Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.)

Plant and Soil 257: 125 131, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands. 125 Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.) Samih M Tamimi 1 & Michael P. Timko Department Of Biology, University Of Virginia, Charlottesville, VA 22903, USA. 1 Corresponding author Received 11 November 2002. Accepted in revised form 2 July 2003 Key words: ethylene, nodulation, Phaseolus vulgaris, Rhizobium Abstract The ethylene releasing compound, 2-chloroethylphosphonic acid (ethephon) inhibited nodule development in common bean (Phaseolus vulgaris L.) plants. In contrast, inhibitors of ethylene synthesis or its physiological activity enhanced nodulation. In a co-culture of bean seeds and rhizobia, ethephon inhibited rhizobial growth while inhibitors of ethylene synthesis or action did not influence the growth and proliferation of rhizobia. These data emphasize the role of ethylene as a regulator of nodulation in determinate nodulators and indicate that the ethylene signaling pathway involved in the nodulation process is not limited to the plant host but also involves the bacterial symbiont. Introduction Bacteria of the genus Rhizobium and leguminous plants have the ability to establish a symbiotic association that result in the formation of nitrogen fixing root nodules. Nodule formation involves signal exchange between the bacterial symbionts and their plant host (for review, see Mylona et al., 1995). The gaseous plant hormone ethylene is involved in nodulation (Hirsch and Fang, 1994). However, ethylene effect has been reported to be most common in legumes that form indeterminate nodules. For instance, ethylene has been shown to inhibit nodule development in Pisum sativum (Goodlass and Smith, 1979; Guinel and Soletjes, 1999; Lee and LaRue, 1992), Trifolium repens (Goodlass and Smith, 1979) and Medicago sativa (Peters and Crist-Estes, 1989). Furthermore, the hypernodulating Medicago truncatula mutant, sickle,has been demonstrated to be ethylene insensitive (Penmetsa and Cook, 1997). On the other hand, the nodulation response of determinate nodulators to ethylene is variable and species dependent. For example, ethylene inhibited nodulation in Phaseolus vulgaris (Grobbe- Permanent address: Department of Biology, University of Jordan, Amman-Jordan. E-mail: tamimi@sci.ju.edu.jo laar et al., 1971), Lotus japonicus and Macroptilium atropurpureum (Nukui et al., 2000). but failed to alter nodulation in Glycine max (Hunter, 1993, Schmidt et al., 1999; Sugamuna et al., 1995) although, the number of nodules on soybean cv. Bragg appear to be reduced by ethylene (Caba et al., 1999). With respect to P. vulgaris, however, the effect of ethylene on nodulation has been demonstrated only in excised roots (Grobbelaar et al., 1971) and no data are available on the nodulation response of the intact plant. Since the response of excised roots to ethylene may differ from that of intact plants, the influence of ethylene on the nodulation of P. vulgaris remains questionable and needs to be re-investigated. While the mechanism by which ethylene controls nodulation is not yet fully known, available evidence suggests that exogenous ethylene blocks the invasion of the infection thread in the root cortex of the plant host (Spaink, 1997). However, other possible effects of ethylene especially its effect on the bacterial symbiont, has not been considered. For instance, during nodule development, rhizobial proliferation is stimulated by compounds released from the seed and roots of the host plant (Kato et al., 1997). Since legume root nodulation is dependent on the density of Rhizobium

126 in the root environment (Francoise and Schmidt, 1982; Kucey and Hynes, 1989; Weaver and Frederick, 1974), it is therefore possible that ethylene controls nodulation by limiting rhizobial multiplication. In the present study, our aim was to re-examine the effect of ethylene on the nodulation response of common bean using intact plants and to examine the effect of ethylene on rhizobial proliferation associated with nodulation. To establish whether nodule formation is influenced by ethylene, nodulation experiments were carried out in the presence of an ethylene releasing compound, ethylene synthesis inhibitors or inhibitor of its physiological activity. To avoid possible effects on plant growth, these compounds were applied during seed germination and early seedling development in the presence of the appropriate rhizobial inoculum. To determine the effect of these compounds on rhizobial growth, the number of colony forming units (cfu) in the rhizobial population was monitored in a co-culture of rhizobia and seedlings in the presence of the test compound. Materials and methods Plant growth Seeds of common bean (cultivar Harvester) were surface sterilized in 70% (v/v) ethanol for 30 s, 5% sodium hypochlorite for 3 min and were then washed several times with sterile distilled water. Surface sterilized seeds were grown individually in 150 25-mm glass tubes using the filter paper bridge method. In this method, 16 1-cm filter paper strips were folded to give a bridge with two supporting arms 6 cm in height. The 4-cm span of the bridge was further folded to give a V shaped depression 2 cm in depth. Individual filter paper bridges were mounted, in an upright position, in 150 25-mm tubes. The tubes were filled with 10 ml of Jensen s nitrogen free medium (Vincent, 1970), covered with metal caps and autoclaved prior to use. The surface sterilized seeds were transferred aseptically into the depression of the bridge, about 2-cm above the level of the medium, and were immediately inoculated with Rhizobium for nodulation testing. Nodulation tests Nodulation tests were performed with a strain of Rhizobium that nodulates common bean, previously isolated from the soils of the Jordan valley (Tamimi, 2002). Before inoculation, rhizobia were grown in TYR medium (Tate et al., 1998), centrifuged at low speed and re-suspended in sterile Jensen s medium to afinalod 590 of 0.1 (10 5 to 10 6 cells ml 1 ). One ml of this suspension was then applied to the surface of the seeds and to the medium. To test for the effect of ethylene on nodulation, seeds were grown and inoculated as described above except that tubes were filled with 10 ml of Jensen s medium containing different concentrations of one of the inhibitors of ethylene synthesis L-α-(2-aminoethoxyvinyl) glycine (AVG ) (Peters and Crist-Estes 1989; Yang and Hoffman, 1984; Yu et al., 1979) or aminooxyacetic acid (AOA) (Amrhein and Wenker, 1979), cobalt chloride (an inhibitor of ethylene action) (Beyer, 1976) or 2- chloroethylphosphonic acid (ethephon) (an ethylene releasing compound). Tubes were made air tight by covering caps with a layer of parafilm and the seeds were allowed to grow in darkness (to provide conditions for the interaction between rhizobia and roots that resemble those existing during their natural interaction in soil) for 6 days at 30 C. The 6-day-old seedlings were removed from test tubes, the root system washed in sterile distilled water and the seedlings were then grown individually in sterile plastic pots filled with sterile vermiculite. Seedlings in pots were grown in the greenhouse under 12 14 h normal light at 27 30 to 17 15 C day-night temperature and 70% relative humidity. Pots were arranged on the benches of the greenhouse in a randomized block design with seven replicates of 20 pots per treatment. Four weeks post inoculation, the number of nodules present on the root system of each plant was recorded. Harvested nodules were dried in an oven at 65 C for 3 days and the average nodule dry weight was estimated. Rhizobial proliferation tests To determine the degree of rhizobial proliferation following inoculation, 0.1-mL aliquots of the inoculated Jensen s medium with or without the test compounds were taken immediately after inoculation and thereafter at 20-h intervals. Rhizobial cell number was then determined by the drop plate method (Somasegaran and Hoben, 1994). Data presented are the mean cell number, measured as cfu, from four replicates.

127 Figure 1. The nodulation response of bean plants to inoculation with rhizobia following treatment with various concentrations of aminoethoxyvinylglycine (AVG) (black columns) or Aminooxyacetic acid (AOA) (grey columns). Nodules formed per plant were scored 40 days after inoculation. Data shown are the mean from seven replicates of 20 plants each ± SE. Results Effect of ethylene on nodulation The data presented in Figure 1 showed that nodule development in intact bean plants is stimulated by the inhibitors of ethylene synthesis. Treatment of plants with 25 and 50 µm AVGorAOAresultedinasignificant increase in the number of nodules formed per plant. However, plants treated with 50 µm AVG or AOA showed the largest response and produced 3- and 2-fold more nodules, respectively, than control plants. At 75 µm, AVG caused a slight increase in the number of nodules while AOA did not influence nodulation. At higher concentrations (100 µm), both compounds inhibited nodulation. However, since plant growth was also inhibited by high concentration of AVG and AOA (data not shown), their effect on nodulation appears to be indirect To further demonstrate the role of ethylene in nodule development, the effect of different concentrations of cobalt chloride (an inhibitor of ethylene action) on nodule formation was investigated. The results presented in Figure 2 showed that treatment of plants with cobalt chloride enhanced nodulation. The enhancement of nodule formation by cobalt chloride was the highest at 10 µm. At this concentration the mean Figure 2. The nodulation response of bean plants to inoculation with rhizobia in the presence of various concentration cobalt chloride. Data are the mean from seven replicates of 20 plants each ± SE. number of nodules formed per plant was 92% greater than in the controls. Treatment of plants with 15µM cobalt chloride increased the mean number of nodules by approximately 40% and further increase in the concentration of cobalt chloride inhibited the nodulation response of plants. The nodulation response of bean plants to ethephon, which (at ph 5 and above) spontaneously decomposes into ethylene, is shown in Figure 3. Treatment of plants with ethephon resulted in a significant reduction in the average number of nodules produced per plant. The inhibitory effect of ethephon on nodulation increased with concentration. At 100 µm ethephon the number of nodules formed per plant was reduced by 80% relative to untreated controls and this concentration was selected as the best concentration for further experiments. Nodule size was also influenced by these compounds. The data presented in Figure 4 showed that nodules on plants treated with 50 µm AOA or AVG and with 10 µm cobalt chloride were noticeably larger in terms of their dry weight than nodules on control plants. The increase in nodule size was the largest in plants treated with 50 µm AOA. The average nodule dry weight on these plants was approximately 2-fold

128 Figure 3. The nodulation response of bean plants to inoculation with rhizobia following treatment with various concentration of ethephon. Data are the mean from seven replicates of 20 plants each ± SE. greater than in the controls. The dry weight of nodules on plants treated with 50 µm AVG and 10 µm cobalt chloride increased by 42 and 35% relative to nodules on control plants, respectively. Ethephon at the concentration of 100 µm, however, resulted in the formation of small nodules with an average dry weight 75% less than those produced by control plants. Effect of ethylene on rhizobial proliferation Rhizobial growth in the presence and in the absence of bean seeds and the effect of AVG, AOA, cobalt chloride and ethephon on the growth and proliferation of rhizobia is presented in Figure 5. Figure 5A shows that 20 h after seed inoculation, the number of rhizobia in Jensen s medium increased from 10 5 to 10 7 cells ml 1 and the population reached a stationary phase thereafter. In the absence of seeds, however, there was little change in the number of rhizobia in the medium within 100 h after inoculation. This enhancement of rhizobial growth after seed inoculation was strongly inhibited by ethephon (Figure 5E). In the presence of 100 µm ethephon, rhizobia showed only a small in- Figure 4. Mean dry weight of individual nodules formed on bean plants inoculated with rhizobia Control plants (treatment 1); plants treated for 6 days during seed germination with 50µM AVG, 50µM AOA, 10µM cobalt chloride or 100µM ethephon, (treatments 2, 3, 4 and 5, respectively). Data are the mean from seven replicates of 20 plants each ± SE. crease in growth at 40 and 60 h after seed inoculation. However, ethephon treatment, did not influence the growth of rhizobia when cultured alone, in the absence of seeds (see Figure 5E and A). Treatment of cultures with AVG, AOA or cobalt chloride, on the other hand, had no effect on the growth profiles of rhizobia after seed inoculation (Figure 5B, C and D, respectively). Discussion Previous studies have demonstrated that ethylene inhibits the formation of nodules on legume roots (Goodlass and Smith, 1979; Grobbelaar et al., 1971; Guinel and LaRue, 1991; Heidstra et al., 1997; Ligero et al., 1991; Van Workum et al., 1995; Zaat et al., 1989) although, differences between species (Hunter, 1993; Schmidt et al., 1999; Suganuma et al., 1995) and cultivars within species (Caba et al., 1998; Zie et al., 1996) have been reported. However, Schmidt et al. (1999) suggested that ethylene inhibition of nodulation probably occurs exclusively in the indeterminate nodulators. In the present investigation we showed that

129 Figure 5. (A) Proliferation of rhizobia in Jensen s medium in the presence ( ) and absence ( ) of bean seeds. Proliferation in Jensen s medium supplemented with 50 µm AVG, 50 µm AOA, 10 µm cobalt chloride or 100 µm ethephon, B, C, D and E, respectively. Data are the mean from four replicates ± SE. nodulation in intact bean plant, a determinate nodulator, is very sensitive to ethylene. Treatment of bean seedlings during early stages of nodulation with AVG or AOA, inhibitors of ethylene synthesis, or with cobalt chloride, which interferes with ethylene action, induced a significant increase in the number of nodules formed by the plants. However, application of ethephon (an ethylene-releasing compound) strongly inhibited their nodulation response. These findings are consistent with recent reports indicating that nodulation in determinate nodulators is sensitive to ethylene (Nukui et al., 2000; Xie et al., 1996), and suggest that

130 the lack of response to ethylene in some soybean cultivars (Schmidt et al. 1999) may not reflect the general response of determinate nodulators. In fact the data of Schmidt et al. (1999) suggesting that nodulation in soybean is independent of ethylene signaling is not conclusive, since the evidence they presented was derived from work conducted on an ethylene insensitive soybean mutant. The lack of response to ethylene in these plants may indicate that either ethylene does not affect their nodulation or that the gene mutated does not play a role in ethylene regulation of nodulation. Seed and root exudates of common bean are known to stimulate rhizobial proliferation (Kato et al., 1997). Consequently, one could expect the negative influence of ethephon and the positive effects of AVG, AOA and cobalt chloride on nodulation to be the result of a corresponding effect of these compounds on the growth and persistence of rhizobia. Our data showed that only ethephon affected rhizobial proliferation and reduced its growth response following inoculation. On the contrary, this compound had no influence on the growth behavior of rhizobia when cultured alone, in the absence of seeds. These observations indicated that the effect of ethephon on rhizobial growth is associated with the nodulation process since the growth of rhizobia on yeast extract mannitol agar (YEM) was not affected when the medium was supplemented with100 µm ethephon (data not shown). While it is difficult to explain the inhibitory effect of ethephon on rhizobial proliferation, it is possible that ethylene evolved from this compound may have inhibited the formation or release of certain growth stimulating seed exudates critical for the proliferation of rhizobia. Alternatively, ethylene may have reduced the responsiveness of rhizobia to these exudates. Nevertheless, the data of this investigation suggest that ethylene acts at an early stage in the plant rhizobial interaction by limiting the number of rhizobial cells that persist to colonize mature nodules. These results correlate with the findings of Oldroyd et al. (2001), which suggested that ethylene modulates an early step in the Nod factor signal transduction pathway. It is therefore concluded that the pathway of ethylene action on nodulation is not limited to the host plant but also involves the bacterial symbiont. References Amrhein N and Wenker D 1979. Novel inhibitors of ethylene production in higher plants. Plant Cell Physiol. 20, 1635 1642. Beyer M E 1976 A potent inhibitor of ethylene action in plants. Plant Physiol. 58, 268 271. Caba J M, Recalde L and Ligero F 1998 Nitrate induced ethylene biosynthesis and the control of nodulation in alfalfa. Plant Cell Environ. 21, 87 93. Dennan D S and Norton C 1972 The effect of ethrel on nodulation in Pisum sativum L. Plant Soil 36, 53 57. Fearn J C and LaRue T A 1991 Ethylene inhibitors restore nodulation by Sym 5 mutant of Pisum sativum L. cv. Sparkle. Plant Physiol. 96, 239 244. Francoise M R and Schmidt E L 1982 Population changes and persistence of Rhizobium phaseoli in soil and rhizospheres. Appl. Environ. Microbiol. 35, 661 667. Goodlass G and Smith K A 1979 Effect of ethylene on root extension and nodulation of pea (Pisum sativum L.) and white clover (Trifolium repens L.). Plant Soil 51, 387 395. Grobbelaar N, Clarke B and Hough MC 1971 The nodulation and nitrogen fixation of isolated roots of Phaseolus vulgaris L. Plant Soil (special volume) 215 223. Guinel F C and LaRue T A 1992 Ethylene inhibitors partly restore nodulation to pea mutant E 107 (brz). Plant Physiol. 99, 515 518 Hirsch M and Fang Y 1994 Plant hormones and nodulation: What s the connection? Plant Mol. Biol. 26, 5 9 Heidstra R, Yang W C, Yalcin Y, Peck S, Emons A M, Van Kammen A and Bisseling T 1997 Ethylen provides positional information on cortical cell division but is not involved in Nod factorinduced root hair tip growth in Rhizobium-legume interaction. Development 124, 1781 1787. Hunter W J 1993 Ethylene production by root nodules and effect of ethylene on nodulation in Glycine max. Appl. Environ. Microbiol. 59, 1947 1950. Kato K, Arima Y and Hirata H 1997 Effect of exudate released from seeds and seedling roots of common bean (Phaseolus vulgaris L.) on proliferation of Rhizobium sp. (Phaseolus). Soil Sci Plant Nutr. 43, 275 283. Kucey R M and Hynes M F 1989 Populations of Rhizobium leguminosarum bv. Phaseoli and viciae in fields of bean and pea in rotation with nonlegumes. Can. J. Microbiol. 35, 661 667. Lee K H and La Rue T A 1992 Exogenous ethylene inhibits nodulation of Pisum sativum L. cv Sparkle. Plant Physiol. 100, 1334 1338. Ligero F, Lluch C and Olivares J 1986 Evolution of ethylene from roots of Medicago sativa plants nodulated with Rhizobium meliloti. J. Plant Physiol. 125, 361 365. Ligero F, Caba J M, Lluch C and Olivares J 1991 Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol. 97, 1221 1225. Long S R 1996 Rhizobium symbiosis: Nod factors in prespective. Plant Cell 8, 1885 1898. Mellor R and Collinge D B 1995 A simple model based on known plant defence reaction is sufficient to explain most aspects of nodulation. J. Exp. Bot. 46, 1 18. Mylona P, Pawlowski K and Bisseling T 1995 Symbiotic nitrogen fixation. Plant Cell 7, 869 885 Nukui N, Ezura H, Yuhashi K-I, Yasuta T and Minamisawa K 2000 Effects of ethylene precursor and inhibitors for ethylene biosynthesis and perception on nodulation in Lotus japonicus and Macroptilium atropurpureum. Plant Cell Physiol. 41, 893 897. Oldroyd G E, Engstrom E M and Long S R 2001 Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula. Plant Cell 13, 1835 1849. Penmetsa R V and Cook D R 1997 A legume ethylene-insensitive mutant hyperinfected by its rhizobial symbiont. Science 257, 527 530.

131 Peters N K and Crist-Estes D K 1989 Nodule formation is stimulated by the ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol. 91, 690 693. Schmidt J S, Harper J E, Hoffman T K and Bent A F 1999 Regulation of soybean nodulation independent of ethylene signaling. Plant Physiol. 119, 951 959. Spaink H P 1997 Ethylene as a regulator of Rhizobium infection. Trends Plant Sci. 2, 203 204. Somasegaran P and Hoben H J 1994 Methods in legume Rhizobium Technology. NifTAL, University of Hawaii, Paia, Hawaii. Suganuma N, Yamauchi H and Yamamoto K 1995 Enhanced production of ethylene by soybean roots after inoculation with Bradyrhizobium japonicum. Plant Sci. 111, 163 168. Tamimi S M 2002 Genetic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of common bean (Phaseolus vulgaris L.) grown in the soils of the Jordan valley. Appl. Soil Ecol. 19, 183 190. Tate R, Riccio A, Merrick M and Patriarca E J 1998 The Rhizobium etli amtb gene coding for an NH4+ transporter is down-regulated during bacteroid differentiation. Mol Plant Microbe Interact. 11, 188 198. Van Workum W A, Van Brussel A N, Tak T, Wijffelman C A and Kijne J W 1995. Ethylene prevents nodulation of Vicia sativa sp. nigra by exopolysaccharide-deficient mutant of Rhizobium leguminosarum bv. Viciae. Molec. Plant Microbe Interact. 8, 278 285. VanRhijn P and Vandeleyden J 1995 The Rhizobium Plant symbiosis. Microbiol. Rev. 59, 124 142. Vincent J M 1970 A Manual for the Practical Sstudy of the Root Nodule Bacteria. Blackwell Scientific Publ., Oxford. Weaver R W and Frederick R L 1974 Effect of inoculum rate on competitive nodulation of Glycine max L.Merrill. I: Greenhouse studies. Agron. J. 66, 229 232. Xie Z P, Staehelin C, Wiemken A and Boller T 1996 Ethylene responsiveness of soy bean cultivars characterized by leaf senescence, chitinase induction and nodulation. J. Plant Physiol. 149, 690 694. Yang S F and Hoffmann N E 1984 Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155 189. Yu Y B and Yang S F 1979 Auxin-induced ethylene production and its inhibition by aminoethoxyvinylglycine and cobalt ion. Plant Physiol. 64, 1074 1077. Zaat S A J, Van Brussel A A, Tak T, Lugtenberg B J and Kijne J W 1989 The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar Viciae on Vicia sativa subsp. nigra by supressing the thick and short roots pheotype. Planta 177, 141 150. Section editor: F. R. Minchin