.3 mM mercaptoethanol, and 10% glycerol). The homogenates were centrifuged at 5000 rpm (5 K) for 10 min at 4 °C. The pellets were discarded, and the supernatants (designated as crude homogenates, containing both soluble and membrane fractions) were collected and stored at –80 °C until further use. A portion of the supernatants were centrifuged again at 15 000 rpm (15 K) for 60 min at 4 °C after adding glycerol to a final concentration of 20%. The resulting pellets (crude microsomal fractions) and supernatants were collected and stored at –80 °C until further use. The protein contents of 5 K and 15 K preparations were determined using the Bradford protein microassay (Bio-Rad, Hercules, CA, USA), and isoflavone synthase activity assays were performed according to Yu et al. (2000). Approximately 150 mg of the above protein fractions were incubated for 12 h at room temperature in a reaction mix containing 80 mM K2HPO4, 0.5 mM glutathione, 20% sucrose, pH 8.0, with 100 µM naringenin or 100 µM liquiritigenin substrate and 0.4 mM of NADPH. Following incubation, reactions were extracted with methanol. The samples were evaporated and resuspended in 80% methanol, and then were separated on a HPLC system as described above.
Measurement of the nod gene-inducing activity
Methanol extracts prepared from untransformed or vector transformed control plants, and 35S-IFS transgenic rice tissues, and the eluents obtained following HPLC separation of the methanol extracts were assessed for their ability to induce nod gene expression in rhizobia harbouring nod::lacZ fusions (Table 1) as follows. Bacteria were grown overnight to mid-log phase and diluted to an OD600 of 0.2 in 2 ml of YM medium containing 50 µl of the rice tissue extracts or the eluents obtained after HPLC separation, together with appropriate antibiotics. All extracts were diluted at 0.01 mg ml–1, in 2 ml of culture. After overnight shaking for 16 h at 30 °C, the cultures were assayed for ß-galactosidase activity (expressed by the nod::lacZ fusion gene) according to the method of Miller (1972) at a temperature of 37 °C with o-nitrophenyl-ß-D-galactopyranoside (ONPG) or chlorophenol red-ß-D-galactopyranoside (CRPG) as a substrate. The OD420 (ONPG), OD574 (CRPG). and OD600 (bacterial cells) readings were obtained using a spectrophotometer (DU800, Beckman, USA). All assays with the extracts were conducted at least four times, and mean and standard error of the data points were calculated using the Microsoft Excel program, with appropriate controls in each experiment. Data obtained with nod gene induction assays were subjected to Student's t test to delineate significance of any differences in ß-galactosidase activities elicited in rhizobia in response to the plant extracts of 35S-IFS plants versus vector-transformed control rice plants. Threshold levels of significance were adjusted for multiple comparisons by Bonferroni's correction. Corrected P-values were then calculated according to sample size. Bonferroni correction for multiple comparisons was applied to enhance stringency level while determining the significance of any differences between the compared groups by the t test. Thus, the significance test performed was at a highly stringent two-tailed alpha level (significance level) of 0.01.
ß-galactosidase assays of rhizobia performed with appropriately diluted methanol (solvent used for the dilution of extracts) served as negative controls, and the assays with the authentic flavonoids (100 µM) apigenin, 7,4-dihydroxyflavone, daidzein, genistein, liquiritigenin, luteolin, and naringenin served as positive controls.
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Results |
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Generation of transgenic rice carrying the soybean IFS gene
Transgenic rice plants carrying the IFS gene fused to the cauliflower mosaic virus 35S promoter were generated using A. tumefaciens-mediated transformation. The plantlets regenerated from the hygromycin-resistant transformed rice calli were analysed for the presence of IFS using gene-specific primers in the polymerase chain reaction. PCR analysis showed that 11% of the putative transgenic plants of rice contained the IFS gene (Table 2), and these were designated as 35S-IFS plants. Southern analyses of the 35S-IFS plants revealed that all of them had a single copy of the IFS gene, and were derived from five independent transformation events (Fig. 2B). Northern analysis of the selected representative 35S-IFS plants revealed that the IFS gene was expressed in the transgenic lines (Fig. 2C). During the generation of the primary transgenic plants, most of the 35S-IFS plants, excepting D121, showed normal growth and development similar to untransformed or vector control plants (Fig. 3A). Representative 35S-IFS plants (T0) derived from each independent line (D90, D93, D95, D114, and D121) were used in nod gene induction studies. Untransformed and/or vector-transformed rice plants served as controls.
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Evaluation of nod gene expression in rhizobia in response to standard flavonoids
Most of the known plant substances that act as signals to rhizobia are flavonoids. Flavonoids selectively activate the expression of the nodulation (nod) genes of symbiotic rhizobia (Broughton et al., 2000). Flavonoids interact with a class of transcriptional activators (NodD) of the LysR family that bind to highly conserved 49 bp DNA motifs (nod boxes) found in the promoter regions of many nodulation loci, thus aiding in the expression of nod genes. Despite the fact that nodD genes are present in all rhizobia, their symbiotic characteristics vary from one species to another, and Rhizobium species respond in different ways to flavonoids. NodD homologues from the same or different strains may have different flavonoid preferences. In the present study, authentic flavonoids such as apigenin, daidzein, 7,4-dihydroxyflavone, genistein, liquiritigenin, luteolin, and naringenin have been tested in order to ascertain flavonoid preferences for nod gene activation in the rhizobial strains used in this study (Table 3). Quantitative measurements of the expression of the nod::lacZ fusions harboured by various rhizobia were made by assaying ß-galactosidase activity in bacteria. The rhizobia tested in the present study exhibited differential responses towards various flavonoids. A comparison of data presented in Table 3 gives a clear picture of potent and weak inducers of nod gene expression in NGR234, ORS571, Rm1021, and USDA110. The most potent inducers that showed half-maximal nod gene induction were 7,4-dihydroxyflavone, apigenin, naringenin, liquiritigenin, daidzein, and genistein in the case of NGR234, naringenin and liquiritigenin in ORS571, luteolin and 7,4-dihydroxyflavone in Rm1021, and genistein and daidzein in USDA110. These results agree with the findings reported earlier for the same strains, reiterating differential flavonoid preferences for the induction of nod gene expression in different rhizobia (Mulligan and Long, 1985; Peters et al., 1986; Banfalvi et al., 1988; Bassam et al., 1988; Goethals et al., 1989; Le Strange et al., 1990; Lewin et al., 1990; Kobayashi et al., 2004).
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Assessment of the ability of the wild-type R86 and vector-transformed control rice plants to induce nod gene expression in rhizobia
Earlier studies showed that root exudates or plant extracts from various indica and japonica rice varieties have only a limited capacity to induce the transcription of nod genes in rhizobia (Reddy et al., 2000b; Rolfe et al., 2000). In order to ascertain the extent of the nod gene-inducing abilities of the R86 rice variety used in the present study, root and leaf extracts prepared from the vector transformed (for example, see Fig. 4) and untransformed wild-type control plants were examined for their ability to induce ß-galactosidase activities in NGR234, ORS571, Rm1021, and USDA110. The study showed that extracts of both vector-transformed and the untransformed control behaved very similarly in inducing nod gene expression in rhizobia (data not shown). Extracts derived from the control rice plants elicited modest levels of nod gene expression in NGR234 and ORS571, while they produced very low levels of induction in Rm1021 and USDA110.
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0.01 after Bonferroni correction) in ß-galactosidase activities induced by the extracts from 35S-IFS plants versus that from vector control plants.Induction of rhizobial nod genes by transgenic rice expressing IFS
To ascertain if the presence of the soybean IFS gene in transgenic rice confers the competence to produce nod gene-activating flavonoids, the methanol extracts generated from leaves and roots of representative 35S-IFS rice plants (D90, D93, D95, D114, and D121) from each independent transformation line were analysed for their ability to induce nod gene expression in NGR234, ORS571, Rm1021, and USDA110 harbouring nod::lacZ fusions. When NGR234 carrying nodSU::lacZ fusion was tested for nod gene induction, the leaf extracts derived from D90, D95, D114, and D121 transgenic lines induced significantly higher ß-galactosidase activity compared with the leaf extract from vector-transformed control plants (Fig. 4A). By contrast, compared with control plants, none of the root extracts generated from various transgenic plants were able to enhance nod gene induction in NGR234. In the case of ORS571, compared with vector control plants, even though leaf extracts derived from all 35S-IFS transgenic plants, in general, were able to promote nod gene expression, the leaf extracts particularly from the D90, D95, D114, and D121 lines produced a significant enhancement in nod gene induction (Fig. 4B). By contrast, only the root extract obtained from D121 could promote a significant increase in ß-galactosidase activity in ORS571. Analysis with Rm1021 revealed that the leaf extracts derived from the 35S-IFS plants of all transgenic lines were able significantly to enhance nod gene expression in this bacterium, compared with the leaf extracts from control transgenic plants transformed with the vector alone (Fig. 4C). However, when the ability of root extracts from the 35S-IFS transgenic plants was assessed, it was found that they were able to induce ß-galactosidase activity in Rm1021 at much lower levels than the leaf extracts from the respective transgenic plants. In the case of USDA110, almost all leaf and root extracts derived from the 35S-IFS plants (excepting the leaf extract from D114 and the root extract from D90, which showed no difference in nod gene induction levels compared with the vector control) stimulated nod gene expression significantly in this bacterium (Fig. 4D). The above results suggest that manipulation by incorporating the IFS gene bestowed rice plants with the ability to produce flavonoids that are able to regulate nod gene expression, albeit to varied degrees, in NGR234, ORS571, Rm1021, and USDA110. Further, it is interesting to note that both leaf and root extracts derived from most of the 35S-IFS transgenic lines were able to stimulate nod gene expression in USDA110, compared with other rhizobia, where leaf extracts were more effective than root extracts (excepting the root extract derived from D121 on nod gene induction in ORS571) in promoting nod activity. This observation is intriguing since nod gene expression in USDA110 is maximally induced by isoflavones, such as genistein (Table 3; Banfalvi et al., 1988), and the present study showed that IFS-transformed rice plants are indeed able to produce genistein (see below).
nod gene induction in B. japonicum USDA110 by the HPLC-separated fractions of methanol extracts derived from transgenic rice expressing IFS
Investigations with the crude methanol extracts revealed that the leaf and root extracts derived from 35S-IFS transgenic plants were generally able to induce greater nod gene expression in USDA110 (see above). In particular, both root and leaf extracts from D93 elicited very high levels of nod gene expression. To investigate the ability of the D93 plant extracts to induce nod gene expression in USDA110 further, the methanol extracts prepared from leaves and roots of D93 were fractionated by the HPLC system equipped with a reverse-phase Luna C18 column, and the fractions collected at 1 min intervals were assessed for their ability to induce nodY::lacZ fusion in USDA110 as against the fractions collected from the extracts derived from untransformed plants. Analysis of various HPLC fractions of leaf and root extracts produced conspicuous differences in their abilities to induce nod gene expression in USDA110 (Fig. 5A, B). The ‘leaf fractions’ of D93 obtained at 6, 8, 13, 14, and 21 min significantly stimulated nod gene induction in USDA110 compared with the ‘leaf fractions’ derived from control plants at similar retention times (Fig. 5A). It is interesting to note that a number of fractions from the wild-type R86 leaf extract produced inhibitory effect on nod gene expression, in particular those with 13, 14, and 21 min retention times exerted a significant inhibition compared with comparable fractions obtained from D93 plants. In the case of D93 transgenic plants, however, only one ‘leaf fraction’ that was eluted at the 10 min retention time elicited reduced nod gene expression compared with a comparable fraction from control plants. Overall, the above results indicated that R86 leaves, besides nod gene-inducing compounds, also produce compounds that are inhibitory to nod gene expression in USDA110, and these inhibitory effects, at least partially, could be alleviated by transforming R86 plants with the IFS gene, which perhaps enables the plants to produce new flavonoid compounds that are conducive to nod gene induction.
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In contrast to the ‘leaf fractions’, none of the HPLC fractions derived from the roots of wild-type R86 plants exerted any inhibitory effect on nod gene expression in USDA110 (Fig. 5B). Nonetheless, expression of IFS in R86 roots, by and large, substantially enhanced their ability to promote nod induction. The ‘root fractions’ of D93 obtained at 5, 6, 7, 8, 14, 16, 21, 22, and 24 min retention times significantly stimulated nod gene expression in USDA110 compared with the ‘root fractions’ derived from control plants at similar retention times (Fig. 5B). The only ‘root fraction’ of D93 that produced a marginal, but significant reduction in nod gene expression (compared with the comparable fraction from control plants) was eluted at a retention time of 12 min. All other ‘root fractions’ of D93 collected at the retention times of 9, 10, 11, 13, 17, and 21 min promoted nod gene induction similar to the corresponding fractions derived from the roots of control plants. Taken together these results suggest that R86 roots inherently produce compounds that are modestly stimulatory to nod gene expression in USDA110, and this ability could be improved by transforming R86 plants with IFS.
Identification of genistein in acid hydrolysed methanol extracts of transgenic rice roots expressing IFS, and determination of the ability of the acid hydrolysed-fractions to induce nod gene expression in B. japonicum
A comparison of the HPLC chromatograms obtained with the leaf and root extracts of IFS-transformed plants with those acquired with R86 control rice plants revealed no new peak with a retention time (RF) 7.3–7.4 min corresponding to the genistein standard, in 35S-IFS plants (for example, see HPLC profiles of D93 extracts in Fig. 5, insets). In legumes, free isoflavones rarely accumulate to high levels in plant tissues. Instead they are usually conjugated to carbohydrates (Graham, 1991). This prompted the hypothesis that the genistein produced in 35S-IFS plants probably converted to a glycosylated form, thus evading elution at an expected retention time (similar to the genistein standard) during HPLC fractionation. Therefore, in order to liberate genistein as an aglycone free-form from any possible glycoconjugates, methanol extracts of the roots of D93 and D121 35S-IFS transgenic rice plants were subjected to acid hydrolysis, and analysed for the presence of genistein by HPLC. Upon acid hydrolysis, a peak corresponding to the RF value of the genistein standard could be detected in the samples of the root extracts from D93 and D121 (Fig. 6B, C, arrows). The UV spectra of the eluents of these peaks (in methanol) showed a 
max of 262 nm (Fig. 6B, C, boxed), which coincided with the published absorption maximum of the aglycone genistein standard (Kosslak et al., 1987), thus further showing that the peaks are that of genistein. In contrast to D93 and D121, samples of the acid hydrolysed root extracts from other 35S-IFS transgenic plants failed to produce such a genistein peak. Untransformed control plants never showed any peak corresponding to the peak of the genistein standard (Fig. 6A). The finding obtained with D93 and D121 suggests that rice is able to synthesize genistein upon transformation with the IFS gene, and that the naringenin intermediate of the anthocyanin pathway is available for the introduced foreign enzyme for the production of genistein. Non-detection of the genistein peak in the samples from the rest of the transgenic rice plants may be due to low levels of its synthesis, or rapid conversion of the generated genistein to other forms of flavonoids, leaving no detectable levels of accumulation of this isoflavonoid in the plant tissues. Indeed drastic variations in the production of genistein were also commonly encountered among independent transgenic Arabidopsis lines expressing soybean IFS (Liu et al., 2002).
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To determine the nod gene-inducing abilities of the fractions derived from the acid-hydrolysed methanol extracts, the individual HPLC eluents belonging to D93 and D121 plants were tested on USDA110 (nodY::lacZ). Most of the HPLC fractions obtained from the acid-hydrolysed methanol extracts of roots of D93 and D121 transgenic plants significantly stimulated nod gene induction in USDA110, compared with the comparable root fractions derived from control plants at similar retention times (Fig. 6D). It is noteworthy that the fractions from the D93 and D121 root extracts that eluted around the retention time of 7 min produced about an 8–20-fold increase in ß-galactosidase activity in the test bacterium. Coincidentally, the retention time span covering elution of the above fractions extended over the genistein peak-retention time observed with the acid-hydrolysed D93 and D121 root extracts (Fig. 6B, C).
Isoflavone synthase activity in leaves and roots of the 35S-IFS transgenic rice
Results of the above study showed that the plant extracts derived from both D93 and D121 were able to elicit high nod gene induction in USDA110 (Fig. 4D), and the HPLC profiles of the acid-hydrolysed root extracts from these plants revealed a new peak corresponding to that of the isoflavone genistein (Fig. 6B, C), an expected product of isoflavone synthase activity in plant tissues. In order to verify the ability of 35S-IFS plants to produce isoflavones, an in vitro enzyme activity assay was performed utilizing the protein fractions derived from both leaves and roots of D93 plants to evaluate the isoflavone synthase enzyme functioning in transgenic tissues. Isoflavone synthase activities of soluble and membrane protein fractions were assayed for their ability to convert either naringenin to genistein or liquiritigenin to daidzein, in the presence of the NADPH cofactor, and the methanol extracts of the reaction mixtures were analysed for any genistein or daidzein formation by HPLC. Figures 7 and 8 show the HPLC profiles of the products derived after in vitro isoflavone synthase assay reactions with the leaf and root protein fractions of D93 transgenic plants compared with untransformed control plants. The enzyme assays with supernatant protein fractions from both roots and leaves of D93 plants did not produce any new compounds (data not shown) indicating the absence of isoflavone synthase activity in these protein fractions. This result was anticipated as isoflavone synthase is a membrane bound protein (Yu et al., 2000). By contrast, the crude homogenate protein fraction (which contained both soluble and membrane components) from leaves and the partially purified microsomal protein fraction (pellet) from roots of D93 plant produced new compounds (as revealed by new peaks in the HPLC profiles) upon incubation in a reaction mix containing naringenin or liquiritigenin together with NADPH (Figs 7E, F, 8B, arrows). RF values of the peaks of the new products corresponded to the RF values of the genistein (7.3 min; upon incubation with the naringenin substrate, see Fig. 7E, arrow) and daidzein (6.3 min; upon incubation with the liquiritigenin substrate; see Figs 7F, 8B, arrows) standards. Untransformed control plants never showed any peaks corresponding to the peaks of the genistein and daidzein standards (Figs 7B, C, 8A). Results obtained with this study showed that D93 plants are able to produce active isoflavone synthase that is able to convert naringenin to genistein (Fig. 7E, arrow) and liquiritigenin to daidzein (Figs 7F, 8B, arrows).
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Discussion |
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In legumes, the molecular dialogue between symbiotic partners is triggered when root-secreted plant flavonoids act as transcriptional activators of the Rhizobium nodulation (nod) genes. The nodD gene product of rhizobia physically binds to flavonoids, and initiates the induction of nodulation genes leading to the formation of nitrogen-fixing nodules (Broughton et al., 2000; Spaink, 2000). Isoflavones, a class of flavonoids, are derived from the flavonone intermediate naringenin that is universally present in plants. This reaction is carried out by the enzyme, isoflavone synthase (Kochs and Grisebach, 1986) (Fig. 1). cDNAs encoding isoflavone synthase had been cloned from soybean and other legume species (Akashi et al., 1999; Steele et al., 1999; Jung et al., 2000). Soybean isoflavone synthase has been expressed in non-legumes such as Arabidopsis thaliana and tobacco (Nicotiana tobaccum), and the maize BMC cell line, leading to the formation of the isoflavonoid genistein (Jung et al., 2000; Yu et al., 2000; Liu et al., 2002).
Cereal crops such as rice do not naturally produce isoflavones. Synthesis of isoflavones in this crop may enhance its nutritional value. In addition, isoflavone production may pave the way for rice plants to enter into a symbiotic relationship with rhizobia. Indeed, there has recently been much interest in elucidating the potential to transfer the symbiotic nitrogen-fixing ability to rice in an effort to reduce chemical nitrogen fertilizer input in rice farming (Reddy et al., 2002; Ladha and Reddy, 2003). Because the secretion of flavonoids is a first step in the legume–Rhizobium interactions, and rice lacks the pathway to form isoflavones from flavanones such as naringenin (Fig. 1), transgenic R86 rice plants that express the soybean isoflavone synthase gene under the control of the 35S promoter were generated using Agrobacterium-mediated transformation, and tested for their ability to produce the isoflavone genistein, and to induce nod gene expression in rhizobia. Isoflavone synthase activity assays and HPLC analysis of the acid-hydrolysed tissue extracts performed with D93 and D121 35S-IFS transgenic rice plants showed that the soybean IFS gene-expressed enzyme is active in R86 and enables the synthesis of the isoflavone genistein in plant tissues. These results suggested that, in rice cells, the naringenin intermediate in anthocyanin synthesis could be captured by isoflavone synthase for genistein synthesis.
Except in D93 and D121 plants, HPLC analyses of the acid-hydrolysed extracts prepared from root tissues of other transgenic lines did not reveal any peak corresponding to the genistein standard. Failure to detect genistein with acid-hydrolysed extracts from some transgenic lines may be due to its low level of production, or due to preferential channelling of naringenin to flavonol biosynthesis, leaving no detectable levels of accumulation of this isoflavonoid in the plant tissues. In an analogous study with IFS-transformed tobacco, the isoflavone genistein was also not detected in acid-hydrolysed extracts, and the more sensitive GC-MS assay was necessary to detect the small amount of genistein produced (Yu et al., 2000). Based on these findings, Yu et al. (2000) speculated that the low level of genistein in tobacco tissues could be due to the low expression of the transgene, or to the unavailability of the naringenin substrate due to inactivity of the flavonoid pathway.
In soybean, isoflavones usually exist as glucosyl and malonyl-glucosyl conjugates (Graham, 1991). In the case of the 35S-IFS transgenic rice plants, the genistein peak could be detected by HPLC analysis only when the isoflavone was liberated as a free aglycone by acid hydrolysis, indicating that genistein produced in rice tissues is converted to a glycosylated form. The presence of genistein in a conjugated form in rice tissues that do not naturally synthesize isoflavones indicates that endogenous conjugation enzymes are able to use this novel compound as a substrate. In the IFS-transformed non-legume dicot plants such as Arabidopsis and tobacco and in the monocot maize BMS cell line, genistein was always found to be present in conjugated forms (Yu et al., 2000; Liu et al., 2002). Since malonyl conjugates of anthocyanins are commonly found in many non-legume dicots and malonyltransferase activities have been studied in flowers of several plants (Yamaguchi et al., 1999), Yu et al. (2000) suggested that these types of activities may be responsible for producing the conjugates in tissues that do not naturally encounter genistein.
Analysis of the 35S-IFS plants for their ability to induce nod gene expression revealed that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to modulate nod gene expression, albeit to varied degrees, in different rhizobia. An interesting observation of the study is that both leaf and root extracts derived from almost all 35S-IFS transgenic lines were able to stimulate nod gene expression in USDA110 (Fig. 4D). This observation is significant because nod gene expression in USDA110 is maximally induced by isoflavones, such as genistein (Table 3), which IFS-transformed rice is able to synthesize due to isoflavone synthase activity (see above; also Fig. 6B, C). These studies also showed that leaf extracts from most of the 35S-IFS rice plants could elicit enhanced nod gene expression in other rhizobia such as NGR234, ORS571, and Rm1021. These results indicated that the expression of isoflavone synthase enables rice to synthesize new compounds that are able to promote nod gene induction in rhizobia. It will be interesting to ascertain the identity of the newly synthesized compounds, and to incorporate appropriate genetic amendments in rice in order to encourage the synthesis of these nod gene-inducing compounds in roots, a suitable site for rhizobial interaction. It is known that the expression of genes encoding certain transcription factors such as C1 myb-type transcription factor of maize and the AmMyb305 of Antirrhinum majus can regulate the induction of various genes that encode enzymes of the phenylpropanoid pathway (Grotewold et al., 1998; Sablowski et al., 1994). Transcription factors such as these may be expressed in host plant cells to activate the expression of genes in the phenylpropanoid pathway, thereby increasing the encoded enzyme activities and the flux of compounds in the desired plant tissues (Jung et al., 2003).
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We gratefully acknowledge the following scientists for kindly providing the seeds, bacterial strains and IFS cDNA: WJ Broughton (Université de Genève, Geneva, Switzerland) for Rhizobium NGR234 (pA27); RE Dewey (North Carolina State University, Raleigh, NC, USA) for cyp93C1 cDNA (pCR2.1/cyp93C1v1); M Holsters (Universiteit Gent, Gent, Belgium) for A. caulinodans ORS571 (T20); H Kouchi (National Institute of Agrobiological Resources, Tsukuba, Japan) for pMSH2 and A. tumefaciens EHA105; SR Long (Stanford University, Stanford, CA, USA) for R. meliloti 1021 (pRM57); G Stacey (University of Missouri, Columbia, MO, USA) for B. japonicum USDA110 (Z977) and Murasaki R86 rice seeds. We thank Rowena Hernandez-Oane for help with microscopy, and John Bennett for permitting the use of a transgenic containment greenhouse. This research was supported by the grants from BMZ/GTZ, Germany and the Japanese Government to the International Rice Research Institute.
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* These authors contributed equally to this work.
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