Journal of Experimental Botany, Vol. 52, No. 360, pp. 1537-1543,
July 1, 2001
© 2001 Oxford University Press
Original Papers |
Specific flavonoids induced nod gene expression and pre-activated nod genes of Rhizobium leguminosarum increased pea (Pisum sativum L.) and lentil (Lens culinaris L.) nodulation in controlled growth chamber environments
BIOS Agriculture Inc. 21,111 Lakeshore Road, Ste Anne de Bellevue, Quebec, Canada H9X 3V9
Received 26 February 2001; Accepted 7 March 2001
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Abstract |
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The gram-negative soil bacteria Rhizobium spp. infect and establish a nitrogen-fixing symbiosis with legume crops which involves the mutual exchange of diffusable signal molecules. In this study, Rhizobium leguminosarum containing a nod-lacZ gene fusion was used to screen the most effective plant-to-bacteria signal molecules for pea and lentil and the induction conditions. Out of a number of signal compounds including apigenin, daidzein, genistein, hesperetin, kaempferol, luteolin, naringenin, and rutin, hesperetin and naringenin were found to be the most effective plant-to-bacteria signal molecules. The induction of nod genes was temperature-dependent, where nod gene induction was decreased with dropping incubation temperature. The combination of hesperetin at 7 µM and naringenin at 3 µM resulted in better induction of nod gene activities compared to either hesperetin or naringenin alone. Nodulation and plant dry matter accumulation of pea and lentil plants receiving preinduced R. leguminosarum were higher than those of plants receiving uninduced R. leguminosarum cells in controlled environment growth chamber conditions. Preinduced Rhizobium with hesperetin at a concentration of 10 µM increased nodule number on average by 60.5% and dry matter accumulation by 14% in field pea at 17 °C, while it was 32% and 9% at 24 °C, respectively. Similarly, averaged over two rhizobial strains, a 59% and 6% increase in nodule number and biomass production at 17 °C, and a 39% and 27% at 24 °C, were obtained from lentil inoculated with hesperetin-induced R. leguminosarum, respectively.
Key words: Flavonoid, nod gene, nodulation, Rhizobium leguminosarum.
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Introduction |
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Strains of the soil bacteria Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium, and Azorhizobium, collectively called rhizobia, can infect plants, leading to a symbiotic interaction resulting in root nodule formation. Within these nodules, bacteria live in a differentiated form, the bacteroid, and fix nitrogen by reducing atmospheric nitrogen to ammonia. Rhizobium leguminosarum forms nitrogen-fixing root nodules on plants of pea (Pisum sativum L.) and lentil (Lens culinaris L.). Nodulation is a process of complex interactions between the partners. First of all, the molecular mechanisms of recognition between the host plant and the bacteria symbionts are considered as a form of cell-to-cell interorganismal communication. A precise exchange of molecular signals between the host plant and rhizobia over space and time is essential for the development of effective root nodules. The first apparent exchange of signals is involved in the secretion of phenolic compounds, flavonoid and/or isoflavonoid, by host plants (Peters and Verma, 1990
). These specific plant exudates activate the nod gene expression of rhizobia mediated by the nodD regulatory gene product (Peters et al., 1986). The capacity of a flavonoid to interact with a nodD gene product is strongly affected by its molecular structure. Specific flavonoid molecules such as naringenin and hesperetin are normally present in the rhizosphere of pea and lentil, and induce nod gene expression of R. leguminosarum. As a result of nod gene induction, a lipochitin oligosaccharide (Nod factor) is produced by the bacterial symbiont which, in turn, elicits root hair deformation and cortical cell division in the plant root, the early steps in nodule formation (Sanjuan et al., 1992).
Plant nodulation and nitrogen fixation processes in nature are affected by the micro-ecology of the plant rhizosphere. Soil temperature, pH, texture, moisture, salinity, and deficiencies in essential elements inhibit all stages of symbiotic establishment investigated to date (root hair curling, infection thread formation and penetration, nodule formation and function) (reviewed by Zahran, 1999). The infection and early nodule development processes are most sensitive to stressful environmental conditions. Although combinations of rhizobia and plants may be compatible, nodulation failure can still occur in the field (Robson and Bottomley, 1991). The exudation of flavonoid compounds from clover roots required for nod gene induction in R. leguminosarum bv. trifolii was reduced when the plants were grown at a pH<5 (Richardson et al., 1988). The presence of nitrogen in the root rhizosphere also limits the nodulation of legumes (Streeter, 1988), while nitrogen (as ammonia) has been shown to limit the induction of the nodABC genes (Dusha et al., 1989). In the case of soybean, the time between inoculation and onset of nitrogen fixation is delayed by 2–3 d for each degree decrease in temperature from 25 °C to 17.5 °C. However, when the root zone temperature drops below 17.5 °C, the onset of nitrogen fixation is sharply delayed by 7 d for each degree decrease (Zhang et al., 1995). Low temperature was also found to decrease both the biosynthesis of isoflavonoids and the excretion of these signal compounds from plant root cells to the rhizosphere (Zhang et al., 1995).
Recently, it was reported that inoculating soybean with preinduced B. japonicum improved soybean nodulation and shortened the time between inoculation and the onset of nitrogen fixation under low root zone temperature conditions (US Patent 5922316) (Smith and Zhang, 1999). However, to date, there have been no investigations of the sensitivity of R. leguminosarum to plant-to-bacteria signal molecules, and whether pretreatment of R. leguminosarum could increase pea and lentil nodulation and nitrogen fixation under different temperatures. Therefore, in this study, (1) the best way to induce nod gene activity of R. leguminosarum, such as most effective signal molecules and its molar concentrations, was monitored and (2) the responses of pea and lentil to preinduced R. leguminosarum cells under two growth temperatures in controlled growth chamber conditions were determined.
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Materials and methods |
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Bacterial strains and growth conditions
In this study, R. leguminosarum pIJ1477 with a plasmid carrying a Rhizobium nodC gene fused with the Escherichia coli lacZ gene was obtained from the John Innes Centre, Norwich, UK (Rossen et al., 1985
), and R. leguminosarum bv. trifolii 5280 that lacks Sym plasmid, but contains lacZ gene fused with nodD1 from R. leguminosarum bv. viceae, was obtained from the Institute of Molecular Plant Science, Leiden University, the Netherlands (Spaink et al., 1987). A locally isolated R. leguminosarum BR1 was also used for plant tests. All the strains were routinely maintained on TY medium.
Bioassay for nod gene-inducing activity
Rhizobium leguminosarum cells were grown at 28 °C on solid YEM medium (Hooykaas et al., 1977). For stable maintenance of the recombinant plasmids, the medium was supplemented accordingly with streptomycin (400 µg ml-1), chloramphenicol (10 µg ml-1) and tetracycline (2 µg ml-1). After growth for 48 h, the plates were stored for a period of 7 d at 4 °C. Fresh cells grown in 5 ml of TY medium with 5% inoculum at 28 °C on a rotary shaker at 180 rpm were used for ß-galactosidase assay. Unless otherwise stated, cultures were induced at the beginning of inoculation, and units of ß-galactosidase were determined as described previously (Miller, 1972). Duplicate samples were diluted in 1 : 9 Z-buffer and the turbidity was measured at 600 nm using a spectrophotometer. Cells were lysed with chloroform/SDS and 1 ml aliquots were assayed at 28 °C for ß-galactosidase using o-nitrophenyl-galactopyranoside (ONPG) as described earlier (Miller, 1972). Before measuring the optical density at 420 nm, the samples were cleared by centrifugation. In the nodC : lacZ assays, control bacteria that had not been exposed to flavonoids were measured against the standard buffer solution.
The origins of the chemicals naringenin, hesperetin, apigenin, luteolin, rutin, kaempferol, genistein, and daidzein that were tested for the nod gene inducing ability were obtained from the Sigma-Aldrich Co., Sigma-Aldrich Canada, Ltd.
nod gene expression test
All the nod gene expression tests were conducted in Trypton Yeast (TY) extract medium and repeated for three times.
Experiment 1: Determination of the best plant to microbial signal compounds to induce R. leguminosarum nod genes: R. leguminosarum pIJ1477 and RBL5280 were grown in the presence of eight different signal compounds including apigenin, daidzein, genistein, hesperetin, kaempferol, luteolin, naringenin, and rutin at 5 µM concentration. ß-galactosidase activities were determined after 24 h of induction.
Experiment 2: Determination of the optimum concentrations of signal compounds to induce R. leguminosarum nod genes: Rhizobium leguminosarum pIJ1477 was grown in the presence of apigenin, hesperetin, luteolin, and naringenin at five different concentrations of 0, 5, 10, 15, and 20 µM at 28 °C. ß-galactosidase activity was determined after 24 h of induction.
Experiment 3: Determination of nod gene induction with combinations of hesperetin and naringenin: nod gene activities of R. leguminosarum pIJ1477 in the presence of a mixture of hesperetin and naringenin were tested at the molar concentrations of 10/10, 5/5, 7/3, 1/9, 3/7, and 9/1. Hesperetin and naringenin alone at 10 µM were used as controls. ß-galactosidase activity was determined after 24 h of induction.
Experiment 4: Determination of the effect of induction time on nod gene activity: Ten µM of either hesperetin or naringenin were added into R. leguminosarum pIJ1477 culture broth at 0, 7, and 16 h after inoculation, and associated ß-galactosidase activities were determined.
Experiment 5: Determination of the effects of incubation temperature on nod gene induction: R. leguminosarum pIJ1477 was induced by 10 µM of apigenin, luteolin, naringenin, and hesperetin at 15 °C and 28 °C. Cell growth and ß-galactosidase activities were determined at different time intervals.
Plant nodulation and dry matter accumulation test
Plant nodulation tests were performed in a controlled environment growth chamber. Seeds of pea and lentil were surface-sterilized by immersion in 95% ethanol for 5 min followed by rinsing in sterile water and then immersion in 5% commercial bleach for 20 min. To remove the bleach, at least five washes with sterile water were carried out. The seeds were allowed to imbibe for 4 h prior to sowing.
The nodulation test with lentil at 17 °C was carried out in test tubes (200x25 mm) on modified Hoagland's agar (Hoagland and Arnon, 1950). Surface-sterilized seeds were germinated in Petri dishes containing 1.5% agar at room temperature. After germination, two seedlings were transferred onto slides of agar in each tube that was placed in the growth chamber (Model GB48, Controlled Environments Ltd., Winnipeg, Manitoba, Canada) at an irradiance of 300 µmol m-2 s-1 for a 16 : 8 h (day : night) period and with temperatures held constant at 17 °C. After another 2 d of growth, plants were inoculated with the test strains.
The nodulation test with pea at 17 °C and 24 °C and lentil at 24 °C were conducted in 5 inch pots containing sand and turface at a 1 : 1 (v/v) ratio in a growth chamber (Model GB48, Controlled Environments Ltd., Winnipeg, Manitoba, Canada) at an irradiance of 300 µmol m-2 s-1 for a 16 : 8 h (day : night) period and with temperatures held constant at their corresponding treatment temperatures. Six surface-sterilized seeds were sown in each pot and germinated at 22 °C. The seedlings were later thinned to two plants per pot. As soon as the seedlings had been thinned, the pots were transferred into different temperature conditions of 24 °C and 17 °C. After 24 h of acclimation, 1 ml of treated R. leguminosarum at a cell density of 1.0x109 cells ml-1 was applied into the plant rhizosphere. Inocula were prepared by growing R. leguminosarum pIJ1477 and BR1 (native strain isolated from soil) in TY medium with 0 and 10 µM of hesperetin. Cultures were grown overnight and were centrifuged and resuspended into 0.5% saline solution at a concentration of 1.0x109 cells ml-1. Plants were supplied with a modified Hoagland's solution (Hoagland and Arnon, 1950) in which the CaNO3 and KNO3 were replaced with 1 mM CaCl2, 1 mM K2HPO4 and 1 mM KH2PO4, to provide a nitrogen-free solution, once a week. The experiment was carried out in four replicates.
Plants were harvested for nodule counts and dry mass measurement at 6 weeks after transplanting. Plant dry matter was determined by drying the plants at 80 °C for 48 h.
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Results |
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The best plant-to-microbial signal compounds to induce R. leguminosarum nod genes
The flavanones, hesperetin and naringenin and the flavones apigenin and luteolin appeared to be the active inducers and their induction abilities varied with the types of signal compounds (Fig. 1
). The flavanol, kaempferol, had very little induction ability for R. leguminosarum 5280. All the other compounds tested in this study were found to be inactive for both strains of R. leguminosarum pIJ1477 and 5280. Among the signal compounds, maximal induction of the nod gene was shown by R. leguminosarum pIJ1477 in the presence of hesperetin, corresponding to 9560 units of ß-galactosidase activity, while R. leguminosarum 5280 showed maximum activity of 4369 units in the presence of apigenin in the medium. The next most effective inducers for R. leguminosarum pIJ1477 and 5280 were naringenin and luteolin, respectively, with corresponding ß-galactosidase activities of 4939 and 4092 units, respectively (Fig. 1). The results indicated that the most effective signal compound was strain dependent.
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The optimum concentrations of inducers to induce R. leguminosarum nod genes
The optimum concentration for maximum expression of nod genes was found to vary with signal compounds. The ß-galactosidase activity increased linearly with increasing concentrations of hesperetin, apigenin and naringenin, and reached their maximum level at a concentration of 10–15 µM (Fig. 2). Maximum induction with luteolin was obtained at 20 µM.
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), luteolin (•), hesperetin (
), and naringenin (
) on the induction of nodC-lacZ fusion containing R. leguminosarum pIJ1477. ß-galactosidase activity was determined after 24 h of induction and each mean and its experimental error had three replications.
nod gene induction with combinations of hesperetin and naringenin
Hesperetin at 7 µM plus naringenin at 3 µM increased ß-galactosidase activity significantly over hesperetin or naringenin alone or over equimolar applications of these inducers. The effectiveness of naringenin increased when it was combined with either low or high concentrations of hesperetin, whereas effectiveness of hesperetin did not increase except when combined with 3 µM of naringenin (Fig. 3).
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Effects of induction time on nod gene activity
A similar pattern of nod gene induction was observed with both hesperetin and naringenin. Higher ß-galactosidase activity was obtained when inducers were added at 7 h of growth compared to those added at 0 and 16 h, where it reached the statistical difference with hesperetin, but not with naringenin. ß-galactosidase activity at 0 and 7 h was found to be significantly higher than that added at 16 h of growth (Fig. 4). Maximum ß-galactosidase activity for all the treatments was obtained at 24 h after induction, and afterwards the activity decreased.
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) additions. Each mean and its experimental error had three replications.
Effects of incubation temperature on nod gene induction
nod gene induction of Rhizobium was significantly affected by incubation temperatures. Averaged over four signal molecules, lower levels of gene expression were observed at 15 °C than at 28 °C. The effect was more pronounced with hesperetin compared to other inducers, whereas the least effect was found with naringenin. Induction with hesperetin in terms of ß-galactosidase activity at 15 °C was only half of that observed at 28 °C; however, the level of induction was still comparable to that obtained in the presence of naringenin, apigenin and luteolin at 28 °C (Fig. 5).
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Cell growth in the presence of 10 µM hesperetin showed lower growth rates at a low incubation temperature of 15 °C compared to 28 °C. Maximum growth at low temperature was found at 60 h of incubation, while at 28 °C it was observed at 24 h (Fig. 6
). This suggests that the lower expression at low temperature might result from slow cell growth under the lower incubation temperature, therefore a longer induction period was needed to reach maximum expression. When R. leguminosarum pIJ1477 cells were induced with different concentrations of hesperetin at 28 °C, the highest expression in terms of ß-galactosidase was 9000 units at 10 µM and at 24 h of induction. However, the maximum ß-galactosidase activity (6000) was obtained at 120 h at 15 °C in the presence of 15 µM hesperetin (data was not shown).
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Plant responses to induced and uninduced rhizobial cells
A significant difference in nodule number was found on both pea and lentil plants that received preinduced or uninduced R. leguminosarum cells and these differences were reflected in shoot dry weight. Depending on the strains used, pea plants inoculated with induced cells and grown at 17 °C showed about 47–75% increased nodulation and 9–18% increased biomass production compared to the plants inoculated with uninduced cells (Table 1). Similarly, plants grown at 24 °C showed a 28–35% increase in nodulation and a 3–16% increase in biomass production. Thus the results indicated that the responses of nodulation and biomass production towards inoculation of plants with preinduced R. leguminosarum were higher when plants grown at low temperature (Table 1).
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Lentil plants also revealed increased nodulation and biomass production with induced cell inoculation and responses varied with the growth temperature. A 52–67% increase in nodulation with a 4–7% increase in biomass production was observed when plants were grown at 17 °C in test tubes. Plants grown at 24 °C in pots showed a 23–54% increase in nodulation and 15–39% increased biomass production (Table 2
).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Using the nodC promoter of R. leguminosarum in an expression vector (Rossen et al., 1985
), the behaviour of some inducing substances to design products for pea and lentil was initially monitored. Of the large number of commercially available flavanones, flavones, isoflavones and other related compounds that were tested, naringenin and hesperetin and, to a lesser extent, apigenin and luteolin, were found to be very powerful inducers of the nodC promoter of R. leguminosarum (Fig. 1
). R. leguminosarum pIJ1477 seems to be able to distinguish among flavonoids and isoflavonoids, so that genistein and daidzein were not able to induce nod genes in R. leguminosarum. In contrast, R. leguminosarum bv. phaseoli did not have that capacity and was induced by genistein and daidzein (Hungria et al., 1992; Dakora et al., 1993). Structural differences in nod gene inducers may be related to their capacity to inhibit instead of induce nod gene expression in different rhizobia. For example, genistein inhibited nod gene induction in R. leguminosarum bv. viceae and trifolii (Firmin et al., 1986) and naringenin inhibited nod gene induction in S. meliloti (Peters and Long, 1988) and some B. japonicum strains (Kosslak et al., 1990). Flavonols and isoflavonols were ineffective for R. leguminosarum (Fig. 1); moreover, some isoflavones were even reported to inhibit induction of R. leguminosarum by pea root exudate (Firmin et al., 1986). As genistein, in contrast to hesperetin or others, was not able to provoke a significant promoter response, it might be that the attachment of the B ring to C-2, as was found in flavones and flavanones, is of crucial importance for induction (Zaat et al., 1989).
It is known that different legume species secrete a number of different inducer compounds. To date there is no clear understanding of how legumes profit by releasing more than one nod gene inducing flavonoid. The presence of more than one nodD gene in S. meliloti or R. leguminosarum (Davis and Johnston, 1990) suggested that various flavonoids released from the host plant might bind different NodD proteins. Common beans released natural nod gene inducers belonging to four different classes of flavonoids (Hungria et al., 1991a, b). Thus R. leguminosarum nod genes may be maximally activated together with a number of different flavonoids in different proportions. This concept supports the findings that R. leguminosarum pIJ1477 showed higher ß-galactosidase activity when both hesperetin and naringenin were present in the culture medium at a ratio of 7 : 3 (Fig. 3).
Nodule formation and biomass production of soybean at suboptimal temperatures were found to be greater when preinduced rhizobial cells were used as inoculant (Smith and Zhang, 1999). Similar results were observed for pea and lentil in this study. However, the extent of increase in nodulation and biomass production over uninduced cell inoculation varied with the rhizobial strains. In terms of temperature, nodulation responses (i.e. difference between nodule and biomass production by preinduced and uninduced cell inoculation) in pea with preinduced cells were higher at the lower temperature of 17 °C than at the optimal temperature of 24 °C. It was demonstrated that nodule occupancy by different rhizobial strains in some cases was determined by certain environmental changes (McKay and Djordjevic, 1993). In addition to the reduction of nodulation at temperature extremes, there were also specific temperature-sensitive legume–Rhizobium combinations as was found for R. leguminosarum bv. trifolii TA1, which formed nodules with T. subterraneum cv. Woogenellup above 25 °C but not below 22 °C, although it nodulated a range of other cultivars at the lower temperatures (Lews-Henderson and Djordjevic, 1991). The ability of different strains to produce and release Nod metabolites was likely to be a major determinant of nodule occupancy. Hence screening of strains suitable for low temperature Rhizobium–legume combinations was necessary and appropriate strains need to be determined for Canadian environments.
In summary, results from this study clearly proved that (1) hesperetin and naringenin were the most effective plant-to-bacteria signal molecules for R. leguminosarum; (2) the combinations of multiple inducers gave better nod gene induction; (3) the most effective concentrations of tested signal molecules were from 10–20 µM; (4) the best inducing time was 7 h after culturing cells and the maximal inductions were obtained at 24 h after induction; (5) the induction of nod gene activities was temperature dependent; and (6) plants receiving preactivated rhizobial cells had better nodulation and dry matter accumulation under optimal and sub-optimal temperature conditions.
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Acknowledgments |
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We thank Dr A Downie, in the John Innes Centre, Norwich, UK for providing the two strains of R. leguminosarum carrying the plasmids pIJ1477 (nodC-lacZ ) and pIJ1478 (nodD-lacZ ). We thank Dr Ine HM Mulders, at the Institute of Molecular Plant Science, the Netherlands for providing the R. trifolii strain RBL5280 carrying a lacZ fusion with a nodD1 gene from R. leguminosarum bv. viceae, R. trifolii strain RBL5283 carrying a lacZ fusion with a nodD1 gene from R. leguminosarum bv. trifolii, and R. trifolii strain RBL5284 carrying a lacZ fusion with a nodD1 gene from S. meliloti.
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Notes |
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1 To whom correspondence should be addressed. Fax: +1 514 398 7616. E-mail: zhang{at}biosagriculture.com
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References |
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