Y. ARIMA, Department of Bioproduction, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaityou, Futyu, Tokyo 183-8509, Japan. Email: AND46529@nifty.com
Results of grafting experiments between super-nodulation (or hyper-nodulation) mutants of soybean and their parents reconfirmed that super nodulation is a shoot-controlled phenomenon, suggesting that a systemic regulatory mechanism acts in soybean plants and a specific nodulation-controlling substance (SNS) is synthesized in the shoot and transported to the roots. To search for the SNS involved in the super-nodulation trait of NOD1-3, a mutant of soybean (Glycine max [L.] Merr. cv. Williams), we adopted a bioassay system using plantlets derived from the first trifoliate leaf of the seedlings; this system enabled us to introduce liquid substances continuously into leaves and to assess their effect on root nodulation. Following the application of leaf extract from Williams82 plants lacking visible root nodules, formation of root nodule meristems in NOD1-3 plantlets was repressed on the sixth day after rhizobial inoculation and the number of visible nodules on the eighth day declined to the same level as that in the Williams82 plantlets. Application of NOD1-3 leaf extract resulted in no significant change in the nodulation of both NOD1-3 and Williams82 plantlets. These results suggested that the SNS is a downregulator of nodulation and is responsible for the wild-type (Williams82) phenotype, and that the super-nodulation phenomenon is caused by a paucity of the SNS. The intensity of the repressive effect of the Williams82 leaf extract was not changed by nodulation of the source plants, thus we conclude that visible nodule formation is not required to induce production of the SNS.
In sustainable agriculture, leguminous plants play an important role because of their ability to fix atmospheric dinitrogen through symbiosis with rhizobia. The timing and intensity of root nodulation are the principal factors that influence nitrogen acquisition in a leguminous symbiotic system. In relation to the regulatory mechanism of root nodulation intensity, it has been reported that the number of primordial root nodules greatly exceeds that of mature nodules (Francisco and Akao 1993), and this suggests that the number of mature soybean nodules is regulated at a later developmental stage rather than at an early stage of nodule formation.
Super-nodulation mutants, which harbor over 5–10-fold the number of root nodules of their wild-type parents, exist in some leguminous plants (Akao and Kouchi 1992; Carroll et al. 1985; Gremaud and Harper 1989; Jacobsen 1984). Regarding the super-nodulation phenomenon, Gresshoff (Caetano-Anolles and Gresshoff 1991; Gresshoff and Delves 1986) proposed an “autoregulation mechanism” that restricts the number of root nodules in wild-type host plants through systemic movement of the key substances; these researchers proposed that the super-nodulation phenomenon develops as a result of a lack of this “autoregulation mechanism”. However, direct evidence supporting this hypothesis is still limited to that presented by Gresshoff et al. (1988), and the identities of the compounds involved remain unknown.
The results of grafting experiments between super-nodulation mutants of soybean and their wild types have revealed that the super-nodulation phenomenon is controlled by the shoot (Delves et al. 1986, 1987a; Francisco and Akao 1993; Francisco and Harper 1995; Lee et al. 1991; Sheng and Harper 1997). This strongly suggests that the nodulation trait of leguminous plants is under the control of a systemic regulatory mechanism and that a specific nodulation-controlling substance (SNS) is synthesized in the shoot and transported to the roots. Based on the results of interspecific grafting experiments between super-nodulation mutants of soybean and Glycine soja (Delves et al. 1987b), mung bean and hyacinth bean (Harper et al. 1997), it has been suggested that the SNS is widely effective among leguminous species from a variety of genera.
In the present study, we used this technique to address two questions concerning the SNS, namely whether the SNS is an upregulator or a downregulator of nodulation and whether rhizobium inoculation is essential for SNS synthesis. A reciprocal grafting experiment was undertaken to address the former question.
Materials and methods
Plant material and preparation of the shoot extracts
Surface-sterilized seeds of the wild-type soybean Glycine max (L.) Merr. cv. Williams82 and its super-nodulation mutant NOD1-3 were sown on a heat-sterilized vermiculite bed, with or without inoculation of Bradyrhizobium japonicum (USDA110), and supplemented with a germfree plant culture solution. Composition of the plant culture solution for the uninoculated plants was as follows: 5 mmol L−1 CaCl2, 2.5 mmol L−1 K2SO4, 2 mmol L−1 MgSO4, 0.034 mmol L−1 ethylenediaminetetraacetic acid-FeNa, 25 μmol L−1 H3BO4, 5 μmol L−1 MnSO4·H2O, 2 μmol L−1 ZnSO4·7H2O, 0.5 μmol L−1 CuSO4·5H2O, 0.014 μmol L−1 (NH4)6 Mo7O24·4H2O, 6 mmol L−1 urea, 0.16 mmol L−1 K2HPO4 and 0.84 mmol L−1 KH2PO4 (pH 5.8). In the case of the inoculated plants, urea was omitted from the solution (N-free culture solution). The plants were grown in a natural light phytotron (day/night temperature = 25/18°C) for 3 weeks.
The shoots were excised 2 cm above the cotyledons and frozen in liquid nitrogen and stored in a refrigerator at −80°C until extraction. The frozen shoots (165 g) were ground into a powder and extracted with 1 L cold distilled water. The plant residue was removed by sequential filtration through a nylon mesh (50 μm pore size), centrifugation for 15 min at 9,000 g and membrane filtration through a mesh (0.45 μm pore size). Ethanol was added to the filtrate to obtain a final concentration of 80% and the precipitate was removed by centrifugal separation (15 min, 9,000 g). The supernatant was concentrated with a rotary evaporator and then centrifugally ultra-filtrated (10,000 MW: Centricon Plus-20; Millipore Corporation, Billerica, MA, USA) to remove substances with high molecular weights. The prepared extracts were diluted with distilled water to produce a concentration approximately one-fifth of that estimated to occur in intact soybean leaves. The diluted extracts were stored at −50°C until use.
Preparation of the plantlets for assessment of SNS activity
Plantlets were prepared from the excised first trifoliate leaves of 3-week-old uninoculated Williams82 and NOD1-3 plants with average fresh weights of 0.48 g and 0.42 g, respectively. Each excised leaf was placed into a small plastic cup filled with heat-sterilized vermiculite holding the germfree and N-free plant culture solution. The cups were kept in a moisture-saturated translucent box at 25°C under fluorescent lamps (light/dark period = 14 h/10 h) for 1 week. The rooting excised leaves (RELs) for Williams82 (REL-W) and NOD1-3 (REL-N) were used for evaluation of SNS activity in the shoot extracts.
Assessment of SNS activity in the shoot extracts
The assay solution was applied to the RELs by excising the central leaflet of the REL at its petiole, and a both-end-open glass tube containing the assay solution or distilled water (control) was brought into contact with the cut surface of the petiolule at one end. Before initiating application of the assay solution, however, the uptake by each REL was checked by feeding distilled water through the glass tube for 2 h. Once the uptake of liquid was established, application of the assay solution was initiated (Fig. 1). The assay solutions (diluted shoot extracts) were continuously applied to the RELs for either 6 or 8 days. The volume of solution taken up by each REL was determined by measuring the reduction in solution volume in the glass tube. Feeding of the assay solutions was carried out in six replicates under sequential light (photon flux density of approximately 140 μmol m−2 s−1) and dark conditions (light/dark = 14 h/10 h) at 25°C. To avoid any microbial effect, the assay solution and glass tube were replaced daily. At 24 h after the introduction of the assay solution, the RELs were inoculated with a suspension of Bradyrhizobium japonicum (USDA110) (108 cells per REL) in the N-free plant culture solution.
At 5 and 7 days after inoculation, the roots of RELs were fixed in FAA solution (70% ethanol : acetic acid : formaldehyde = 90:5:5 v/v/v) and then stained with 0.03% toluidine blue. The stained root nodule primordia number was measured under an optical microscope and the developmental stage was classified as follows: Stage 1, the meristem was formed, but no root cortical swelling was observed; Stage 2, the meristem showed root cortical swelling, but no stricture was observed at the root nodule connection; Stage 3, mature root nodule showing a stricture at the root nodule connection. Stage 3 nodules were first observed on the seventh day after inoculation on each REL root. The SNS activity of the applied shoot extracts was evaluated based on the number of primordial root nodules.
Statistical analysis of the data was carried out using Tukey–Kramer tests as implemented in KyPlot 4.0 (Kyens Laboratory, Tokyo, Japan).
Comparison of root nodule formation in RELs treated with shoot extracts from uninoculated Williams82 and NOD1-3
The effects of the introduction of Williams82 and NOD1-3 shoot extracts for 6 days on the formation of primordial root nodules in RELs are shown in Fig. 2. The number of primordial nodules (Stage 1 and Stage 2) on REL-N roots was reduced by the introduction of the Williams82 extract compared with the introduction of distilled water. The Williams82 extract introduced to REL-N inhibited the developmental process of nodule primordia and reduced the total number of nodule primordia. In REL-W, both the Williams82 and NOD1-3 extracts induced no evident change in the total number of primordial nodules.
The rate of water uptake by REL-W was higher than that by REL-N (Fig. 3). This phenomenon probably resulted from the genetically larger leaflet area of REL-W compared with REL-N (the average [mean ± standard deviation] leaflet areas were: 34.8 ± 3.68 cm2 [REL-W] and 24.8 ± 0.45 cm2 [REL-N], n = 9). The larger leaflet area might result in greater transpiration and water uptake. The total volumes of extracts or distilled water introduced into the RELs during the 6-day treatment are shown in Table 1. There was no significant difference between the intakes of the two extracts, although the intake of distilled water exceeded the intake of the extracts.
Table 1. Total volume (mL per leaf) of solutions taken up by the rooting excised leaves (RELs) treated for 6 days
Data represent the mean values from six replicates. Superscript letters indicate the results of the Tukey–Kramer test. Means followed by different letters are significantly different (P ≤ 0.05).
The effects of the introduction of the Williams82 and NOD1-3 shoot extracts for 8 days on the formation of mature root nodules (Stage 3) are shown in Fig. 4. In REL-N, the introduction of Williams82 extract reduced the nodule number to one-third of that recorded in the distilled water treatment, but the introduction of NOD1-3 extract did not show a significant effect. In REL-W, the formation of Stage 3 nodules was not significantly affected by either extract.
Comparison of root nodule formation in RELs treated with shoot extracts from rhizobium-inoculated and uninoculated plants
To examine whether SNS production is increased by rhizobium inoculation, primordial root nodule numbers were compared between RELs treated for 6 days with shoot extracts prepared from rhizobium-inoculated (with visible nodules) and uninoculated (without nodules) William82 plants. As shown in Fig. 5, the root nodule number of REL-N was clearly reduced by extracts from both inoculated and uninoculated William82, with no significant difference in repressive intensity between the two extracts. Nodule formation of REL-W is shown in Fig. 6. The extracts from both inoculated and uninoculated NOD1-3 showed no significant effect on the primordial root nodule number.
Super-nodulation (or hyper-nodulation) mutants are extremely useful for clarifying the regulatory mechanism determining root nodulation in leguminous plants. To reconfirm the existence of SNS, reported by Gresshoff et al. (1988), we attempted extract-injection methods using a number of injectors. However, it was very hard to inject a reasonable amount of the extracts into the cotyledons or stems of the young soybean plants, and we were unsuccessful in reconfirming the events reported by Gresshoff et al. (1988). Thus, we established a reliable bioassay method for SNS without the use of injectors. In the present study, we applied a unique technique to assess the effects of shoot-derived substances on root nodulation using plantlets prepared from rooting soybean leaves (Yamaya and Arima 2004; Arima et al. 2005).
The first aim of the present study was to evaluate whether SNS is a downregulator or an upregulator of nodulation or, in other words, which of the wild-type (Williams82) or super-nodulation mutant (NOD1-3) genotypes produces SNS. Only the Williams82 shoot extract affected root nodule formation in REL-N (Figs 2,4), and this extract appeared to suppress root nodulation. In these REL-Ns, the number of mature nodules at Stage 3 was more clearly reduced than the number of immature nodules at Stages 1 and 2 (Figs 2,4). This phenomenon coincides with a quantitative change in extract uptake during the assay period, in which a larger amount of the extract was taken up during the first 48 h compared with the following 4 days (Fig. 3). The total urea-N applied over the entire cultivation period was limited to less than approximately 2.5% of the dry weight of the soybean plants harvested for extraction; thus, it is unlikely that excessive nitrogen in the shoot extracts repressed root nodulation. Only shoot extracts prepared from Williams82 showed a repressive effect on root nodulation, although it was assumed that the nitrogen concentration was not distinctively different between the shoot extracts of Williams82 and NOD1-3. These results suggest that the Williams82 shoot extract has a suppressive effect on NOD1-3 nodulation, resulting in a normal nodule number.
The reciprocal grafting experiment showed that the scion’s nodulation phenotype dominated the stock’s phenotype (Fig. 7a). This result is in accordance with previous reports (Francisco and Harper 1995; Sheng and Harper 1997), but we cannot determine from the grafting results whether a gain of activator or a lack of inhibitor is the cause of the super-nodulation phenotype.
In the shoot-extract application experiment, roots of the respective REL should receive both component(s) of the applied shoot extract and substance(s) should be translocated from the leaflets of the REL. When Williams82 extract is applied to REL-N, roots of the REL-N should receive shoot products of both REL-N (NOD1-3) and Williams82. In addition, when NOD1-3 extract is applied to REL-W, roots of the REL-W should receive shoot products of both NOD1-3 and REL-W (Williams82). In these RELs receiving two different types of shoot products, normal nodulation commonly resulted, suggesting that the effect on nodulation of Williams82 shoot products was superior to NOD1-3 shoot products (Fig. 7). Thus, we conclude that a nodulation inhibitor produced in Williams82 shoots is the cause of the extreme difference in nodulation number between Williams82 and NOD1-3. The SNS is a downregulator of nodulation and is produced by the wild-type plant (Williams82). We suggest that a new term, “shoot-synthesized nodulation-restricting substance’ (SNRS), is more appropriate for the nodulation inhibitor than SNS. Our results provide the second report of direct evidence of SNRS, following on from the data reported by Gresshoff et al. (1988). In their experiment, injection of Bragg (wild-type) extract into its super-nodulation mutant (nts382) suppressed their nodulation. In our experiments, owing to the continuous application of the shoot extracts to RELs, very clear statistically significant evidence of SNRS in Williams82 was obtained.
The second objective of the present study was to determine whether rhizobium inoculation is required for SNRS production. The soybean genotypes (Williams82 or NOD1-3), which were used for extraction, were grown in the presence of urea fertilizer and without inoculation by B. japonicum so that the plants lacked visible nodules and nodule meristems. The effect of shoot extracts from rhizobium-inoculated (with visible nodules) and uninoculated (without visible nodule and nodule meristem) William82 plants on the formation of root primordial nodules was almost identical (Fig. 5). Based on our results, it is clear that both rhizobium inoculation and visible nodule formation are not essential for SNRS synthesis. However, we cannot deny the possibility of low level rhizobium infection by contamination in our experiments. Thus, the essentiality of rhizobium infection for SNRS synthesis is still an open question.
Evidence from the present study provides a substantial foothold for future identification of the SNRS produced in shoots of wild-type soybean.
We sincerely thank Dr J. E. Harper for kindly providing us with seeds of the Williams82 and NOD1-3 soybean genotypes.