Terra Latinoamericana E-ISSN: 2395-8030 [email protected] Sociedad Mexicana de la Ciencia del Suelo, A.C. México

Espinosa Victoria, David; Graham, Peter H. Accumulation of coumestrol, , and is not related to crown nodule senescence Terra Latinoamericana, vol. 19, núm. 4, octubre-diciembre, 2001, pp. 345-351 Sociedad Mexicana de la Ciencia del Suelo, A.C. Chapingo, México

Available in: http://www.redalyc.org/articulo.oa?id=57319407

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative TERRA VOLUMEN 19 NUMERO 4, 2001

ACCUMULATION OF COUMESTROL, DAIDZEIN, AND GENISTEIN IS NOT RELATED TO SOYBEAN CROWN NODULE SENESCENCE Acumulación de Coumestrol, Daidzeina y Genisteina no está Relacionada con la Senescencia de los Nódulos de la Corona de la Soya

David Espinosa-Victoria1 and Peter H. Graham2

SUMMARY cases, levels peaked 38 to 45 DAE, with levels of coumestrol and daidzein in 'Hardin' and Greenhouse experiments were performed to 'Hodgson-78' statistically higher than in 'Alpha' and determine if coumestrol, daidzein, and genistein 'Chippewa'. There were no consistent differences in accumulation was related to soybean crown nodule accumulation of genistein. We did not find any senescence. Soybean cultivars exhibiting delayed relation between the accumulation of the three tested ('Hardin' and 'Hodgson-78') and early ('Chippewa' and and the onset of nodule senescence 'Alpha') decrease in nitrogenase activity, as well as the (decrease of nitrogenase activity). We assumed that natural hypernodulating genotype 'Nod 1-3' were used the three tested isoflavonoids would lack antibiotic in the present study. Plastic pots containing a sterile activity since their high accumulation levels did not 1:1 mixture of silica sand and Sunshine mix # 2 affect the bacteroid nitrogenase activity. sterilized by autoclaving 90 min at 121 °C were used as experimental units. Before direct soaking, all pots Index words: Isoflavonoids, PAL (phenylalanine were watered with Bradyrhizobium japonicum strain ammonia lyase), CHS (chalcone synthase). UMR 161, providing an initial population of 105 cells g-1 of mixture. Sufficient experimental units RESUMEN (three replicates cultivar-1 harvest-1) were set up to allow weekly destructive sampling. We emphasized Se realizaron diferentes experimentos en on crown nodules because they are basically induced invernadero para determinar la posible relación entre by inoculant rhizobia and they are uniform in terms of la acumulación de coumestrol, daidzeina y genisteina age. 'Hardin' and 'Hodgson-78' induced more nodules y la senescencia de los nódulos formados en la corona at crown-root zone than 'Chippewa' and 'Alpha'. de la raíz de la soya. Se emplearon variedades de soya Significant differences in fresh weight of nodules con reducción temprana ('Chippewa' y 'Alpha') y between both soybean groups as early as 17 days after tardía ('Hardin' y 'Hodgson-78') de la actividad emergence (DAE) were observed. Crown nodule nitrogenasa, así como el mutante natural mRNA showed a more intense hybridization with supernodulante 'Nod 1-3'. Como unidades PAL (phenylalanine ammonia lyase) rather than CHS experimentales se emplearon macetas de plástico (chalcone synthase) probe. PAL and CHS transcript conteniendo una mezcla 1:1 de arena de sílica con levels were higher in 'Chippewa', 'Alpha' and materia orgánica Sunshine mix # 2, esterilizada a 'Nod 1-3' than in 'Hardin' and 'Hodgson-78' nodules 121 °C durante 90 min. Todas las macetas se throughout the experiment. Levels of daidzein were inocularon mediante el riego con una población inicial higher than coumestrol or genistein in all five de 105 células de Bradyrhizobium japonicum cultivars during the lifespan of the symbiosis. In all UMR 161 g-1 de mezcla antes de la siembra directa. Se instalaron tres repeticiones variedad-1 cosecha-1 para 1 Instituto de Recursos Naturales, Colegio de Postgraduados. realizar muestreos destructivos semanales. Se hizo 56230 Montecillo, Texcoco, México. énfasis en los nódulos de la corona de la raíz debido a ([email protected]) que éstos son básicamente formados por la Benemérita Universidad Autónoma de Puebla. Instituto de Ciencias. Edif. Complejo de Ciencias No. 76, 3er piso, Ciudad inoculada y porque representan la población más Universitaria. 72570 Puebla, Puebla. uniforme en términos de edad. Las variedades 'Hardin' 2University of Minnesota, Department of Soil, Water, and y 'Hodgson-78' indujeron más nódulos en la corona Climate..Saint Paul, MN 55108. que 'Chippewa' y 'Alpha'. Se presentaron diferencias

Recibido: Enero de 2001. Aceptado: Agosto de 2001. significativas en el peso fresco de nódulos entre los Publicado en Terra 19: 345-351. dos grupos de soya a los 17 días después de la

345 TERRA VOLUMEN 19 NUMERO 4, 2001

emergencia (DDE). El ARNm nodular presentó mayor N2 fixation (Pazdernik et al., 1996), as well as intensidad de hibridación con la sonda PAL differences in the onset of crown nodule senescence (fenilalanina amonia liasa) que con la sonda CHS (Espinosa-Victoria et al., 2000). Crown nodules in (chalcona sintasa). Los valores de transcriptos de PAL are predominantly formed by the inoculant y CHS fueron superiores en los nódulos 'Chippewa', strain, and they contribute essentially 100% of the N2 'Alpha' y 'Nod 1-3' que en los de 'Hardin' y fixed during early vegetative growth (McDermott and 'Hodgson-78' durante el experimento. Los valores de Graham, 1989). Senescence of soybean nodules has daidzeina fueron mayores que los de coumestrol y been directly associated with elevated proteolytic genisteina en los cinco cultivares a través del tiempo activity, decreased of soluble protein content, que duró la simbiosis. En todos los casos, los including the disappearance of leghemoglobin (Malik isoflavonoides alcanzaron su máxima acumulación et al., 1981; Pfeiffer et al., 1983a,b; Espinosa-Victoria entre 38 y 45 DDE, y los valores de coumestrol y et al., 2000), and antibiotic effect of polyamines daidzeina fueron estadísticamente superiores en (Osawa and Tsuji, 1992, 1993). It has been 'Hardin' y 'Hodgson-78' que en 'Alpha' y 'Chippewa'. demonstrated that senescence is associated with the No se detectó relación entre la acumulación de alguno accumulation of isoflavonoid I. Espinosa– de los tres isoflavonoides evaluados y el Victoria et al. (2000) suggested that measurement of establecimiento de la senescencia nodular levels in crown nodules at, or soon after, (disminución de la actividad nitrogenasa). Se asume flowering could be a useful parameter for selecting que los tres isoflavonoides evaluados carecen de cultivars which show delayed nodule senescence. actividad antibiótica, ya que sus altas concentraciones However, it has not been evaluated if phenyl no afectaron la actividad nitrogenasa de los propanoid compounds different from glyceollin I are bacterioides. involved in the senescence of crown soybean nodules. Thus, the objectives of the present study were to: Palabras clave: Isoflavonoides, PAL (fenilalanina a) determine if the accumulation of isoflavonoids amonia liasa), CHS (chalcona sintasa). coumestrol, daidzein and genistein is associated with nodule senescence, and b) follow the expression of isoflavonoid-encoded multigenes PAL (phenylalanine INTRODUCTION ammonia-lyase) and CHS (chalcone synthase) in crown nodules of soybean cultivars exhibiting early Symbiotic nitrogen (N2) fixation by soybean and delayed senescence. (Glycine max (L.) Merril) can decrease the need for expensive nitrogenous fertilization of this crop, as MATERIALS AND METHODS well as minimize environmental pollution. However, the contribution of biological nitrogen (N2) fixation to Plant Growth and Inoculation N accumulation and yield by this crop can vary markedly. In Brazil where N fertilization of soybean Plastic pots (16.25 x 15 cm) containing a is rare, biological N2 fixation contributes the 1:1 sterile mixture of silica sand and Sunshine-mix # 2 9 -1 equivalent of 2.5 x 10 kg of N2 yr , and represents a (J.R. Johnson, Roseville, MN), that had been sterilized saving of 1.8 billion US dollars (Dobereiner et al., by autoclaving 90 min at 121 °C, were used as 1995). Less priority has been given to the experimental units. One day before planting, all pots improvement in the N2-fixing ability of soybeans in were inoculated by watering with Bradyrhizobium US agriculture (Vance, 1997). Some gains in N2 japonicum strain UMR 161, grown in YEM broth fixation by this crop have been obtained (Coale et al., (Graham, 1963) for eight days, providing an initial 1985; Creagan and Yaklich, 1986), but mainly population of 105 cells g-1 of mixture. Seeds of four through the study of cultivar variation of seasonal soybean (Glycine max (L.) Merrill) cultivars ('Alpha', differences in N accumulation under low N soil 'Chippewa', 'Hardin', and 'Hodgson-78') of Maturity conditions (Neuhausen et al., 1988; Sinclair et al., Group I and the hypernodulating genotype 'Nod 1-3' 1991; Herridge et al., 1994). We look for different (generously provided by Dr. J.E. Harper, USDA-ARS, traits that contribute to genetic variation for University of Illinois) were surface sterilized using nodulation and nitrogen fixation. Recent papers have 95% (v/v) ethanol and 3% (v/v) sodium hypochlorite reported cultivar variation in early nodulation and (Vincent, 1970), and rinsed repeatedly in sterile water.

346 ESPINOSA-VICTORIA Y GRAHAM. ACCUMULATION OF COUMESTROL, DAIDZEIN, AND GENISTEIN IN SOYBEAN

Ten seeds were planted per pot and thinned to two NM) were 32P-labeled using a Prep-A-Gene Kit seedlings per pot after emergence. Plants were grown (Bio-Rad Laboratories, Hercules, CA), according to in a greenhouse with a 16 h photoperiod and 26/21 °C the manufacturers' recommendation. Hybridization day/night temperature, and watered alternately with was performed overnight at 42 °C, then nitrocellulose sterile water and sterile low N plant nutrient solution was exposed to x-ray film and incubated overnight (McDermott and Graham, 1990) as needed. Sufficient at -40 °C. Finally, after developing, x-ray film was experimental units (three replicates cultivar-1 photographed. harvest-1) were prepared to allow weekly destructive sampling throughout nine weeks. Accumulation of Coumestrol, Daidzein, and Genistein in Crown Nodules Crown Nodulation and Shoot Biomass A third experiment, performed under the same Plants were harvested every week beginning greenhouse conditions, determined variation in 10 days after emergence (DAE). At each harvest, the coumestrol, daidzein, and genistein accumulation of root system was cleaned, rinsed and nodules located at taproot nodules of each soybean cultivar through the the first 4-cm segment of the taproot, including the lifespan of the symbiosis. At each harvest date, first 1 to 4 cm of lateral roots arising from this nodules located at the 4-cm segment of the taproot, segment, were picked and weighed. Shoots were dried including the first 1 to 4 cm of lateral roots arising at 70 °C for 72 h and then weighed. The experiment from this segment, were removed and immediately was analyzed as a two-factor (DAE and cultivars), frozen in liquid nitrogen. Nodules were ground in randomized complete block design having three liquid nitrogen, and 0.2 g of the fine powder was replicates, each of which consisted of two plants. Each mixed with the 500 µL extraction buffer containing replicate was grown separately in the greenhouse with 5:1 acetonitrile: 0.1 N HCl. The extraction was an analysis of variance computed across DAE for all performed at 4 °C overnight, then samples centrifuged parameters using the General Linear Models (GLM) and the supernatant dried in a speed vacuum at 40 °C. Procedure within SAS, version 6.03 (1992). Samples were suspended in minimal amounts of HPLC-grade methanol then filtered through a 0.2 µm Expression of PAL and CHS Multigenes in Crown nylon acrodisc filter (Gelman Sciences, Ann Arbor, Nodules MI). Analytical HPLC separations were performed in a Hewlett Packard 1090 (Waldbronn, FRG) system A second experiment was performed in the equipped with a scanning UV (287 nm) detector and, greenhouse to study the expression of PAL and CHS a Phenomenex Maxil 5 C1B (Torrance, CA) multigenes in the crown nodules of cultivars reverse-phase analytical column (4.6 x 250 mm). 'Chippewa', 'Alpha', 'Hardin', 'Hodgson-78', and Isoflavonoids were identified by comparing their 'Nod 1-3' through the lifespan of the symbiosis. retention times with those of known standards (Sigma Nodules of plants harvested every week were picked Chemical Co., St. Louis, MO) and the identification and immediately frozen in liquid nitrogen. Nodules was confirmed by coinjections with known standards. were ground in liquid nitrogen and 1 g of the This experiment was also analyzed as a two factor powdered tissue was used to extract RNA (De Vries et (DAE and cultivars), randomized complete block al., 1988). The dot blot technique was used for design having three replicates, each of which detecting the nodule expression of PAL and CHS consisted of two plants. An analysis of variance genes. Samples of 8 µg of RNA were loaded on a computed across DAE for all three isoflavonoids nitrocellulose membrane ensembled in a using the GLM procedure within SAS, version 6.03 BRL HybriII-Dot Manifold (No. 1050MM Life (1992) was performed. Technologies, Inc., Gaithersburg, MD), then the nitrocellulose was removed and baked for 2 h at 80 °C RESULTS AND DISCUSSION and prehybridized for 2 h at 42 °C. Positive and negative controls for hybridization were loaded on the In a previous paper we reported genetic variation nitrocellulose under the same conditions. in terms of onset of crown nodule senescence in Soybean-nodule specific cDNA probes for PAL and soybean cultivars within the Maturity Group I CHS (generously provided by Dr. Sengupta-Gopalan, (Espinosa-Victoria et al., 2000). Cultivars 'Hardin' and

347 TERRA VOLUMEN 19 NUMERO 4, 2001

'Hodgson-78' were fixing nitrogen one and two weeks nodulation. Because nodules formed by this strain after flowering, showing delayed decrease in practically do not fix nitrogen (Pazdernik et al., 1996), nitrogenase activity 52 and 59 DAE, respectively. In there was no contribution of this element for contrast, cultivars Chippewa and Alpha with early increasing shoot biomass. onset of nodule senescence, exhibited a conspicuous Crown nodule mRNA showed a more intense decrease in nitrogenase activity as early as 38 and hybridization with PAL (Figure 1) rather than CHS 45 DAE, respectively. (Figure 2) probe. PAL and CHS transcript levels were Our data on fresh weight of crown nodules higher in 'Chippewa', 'Alpha', and 'Nod 1-3' than in (Table 1) show significant differences between both 'Hardin' and 'Hodgson-78' nodules throughout the soybean groups as early as 17 DAE. 'Hardin' and experiment. 'Hodgson-78' formed more nodules at crown-root The PAL transcripts level remained without zone than 'Chippewa' and 'Alpha', with 'Hardin' change in 'Chippewa', 'Alpha' and 'Nod 1-3' nodules showing the highest values. through the lifespan of the symbiosis. Instead, same In the same way, 'Hardin' and 'Hodgson-78' transcripts exhibited a strong hybridization signal in obtained the highest values in shoot dry weight, which 'Hardin' and 'Hodgson-78' 38 DAE, coinciding with were significantly different to 'Chippewa' and 'Alpha' their period of maximum nitrogenase activity (Table 2). We assume early crown nodulation (Espinosa-Victoria et al., 2000). In contrast, the level triggered the increase in shoot biomass during of CHS transcripts was not consistent for 'Chippewa', vegetative stage, because at this time nodules located 'Alpha' and 'Nod 1-3' crown nodules, showing in this zone contribute essentially 100 % of the fluctuations throughout the assay, with an intense N2 fixation activity (Kamicker and Bill, 1987; hybridization for 'Alpha' nodules 38 DAE. Decrease McDermott and Graham, 1989). Because secondary of PAL and CHS transcripts occurred about 45 to nodulation (nodule formation at lateral roots) 52 DAE in all cultivars. significantly contribute to nitrogen fixation at, or soon Subsets of the PAL and CHS gene families after, flowering, we assumed that the amount of fixed specifically induced in soybean roots after infection nitrogen in 'Hardin' and 'Hodgson-78' must be higher with B. japonicum have been studied during early because their crown nodules continue to fix nitrogen stages of the symbiosis (Sengupta-Gopalan et al., after the reproductive stage. 1990; Estabrook and Sengupta-Gopalan, 1991). The hypernodulating genotype 'Nod 1-3', used as reference, exhibited the highest values for crown Our data show that PAL and CHS transcripts are also

Table 1. Crown nodule fresh weight of soybean cultivars within the Maturity Group I at different harvesting dates.

Soybean Days after emergence cultivars 10 17 24 31 38 45 52 59 66 ------mg ------Alpha 140.00 b* 354.00 b 645.00 b 887.00 c 969.00 c 822.33 c 748.67 c 631.67 d 448.33 e Chippewa 119.00 b 363.00 b 554.33 c 805.33 cd 997.00 bc 860.67 c 788.33 c 686.00 d 655.00 d Hardin 119.00 b 334.67 bc 542.67 c 960.67 b 1045.00 b 1174.33 b 1235.33 b 1115.67 b 996.33 b Hodgson-78 130.00 b 306.00 c 512.67 d 794.33 d 986.67 b 1081.67 b 1138.33 b 1036.00 c 892.00 c Nod 1-3 185.00 a 410.00 a 710.33 a 1020.67 a 1370.44 a 1650.33 a 1545.44 a 1388.33 a 1200.44 a *Means followed by the same letter within columns are not significantly different (P ≤ 0.05) using T test (LSD).

Table 2. Shoot dry weight of soybean cultivars within the Maturity Group I at different harvesting dates.

Soybean Days after emergence cultivars 10 17 24 31 38 45 52 59 66 ------g ------Alpha 0.285 b* 0.560 c 1.125 c 2.107 c 3.930 b 5.638 c 6.087 c 8.273 b 10.156 b Chippewa 0.313 a 0.495 d 0.915 d 1.920 d 3.123 c 4.787 d 5.826 d 8.574 b 10.114 b Hardin 0.315 a 0.786 b 1.879 a 3.266 a 4.453 a 6.562 a 7.905 a 9.117 a 11.477 a Hodgson-78 0.272 b 0.819 a 1.555 b 2.536 b 4.504 a 5.586 b 7.255 b 9.041 a 11.138 a Nod 1-3 0.110 c 0.350 e 0.799 e 1.012 e 2.905 d 3.565 e 4.022 e 6.121 c 7.055 c *Means followed by the same letter within columns are not significantly different (P ≤ 0.05) using T test (LSD).

348 ESPINOSA-VICTORIA Y GRAHAM. ACCUMULATION OF COUMESTROL, DAIDZEIN, AND GENISTEIN IN SOYBEAN

10 17 24 31 38 45 52 59 C

Chippewa

Alpha

Hardin

Hodgson-78

Nod 1-3

Figure 1. Expression of PAL (Phenylalanine ammonia lyase) multigene family in crown nodules induced by Bradyrhizobium japonicum strain UMR161 on five soybean cultivars at different days after emergence. Dot blots (8 µg of total nodule mRNA) were hybridized with soybean-nodule specific PAL probe. C = positive control.

10 17 24 31 38 45 52 59 C

Chippewa

Alpha

Hardin

Hodgson-78

Nod 1-3

Figure 2. Expression of CHS (Chalcone synthase) multigene family in crown nodules induced by Bradyrhizobium japonicum strain UMR161 on five soybean cultivars at different days after emergence. Dot blots (8 µg of total nodule mRNA) were hybridized with soybean-nodule specific CHS probe. C = positive control. expressed in soybean crown nodules at vegetative, We found a high expression of PAL and CHS reproductive and maturity stages. multigenes in crown nodules of the hypernodulating Variation in levels of coumestrol, daidzein, and 'Nod 1-3' genotype and it was related to high levels of genistein in crown nodules of these cultivars are coumestrol, daidzein, and genistein throughout the shown in Tables 3 to 5. Levels of daidzein were symbiosis. higher than coumestrol or genistein in all five The tested isoflavonoids reached their highest cultivars during the lifespan of the symbiosis. In all accumulation values near the flowering season cases, isoflavonoid levels peaked 38 to 45 DAE, with (38 DAE). The pattern of coumestrol, daidzein, and levels of coumestrol and daidzein in 'Hardin' and genistein accumulation in 'Alpha', 'Chippewa', and 'Hodgson-78' statistically higher than in 'Alpha' and 'Hodgson-78' nodules exhibited maximum values one 'Chippewa'. There were no consistent differences in and two weeks (38 and 45 DAE) after their accumulation of genistein. nitrogenase peaks (Espinosa-Victoria et al., 2000). In It has been reported that the expression of PAL contrast, coumestrol and daidzein maximum and CHS in hypernodulating genotype nodules tends accumulation in 'Hardin' occurred one week to be higher (Estabrook and Sengupta-Gopalan, 1990). before (38 DAE) the nitrogenase peak. There was no

349 TERRA VOLUMEN 19 NUMERO 4, 2001

Table 3. Coumestrol accumulation in crown nodules of five cultivars of soybean (Glycine max. (L.) Merr.) formed by Bradyrhizobium japonicum strain UMR161.

Soybean Days after emergence line 10 17 24 31 38 45 52 59 ------μg/fgw ------Alpha 1.622 c* 17.82 d 18.26 d 218.48 d 576.57 d 607.60 c 163.13 c 109.49 c Chippewa 14.389 b 18.02 d 17.89 d 33.06 e 528.92 e 519.77 e 164.43 c 151.24 b Hardin 9.467 b 158.77 c 210.63 c 254.48 c 613.70 c 555.79 d 102.87 d 88.84 c Hodgson-78 10.736 b 252.54 b 306.52 b 382.58 b 813.43 b 735.51 b 190.98 b 147.52 b Nod 1-3 72.596 a 299.39 a 384.37 a 480.71 a 1075.64 a 886.05 a 358.77 a 272.29 a *Means followed by the same letter within columns are not significantly different (P ≤ 0.05) using T test (LSD).

Table 4. Daidzein accumulation in crown nodules of five cultivars of soybean (Glycine max (L.) Merr.) formed by Bradyrhizobium japonicum strain UMR161.

Soybean Days after emergence line 10 17 24 31 38 45 52 59 ------μg/fgw ------Alpha 118.71 d* 113.93 e 154.61 c 187.37 e 476.14 d 556.22 d 202.42 c 133.97 c Chippewa 155.63 c 167.97 d 187.13 c 250.92 d 626.67 c 753.63 c 251.60 c 230.58 b Hardin 116.62 d 202.66 c 259.85 b 317.07 c 645.64 c 543.91 d 342.42 b 220.04 b Hodgson-78 260.12 b 314.55 b 391.04 b 391.94 b 903.88 b 1117.63 b 317.28 b 249.25 b Nod 1-3 307.62 a 382.38 a 441.76 a 591.68 a 993.51 a 1349.90 a 512.46 a 368.50 a *Means followed by the same letter within columns are not significantly different (P ≤ 0.05) using T test (LSD).

Table 5. Genistein accumulation in crown nodules of five cultivars of soybean (Glycine max (L.) Merr.) formed by Bradyrhizobium japonicum strain UMR161.

Soybean Days after emergence line 10 17 24 31 38 45 52 59 ------μg/fgw ------Alpha 19.18 c* 39.78 d 73.08 c 91.11 d 315.99 c 408.13 b 138.50 b 82.18 c Chippewa 24.30 b 64.02 b 122.02 a 142.64 c 251.29 e 356.52 d 139.18 b 132.75 b Hardin 24.29 b 61.55 b 90.18 b 104.97 d 295.70 d 418.60 b 80.81 d 26.37 d Hodgson-78 22.84 b 54.31 c 133.94 a 176.47 b 364.69 b 381.35 c 98.85 c 71.04 c Nod1-3 45.42 a 83.80 a 104.62 b 294.99 a 440.71 a 894.00 a 358.76 a 204.37 a *Means followed by the same letter within columns are not significantly different (P ≤ 0.05) using T test (LSD). difference in isoflavonoid accumulation between Due to its antibiotic activity, glyceollin I has been soybean cultivars exhibiting early and delayed crown associated with Bradyrhizobium bacteroid viability nodule senescence. In a previous report (Espinosa- (Werner et al., 1985; Karr et al., 1992). We Victoria et al., 2000) we showed no glyceollin I hypothesized the three tested isoflavonoids lack this accumulation in crown nodules of soybean cultivars property, since their high accumulation did not affect with late nodule senescence. In comparison, crown nodules of soybean cultivars exhibiting early nodule the nitrogenase activity of any of the four soybean senescence accumulated high levels of this compound. cultivars. In the present study, we did not find relation between Further research must consider the evaluation of the accumulation of any of the three tested additional Bradyrhizobium japonicum strains and isoflavonoids and the onset of the nodule senescence. soybean cultivars belonging to different Maturity It has been reported that coumestrol, daidzein, and Groups, field performance of soybean cultivars genistein are naturally accumulated in plant root differing in crown nodule senescence, and the impact tissues (Dewick, 1988); however, there is no of delayed crown nodule senescence on secondary information related to their nodule accumulation nodulation. pattern and biological significance.

350 ESPINOSA-VICTORIA Y GRAHAM. ACCUMULATION OF COUMESTROL, DAIDZEIN, AND GENISTEIN IN SOYBEAN

REFERENCES enzymes from bacteroid-free extracts. Plant Physiol. 68: 386-392. McDermott, T. and P.H. Graham. 1989. Bradyrhizobium Coale, F.J., J.J. Meisingeer, and W.J. Wiebold. 1985. Effects of japonicum inoculant mobility, nodule occupancy, and plant breeding and selection on yields and nitrogen fixation in acetylene reduction in the soybean root systems. Appl. soybean under two soil nitrogen regimens. Plant Soil 86: Environ. Microbiol. 55: 2493-2498. 357-367. McDermott, T. and P.H. Graham. 1990. Competitive ability and Creagan, P.B. and R.W. Yaklich. 1986. Dry matter and nitrogen efficiency in nodule formation strains of Bradyrhizobium accumulation and partitioning in selected soybean genotypes japonicum. Appl. Environ. Microbiol. 56: 3035-3039. of different derivation. Theor. Appl. Genet. 72: 782-786. Neuhausen, S.L., P.H. Graham, and J.H. Orf. 1988. Genetic De Vries, S., H. Hoge, and T. Bisseling. 1988. Isolation of total variation for dinitrogen fixation. CRC Critical Rev. Plant Sci. and polysomal RNA from plant tissues. pp. B6-13 In: S.B. 6-3: 267-321. Galvin and R.A. Schilperrot. Plant molecular biology manual. Osawa, T. and T. Tsuji. 1992. Inhibition of growth of Kluwer. Dordrecht, Boston, London. Bradyrhizobium japonicum bacteroid by spermidine and Dewick, P.M. 1988. Isoflavonoids. pp. 125-209. In: The spermine in yeast extract. Soil Sci. Plant Nutr. 38: 375-379. : Advances in research since 1980. J.B. Harborne Osawa, T. and T. Tsuji. 1993. A possible role for polyamines in (ed.). Chapman and Hall. London, New York. the repression of growth of Bradyrhizobium japonicum Dobereiner, J., S. Urquiaga and M. Boddey. 1995. Alternatives for bacteroids in soybean nodules. Plant Cell Physiol. 34: nitrogen nutrition of crop in tropical agriculture. Fertil. Res. 899-904. 42: 339-346. Pazdernik, D.L., P.H. Graham, C.P. Vance, and J.H. Orf. 1996. Espinosa-Victoria, D., C.P. Vance, and P.H. Graham. 2000. Host Host genetic variation in the early nodulation and dinitrogen variation in traits associated with crown nodule senescence in fixation of soybean. Crop Sci. 36: 1102-1107. soybean. Crop Sci. 40: 103-109. Pfeiffer, N.E., C.M. Torres, and F.W. Wagner. 1983a. Proteolytic Estabrook, E.M. and C. Senguta-Gopalan. 1990. Expression of activity in soybean root nodules. Activity in host cell cytosol genes encoding for phenylalanine ammonia lyase and and bacteroids throughout physiological development and chalcone synthase and their possible role during nodule senescence. Plant Physiol. 71: 797-802. development in soybean. pp. 735-739. In: Gresshoff, Roth, Pfeiffer, N.E., N.S.A. Malik, and F.W. Wagner. 1983b. Reversible Stacey, and Newton (eds.). Nitrogen fixation: Achievements dark-induced senescence of soybean root nodules. Plant and objectives. Chapman and Hall. New York, London. Physiol. 71: 393-399. Estabrook, E.M. and C. Senguta Gopalan. 1991. Differential Sengupta-Gopalan, C., E.M. Estabrook, H. Gambliel, expression of Phenylalanine ammonia-lyase and chalcone W. Nirunsuksiri, and H. Richter. 1990. Regulation of host synthase during soybean nodule development. Plant Cell 3: gene expression during nodule development in soybeans. pp. 299-308. 701-707. In: Gresshoff, Roth, Stacey and Newton (eds). Graham, P.H. 1963. Antibiotic sensitivities of the root nodule Nitrogen fixation: Achievements and objectives. Chapman bacteria. Aust. J. Biol. Sci. 16: 557-560. and Hall. New York, London. Herridge, D.F., O.P. Rupela, R. Serraj, and D.P. Beck. 1994. Sinclair, T.R., A.R. Soffes, K. Hinson, S.L. Albretch, and Screening techniques and improved biological nitrogen P.L. Pfahler. 1991. Genotypic variation in soybean nodule fixation in cool season food . Euphytica 73: 95-108. number and weight. Crop Sci. 31: 301-304. Kamicker, B.J. and W.J. Brill. 1987. Methods to alter the recovery and nodule locations of Bradyrhizobium japonicum inoculant Vance, C.P. 1997. symbiotic nitrogen fixation: strain in the field-grow soybean. Appl. Environ. Microbiol. Agronomic aspects. CRC Critical Rev. Plant Sci. 5-2: 53: 1737-1742. 120-131. Karr, D.B., D.W. Emerich, and A.L. Karr. 1992. Accumulation of Vincent, J.M. 1970. A manual for practical study of root-nodule the phytoalexin, glyceollin, in root nodules of soybean bacteria. IBP Handbook 15. Blackwell. Oxford, England. formed by effective and ineffective strains of Bradyrhizobium Werner, D., R.B. Mellor, M.G. Hahn, and H. Grisebach. 1985. japonicum. J. Chem. Ecol. 18: 997-1008. Soybean root response to symbiotic interactions with Malik, N.S.A., N.E. Pfeiffer, D.R. Williams, and F.D. Wagner. glyceollin I. Accumulation in an ineffective type of soybean 1981. Peptidohydrolases of soybean root nodules. nodules with an early loss of the peribacteroid membrane. Identification, separation, and partial characterization of Z. Naturforsch. 40c: 179-181.

351