sign in sign up
<<
Home , Hippophae, Rhamnaceae

J. Japan. Soc. Hort. Sci. 76 (3): 185–190. 2007. Available online at www.jstage.jst.go.jp/browse/jjshs JSHS © 2007

Nitrogen Fixation in Seabuckthorn ( rhamnoides L.) Root Nodules and Effect of Nitrate on Nitrogenase Activity

Kazuhisa Kato**, Yoshinori Kanayama*, Wataru Ohkawa and Koki Kanahama

Graduate School of Agricultural Science, Tohoku University, Sendai 981–8555, Japan

Seabuckthorn ( L.) is a small tree belonging to the family . Because seabuckthorn fruit is rich in unsaturated fatty acids and vitamins, this has potential as a food and medicinal crop. Here, we focused on symbiotic that could aid in the cultivation of this . Microscopic observations showed that seabuckthorn root nodules have a standard morphology characteristic of - actinorhizal root nodules. Under nitrogen-free conditions, seabuckthorn seedlings inoculated with a homogenate of root nodules grew normally, and the fresh weight of root nodules was positively correlated with plant growth. In the field, nitrogenase activity in root nodules was high from May to September, when air temperatures were high and photosynthesis was active. Inhibition of nitrogen fixation by nitrate has been well documented in the root nodules of legumes. Therefore, we investigated the effect of nitrate on nitrogenase activity in seabuckthorn root nodules. Nitrogenase activity in seabuckthorn root nodules was not inhibited by the addition of high concentrations (up to 30 mM) of nitrate over a short term (5 days), but was apparently inhibited by long-term (20–30 days) treatments with 5 and 10 mM of nitrate.

Key Words: Frankia, Hippophae rhamnoides L., nitrate, nitrogen fixation, seabuckthorn.

Introduction seabuckthorn (Hippophae rhamnoides L.) of the family Elaeagnaceae is also a small fruit tree that has great Frankia is an actinomycetic, nitrogen-fixing, nodule- potential as a horticultural crop. Seabuckthorn is a hardy, forming endophyte of woody trees and . Symbiotic , dioecious, and spinescent with berries. infected with Frankia are termed actinorhizal The fruit flesh is rich in vitamins A and C, and the flesh plants and are classified into eight families and 25 genera and seeds contain oil rich in vitamin E, which is generally comprising more than 220 species (Wall, 2000). Five of scarce in horticultural . Furthermore, seabuckthorn these families, the Betulaceae, Myricaceae, Coriariaceae, is drought- and cold-tolerant and is a symbiotic fixer of Elaeagnaceae, and Casuarinaceae, are native to Japan. nitrogen. The seabuckthorn subspecies mongolica used Because they can utilize nitrogen gas from the in this study has promising fruit characters and is atmosphere by symbiotic nitrogen fixation, plants of cultivated in Siberia. Seabuckthorn cultivation has these families can be used as pioneers. Although recently extended to Canada and the USA and is newly Myricaceae and Elaeagnaceae plants bear fruits, they are developing in Japan (Kanahama, 2001). not popular as horticultural crops. Symbiotic nitrogen fixation could be important to Recently, small fruit trees like blueberry have become develop seabuckthorn production as a horticultural crop popular because of functional components in their fruit in Japan. However, insufficient physiological research and are studied to improve their productivity (Piller et has been conducted on the symbiosis of seabuckthorn al., 2003; Ye et al., 2005). The and Frankia. Information is lacking on the inhibition of nitrogen fixation by nitrate, which has been intensively studied as an important agricultural problem in legume Received; June 23, 2006. Accepted; December 25, 2006. crops. In this study, we investigated the capacity of This study was partly supported by Research Project for Utilizing symbiotic nitrogen fixation in seabuckthorn root nodules Advanced Technologies in Agriculture, Forestry and Fisheries from and the effects of nitrate on nitrogenase activity. the Ministry of Agriculture, Forestry and Fisheries of Japan. * Corresponding author (E-mail: [email protected]). ** Present address: Horticultural Research Institute, Ibaraki Agricul- tural Center.

185 186 K. Kato, Y. Kanayama, W. Ohkawa and K. Kanahama

Materials and Methods solution once per week. Three months after inoculation, the fresh weights of plants and root nodules were Plant material investigated. Seabuckthorn (Hippophae rhamnoides L. ssp. mongolica) seeds were provided by the Siberian branch Seasonal changes in nitrogenase activity of the Russian Academy of Sciences. We used trees and Root nodules of 4-year-old mature trees were sampled seedlings grown from these seeds as well as rooted every 2 months from November 2003 to November 2004 cuttings from the mature trees. Plants were grown in a at about 14:00 on days when the weather was good. field of the Graduate School of Agricultural Science, Sampling dates are indicated in Figure 4. Nitrogenase Tohoku University, Japan. activity was measured by an acetylene reduction assay (Hardy et al., 1968; Suganuma et al., 2003). Detached Morphology of root nodules and Frankia 16S ribosomal root nodules were placed in 50-mL vials containing 10% DNA sequence acetylene and incubated at 25°C. After 30 min, the Root nodules of rooted cuttings were used for the amount of ethylene produced was determined by gas observation of histological sections. One-year-old chromatography. branches from 4-year-old mature trees were planted as cuttings in vermiculite in April 2003. The rooted cuttings Influence of nitrate treatments on nitrogenase activity were grown in a non-heated glass greenhouse under a Rooted cuttings with root nodules were used. In natural photoperiod and inoculated with Frankia 2 February 2004, 1-year-old branches of 5-year-old mature months later, as follows. Root nodules of 4-year-old trees were planted as cuttings in soil taken from a field mature trees were sampled, and surface-sterilized in (fine-textured yellow soil; Cultivated soil classification 2%(v/v) sodium hypochlorite for 5 min, followed by committee, 1995) and grown in a heated greenhouse rinses in water. The root nodules were crushed with maintained at above 15°C. In May, rooted cuttings with nitrogen-free Evans solution (Huss-Danell, 1978) root nodules were selected, repotted in vermiculite, and containing 1% (v/v) polyvinylpyrrolidone, and the roots grown in a non-heated greenhouse under a natural of rooted cuttings were inoculated with the homogenate. photoperiod. The plants were supplied with nitrogen- After inoculation, the plants were transplanted to pots free Evans solution once per week. In October, the plants containing vermiculite and grown further. The plants were supplied with KNO3 once a day for 5 days (short- were supplied with a nitrogen-free Evans solution once term treatment) or for 10–30 days (long-term treatment). per week. After 3 months, root nodules were sampled Control plants were supplied with KCl. Nitrogenase and fixed in FAA (70% (v/v) ethanol : acetic activity was investigated as described above. = acid : formalin 18:1:1). The nodules were then Results and Discussion dehydrated in a graded butanol series, substituted with xylene, and embedded in histoparaffin (Wako, Osaka, Morphology of root nodules and Frankia 16S rDNA Japan). Microtome sections (10 µm thick) were mounted sequence onto silane-coated glass slides (Dako, Kyoto, Japan). Root nodules with many lobes were found in mature Tissues were deparaffinized with xylene, rehydrated seabuckthorn trees grown in the field (Fig. 1A). For through a graded series of butanol, and stained with detailed investigation, we examined the histological 0.05% (w/v) toluidine blue. sections of a lobe. The diameter of the lobe DNA was extracted from the hyphal clusters of was approximately 1 mm, and the lobe contained a Franakia microsymbionts living in root nodules for the central vascular bundle and infected cells in the cortex sequence analysis of 16S ribosomal DNA (rDNA), as (Fig. 1B, C). These morphological properties were described previously (Benson et al., 1996). Franakia consistent with those of Frankia-actinorhizal root 16S rDNA was amplified by PCR using primers fD1 nodules (Bond, 1976; Wall, 2000). and rDB1 in Clawson et al. (1998). A PCR product was In this study, we used root nodules formed by native purified and sequenced for phylogenetic analysis. Frankia in field of the Graduate School of Agricultural Science, Tohoku University. Phylogenetic analysis was Influence of nodulation on plant growth carried out using 16S rDNA sequences, as described Seeds were peeled from their coats and sterilized with previously (Clawson et al., 1998). The results in Figure 2 2% (v/v) sodium hypochlorite for 5 min, followed by show that Frankia 16S rDNA sequences cluster into four rinses in water. They were germinated on sterile filter host plant-specificity groups (Clawson et al., 1998), and paper and grown at 25°C under an 18-h photoperiod in the Frankia used in this study belongs to the elaeagnus May 2004. After 2 weeks, the seedlings were inoculated group. with Frankia using the homogenate prepared as described above, planted in vermiculite, and grown in a Influence of inoculation on plant growth non-heated greenhouse under a natural photoperiod. The Symbiotic nitrogen-fixing plants can be supplied by seedlings were supplied with a nitrogen-free Evans three different nitrogen sources: symbiotically fixed J. Japan. Soc. Hort. Sci. 76 (3): 185–190. 2007. 187

Fig. 1. Morphology of seabuckthorn root nodules. A root nodule of a 4-year-old mature tree was viewed using a stereomicroscope (A). Longitudinal (B) and transverse (C) sections of a lobe of a root nodule from a rooted cutting stained with 0.05% (w/v) toluidine blue. The bars represent 1 cm (A) and 500 µm (B, C). V: vascular bundle; IC: infected cells.

Fig. 3. Influence of inoculation on plant growth in seabuckthorn. A: Correlations between the fresh weight of root nodules and plant fresh weight. ** Significant at P < 0.01 by t-test. B: Typical examples of inoculated (left) and non-inoculated (right) seedlings are shown. The bar represents 5 cm.

nitrogen, nitrogen from fertilizer, and nitrogen from the soil. In the actinorhizal plant velutinus, Busse (2000) showed that the percentage of nitrogen fixed in root nodules exceeded 80% of the total nitrogen accumulated in the plants. Further, Wall (2000) reported that the annual amount of nitrogen (240–350 kg·ha−1· year−1) fixed in Frankia-actinorhizal root nodules was similar to that in Rhizobium-legume root nodules. These reports suggest that actinorhizal root nodules have a strong ability to fix nitrogen. However, Uemura (1980) Fig. 2. A phylogenetic tree based on the nucleic acid sequences of reported that the amount of nitrogen fixed in actinorhizal Frankia 16S rDNA that are found in the root nodule inhabitants root nodules was very different among species, ranging of Alnus cordata, Alnus rugosa (Betulaceae); Myrica nagi from 200–300 kg·ha−1·year−1. Therefore, the effect of (Myricaceae); Dryas drummondii (Rosaceae); Coriaria arborea (Coriariaceae); Hippophae rhamnoides and Shepherdia inoculation on plant growth was investigated in canadensis (Elaeagnaceae); Talguenea quinquenervia, symbiosis between seabuckthorn and Frankia in this trinervis (); , Purshia triden- study. Figure 3A shows the correlations between the tata (Other). The sequences were chosen from Clawson et al. fresh weight of root nodules and plant growth. A strong (1998) except that of H. rhamnoides FE245 which is the Frankia positive correlation was observed with regard to the fresh strain infecting H. rhamnoides with the highest score in the BLAST search. The sequence from Frankia that infected weight of plants. These findings suggest that the increase seabuckthorn (H. rhamnoides) in this study is underlined. The of nodulation could accelerate plant growth. Seabuck- tree was constructed by the neighbor-joining method after thorn seedlings that were well nodulated showed normal sequence alignment using the ClustalW program. The scale bar growth, even though they were grown in a nitrogen-free corresponds to 0.01 substitutions/site. nutrient solution (Fig. 3B).

Seasonal changes in nitrogenase activity To cultivate seabuckthorn, it is important to determine 188 K. Kato, Y. Kanayama, W. Ohkawa and K. Kanahama the appropriate timing of fertilizer application based on nitrate inhibition (Streeter, 1988; Vessey and Waterer, the seasonal changes in nitrogenase activity. We, 1992). Thus, we first investigated the influence of short- therefore, investigated seasonal changes in nitrogenase term nitrate treatment on nitrogenase activity in activity in mature seabuckthorn trees in the field (Fig. 4). seabuckthorn root nodules (Fig. 5). Nitrogenase activity was low in November when To investigate the influence of short-term treatment yellowing and defoliation began. Nitrogenase activity with comparatively high concentrations of nitrate, rooted was not detected in January when trees had no . cuttings with root nodules were supplied with 10 and The highest activity was shown in May when leaves 30 mM of nitrate for 5 days. As shown in Figure 5, these opened and current shoots grew. Activity gradually treatments did not inhibit nitrogenase activity. In the decreased from July to September when the growth of root nodules of legumes (soybean and cowpea), the current shoots was ending. In the root nodules of nitrogenase activity decreased to approximately 50% Alnus incana (Betulaceae), nitrogenase activity starts to with the addition of 10 and 20 mM of nitrate for 2 and increase when the leaves open in the spring and decreases 3 days, respectively (Kanayama and Yamamoto, 1991; with leaf fall (Huss-Danell et al., 1992). Nitrogenase Kanayama et al., 1990). Compared to legume root activity in legume root nodules depends on temperature nodules, seabuckthorn root nodules are apparently and current photosynthate levels (Guy et al., 1997; Ryle nitrate-tolerant. In legume root nodules, the nitrogenase et al., 1989). The high nitrogenase activity in root nodules is protected from oxygen by an oxygen diffusion barrier between May and September may be related to in the nodule cortex (Minchin, 1997). In addition, appropriate temperature and active photosynthesis in symbiotic haemoglobin (leghaemoglobin) enhances the leaves. oxygen supply to bacteroids in the infected region. The oxygen diffusion barrier and leghaemoglobin are Influence of nitrate treatments on nitrogenase activity probably related to the nitrate inhibition of nitrogenase The positive correlation between the fresh weight of activity in legume root nodules (Kanayama and root nodules and plant growth suggests the enhancement Yamamoto, 1990; Kato et al., 2003; Vessey and Waterer, of plant growth by nodulation. Symbiosis between 1992). On the other hand, in actinorhizal root nodules, seabuckthorn and Frankia likely guarantees normal plant the envelope surrounding Frankia vesicles in infected growth in infertile soil. On the other hand, because plant cells is assumed to retard the diffusion of oxygen into growth does not seem to reach a maximum, even with the nitrogenase-containing vesicle (Huss-Danell, 1997). a high fresh weight of root nodules in Figure 3A, it is Symbiotic haemoglobin was not observed in seabuck- possible that growth may be further accelerated by the thorn root nodules based on the color of the infected application of nitrogen fertilizer. In this case, the region of the root nodules; the pink color of application of nitrogen fertilizer would be necessary for leghaemoglobin was observed in the infected region of high yields. However, previous studies (Arnone et al., legume root nodules. Thus, the different responses in 1994; Bond and Mackintosh, 1975) have reported that nitrogenase activity to nitrate between legumes and nitrogenase activity in actinorhizal root nodules is seabuckthorn are presumably due to differences in the inhibited by nitrate treatments such as in legume root mechanism of oxygen diffusion and the existence of nodules. Nitrate inhibition of nitrogenase activity could symbiotic haemoglobin. hinder high-yield cultivation of seabuckthorn. The above Bond and Mackintosh (1975) reported that nitrogenase previous studies examined low-concentration nitrate activity per plant decreased to 31% in Coriaria arborea treatments for long-term periods of more than one month. root nodules and 61% in seabuckthorn root nodules with In legume root nodules, the mechanism of short-term the long-term (2.5 months) addition of 1.8 mM nitrate. nitrate inhibition, which is induced by comparatively However, because Bond and Mackintosh (1975) showed high concentrations of nitrate, differs from long-term nitrogenase activity on a whole-plant basis, it is unclear

Fig. 5. Influence of short-term nitrate treatment on nitrogenase activity Fig. 4. Seasonal changes in nitrogenase activity measured in the root in seabuckthorn root nodules. Rooted cuttings with root nodules nodules of mature seabuckthorn trees in the field. Vertical bars were supplied once a day with 10 and 30 mM KCl (Control) or indicate SE (n ≥ 3). KNO3 (Nitrate) for 5 days. Vertical bars indicate SE (n = 4). J. Japan. Soc. Hort. Sci. 76 (3): 185–190. 2007. 189 whether the decreases in nitrogenase activity were due characterized the response of nitrogenase activity to to reductions in the fresh weight of root nodules or in nitrate in both short- and long-term treatments. The the activity per unit fresh weight of nodules. Generally, findings will aid in further investigations of the nitrate inhibits nodulation, nodule growth, and nitroge- mechanism of inhibition of nitrogen fixation by nitrate nase in legume root nodules (Streeter, 1988). Gentili and and the establishment of a method of fertilizer application Huss-Danell (2002) reported an inhibitory effect of the in seabuckthorn cultivation. long-term addition of nitrogen fertilizer on nodule Literature Cited number and biomass in seabuckthorn and indicated that whether nitrogen fertilizer also alters nitrogenase activity Arnone, J. A., S. J. Kohls and D. D. Baker. 1994. Nitrate effects requires further investigation. Therefore, in this study, on nodulation and nitrogenase activity of actinorhizal we focused on the nitrate inhibition of nitrogenase Casuarina studied in split-root systems. Soil Biol. Biochem. 26: 599–606. activity per unit fresh weight of nodules. In 5 mM nitrate Benson, D. R., D. W. Stephens, M. L. Clawson and W. B. Silvester. treatment, nitrogenase activity was equivalent to that in 1996. Amplification of 16S rRNA genes from Frankia strains the control 10 days after treatment but then decreased in root nodules of , Coriaria arborea, to 62% and 22% after 20 and 30 days, respectively Coriaria plumose, toumatou, and Purshia tridentata. (Fig. 6A). In 10 mM nitrate treatment, the nitrogenase Appl. Environ. Microbiol. 62: 2904–2909. activity decreased after 10 days and was almost Bond, G. 1976. The results of the IBP survey of root-nodule undetectable after 30 days (Fig. 6B). These results formation in non-leguminous angiosperms. p. 443–474. In: P. S. Nutman (ed.). Symbiotic nitrogen fixation in plants. indicate that nitrogenase in seabuckthorn root nodules Cambridge University Press. Cambridge. is inactivated by the long-term application of nitrate, Bond, G. and A. H. Mackintosh. 1975. Effect of nitrate-nitrogen although nitrogenase is nitrate-tolerant to short-term on the nodule symbioses of Coriaria and Hippophae. Proc. application. In legume root nodules, while short-term Royal Soc. London Series B; Biol. Sci. 190: 199–209. inhibition is associated with a decline in the diffusion Busse, M. D. 2000. Suitability and use of the 15N-isotope dilution of oxygen, as described above, long-term treatment of method to estimate nitrogen fixation by actinorhizal shrubs. nitrate induces premature nodule senescence with Forest Ecol. Manag. 136: 85–95. Clawson, M. L., M. Caru and D. R. Benson. 1998. Diversity of oxidative stress (Matamoros et al., 1999). Further work Frankia strains in root nodules of plants from the families is needed to determine whether a similar phenomenon Elaeagnaceae and Rhamnaceae. Appl. Environ. Microbiol. occurs in seabuckthorn root nodules. 64: 3539–3543. In this study, we showed an efficient use of symbiotic Cultivated soil classification committee. 1995. Classification of nitrogen fixation in seabuckthorn plants and marked cultivated soils in Japan (3rd approximation). Miscellaneous seasonal changes in nitrogenase activity. Besides, we Publication of the National Institute of Agro-Environmental Sciences 17: 79. Gentili, F. and K. Huss-Danell. 2002. Phosphorus modifies the effect of nitrogen on nodulation in split-root systems of Hippophae rhamnoides. New Phytol. 153: 53–61. Guy, S., M. Berger and C. Planchon. 1997. Response temperature in dinitrogen fixing soybeans. Plant Sci. 123: 67–75. Hardy, R. W. F., R. D. Holsten, E. K. Jackson and R. C. Burns. 1968. The acetylene-etylene assay for N2 fixation: Laboratory and field evaluation. Plant Physiol. 43: 1185–1207 Huss-Danell, K. 1978. Nitrogenase activity measurements in intact plants of Alnus incana. Physiol. Plant. 43: 372–376. Huss-Danell, K. 1997. Actinorhizal symbioses and their N2 fixation. New Phytol. 136: 375–405. Huss-Danell, K., P. O. Lundquist and H. Ohlsson. 1992. Nitrogen fixation in a young Alnus incana stand, based on seasonal and diurnal variation in whole plant nitrogenase activity. Can. J. Bot. 70: 1537–1544. Kanahama, K. 2001. Production and utilization of a multipurpose new crop, oblepikha. Agric. Hortic. 76: 469–474 (In Japanese). Kanayama, Y. and Y. Yamamoto. 1990. Inhibition of nitrogen fixation in soybean plants supplied with nitrate. II. Accumulation and properties of nitrosylleghemoglobin in nodules. Plant Cell Physiol. 31: 207–214. Kanayama, Y., I. Watanabe and Y. Yamamoto. 1990. Inhibition of nitrogen fixation in soybean plants supplied with nitrate Fig. 6. Influence of long-term nitrate treatment on nitrogenase activity in seabuckthorn root nodules. Rooted cuttings with root nodules I. Nitrite accumulation and formation of nitrosylleghemoglo- were supplied with 5 mM (A) or 10 mM (B) of KCl (Control) bin in nodules. Plant Cell Physiol. 31: 341–346. or KNO3 (Nitrate) for 10, 20, and 30 days. Vertical bars indicate Kanayama, Y. and Y. Yamamoto. 1991. Formation of SE (n = 4). nitrosylleghemoglobin in nodules of nitrate-treated cowpea 190 K. Kato, Y. Kanayama, W. Ohkawa and K. Kanahama

and pea plants. Plant Cell Physiol. 32: 19–24. Suganuma, N., Y. Nakamura, M. Yamamoto, T. Ohta, H. Koiwa, Kato, K., Y. Okamura, K. Kanahama and Y. Kanayama. 2003. S. Akao and M. Kawaguchi. 2003. The Lotus japonicus Sen1 Nitrate-independent expression of plant nitrate reductase in gene controls rhizobial differentiation into nitrogen-fixing Lotus japonicus root nodules. J. Exp. Bot. 54: 1685–1690. bacteroids in nodules. Mol. Gen. Genom. 269: 312–320. Matamoros, M. A., L. M. Baird, P. R. Escuredo, D. A. Dalton, Streeter, J. G. 1988. Inhibition of legume nodule formation and F. R. Minchin, I. Iturbe-Ormaetxe, M. C. Rubio, J. F. Moran, N2 fixation by nitrate. Crit. Rev. Plant Sci. 7: 1–23. A. J. Gordon and M. Becana. 1999. Stress-induced legume Uemura, S. 1980. Symbiotic nitrogen fixation in non-legume root root nodule senescence. physiological, biochemical, and nodules. p. 222–231 (In Japanese). In M. Nakamura (ed.). structural alterations. Plant Physiol. 121: 97–112. Biological nitrogen fixation. Gakkaishuppan Center, Tokyo. Minchin, F. R. 1997. Regulation of oxygen diffusion in legume Vessey, J. K. and J. Waterer. 1992. In search of the mechanism nodules. Soil Biol. Biochem. 29: 881–888. of nitrate inhibition of nitrogenase activity in legume nodules. Piller, G., M. Fukushima and S. Iwahori. 2003. Growth responses Recent developments. Physiol. Plant. 84: 171–176. of three New Zealand northern highbush blueberry cultivars Wall, L. G. 2000. The actinorhizal symbiosis. J. Plant Growth (Vaccinium corymbosum) to nutrient availability. J. Japan. Reg. 19: 167–182. Soc. Hort. Sci. 72: 13–17. Ye, Y-H., N. Aoki, N. Nonishi and N. Patel. 2005. Patterns of Ryle, G. J., C. E. Powell, M. K. Timbrell and A. J. Gordon. 1989. -bud differentiation, development, and growth habits Effect of temperature on nitrogenase activity in white clover. of several blueberry (Vaccinium spp.) cultivar types grown J. Exp. Bot. 40: 733–739. in Japan and New Zealand. J. Japan. Soc. Hort. Sci. 74: 1–10.