Plant Physiology and Biochemistry 80 (2014) 53e59
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Research article
The nodule conductance to O2 diffusion increases with phytase activity in N2-fixing Phaseolus vulgaris L Mohamed Lazali a, b, *, Jean Jacques Drevon b a
Université de Khemis Miliana, Faculté des Sciences de la Nature et de la Vie & des Sciences de la Terre, Route Theniet El Had, 44225 Khemis Miliana, Algerie Institut National de la Recherche Agronomique, UMR Ecologie Fonctionnelle & Biogéochimie des Sols et Agroécosystèmes, INRA-IRD-CIRAD-SupAgro, Place Pierre Viala, 34060 Montpellier, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2013 Accepted 22 March 2014 Available online 1 April 2014
To understand the relationship between phosphorus use efficiency (PUE) and respiration for symbiotic nitrogen fixation (SNF) in legume nodules, six recombinant inbred lines of common bean (RIL Phaseolus vulgaris L.), contrasting in PUE for SNF, were inoculated with Rhizobium tropici CIAT899, and grown under hydroaeroponic culture with sufficient versus deficient P supply (250 versus 75 mmol P plant1 week1). At the flowering stage, the biomass of plants and phytase activity in nodules were analyzed after measuring O2 uptake by nodulated roots. Our results show that the P-deficiency significantly increased the phytase activity in nodules of all RILs though with highest extent for RILs 147, 29 and 83 (ca 45%). This increase in phytase activity was associated with an increase in nodule respiration (ca 22%) and in use of the rhizobial symbiosis (ca 21%). A significant correlation was found under P-deficiency between nodule O2 permeability and phytase activity in nodules for RILs 104, 34 and 115. This observation is to our knowledge the first description of a correlation between O2 permeability and phytase activity of a legume nodule. It is concluded that the variation of phytase activity in nodules can increase the internal utilization of P and might be involved in the regulation of nodule permeability for the respiration linked with SNF and the adaptation to P-deficiency. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Legume Phaseolus vulgaris Phosphorus deficiency Phytase Respiration Root-nodule Symbiosis
1. Introduction The formation of N2-fixing nodules on roots is a feature shared by many legume species. These nodules are the result of a symbiosis with soil bacteria called rhizobia, resulting in the ability of the plant to use atmospheric N2 (Patriarca et al., 2004). The symbiotic nitrogen fixation (SNF) can act as a renewable and environmentally sustainable source of nitrogen, and complement or replace fertilizer inputs (Peoples et al., 1995; Herridge et al., 2008). However, such environmental factors as drought, salinity, and low soil P (Graham and Vance, 2003) are major constraints worldwide for legumes production, especially in arid, semi-arid, and tropical
Abbreviations: Conr, consumption of oxygen by the nodulated-roots; COP, critical oxygen pressure; DAT, days after transplantation; N, nitrogen; nDW, nodule dry weight; P, Phosphorus; pO2, partial pression of oxygen; RIL, recombinant inbred line; sDW, shoot dry weight. * Corresponding author. Institut National de la Recherche Agronomique, UMR Ecologie Fonctionnelle & Biogéochimie des Sols et Agroécosystèmes, Place Pierre Viala, 34060 Montpellier, France. Tel.: þ33 (0) 4 99 61 23 32; fax: þ33 (0) 4 99 61 21 19. E-mail address: [email protected] (M. Lazali). http://dx.doi.org/10.1016/j.plaphy.2014.03.023 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.
ecosystems, and particularly for common bean (Phaseolus vulgaris L.), often in developing countries (Zahran, 1999). The sensitivity of N2-fixing plants to P-deficiency has been widely documented. It is generally attributed to the large amount of P-dependent carbon and energy turnover required in nodule metabolism (Schulze and Drevon, 2005). The impaired SNF in Pdeficient plants is usually explained by an effect of the low P supply on the growth of the host plant or the rhizobia and their interactions (Almeida et al., 2000). Thus, plant growth requires more P with N2 fixation than with nitrate assimilation for most legume species. This is attributed to the high energy requirements for reduction of atmospheric N2 by nitrogenase (Rotaru and Sinclair, 2009) which is presumed to be regulated by nodule O2 permeability (Schulze and Drevon, 2005). Nodule O2 permeability is though to be involved in the regulation of N2 fixation (Hunt and Layzell, 1993). Since nitrogenase, the enzyme responsible for N2 fixation, is O2 labile, legume nodules have evolved mechanisms to down regulate their O2 content, including oxygenated leghemoglobin gradients for the fine control of O2 levels in infected cells (Thumfort et al., 1999), though a physical barrier to gas diffusion in the inner cortex seems to be the primary site for the regulation of nodule permeability to gas
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diffusion (Hunt and Layzell, 1993; Minchin, 1997; Galvez et al., 2000). Moreover, nodule O2 permeability was reported to increase under P-deficiency in soybean (Ribet and Drevon, 1995) and common bean (Vadez et al., 1996). Alfalfa also adapts to P-deficiency by forming small nodules with high O2 permeability which enables an increase in O2 consumption per unit N2 fixed and P content (Schulze and Drevon, 2005). In N2-fixing nodules, recent studies have suggested that bean plants subjected to P-deficiency increased the activities of phosphatases (APases) and phytase in nodules (Araujo et al., 2008; Kouas et al., 2009; Bargaz et al., 2013). In this context, Li et al. (2012) also reported high expression of large number of purple acid phosphatases genes in the nodules of soybean under low-P conditions. These results suggest that this response could contribute to improved nutrition of legumes under these limiting conditions. Indeed, phytase is one of the most active APases for the sequential hydrolysis of phytate to a series of myo-inositol phosphate derivatives and inorganic phosphate (Pi) (Rodríguez and Fraga, 1999; Yadav and Tarafdar, 2003). It might play a major role for internal plant metabolism and thus may contribute to P use efficiency (PUE) for SNF. The involvement of APases in the tolerance of various abiotic and biotic constraints, led us to an assumption that phytase may have a multiplicity of functions as well as those related to phosphate metabolism and should provide new insights into adaptation to P-deficiency. Thus, this study is among the first reports investigating the correlation between nodule respiration and phytase activity of a legume. It is also hypothesized that increase in nodule phytase activity of common bean regulates nodule respiration and plays a key role in P-deficiency tolerance for N2 fixation of the legume-rhizobia symbiosis. 2. Methods 2.1. Plant growth and nodulation conditions This study was carried out with six recombinant inbred lines, namely RILs 147, 115, 104, 83, 34 and 29, originating from the International Center of Tropical Agriculture (CIAT, Cali, Colombia). These RILs were selected from a previous screen among 100 progenies of the crossing, in 1996, of two parental RILs, namely BAT477 and DOR364. Under conditions of P deficiency, RILs 115, 104 and 34 were characterized as P-efficient genotypes, while RILs 147, 83 and 29 as P-inefficient genotypes respectively, based on plant growth and seed yield in relation of SNF-derived to P availability (Drevon et al., 2011). Seeds were surface sterilized with 3% calcium hypochlorite for 10 min and rinsed with 5 washings of sterile distilled water before germination. Five days after sowing, the roots of selected uniform seedlings were inoculated with the Rhizobium tropici CIAT899 grown in liquid yeast extract mannitol medium at 28 C for 3 days to an approximate cell density of 109 mLe1. Thereafter, the seedlings were transferred into hydroaeroponic culture consisting of vats filled with 40 L of nutrient solution, which were aerated intensely and arranged in a fully randomized block design. Cotton wool was fitted at the hypocotyl level to maintain the root system suspended in the nutrient solution with either 75 or 250 mmol P per plant per week, defined as P-deficient or P-sufficient supplies, respectively (Lazali et al., 2013a). Urea was supplied as 2 mM per plant into the nutrient solution during the initial 2 weeks of growth in order to optimize nodulation (Hernandez and Drevon, 1991). Thereafter, the plants were grown in N-free nutrient solution. At the beginning of the third week of culture, plants were transferred to 1 L glass bottles containing nutrient solution and wrapped in aluminum foil to ensure a dark plantrooting environment. The nutrient solution was first replaced at 15 days after transplantation (DAT) and each week, subsequently.
The nutrient solution pH was adjusted to around 7 by adding 0.2 g L1 CaCO3 and the medium was aerated by an air flow of ambient air compressed to 400 mL min1 plant1. The experiment was carried out in a glasshouse under natural light with day/night temperatures 28/20 C and 16 h photoperiod with 70% relative humidity and additional illumination of 400 mmol photons m2 s1 during the day. 2.2. Nodulated-root O2 uptake measurements The in situ measurement of O2 uptake by the nodulated roots (Conr) was performed with a Witt Logger Oxymeter (Abiss, Villemoisson, France) between 35 and 42 DAT, before their harvest. One day before the measurement of Conr, the level of the nutrient solution in the bottle was reduced to one-third of the volume so that the whole nodule population was in direct contact with the gas phase. The circulation of the gaseous phase in the circuit from the nodulated-root environment through the oxymeter was driven by peristaltic pump with a flow rate of 400 mL min1. Successive measurements were performed with an initial pO2 of 21, 25, 30, 35, 40, 45 and 50 kPa O2 corresponding to 250, 298, 358, 417, 476, 536 and 596 mmol O2 L1, respectively. Nodulated root O2 uptake was determined by measuring the rate of decline in O2 concentration in the gas surrounding the nodulated roots during a 30 min period after closing the system. The consumption of O2 was calculated as Conr ¼ [(initial - final) pO2] [V/24.2] [60/t] with : t in min, duration between initial and final O2 measurement; pO2 in % of the atmospheric pressure; V in L, volume of gas phase; 24.2 in L, volume of 1 mol pure gas in experimental conditions (Schulze and Drevon, 2005). The Conr was calculated in mmol O2 consumed h1 plant1 and nodule permeability (mm3 h1) was defined as the slope of the linear part of the regression of Conr (mmol O2 h1) as a function of external O2 concentrations (mmol O2 L1). The critical oxygen pressure (COP) was calculated as the first derivative of the curvilinear regression of Conr as a function of pO2. 2.3. Phytase activity assay Samples of nodules of 3 mm diameter of each plant corresponding to 60 mg of nodules fresh weight were carefully detached from roots at 42 DAT. Each nodule sample was ground in vibrating mill (FastPrepÒe24) with an extraction mixture consisting of 900 mL sodium acetate-buffer (50 mM pH 5.5) containing 5 mM dithiotrietol. After centrifugation at 22 000 g during 6 min, 3 aliquots of 100 mL of the supernatant were incubated during 90 min at 37 C with a mixture of 300 mL sodium acetate-buffer (0.2 M pH 5.5) and 100 mL of phytic acid (10 mM pH 5.5). The reaction was stopped by the 0.5 mL addition of 10% trichloroacetic acid and centrifugation at 20 000 g during 6 min. Another 100 mL aliquot received the same buffer and amount of phytic acid substrate, but the reaction was stopped immediately without incubation and the mixture centrifuged. Concentration of Pi in the extracts was measured spectrophotometrically at 630 nm using malachite green. The phytase activity was calculated as the difference between the Pi in the extracts with and without incubation and expressed in nmol of Pi released per min per g of nodule fresh mass. 2.4. Biomass measurements The plants were harvested at 42 DAT after gas exchange measurements and separated in shoots, roots and nodules. After drying for 72 h at 65 C, plant and nodule dry weight (DW) was measured and used to determine the efficiency in use of the rhizobial symbiosis (EURS) estimated as the slope of the regression model i.e.,
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y ¼ ax þ b of plant biomass as a function of nodule biomass (Drevon et al., 2011). 2.5. Statistical analysis Experimental design was a randomized complete block. The R software (2.14.1) was used to perform the statistical analyses of biomass, correlation analyses, and regressions of gas exchange parameters as a function of external pO2. The analysis of variance and the standard deviation of the means were used to determine the significance (P < 0.05) of differences in symbiotic effectiveness. 3. Results 3.1. Phytase activity and nodule Pi concentration Under P-sufficiency the phytase activity in nodules was significantly higher for RILs 115, 104 and 34 (ca 21 nmol min1 g1 nFW) than for RILs 147, 83 and 29 (ca 14 nmol min1 g1 nFW) (Fig. 1A). Pdeficiency increased significantly the phytase activity, though to a higher extent for RILs 147 and 29 (ca 44%) than for RILs 115 and 34 (ca 27%). Under P-sufficiency the P-efficient RIL115 had the significantly highest nodule Pi concentration (13 mmol Pi g1 nFW), whereas the
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significantly lowest value was observed for P-inefficient RILs 147 and 83 (9 mmol Pi g1 nFW) (Fig. 1B). Under P-deficiency, the nodule Pi concentration decreased for all RILs though this decrease was significantly the highest for the P-inefficient RILs 29 and 83 (ca 40%). 3.2. Nodule permeability to O2 diffusion The response of O2 uptake by nodulated roots to variation of rhizospheric pO2 was measured in order to assess the effect of Pdeficiency on nodule respiration, following the protocol described by Jebara and Drevon (Jebara and Drevon, 2001). Data in Fig. 2 show that Conr increased with an increase in rhizospheric pO2 until a COP beyond which the O2 diffusion within nodules was no longer stimulatory for respiration. Independently of P supplies, the COP was the highest (536 mmol O2 L1) for the RILs 147 and 34 whereas it was the lowest (476 mmol O2 L1) for RILs 104, 83 and 29. However for the RILs 115 and 83 the COP was the highest under Pdeficiency (Fig. 2). The maximal Conr was the highest for the RILs 115 and 104 with more than 1000 mmol O2 h1 plant1 whereas it was the lowest for the RILs 147 and 83 with about 945 mmol O2 h1 plant1 under P-sufficiency. However, P-deficiency induced a large decrease in Corn for all RILs though to most extent for the RILs 147 and 83. At ambient pO2 (250 mmol O2 L1), the Conr varied between both RILs and P supplies. It was increased under P-sufficiency for the P-efficient RILs 115 (4%) and 104 (13%) and decreased under Pdeficiency for the P-inefficient RILs 147 (11%) and 83 (9%) whereas for the P-inefficient RIL29 there were no differences in Conr between P supplies. The nodule permeability is calculated as the slope of the response of Conr as a function of pO2 as explained in Jebara et al. (2005). P deficiency decreased significantly the nodule permeability for the P-inefficient RILs 147 (23%) and 29 (26%), and by contrast this parameter was not significantly affected for the Pefficient RILs 115 (10%), 104 (6%) and 34 (8%). 3.3. Relationship between nodule phytase activity and permeability to O2 diffusion In order to assess whether phytase activity was involved in the regulation of the nodule respiration linked with SNF, the correlation between nodule phytase activity and the nodule permeability to O2 diffusion was examined (Fig. 3). Under P-sufficiency, a significant correlation between nodule permeability and phytase activity was found for the P-efficient RILs 115 (R2 ¼ 0.73*), 34 (R2 ¼ 0.74*) and 104 (R2 ¼ 0.96***) and for the P-inefficient RILs 147, 83 and 29 (Fig. 3). Although both nodule phytase activity and respiration were significantly increased under P-deficiency, the relation between these two parameters varied among RILs. The phytase activity in nodules of the P-efficient RILs 104 (R2 ¼ 0.91***), 34 (R2 ¼ 0.72*) and 115 (R2 ¼ 0.61*) was significantly correlated with their nodule O2 permeability but this was not the case for the Pinefficient RILs 147 (R2 ¼ 0.32), 83 (R2 ¼ 0.33) and 29 (R2 ¼ 0.25). 3.4. Efficiency in use of the rhizobial symbiosis
Fig. 1. Phytase activity (A) and inorganic P concentration (B) in nodules of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white) versus deficient (gray) P supply. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. Mean values labeled with the same letter were not significantly different at P < 0.05.
To assess the efficiency in use of the rhizobial symbiosis (EURS), i.e., the ratio of SNF-dependent growth of shoot per unit of nodule biomass, the values of the shoot biomass were plotted as a function of those of nodule biomass, with the slope of the linear regression of the curves shown in Fig. 4 being considered as an estimate of the EURS.
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Fig. 2. Nodule O2 consumption (Conr) of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white circle) versus deficient (gray circle) P supply. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. *Significant difference at P < 0.05.
Under P-sufficiency, the EURS ranged between 9.6 g sDW g1 nDW and 11.4 g sDW g1 nDW for the RILs 34 and 147, respectively. However, it was comparable for the RILs 83 and 29 with a mean of 9.6 g sDW g1 nDW. Under P-deficiency, the EURS increased for all RILs, although this increase was significantly higher for the P-efficient RILs 115 and 104 (ca 22%) than for the P-inefficient RILs 147 and 83 (ca 15%). 4. Discussion Although recent studies have addressed the effects of P-deficiency on nodule O2 permeability, little attention has been given to the relationship between phytase activity and nodule respiration. In this study, the observation of an increase in nodule O2 permeability for each increase of phytase activity in nodules of common bean (Fig. 3) opens new insights into understanding the physiology of N2-fixing legumes as well as requirements for N2 fixation and regulation of nodule permeability to O2 diffusion. Indeed, the nodule permeability controls the nodule respiration that supplies ATP for N2 reduction catalyzed by the bacteroidal nitrogenase within the nodule infected zone (Hunt and Layzell, 1993; Kouas et al., 2008). The response-curves in Fig. 2 confirm that the nodule energetic metabolism supporting N2-fixation is O2 limited (Ribet and Drevon, 1995; Serraj et al., 1994). This O2 limitation is attributed to the physical limitation to the O2 diffusion through the nodule cortex of which the variations would be dependent on cell
deformation of external or internal nodule cortex (Vadez et al., 1996; Serraj et al., 1995). Increased nodule permeability to O2 might be a response to enhanced O2-limitation due to wasteful O2 alternative respiration in the nodule under P-deficiency (Millar et al., 1997), or the result of a direct effect of P-deficiency on nodule permeability to O2 which subsequently induces alternative respiratory pathways to scavenge excess O2 that would inactivate nitrogenase. In P-deficient nodules the adenylate charge, at least of the plant fraction, appears to decrease (Sa and Israel, 1991). Thus the high O2 uptake might contribute to maintaining a sufficient adenylate charge for high N2 fixation rates. Based on the fact that nodule O2 permeability was reported to increase under P-deficiency in soybean (Ribet and Drevon, 1995) and common bean (Vadez et al., 1996), and the ability of alfalfa to tolerate P-deficiency by forming small nodules with high O2 permeability (Schulze and Drevon, 2005), this supports an increase in O2 consumption per unit N2 fixed and P content. The positive correlation between phytase enzyme activity and nodule respiration (Fig. 3) substantiates a physiological role of phytase in regulating nodule O2 diffusion. This may suggest a role of phytase in the accumulation of Pi in vacuoles of the inner cortex that would induce an increase in the size of these cells which has been associated with an increase in nodule respiration (Drevon et al., 1998). This is in agreement with nodule enzyme APases stimulation and especially the elevated levels of ascorbate peroxidase transcript in nodule cortex of common bean, chickpea and
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Fig. 3. Relation of phytase activity with O2 permeability in nodules of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white circle) versus deficient (gray circle) P supply. The nodule O2 permeability is calculated as the ratio of the slope of the regression between Conr and external pO2. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. *Significant difference at P < 0.05; **significant difference at P < 0.01; ***significant difference at P < 0.001.
alfalfa (Bargaz et al., 2013; Molina et al., 2011; Dalton et al., 1998). The elevated APases activities may likely coincide with high nodule P requirement and depletion of the vacuolar Pi pool. This points to an intracellular mechanism of Pi utilization. Furthermore, phytase gene expression in nodule cortex (Lazali et al., 2013b) and the regulation of the nodule respiration since particularly the inner cortex is postulated to be a physical barrier for the regulation of nodule permeability to gas diffusion (Witty and Minchin, 1998) and osmotic conditions (Schulze and Drevon, 2005). In addition, the higher EURS under P-deficiency for the P-efficient RILs 115 and 104 than for the P-inefficient RILs 147 and 83 (Fig. 4) may correspond to higher nodule demands for respiration, in relation to nodulation and biomass yield with the increase in nodule conductance to O2 diffusion for N2 fixation. Those genotypes having higher EURS were most efficient in PUE for SNF. Indeed, an interactive effect between nodulation, nodule respiration and SNF could exist based on the hypothesis assuming that O2 diffusion into the nodules is tightly regulated and is the principal regulatory factor for N2 fixation (Schulze, 2004). Nevertheless, the increases in phytase enzyme activity was linked to a decrease in nodule P content, which may subsequently serve for ATP generation via O2 respiration, with at least 16 ATPs consumed per N2 reduced (Salsac et al., 1984). Recent studies have reported that stimulation of several APase, such as phytase (Lazali et al., 2013b) and
phosphoenolpyruvate phosphatase (Bargaz et al., 2012) within common bean nodules is considered as an adaptive mechanism to tolerate P-deficient conditions. This suggest that phytase may be involved in the mobilization of the internal P and its utilization for SNF. The link of phytase activity to EURS, particularly for the Pefficient RILs 115 and 34 under P-deficiency (Fig. 4), suggests tight regulation between EURS and the nodule Pi requirement (Fig. 1B), probably in relation with the high energy requirement of the SNF process. However, the efficient RILs that were able to moderate their decrease in P contents under P-deficiency, and allocate a larger percentage of plant P to the nodules, also maintained their nodule permeability to O2 diffusion (Fig. 2). This is consistent with the correlation between the P content of nodules and the nodule permeability to O2 diffusion (Bargaz et al., 2011). In conclusion, the increase of phytase activity in nodules of common bean under P-deficiency could be attributed to the maintenance of high nodule respiration. Differences in phytase activity among RILs points to the need for further investigations of mechanisms responsible for Pi distribution in nodule and its relation with nodule O2 permeability that is assumed to regulate the N2 fixation process. Understanding the role of nodule respiration and phytase activity in response to environmental constraints may be important in developing strategies to improve the tolerance of nodulated beans to low P soils.
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Fig. 4. Efficiency in use of the rhizobial symbiosis of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white circle) versus deficient (gray circle) P supply. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. *Significant difference at P < 0.05; **significant difference at P < 0.01; ***significant difference at P < 0.001.
Acknowledgments This work was supported by the Great Federative Project FABATROPIMED, financed by Agropolis Fondation under the reference ID 1001-009 and the framework of Algeria-French cooperation AUF-PCSI 63113PS012. The authors are grateful to Catherine Pernot and Josiane Abadie (INRA Montpellier, France) for their technical assistance. Contributions Mohamed Lazali developed the experimental design, and was responsible for the preparation of the manuscript. Jean-Jacques Drevon conceived the study, coordinated its realization and supervised the preparation of the manuscript. All authors of the present work have read and approved the final manuscript. References Almeida, J.P.F., Hartwig, U.A., Frehner, M., Nosberger, J., Luscher, A., 2000. Evidence that P deficiency induces N feedback regulation of symbiotic N2 fixation in white clover (Trifolium repens L.). J. Exp. Bot. 51, 1289e1297. Araujo, A.P., Plassard, C., Drevon, J.J., 2008. Phosphatase and phytase activities in nodules of common bean genotypes at different levels of phosphorus supply. Plant Soil. 312, 129e138.
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M. Lazali, J.J. Drevon / Plant Physiology and Biochemistry 80 (2014) 53e59 Hunt, S., Layzell, D.B., 1993. Gas exchange of legume nodules and the regulation of nitrogenase activity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 483e511. Jebara, M., Drevon, J.J., 2001. Genotypic variation in nodule conductance to the oxygen diffusion in common bean (Phaseolus vulgaris). Agronomie 21, 667e674. Jebara, M., Aouani, M.E., Payre, H., Drevon, J.J., 2005. Nodule conductance varied among common bean (Phaseolus vulgaris) genotypes under phosphorus deficiency. J. Plant Physiol. 162, 309e315. Kouas, S., Alkama, N., Abdelly, C., Drevon, J.J., 2008. Proton efflux by nodulated roots varies among common-bean genotypes (Phaseolus vulgaris) under phosphorus deficiency. J. Plant Nutr. Soil. Sci. 171, 242e248. Kouas, S., Louche, J., Debez, A., Plassard, C., Drevon, J.J., Abdelly, C., 2009. Effect of phosphorus deficiency on acid phosphatase and phytase activities in common bean (Phaseolus vulgaris L.) under symbiotic nitrogen fixation. Symbiosis 47, 141e149. Lazali, M., Bargaz, A., Carlsson, G., Ounane, S.M., Drevon, J.J., 2013. Discrimination against 15N among recombinant inbred lines of Phaseolus vulgaris L. contrasting in phosphorus use efficiency for nitrogen fixation. J. Plant Physiol.. http:// dx.doi.org/10.1016/j.jplph.2013.07.009. Lazali, M., Zaman-Allah, M., Amenc, L., Ounane, G., Abadie, J., Drevon, J.J., 2013. A phytase gene is over-expressed in root nodules cortex of Phaseolus vulgarisrhizobia symbiosis under phosphorus deficiency. Planta 238, 317e324. Li, C., Gui, S., Yang, T., Walk, T., Wang, X., Liao, H., 2012. Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis. Ann. Bot. 109, 275e285. Millar, A.H., Finnegan, P.M., Whelan, J., Drevon, J.J., Day, D.A., 1997. Expression and kinetics of the mitochondrial alternative oxidase in nitrogen-fixing nodules of soybean roots. Plant Cell. Environ. 20, 1273e1282. Minchin, F.R., 1997. Regulation of oxygen diffusion in legume nodules. Soil. Biol. Biochem 29, 881e888. Molina, C., Zaman-Allah, M., Khan, F., Fatnassi, N., Horres, R., Rotter, B., Steinhauer, D., Amenc, L., Drevon, J.J., Winter, P., Kahl, G., 2011. The saltresponsive transcriptome of chickpea roots and nodules via deepSuperSAGE. BMC Plant Biol. 11, 31. Patriarca, E.J., Tate, R., Ferraioli, S., Iaccarino, M., 2004. Organogenesis of legume root nodules. Int. Rev. Cytol. 234, 201e262. Peoples, M.B., Herridge, D.F., Ladha, J.K., 1995. Biological nitrogen fixation: an efficient source of nitrogen for sustainable agricultural production. Plant Soil. 174, 3e28.
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Ribet, J., Drevon, J.J., 1995. Increase in permeability to oxygen and in oxygen uptake of soybean nodules under limiting phosphorus nutrition. Physiol. Plant. 94, 298e304. Rodríguez, H., Fraga, R., 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol. Adv. 17, 319e339. Rotaru, V., Sinclair, T.R., 2009. Interactive influence of phosphorus and iron on nitrogen fixation by soybean. Environ. Exp. Bot. 66, 94e99. Sa, T., Israel, D.W., 1991. Energy status and functioning of phosphorus deficient soybean nodules. Plant Physiol. 97, 928e935. Salsac, L., Drevon, J.J., Zengbe, M., Cleyet-Marel, J.C., Obaton, M., 1984. Energy requirement of symbiotic nitrogen fixation. Physiol. Veg. 22, 509e521. Schulze, J., 2004. How are nitrogen fixation rates regulated in legumes? J. Plant Nutr. Soil. Sci. 167, 125e137. Schulze, J., Drevon, J.J., 2005. P-deficiency increases the O2 uptake per N2 reduced in alfalfa. J. Exp. Bot. 56, 1779e1784. Serraj, R., Roy, G., Drevon, J.J., 1994. Salt stress induces a decrease in the oxygen uptake of soybean nodules and in their conductance to oxygen diffusion. Physiol. Plant. 91, 161e168. Serraj, R., Fleurat-Lessard, P., Jaillard, B., Drevon, J.J., 1995. Structural changes in the inner cortex cells of soybean root nodules are induced by short-term exposure to high salt or oxygen concentrations. Plant Cell. Environ. 18, 455e462. Thumfort, P.P., Layzell, D.B., Atkins, C.A., 1999. Diffusion and reaction of oxygen in the central tissue of ureide producing legume nodules. Plant, Cell. Environ. 22, 1351e1363. Vadez, V., Rodier, F., Payre, H., Drevon, J.J., 1996. Nodule permeability to O2 and nitrogenase-linked respiration in bean genotypes varying in the tolerance of N2 fixation to P deficiency. Plant Physiol. Biochem 34, 871e878. Witty, J.F., Minchin, F.R., 1998. Hydrogen measurements provide direct evidence for a variable physical barrier to gas diffusion in legume nodules. J. Exp. Bot. 49, 1015e1020. Yadav, R.S., Tarafdar, J.C., 2003. Phytase and phosphatase producing fungi in arid and semi-arid soils and their efficiency in hydrolyzing different organic P compounds. Soil. Biol. Biochem 35, 745e751. Zahran, H.H., 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol. Mol. Biol. Rev. 63, 968e989.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
The nodule conductance to O2 diffusion increases with phytase activity in N2-fixing Phaseolus vulgaris L Mohamed Lazali a, b, *, Jean Jacques Drevon b a
Université de Khemis Miliana, Faculté des Sciences de la Nature et de la Vie & des Sciences de la Terre, Route Theniet El Had, 44225 Khemis Miliana, Algerie Institut National de la Recherche Agronomique, UMR Ecologie Fonctionnelle & Biogéochimie des Sols et Agroécosystèmes, INRA-IRD-CIRAD-SupAgro, Place Pierre Viala, 34060 Montpellier, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2013 Accepted 22 March 2014 Available online 1 April 2014
To understand the relationship between phosphorus use efficiency (PUE) and respiration for symbiotic nitrogen fixation (SNF) in legume nodules, six recombinant inbred lines of common bean (RIL Phaseolus vulgaris L.), contrasting in PUE for SNF, were inoculated with Rhizobium tropici CIAT899, and grown under hydroaeroponic culture with sufficient versus deficient P supply (250 versus 75 mmol P plant1 week1). At the flowering stage, the biomass of plants and phytase activity in nodules were analyzed after measuring O2 uptake by nodulated roots. Our results show that the P-deficiency significantly increased the phytase activity in nodules of all RILs though with highest extent for RILs 147, 29 and 83 (ca 45%). This increase in phytase activity was associated with an increase in nodule respiration (ca 22%) and in use of the rhizobial symbiosis (ca 21%). A significant correlation was found under P-deficiency between nodule O2 permeability and phytase activity in nodules for RILs 104, 34 and 115. This observation is to our knowledge the first description of a correlation between O2 permeability and phytase activity of a legume nodule. It is concluded that the variation of phytase activity in nodules can increase the internal utilization of P and might be involved in the regulation of nodule permeability for the respiration linked with SNF and the adaptation to P-deficiency. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Legume Phaseolus vulgaris Phosphorus deficiency Phytase Respiration Root-nodule Symbiosis
1. Introduction The formation of N2-fixing nodules on roots is a feature shared by many legume species. These nodules are the result of a symbiosis with soil bacteria called rhizobia, resulting in the ability of the plant to use atmospheric N2 (Patriarca et al., 2004). The symbiotic nitrogen fixation (SNF) can act as a renewable and environmentally sustainable source of nitrogen, and complement or replace fertilizer inputs (Peoples et al., 1995; Herridge et al., 2008). However, such environmental factors as drought, salinity, and low soil P (Graham and Vance, 2003) are major constraints worldwide for legumes production, especially in arid, semi-arid, and tropical
Abbreviations: Conr, consumption of oxygen by the nodulated-roots; COP, critical oxygen pressure; DAT, days after transplantation; N, nitrogen; nDW, nodule dry weight; P, Phosphorus; pO2, partial pression of oxygen; RIL, recombinant inbred line; sDW, shoot dry weight. * Corresponding author. Institut National de la Recherche Agronomique, UMR Ecologie Fonctionnelle & Biogéochimie des Sols et Agroécosystèmes, Place Pierre Viala, 34060 Montpellier, France. Tel.: þ33 (0) 4 99 61 23 32; fax: þ33 (0) 4 99 61 21 19. E-mail address: [email protected] (M. Lazali). http://dx.doi.org/10.1016/j.plaphy.2014.03.023 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.
ecosystems, and particularly for common bean (Phaseolus vulgaris L.), often in developing countries (Zahran, 1999). The sensitivity of N2-fixing plants to P-deficiency has been widely documented. It is generally attributed to the large amount of P-dependent carbon and energy turnover required in nodule metabolism (Schulze and Drevon, 2005). The impaired SNF in Pdeficient plants is usually explained by an effect of the low P supply on the growth of the host plant or the rhizobia and their interactions (Almeida et al., 2000). Thus, plant growth requires more P with N2 fixation than with nitrate assimilation for most legume species. This is attributed to the high energy requirements for reduction of atmospheric N2 by nitrogenase (Rotaru and Sinclair, 2009) which is presumed to be regulated by nodule O2 permeability (Schulze and Drevon, 2005). Nodule O2 permeability is though to be involved in the regulation of N2 fixation (Hunt and Layzell, 1993). Since nitrogenase, the enzyme responsible for N2 fixation, is O2 labile, legume nodules have evolved mechanisms to down regulate their O2 content, including oxygenated leghemoglobin gradients for the fine control of O2 levels in infected cells (Thumfort et al., 1999), though a physical barrier to gas diffusion in the inner cortex seems to be the primary site for the regulation of nodule permeability to gas
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diffusion (Hunt and Layzell, 1993; Minchin, 1997; Galvez et al., 2000). Moreover, nodule O2 permeability was reported to increase under P-deficiency in soybean (Ribet and Drevon, 1995) and common bean (Vadez et al., 1996). Alfalfa also adapts to P-deficiency by forming small nodules with high O2 permeability which enables an increase in O2 consumption per unit N2 fixed and P content (Schulze and Drevon, 2005). In N2-fixing nodules, recent studies have suggested that bean plants subjected to P-deficiency increased the activities of phosphatases (APases) and phytase in nodules (Araujo et al., 2008; Kouas et al., 2009; Bargaz et al., 2013). In this context, Li et al. (2012) also reported high expression of large number of purple acid phosphatases genes in the nodules of soybean under low-P conditions. These results suggest that this response could contribute to improved nutrition of legumes under these limiting conditions. Indeed, phytase is one of the most active APases for the sequential hydrolysis of phytate to a series of myo-inositol phosphate derivatives and inorganic phosphate (Pi) (Rodríguez and Fraga, 1999; Yadav and Tarafdar, 2003). It might play a major role for internal plant metabolism and thus may contribute to P use efficiency (PUE) for SNF. The involvement of APases in the tolerance of various abiotic and biotic constraints, led us to an assumption that phytase may have a multiplicity of functions as well as those related to phosphate metabolism and should provide new insights into adaptation to P-deficiency. Thus, this study is among the first reports investigating the correlation between nodule respiration and phytase activity of a legume. It is also hypothesized that increase in nodule phytase activity of common bean regulates nodule respiration and plays a key role in P-deficiency tolerance for N2 fixation of the legume-rhizobia symbiosis. 2. Methods 2.1. Plant growth and nodulation conditions This study was carried out with six recombinant inbred lines, namely RILs 147, 115, 104, 83, 34 and 29, originating from the International Center of Tropical Agriculture (CIAT, Cali, Colombia). These RILs were selected from a previous screen among 100 progenies of the crossing, in 1996, of two parental RILs, namely BAT477 and DOR364. Under conditions of P deficiency, RILs 115, 104 and 34 were characterized as P-efficient genotypes, while RILs 147, 83 and 29 as P-inefficient genotypes respectively, based on plant growth and seed yield in relation of SNF-derived to P availability (Drevon et al., 2011). Seeds were surface sterilized with 3% calcium hypochlorite for 10 min and rinsed with 5 washings of sterile distilled water before germination. Five days after sowing, the roots of selected uniform seedlings were inoculated with the Rhizobium tropici CIAT899 grown in liquid yeast extract mannitol medium at 28 C for 3 days to an approximate cell density of 109 mLe1. Thereafter, the seedlings were transferred into hydroaeroponic culture consisting of vats filled with 40 L of nutrient solution, which were aerated intensely and arranged in a fully randomized block design. Cotton wool was fitted at the hypocotyl level to maintain the root system suspended in the nutrient solution with either 75 or 250 mmol P per plant per week, defined as P-deficient or P-sufficient supplies, respectively (Lazali et al., 2013a). Urea was supplied as 2 mM per plant into the nutrient solution during the initial 2 weeks of growth in order to optimize nodulation (Hernandez and Drevon, 1991). Thereafter, the plants were grown in N-free nutrient solution. At the beginning of the third week of culture, plants were transferred to 1 L glass bottles containing nutrient solution and wrapped in aluminum foil to ensure a dark plantrooting environment. The nutrient solution was first replaced at 15 days after transplantation (DAT) and each week, subsequently.
The nutrient solution pH was adjusted to around 7 by adding 0.2 g L1 CaCO3 and the medium was aerated by an air flow of ambient air compressed to 400 mL min1 plant1. The experiment was carried out in a glasshouse under natural light with day/night temperatures 28/20 C and 16 h photoperiod with 70% relative humidity and additional illumination of 400 mmol photons m2 s1 during the day. 2.2. Nodulated-root O2 uptake measurements The in situ measurement of O2 uptake by the nodulated roots (Conr) was performed with a Witt Logger Oxymeter (Abiss, Villemoisson, France) between 35 and 42 DAT, before their harvest. One day before the measurement of Conr, the level of the nutrient solution in the bottle was reduced to one-third of the volume so that the whole nodule population was in direct contact with the gas phase. The circulation of the gaseous phase in the circuit from the nodulated-root environment through the oxymeter was driven by peristaltic pump with a flow rate of 400 mL min1. Successive measurements were performed with an initial pO2 of 21, 25, 30, 35, 40, 45 and 50 kPa O2 corresponding to 250, 298, 358, 417, 476, 536 and 596 mmol O2 L1, respectively. Nodulated root O2 uptake was determined by measuring the rate of decline in O2 concentration in the gas surrounding the nodulated roots during a 30 min period after closing the system. The consumption of O2 was calculated as Conr ¼ [(initial - final) pO2] [V/24.2] [60/t] with : t in min, duration between initial and final O2 measurement; pO2 in % of the atmospheric pressure; V in L, volume of gas phase; 24.2 in L, volume of 1 mol pure gas in experimental conditions (Schulze and Drevon, 2005). The Conr was calculated in mmol O2 consumed h1 plant1 and nodule permeability (mm3 h1) was defined as the slope of the linear part of the regression of Conr (mmol O2 h1) as a function of external O2 concentrations (mmol O2 L1). The critical oxygen pressure (COP) was calculated as the first derivative of the curvilinear regression of Conr as a function of pO2. 2.3. Phytase activity assay Samples of nodules of 3 mm diameter of each plant corresponding to 60 mg of nodules fresh weight were carefully detached from roots at 42 DAT. Each nodule sample was ground in vibrating mill (FastPrepÒe24) with an extraction mixture consisting of 900 mL sodium acetate-buffer (50 mM pH 5.5) containing 5 mM dithiotrietol. After centrifugation at 22 000 g during 6 min, 3 aliquots of 100 mL of the supernatant were incubated during 90 min at 37 C with a mixture of 300 mL sodium acetate-buffer (0.2 M pH 5.5) and 100 mL of phytic acid (10 mM pH 5.5). The reaction was stopped by the 0.5 mL addition of 10% trichloroacetic acid and centrifugation at 20 000 g during 6 min. Another 100 mL aliquot received the same buffer and amount of phytic acid substrate, but the reaction was stopped immediately without incubation and the mixture centrifuged. Concentration of Pi in the extracts was measured spectrophotometrically at 630 nm using malachite green. The phytase activity was calculated as the difference between the Pi in the extracts with and without incubation and expressed in nmol of Pi released per min per g of nodule fresh mass. 2.4. Biomass measurements The plants were harvested at 42 DAT after gas exchange measurements and separated in shoots, roots and nodules. After drying for 72 h at 65 C, plant and nodule dry weight (DW) was measured and used to determine the efficiency in use of the rhizobial symbiosis (EURS) estimated as the slope of the regression model i.e.,
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y ¼ ax þ b of plant biomass as a function of nodule biomass (Drevon et al., 2011). 2.5. Statistical analysis Experimental design was a randomized complete block. The R software (2.14.1) was used to perform the statistical analyses of biomass, correlation analyses, and regressions of gas exchange parameters as a function of external pO2. The analysis of variance and the standard deviation of the means were used to determine the significance (P < 0.05) of differences in symbiotic effectiveness. 3. Results 3.1. Phytase activity and nodule Pi concentration Under P-sufficiency the phytase activity in nodules was significantly higher for RILs 115, 104 and 34 (ca 21 nmol min1 g1 nFW) than for RILs 147, 83 and 29 (ca 14 nmol min1 g1 nFW) (Fig. 1A). Pdeficiency increased significantly the phytase activity, though to a higher extent for RILs 147 and 29 (ca 44%) than for RILs 115 and 34 (ca 27%). Under P-sufficiency the P-efficient RIL115 had the significantly highest nodule Pi concentration (13 mmol Pi g1 nFW), whereas the
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significantly lowest value was observed for P-inefficient RILs 147 and 83 (9 mmol Pi g1 nFW) (Fig. 1B). Under P-deficiency, the nodule Pi concentration decreased for all RILs though this decrease was significantly the highest for the P-inefficient RILs 29 and 83 (ca 40%). 3.2. Nodule permeability to O2 diffusion The response of O2 uptake by nodulated roots to variation of rhizospheric pO2 was measured in order to assess the effect of Pdeficiency on nodule respiration, following the protocol described by Jebara and Drevon (Jebara and Drevon, 2001). Data in Fig. 2 show that Conr increased with an increase in rhizospheric pO2 until a COP beyond which the O2 diffusion within nodules was no longer stimulatory for respiration. Independently of P supplies, the COP was the highest (536 mmol O2 L1) for the RILs 147 and 34 whereas it was the lowest (476 mmol O2 L1) for RILs 104, 83 and 29. However for the RILs 115 and 83 the COP was the highest under Pdeficiency (Fig. 2). The maximal Conr was the highest for the RILs 115 and 104 with more than 1000 mmol O2 h1 plant1 whereas it was the lowest for the RILs 147 and 83 with about 945 mmol O2 h1 plant1 under P-sufficiency. However, P-deficiency induced a large decrease in Corn for all RILs though to most extent for the RILs 147 and 83. At ambient pO2 (250 mmol O2 L1), the Conr varied between both RILs and P supplies. It was increased under P-sufficiency for the P-efficient RILs 115 (4%) and 104 (13%) and decreased under Pdeficiency for the P-inefficient RILs 147 (11%) and 83 (9%) whereas for the P-inefficient RIL29 there were no differences in Conr between P supplies. The nodule permeability is calculated as the slope of the response of Conr as a function of pO2 as explained in Jebara et al. (2005). P deficiency decreased significantly the nodule permeability for the P-inefficient RILs 147 (23%) and 29 (26%), and by contrast this parameter was not significantly affected for the Pefficient RILs 115 (10%), 104 (6%) and 34 (8%). 3.3. Relationship between nodule phytase activity and permeability to O2 diffusion In order to assess whether phytase activity was involved in the regulation of the nodule respiration linked with SNF, the correlation between nodule phytase activity and the nodule permeability to O2 diffusion was examined (Fig. 3). Under P-sufficiency, a significant correlation between nodule permeability and phytase activity was found for the P-efficient RILs 115 (R2 ¼ 0.73*), 34 (R2 ¼ 0.74*) and 104 (R2 ¼ 0.96***) and for the P-inefficient RILs 147, 83 and 29 (Fig. 3). Although both nodule phytase activity and respiration were significantly increased under P-deficiency, the relation between these two parameters varied among RILs. The phytase activity in nodules of the P-efficient RILs 104 (R2 ¼ 0.91***), 34 (R2 ¼ 0.72*) and 115 (R2 ¼ 0.61*) was significantly correlated with their nodule O2 permeability but this was not the case for the Pinefficient RILs 147 (R2 ¼ 0.32), 83 (R2 ¼ 0.33) and 29 (R2 ¼ 0.25). 3.4. Efficiency in use of the rhizobial symbiosis
Fig. 1. Phytase activity (A) and inorganic P concentration (B) in nodules of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white) versus deficient (gray) P supply. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. Mean values labeled with the same letter were not significantly different at P < 0.05.
To assess the efficiency in use of the rhizobial symbiosis (EURS), i.e., the ratio of SNF-dependent growth of shoot per unit of nodule biomass, the values of the shoot biomass were plotted as a function of those of nodule biomass, with the slope of the linear regression of the curves shown in Fig. 4 being considered as an estimate of the EURS.
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Fig. 2. Nodule O2 consumption (Conr) of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white circle) versus deficient (gray circle) P supply. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. *Significant difference at P < 0.05.
Under P-sufficiency, the EURS ranged between 9.6 g sDW g1 nDW and 11.4 g sDW g1 nDW for the RILs 34 and 147, respectively. However, it was comparable for the RILs 83 and 29 with a mean of 9.6 g sDW g1 nDW. Under P-deficiency, the EURS increased for all RILs, although this increase was significantly higher for the P-efficient RILs 115 and 104 (ca 22%) than for the P-inefficient RILs 147 and 83 (ca 15%). 4. Discussion Although recent studies have addressed the effects of P-deficiency on nodule O2 permeability, little attention has been given to the relationship between phytase activity and nodule respiration. In this study, the observation of an increase in nodule O2 permeability for each increase of phytase activity in nodules of common bean (Fig. 3) opens new insights into understanding the physiology of N2-fixing legumes as well as requirements for N2 fixation and regulation of nodule permeability to O2 diffusion. Indeed, the nodule permeability controls the nodule respiration that supplies ATP for N2 reduction catalyzed by the bacteroidal nitrogenase within the nodule infected zone (Hunt and Layzell, 1993; Kouas et al., 2008). The response-curves in Fig. 2 confirm that the nodule energetic metabolism supporting N2-fixation is O2 limited (Ribet and Drevon, 1995; Serraj et al., 1994). This O2 limitation is attributed to the physical limitation to the O2 diffusion through the nodule cortex of which the variations would be dependent on cell
deformation of external or internal nodule cortex (Vadez et al., 1996; Serraj et al., 1995). Increased nodule permeability to O2 might be a response to enhanced O2-limitation due to wasteful O2 alternative respiration in the nodule under P-deficiency (Millar et al., 1997), or the result of a direct effect of P-deficiency on nodule permeability to O2 which subsequently induces alternative respiratory pathways to scavenge excess O2 that would inactivate nitrogenase. In P-deficient nodules the adenylate charge, at least of the plant fraction, appears to decrease (Sa and Israel, 1991). Thus the high O2 uptake might contribute to maintaining a sufficient adenylate charge for high N2 fixation rates. Based on the fact that nodule O2 permeability was reported to increase under P-deficiency in soybean (Ribet and Drevon, 1995) and common bean (Vadez et al., 1996), and the ability of alfalfa to tolerate P-deficiency by forming small nodules with high O2 permeability (Schulze and Drevon, 2005), this supports an increase in O2 consumption per unit N2 fixed and P content. The positive correlation between phytase enzyme activity and nodule respiration (Fig. 3) substantiates a physiological role of phytase in regulating nodule O2 diffusion. This may suggest a role of phytase in the accumulation of Pi in vacuoles of the inner cortex that would induce an increase in the size of these cells which has been associated with an increase in nodule respiration (Drevon et al., 1998). This is in agreement with nodule enzyme APases stimulation and especially the elevated levels of ascorbate peroxidase transcript in nodule cortex of common bean, chickpea and
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Fig. 3. Relation of phytase activity with O2 permeability in nodules of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white circle) versus deficient (gray circle) P supply. The nodule O2 permeability is calculated as the ratio of the slope of the regression between Conr and external pO2. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. *Significant difference at P < 0.05; **significant difference at P < 0.01; ***significant difference at P < 0.001.
alfalfa (Bargaz et al., 2013; Molina et al., 2011; Dalton et al., 1998). The elevated APases activities may likely coincide with high nodule P requirement and depletion of the vacuolar Pi pool. This points to an intracellular mechanism of Pi utilization. Furthermore, phytase gene expression in nodule cortex (Lazali et al., 2013b) and the regulation of the nodule respiration since particularly the inner cortex is postulated to be a physical barrier for the regulation of nodule permeability to gas diffusion (Witty and Minchin, 1998) and osmotic conditions (Schulze and Drevon, 2005). In addition, the higher EURS under P-deficiency for the P-efficient RILs 115 and 104 than for the P-inefficient RILs 147 and 83 (Fig. 4) may correspond to higher nodule demands for respiration, in relation to nodulation and biomass yield with the increase in nodule conductance to O2 diffusion for N2 fixation. Those genotypes having higher EURS were most efficient in PUE for SNF. Indeed, an interactive effect between nodulation, nodule respiration and SNF could exist based on the hypothesis assuming that O2 diffusion into the nodules is tightly regulated and is the principal regulatory factor for N2 fixation (Schulze, 2004). Nevertheless, the increases in phytase enzyme activity was linked to a decrease in nodule P content, which may subsequently serve for ATP generation via O2 respiration, with at least 16 ATPs consumed per N2 reduced (Salsac et al., 1984). Recent studies have reported that stimulation of several APase, such as phytase (Lazali et al., 2013b) and
phosphoenolpyruvate phosphatase (Bargaz et al., 2012) within common bean nodules is considered as an adaptive mechanism to tolerate P-deficient conditions. This suggest that phytase may be involved in the mobilization of the internal P and its utilization for SNF. The link of phytase activity to EURS, particularly for the Pefficient RILs 115 and 34 under P-deficiency (Fig. 4), suggests tight regulation between EURS and the nodule Pi requirement (Fig. 1B), probably in relation with the high energy requirement of the SNF process. However, the efficient RILs that were able to moderate their decrease in P contents under P-deficiency, and allocate a larger percentage of plant P to the nodules, also maintained their nodule permeability to O2 diffusion (Fig. 2). This is consistent with the correlation between the P content of nodules and the nodule permeability to O2 diffusion (Bargaz et al., 2011). In conclusion, the increase of phytase activity in nodules of common bean under P-deficiency could be attributed to the maintenance of high nodule respiration. Differences in phytase activity among RILs points to the need for further investigations of mechanisms responsible for Pi distribution in nodule and its relation with nodule O2 permeability that is assumed to regulate the N2 fixation process. Understanding the role of nodule respiration and phytase activity in response to environmental constraints may be important in developing strategies to improve the tolerance of nodulated beans to low P soils.
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Fig. 4. Efficiency in use of the rhizobial symbiosis of six common bean recombinant inbred lines inoculated with R. tropici CIAT899 and grown under sufficient (white circle) versus deficient (gray circle) P supply. Data are mean and standard deviation of seven replicates harvested at 42 days after transplantation. *Significant difference at P < 0.05; **significant difference at P < 0.01; ***significant difference at P < 0.001.
Acknowledgments This work was supported by the Great Federative Project FABATROPIMED, financed by Agropolis Fondation under the reference ID 1001-009 and the framework of Algeria-French cooperation AUF-PCSI 63113PS012. The authors are grateful to Catherine Pernot and Josiane Abadie (INRA Montpellier, France) for their technical assistance. Contributions Mohamed Lazali developed the experimental design, and was responsible for the preparation of the manuscript. Jean-Jacques Drevon conceived the study, coordinated its realization and supervised the preparation of the manuscript. All authors of the present work have read and approved the final manuscript. References Almeida, J.P.F., Hartwig, U.A., Frehner, M., Nosberger, J., Luscher, A., 2000. Evidence that P deficiency induces N feedback regulation of symbiotic N2 fixation in white clover (Trifolium repens L.). J. Exp. Bot. 51, 1289e1297. Araujo, A.P., Plassard, C., Drevon, J.J., 2008. Phosphatase and phytase activities in nodules of common bean genotypes at different levels of phosphorus supply. Plant Soil. 312, 129e138.
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