Scientific Registration Number : 1074 Symposium n : 10 Presentation : Poster. and PEÑA-CABRIALES

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1 Scientific Registration Number : 1074 Symposium n : 10 Presentation : Poster Other benefits apart from N 2 fixation in the Rhizobium/legume symbiosis Autres bénéfices issus de la fixation de N 2 par la symbiose Rhizobium/légumineuse FARÍAS-RODRÍGUEZ Rodolfo 1,3, MELLOR Robert B. 2 Juan José 3 and PEÑA-CABRIALES 1. Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3 Ciudad Universitaria Morelia, Mich., México. 2. Biology Experts Ltd. Strandboulevarden 133.1tv, 2100 Copenhagen, Denmark. 3. Centro de Investigación y Estudios Avanzados del IPN Unidad Irapuato. Apdo. Postal 629, Irapuato 36500, Gto., México. INTRODUCTION In the Rhizobium-legume interaction the bacterial partner stimulates the formation of root nodules, which the bacteria subsequently inhabit, and a metabolic cooperation is established (Lomg 1989). The symbiosis has hitherto almost exclusively been studied under aspects pertaining to N fixation, however other factors are attracting attention, amongst which are Fe, K and P assimilation, the synthesis of antibiotics, siderophores, hormones and trehalose. Trehalose has been detected in nodules of all legumes studied, where it is synthesized, but not retained, by the bacteroids, and finally accumulates in the plant cytosol. The role of trehalose is open to discussion (Mellor 1992) but it is worth noticing that trehalose is connected with tolerance to elevated temperatures and dessication (Crowe et al 1984). In a summation of all Mexican field trials between 1976 and 1990, Castellanos et al (1995) found that only 13% of nodules recovered were inhabited by selected high-performing Rhizobium strains when these were used as inoculum. This is because local strains are inefficient in fixing N, but are highly competitive in infection (Aguilar and Peña-Cabriales 1996). Additionally, common bean is a C3 plant and production occurs mostly under rainfed conditions, factors pointing towards a high susceptibility to drought. However various reports from Mexico suggest that this plants carries a novel undefined drought resistance mechanism (Perez-Trujillo et al 1992). Since nodulated common bean plants are more drought-resistant than non-nodulated (Farias-Rodrigues et al 1998) we initiated an investigation to see if these two factors, trehalose and drought resitance, are linked. 1

2 MATERIALS AND METHODS Trehalose in nodules of Vicia faba, Cicer arietinum, Pisum sativum. Legume plants were grown under field conditions in the state of Guanajuato, Mexico, and the trehalose concentration in nodules was determined 30 and 90 days after planting. Pooled plant tissue (Breedveld et al., 1991) were extracted using hot ethanol as described by Streeter (1985) and the trehalose content determined by high-performance ion-exchange chromatography (Carbopak PA-1) with a Dionex pulse electrochemical detector and using 4 mm NaOH as elution buffer. Trehalose in common bean nodules Seeds of common bean were grown in 1 Kg of sand:soil (1:1) mixture, where the Vertisol soil was previously obtained from a bean producing site in Guanajuato, México. Plants grew in a greenhouse with a day-night fluctuation of 30 o C-20 o C and natural illumination with an 11 hr photoperiod and received 100 ml water per plant daily. Drought treatment was effected by suspending watering after 20 th day. Nodules were harvested 30 days post planting and the trehalose concentration determined likewise above. Reinfection of cultivars Canario, FMB and F-38 was by application ( cells/ml per seed) of mixture of homologous Rhizobium strains and growth under axenic conditions. Drought resistance was assayed visually and the relative water content (RWC) of leaf disks determined by the method of Turner (1981). RESULTS Trehalose in nodules of various legumes Trehalose concentrations were similar in the nodules of C. arietinum, P. sativum and V. faba 30 days post planting, but increased to radically different levels 90 days post planting (table 1). Trehalose in nodules of different P. vulgaris cultivars Trehalose accumulation was host determined since trehalose concentrations were similar in plants of each cultivar despite infection by a range of bacterial strains, but was variable between cultivars (table 2). Effects of drought Non-nodulated cultivars of P. vulgaris exhibited a uniformly poor resistance to drought. In nodulated plants, drought brought about a dramatic rise in nodule trehalose content in all cultivars (table 2). Trehalose rose by 600% in nodules of cv Canario, by 300% in nodules of cv F-38, but by less than 100% in nodules of cv FMB. The drought resistance of these cultivars was clearly Canario > FM-38 >> FMB. The RWC of leaves of well watered non-nodulated plants was 87% (av), dropping to 81% (av) after drought stress. Conversely the leaves of nodulated plants was 89% (av), rising to 92% (av) after drought stress. DISCUSSION A causal relationship exists between trehalose production and drought tolerance in Phaseolus nodules. Sterile plants of cvs Canario, F38 and FMB exhibit similar, poor, tolerances to drought stress (Peña-Cabriales and Castellanos 1993). However nodulation with 2

3 natural flora, cvs Canario and F38 nodules exhibit high trehalose concentrations, these nodule trehalose concentrations increase further under drought conditions and nodulated Canario and F38 exhibit an excellent resistance to drought stress. Conversely FMB nodules exhibit low trehalose concentrations, these levels hardly doubled under drought conditions, and indeed nodulated FMB is highly sensitive to drought stress. Our preliminary working theory is that even under normal water regimes the cytoplasm of infected cells in nodules may contain high concentrations of solutes, ions and proteins and may thus be verging on water deficiency (see Mellor 1992) and that bacteria synthesise trehalose as a response to this situation (Breedveld et al 1991). A further decrease in water potential may increase trehalose synthesis either directly via bacterial osmoregulatory mechanisms or indirectly via oxygen partial pressure. Water limitation reduced N-fixation by 25%, but this was reversed by simultaneously increasing po 2 (Guerin et al 1990). Indeed limiting po 2 has been suggested during periods of environmental stress (Walsh 1995). Under similar conditions trehalose synthesis is triggered in cultured rhizobia (Hoelzle and Streeter 1990). If the trehalose concentration is high enough, i.e. if synthesis is high and hydrolysis low, then some trehalose may escape the confines of the nodule (Streeter 1980) and may be able to act as osmoprotectant in other parts of the plant. Thus plants subjected to drought stress could experience a selective advantage if they are nodulated by bacteria which are high trehalose producers in planta. Unfortunately good trehalose production in nodules is inversely correlated to N-fixing ability (Streeter and Salminen 1988) and even ineffective (Fix - ) bacteria can accumulate significant amounts of trehalose in nodules (Müller et al 1994). This could account for the predominance of poor N- fixing rhizobia found in Mexican soils (Castellanos et al 1995, Aguilar and Peña-Cabriales 1996). REFERENCES Aguilar Z. C. and J. J. Peña-Cabriales Diversidad y caracterización de la población nativa de Rhizobium sp. que nodula frijol (Phaseolus vulgaris L.) en el estado de Zacatecas, México. In Aumento de la Fijación Biológica de Nitrógeno en el Frijol Común en América Latina. Eds. F. Zapata and J. J. Peña-Cabriales. (In press). Breedveld M. W., Zevenhuizen L. P. and J. B. Zehnder Osmotically-regulated trehalose accumulation and cyclic ß-(1,2) glucan excretion by Rhizobium leguminosarum biovar trifolii TA-1. Arch. Microbiol. 156: Castellanos J. Z., Peña-Cabriales J. J. and I. Rojas-Martínez Análisis retrospectivo del uso de inoculantes con cepas elite en el cultivo de frijol (Phaseolus vulgaris L.) en México. Turrialba 45: Crowe J. H., Hoekstra F. A. S. and L. M. Crowe Anhydrobiosis. Annu. Rev. Physiol. 54: Farías-Rodríguez R., Mellor R. B., Arias C. and J. J. Peña-Cabriales The accumulation of trehalose in nodules of several cultivars of common bean (Phaseolus vulgaris L.) and its correlation with resistance to drought stress. (In press). Guerin V., Trichant J. C. and J. Rigaud Nitrogen fixation (C 2 H 2 reduction) by broad bean (Vicia faba L.) nodules and bacteroids under water-restricted conditions. Plant Physiol. 91: Long S. R Rhizobium-legume nodulation: life together in the underground. Cell 56:

4 Mellor R. B Is trehalose a symbiotic determinant in symbioses between higher plants and microorganisms? Symbiosis 12: Müller J., Xie Z-P., Staehelin C., Mellor R. B., Boller T. and A. Wiemken Trehalose and trehalase in root nodules from various legumes. Physiol. Plant 90: Peña-Cabriales J. J. and J. Z. Castellanos Effects of water stress on N 2 -fixation and grain yield of Phaseolus vulgaris L. Plant Soil 152: Pérez-Trujillo H., Ochoa-Márquez R., Grageda-Cabrera O., Nuñez-Gonzalez S. and R. Rosales-Serna Respuesta de genotipos indeterminados de frijol a dos condiciones de humedad en la región semiárida. In Resultados de Investigación sobre Frijol 1990, Proyecto Colaborativo INIFAP-MSU (B/C-CRSP). Ed J. A. Acosta-Gallegos, pp SARH México. Streeter J. G Carbohydrates in soybean nodules. II Distribution of compounds in seedling during the onset of nitrogen fixation. Plant Physiol. 66: Streeter J. G Accumulation of alpha,alpha trehalose by Rhizobium bacteria and bacteroids. J. Bacteriol. 164: Streeter J. G. and S. O. Salminen Carbon metabolism and the exchange of metabolites between symbionts in legume nodules. In Physiological Limitations and the Genetic Improvement of Symbiotic Nitrogen Fixation. Eds. F. O Gara, S. Manian and J.J. Drevon, pp Turner N. C Techniques and experimental approaches for the measurement of plant water status. Plant Soil 58: Walsh K. B Physiology of the legume nodule and its response to stress. Soil Biol. Biochem. 27: Keywords : Common bean, drought resistance, legumes, nodules, Phaseolus vulgaris, Rhizobium, trehalase, trehalose Mots clés : haricot, résistance à la sécheresse, légumineuse, nodules, Phaseolus vulgaris, Rhizobium, trehalase, trehalose Table 1. Trehalose accumulation in nodules of selected legume species infected with natural rhizobial flora. DAYS AFTER PLANTING LEGUME Trehalose (mg/ DW) Cicer arietinum Pisum sativum Vicia faba Values represent the average of three replicates 4

5 Table 2. Trehalose accumulation in nodules of P. vulgaris cultivars 30 days post planting, with and without exposure to drought stress. Trehalose (mg/g DW) Cultivar Control Drought Stress Canario F FMB 0.9 Values represent the average of three replicates 1.6 5