Effect of Water Stress on Nodule Physiology and Biochemistry of a Drought Tolerant Cultivar of Common Bean (Phaseolus vulgaris L.)

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1 Annals of Botany 83: 57 63, 1999 Article No. anbo , available on line at Effect of Water Stress on Nodule Physiology and Biochemistry of a Drought Tolerant Cultivar of Common Bean (Phaseolus vulgaris L.) M. L. G. RAMOS*, A. J. GORDON, F. R. MINCHIN, J. I. SPRENT and R. PARSONS * EMATER-GO, CP 331, , Goiania-GO, Brasil, Institute of Grassland and Enironmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK and Uniersity of Dundee, Department of Biological Sciences, Dundee, DD1 4HN, UK Received: 7 July 1998 Returned for revision: 19 August 1998 Accepted: 27 September 1998 Plants of EMGOPA-21, a drought tolerant cultivar of common bean (Phaseolus ulgaris), were maintained either at 9% soil field capacity () or stressed by reducing to 7, 5 or 3% over a 1 d period. Plant dry weight was not affected by any of these treatments although the number and weight of nodules was reduced at 5 and 3%. Nitrogenase activity, determined by the acetylene reduction assay (ARA), was also reduced, on a plant basis, at 5% and was almost stopped at 3%. The latter treatment caused a marked increase in nodule O diffusion resistance and induced nodule senescence. A time-course analysis of the 1 d 3% treatment showed a decrease in leaf water potential from 5 to87 MPa by 8 d, with a cessation of dry weight increase after 3 d, when leaf water potential was 65 MPa. Proteins in the host plant fraction of nodules decreased to 5% of control values by 1 d and leghaemoglobin (Lb) content was also lower at this stage. The activity of sucrose synthase (SS) showed a 76% reduction between 3 and 6 d, whilst glutamate synthase (GOGAT) activity showed a 4% reduction. The activity of other key enzymes of carbon metabolism was also reduced after 1 d. Nodule sucrose content increased to double that of control nodules by 6 d, before declining back to control levels at 1 d. Starch content fell by 3 d and continued to fall throughout the stress period. The results are discussed in terms of drought tolerance strategies in relation to growth and metabolism in whole plants and nodules Annals of Botany Company Key words: Phaseolus ulgaris, common bean, water stress, nitrogen fixation, oxygen diffusion, acetylene reduction, enzyme activity, carbon metabolism. INTRODUCTION Common bean (Phaseolus ulgaris L.) is a major source of protein in the developing world, but plant growth and yield are often reduced by periods of water stress. There are now studies showing different tolerances to water stress amongst common bean cultivars (e.g. Pimentel et al.,199). Nitrogen fixation appears to be particularly sensitive to this stress (Kirda, Danso and Zapata, 1989). It has been known for several years that water stress induces a decrease in O diffusion in legume nodules (Pankhurst and Sprent, 1975, 1976; Durand, Sheehy and Minchin, 1987; Davey and Simpson, 199), but more recent studies have also suggested the involvement of a metabolic component (Guerin, Trichant and Rigaud, 199; Diaz del Castillo, Hunt and Layzell, 1994; Diaz del Castillo and Layell, 1995 Purcell and Sinclair, 1995; Serraj and Sinclair, 1996) which may be related to the activity of sucrose synthase (SS; Gonza lez et al., 1995, Gordon et al., 1997). However, when nitrogen fixing legumes are subjected to drought the leaves normally show a marked decline in water potential and it is not obvious which function of the stressed plant is actually affecting the nodule (cf. Streeter, 1993). Indeed, with soybean, leaves seemed to suffer more from water stress than nodules (Gonza lez et al., 1995). Fur- For correspondence. Fax (44) , r.parsonsdundee.ac.uk $3. thermore, none of these studies have been conducted with a legume cultivar which is recognized as being drought tolerant. The Phaseolus ulgaris cultivar EMGOPA-21 has moderate drought tolerance and in an experimental comparison with cultivars Turrialba 4 and Rio Tibagi, EMGOPA-21 had a significantly lower reduction in yield when exposed to conditions of moderate water deficit during cultivation (Fiegenbaum et al., 1991). EMGOPA- 21 showed comparatively lower reduction in plant height, pod length, number of seeds per pod and mean seed weight (Fiegenbaum et al., 1991). These authors did not examine the role of nitrogen fixation in this drought response. The object of this paper is to examine the effects of water stress on the physiology and biochemistry of nodules of EMGOPA-21 as a drought tolerant cultivar of Phaseolus ulgaris and to compare strategies of nodule protection with those previously reported for soybean. MATERIALS AND METHODS Seeds and inoculum Seeds of common bean, cultivar EMGOPA-21 were surface sterilized in 8% ethanol for 3 sec, 5% sodium hypochlorite for 2 min and then washed ten times in sterilized distilled water. Seeds were pre-germinated in Petri dishes lined with sterile, wet filter paper for 2 d and transferred to pots containing a 1:1 mixture of sand and vermiculite. Pots 1999 Annals of Botany Company

2 58 Ramos et al. Nodule Physiology of a Water Stress Tolerant Cultiar of Phaseolus were 124 cm high and 91 cm in diameter, giving a total volume of 8 cm with a free air volume of 3 cm. Seeds were inoculated with Rhizobium tropici, strain CIAT 899. Plant growth conditions Plants were grown in Saxcil controlled environment cabinets with a 14 h photoperiod, a quantum irradiance of approx. 6 µmol photon m s (PAR) and daynight temperatures of 2515 C. Each day plants received a nutrient solution (which contained no mineral N) until the onset of the drought treatments. Initially, two seedlings were planted per pot and these were thinned to one seedling after 5 d. The soil field capacity () of the sand: vermiculite mixture was determined by weighing. Values of per cent were calculated for each pot by determining as the pot mass when saturated with water and the total available water by subtracting the mass of the dry pot and rooting media. Pots were kept at 9% for 25 d until four drought treatments were started: control (9% ), 7, 5 and 3%, with six replicates for each. Drought was initiated by withholding water, the pots were then weighed daily and, once they had reached the required, they were maintained at that level by the addition of appropriate volumes of nutrient solution. The length of time required to reach the for each treatment was 2 d for 7%, 5 d for 5% and 8 d for 3%. Physiological measurements and plant harvesting were conducted 1 d after the start of the drought stress. A parallel set of plants (six replicates per treatment) was used to determine leghemoglobin and soluble protein in the nodules. In a second experiment the time-course of the 3% treatment was followed, with plants being harvested for leaf water potential determinations and nodule analyses after 3, 6, 8 and 1 d. Control plants (9% ) were harvested at and 1 d. Harvested nodules (1 3 g f. wt) were removed from the plants, stored briefly on ice, surface-dried with tissue paper, weighed, frozen in liquid N and stored at 8 C. Leaf water potential of the second youngest fully expanded leaf was measured 8 h after the beginning of the photoperiod with a pressure chamber (Scholander et al., 1965). Shoots, roots and the remaining nodules were weighed after drying for 48 h at 7 C. Gas exchange measurements Acetylene reduction activity (ARA) and root respiration of intact roots were measured using a flow-through gas system (Minchin et al., 1983) housed in a Fisons cabinet, which provided the same light and temperature conditions as described earlier. Root systems were sealed into the growth pots, allowed to stabilize for h in a gas stream of air enriched to 5 cm m CO, then exposed to a gas stream containing 1% (vv) C H and 21% (wv) O. Respiratory CO production was measured by infra-red gas analysis and C H production was measured by gas chromatography using a flame ionization detector. Maximum rates of ARA were determined from the maximum rate of C production prior to the C -induced decline. The extent of this decline was reduced with increasing periods of drought. After 45 min, O levels were altered in stepped decreases of approx. 2% (i.e. 19, 17, 15, 13 and 11%) and measurements were taken after 6 min, i.e. at the maximum effect of the 2% change. Acetylene was present in the gas stream throughout these determinations. This allowed sufficient variation in CO and C production for determination of nitrogenase-linked respiration (see below). Following this treatment O levels were reduced to 1% and after steady-state conditions had been reached were then increased over the range 1 to 3% (49 to 1227 mol O m ) in steps of 5% O to permit the investigation of the response of the nodule oxygen diffusion barrier. Following each increase in O concentration, there was a 25 to 3 min equilibration period to allow new steadystate conditions to be reached. Carbon costs of nitrogenase activity and nitrogenaselinked respiration (NLR) were determined from the slope of the linear relationship between nodulated-root respiration and C reduction. This was based on changes which occurred in both parameters during the C -induced decline, the O down-stepping and the O up-stepping (Witty, Minchin and Sheehy, 1983). These data were then used to calculate resistance values (R) using an exponential curve-fitting routine for NLR against external oxygen concentration which involves a modified equation for Fick s first law of diffusion (Minchin et al., 1992). This allows for the calculation of an additional respiration factor which, when added to NLR, represents the total flux of oxygen across the diffusion barrier (F, see Minchin et al., 1992 for full details). Root respiration data obtained immediately before exposure to C were used for calculation of the oxygen diffusion resistance in air (R-C ). Extraction and assay of enzymes Nodules were homogenized in a mortar and pestle with 5 mol m MOPS, 1 mol m DTT, 4 mol m MgCl, ph 7 at 2 C (5 cm g f. wt). The homogenate was centrifuged for 3 min at 2 g at 2 C. Samples of the supernatant were immediately assayed for phosphoenol pyruvate carboxylase (PEPC) activity (Gonza lez et al., 1995), and 1 cm aliquots were desalted by low speed centrifugation (18 g, 1 min) through 5 cm columns of Bio Gel P6DG (BioRad) equilibrated with 5 mol m MOPS ph 7, 4 mol m MgCl buffer. The desalted extract was used to assay protein (Lowry et al., 1951), leghaemoglobin (LaRue and Child, 1979) and the following enzymes: aspartate amino transferase (AAT), sucrose synthase (SS), alkaline invertase (AI), aldolase, phosphofructokinase (PFK), pyrophosphate fructose-6-p phosphotransferase (PFP), glutamate synthase (GOGAT) and glutamine synthetase (GS) (Gordon, 1991; Gonza lez et al., 1995; Romanov et al., 1995). The extraction of bacteroid proteins, western immunoblotting of SS, leghaemoglobin (Lb) and nitrogenase components 1 and 2, and the extraction and analyses of carbohydrates and nitrogenous compounds were carried out as described in Gonza lez et al. (1995).

3 Ramos et al. Nodule Physiology of a Water Stress Tolerant Cultiar of Phaseolus 59 Statistical analysis Results were examined by one-way analysis of variance. All effects discussed in this study were significant at P 5 using Duncan s multiple range test. RESULTS Decreasing the soil field capacity () to 7 or 5% for 1 d had no effect on shoot or total plant d. wt, whilst root d. wt was higher in comparison with control plants maintained at 9% (Table 1). A reduction of to 3% resulted in a lower shoot d. wt, but greater root d. wt, such that total plant d. wt was not significantly different from control plants. Nodule d. wt, f. wt and total numbers were lower due to exposure of the plants to s of 5 and 3% for 1 d. Nitrogenase activity (ARA) was decreased, on a plant basis, by a 5% and was almost stopped by a 3% (Table 2). The decrease at 5% was mainly due to a reduction in number and weight of nodules compared to the controls (Table 1) as ARA per g nodule d. wt was not significantly different (Table 2). At 3%, both number TABLE 1. Effects of different water stress regimes on plant growth parameters of common bean c. EMGOPA-21 Parameter 9% * Water stress treatment 7% 5% 3% Plant d. wt (g) 179b 222a 187ab 15b Shoot d. wt (g) 115ab 139a 113ab 88b Root d. wt (g) 42b 64a 59a 64a Nodule d. wt (g) 21a 19a 15b 1b Nodule f. wt (g) 172a 148a 16b 68c Number of nodules 517a 452a 361b 234c *, Soil field capacity. Measurements were made at 1 d. Results are means of six replicates. Values in the same row with the same superscript do not differ statistically at 5% by Duncan s multiple range test. T ABLE 2. Effects of different water stress regimes on nitrogenase actiity and O diffusion resistance of nodules of common bean c. EMGOPA-21. Parameter* 9% Water stress treatment 7% 5% 3% ARA per plant min 71a 75a 41b 5c ARA g nodule d. wt 332ab 391a 279b 5c min R(-CH ) 121a 129a 161a 49b * ARA, Acetylene reduction activity; R, O diffusion resistance., Soil field capacity. R values for 3% are means of three replicates only. Measurements were made at 1 d. Results are means of six replicates. Values in the same row with the same superscript do not differ statistically at 5% by Duncan s multiple range test. of nodules and nodule specific activity were markedly lower than in control plants. Visual observations of the nodules from 3% plants showed that the majority appeared green or brown in colour. Oxygen diffusion resistance in air (R-C ) was not significantly affected by 7 or 5% but showed a marked increase at 3%. In a separate experiment, plants exposed to a water stress down to 3% were examined after, 3, 6, 8 and 1 d. Leaf water potential in droughted plants declined from 5 MPa (controls) to 87 MPa after 8 d at 3% (Fig. 1). Furthermore, in droughted plants there was no increase in total d. wt after day 3, in contrast with control plants which increased from 125 to 35 g over the 1 d period (Fig. 1). Cessation of growth was attributable entirely to the effect on shoot d. wt, which occurred when leaf water potential was only decreased to 65 MPa. Drought caused a gradual decline in the nodule host plant protein content, although this was only significantly different from control values at day 1 (Fig. 2). At this stage, protein was reduced by 5% in comparison with controls. The level of leghaemoglobin per mg protein remained more-or-less constant for most of the time course but was significantly lower at day 1. Although the specific activity of a number of host plant enzymes was not markedly affected by drought (for example, AAT, PFP, PFK and aldolase), others were (Fig. 2). Sucrose synthase declined by 76% between 3 and 6 d, and GOGAT by 4%. Others (PEPC, GS and AI) were only significantly lower than controls between days 8 and 1. Immunoblotting confirmed that these changes in enzyme activities were the result of declines in the amounts of enzyme proteins (Fig. 3). Thus, SS protein was markedly lower at day 6 in comparison with day 3, and GS protein was noticeably lower only at day 1. In the case of nitrogenase, component 2 was more sensitive to the stress treatment than component 1, since it was hardly detectable after 6 d of drought (Fig. 3). Component 1, although present at day 1 was noticeably reduced. The most significant changes in nodule metabolites as a result of drought stress were in the levels of sucrose and starch (Fig. 4). Sucrose increased significantly in the first 3 d, and more than doubled in amount by 6 d, before declining again to control levels. Starch levels fell dramatically within 3 d and continued to decline throughout the 1 d of drought. Neither of the hexoses was significantly affected by the drought treatment and although total amino acids increased somewhat this was not significantly greater than control levels (data not shown). DISCUSSION In previous experiments it was observed that the drought tolerant cultivar EMGOPA-21 showed no alterations in relative water content or leaf area when leaf water potential was reduced to 78 MPa and leaves showed no visible signs of wilt. In general, plants at 9 and 7% soil field capacity () had similar stomatal conductances, whilst plants at 3% showed a sharp decrease to values two to three times lower than those of well watered plants (Ramos, 1996).

4 6 Ramos et al. Nodule Physiology of a Water Stress Tolerant Cultiar of Phaseolus A 3.5 B Leaf water potential (MPa) Total d. wt (g) C 2.5 D Shoot d. wt (g) Root d. wt (g) FIG. 1. Time course of leaf water potential (A) and total (B), shoot (C) and root plus nodule (D) dry weight of control () and drought stressed plants () throughout the 1 d period during which plants were exposed to water stress down to 3%. Results are meanss.e. (n 3). As shown by this study, a 1 d exposure to 5% had little effect on plant growth or nodule activity. Indeed, when plants of this cultivar were exposed to 3% the effects on leaf water potential and plant d. wt were still relatively small. However, 3% had marked effects on number and activity of nodules, with many appearing to be senescent. The time-course study of the 3% drought stress period showed that, in comparison with data for soybean (Gonza lez et al., 1995; Gordon et al., 1997), the decrease in leaf water potential would indicate a relatively mild stress. Indeed, in contrast to soybean, leaves of EMGOPA-21 did not show evidence of wilting during the 1 d stress period. Given this apparently mild effect on the leaves, and the ability of the 5% plants to maintain nodule activity, the most surprising results for the 3% plants concern the rapid loss of nodule function. In particular, SS activity showed marked declines between 3 and 6 d, whilst PEPC showed a gradual decline throughout the 1 d period. This is in contrast to the effects of a more severe water stress on soybean (in terms of leaf water potential) where SS activity showed a gradual decline and PEPC was unaffected (Gonza lez et al., 1995; Muller, Boller and Wiemken, 1996; Gordon et al., 1997). The declineinss activity for Phaseolus nodules was mirrored by a loss of nitrogenase component 2, despite the fact that overall bacteroid protein was not affected and host plant protein, including Lb, only declined between days 8 and 1. These results are in accordance with the measured decline in N fixation and indicate that the effect of 3% probably occurred between 3 and 6 d after beginning the treatment. This preceded the decline in Lb, which Guerin et al. (199, 1991) proposed to be the cause of the decline in N fixation in Vicia faba. It is possible that the decline in N fixation, and in particular nitrogenase component 2, is related to the biochemical dysfunction caused by the down regulation of SS and the consequent reduced ability of the nodules to metabolize sucrose. This is in accord with the increase in nodule sucrose content during the first 6 d.

5 Ramos et al. Nodule Physiology of a Water Stress Tolerant Cultiar of Phaseolus PROTEIN.3 Lb mg g 1 f. wt 8 mg mg 1 protein PEPC.4 GOGAT Units mg 1 protein SS.3 Al Units mg 1 protein GS Units mg 1 protein FIG. 2. Time course of nodule plant protein content, leghaemoglobin (Lb) content and the activities of phosphoenolpyruvate (PEPC), glutamate synthase (GOGAT), sucrose synthase (SS), alkaline invertase (AI) and glutamine synthetase (GS) in nodules of control () and drought stressed plants () throughout the 1 d period during which plants were exposed to water stress down to 3%. One unit 1 µmol of enzymic substrate or product. Results are meanss.e. (n 3). The results from the first experiment also indicate that nodule resistance to gaseous diffusion was much higher at the 3% level of water stress. This would have had a significant effect on internal O concentrations. However, as noted by Gordon et al. (1997), anaerobiosis in legume nodules does not result in increased expression of SS, which is often the case in other plant systems at low O concentrations. Nevertheless, the combination of reduced

6 62 Ramos et al. Nodule Physiology of a Water Stress Tolerant Cultiar of Phaseolus SS GS Lb N2ase2 N2ase1 C D3 D6 D8 D1 C1 FIG. 3. Western immunoblots of host plant (SS, GS and Lb) and bacteroid (nitrogenase 1 and 2) nodule protein from control (C) and drought stressed (D) plants at days, 3, 6, 8 and 1 throughout the period during which plants were exposed to water stress down to 3%. Equal amounts of protein were loaded in each lane. O flux and reduced ability to metabolize sucrose is the likely cause of reduced N fixation and nitrogenase content. The lower GOGAT activities at day 6 could also be a contributory factor in reducing nodule metabolism. This decline was also greater than that in soybean nodules which only showed a 14% decrease after 6 d drought (Gordon et al., 1997). However, in agreement with soybean nodules (Gonza lez et al., 1995), the activity of GS, which is the primary enzyme of ammonia assimilation, was not affected by drought. The response of Phaseolus nodules to 3% was also similar to that of soybean in relation to sucrose and starch levels (Gonza lez et al., 1995; Gordon et al., 1997). In both cases, the remobilization of starch and the increase in sucrose content of nodules were some of the earliest indicators of stress over the first 3 d, and possibly represented an osmotic adjustment. During this period starch mobilization could account for the increase in sucrose levels. Thereafter, however, sucrose levels rose as a result of other factors. The most obvious explanation is that sucrose continued to be imported into nodules but, because SS activity was much reduced, it could not be effectively metabolized. Different stresses can cause different responses in carbohydrate levels of Phaseolus nodules. For example, in another cultivar, dark treatment brought about rapid, major declines in sucrose and hexose levels and only a gradual decline in starch content (Gogorcena et al., 1997). In that case, however, the dramatic reduction in N fixation appeared to be largely due to a rapid depletion in sucrose. With drought, carbohydrate supply appears to have continued, but the primary effect on nodule function may have been the increase in the resistance to gaseous diffusion and the down regulation of key genes encoding enzymes vital to nodule metabolism (cf. Gordon et al.,1997). The signalsinvolved in these processes remain to be identified. In conclusion, accepting that Phaseolus ulgaris cultivar EMGOPA-21 is drought tolerant, our results clearly show that it is able to maintain both leaf and nodule integrity over a 1 d period of water stress down to 5%. However, under more severe water stress conditions, leaf integrity is maintained but shoot growth ceases after 3 d and at this stage nodule integrity also begins to be lost. This response to more severe drought may well be a programmed survival strategy evolved to retain leaf activity but reduce the costs of nodule activity during the recovery period. ACKNOWLEDGEMENTS We thank C. L. James (IGER, Aberystwyth) for excellent technical support and Dr Euan James for valuable suggestions. Maria Lucrecia Ramos was a recipient of a grant from CNPq Brasil. IGER is funded through BBSRC. 2 A 8 B Sucrose (mg g 1 d. wt) 1 Starch (mg g 1 d. wt) FIG. 4. Time course of sucrose (A) and starch (B) content in nodules of control () and drought stressed plants () throughout the 1 d period during which plants were exposed to water stress down to 3%. Results are meanss.e. (n 3).

7 Ramos et al. Nodule Physiology of a Water Stress Tolerant Cultiar of Phaseolus 63 LITERATURE CITED Davey AG, Simpson RJ Nitrogen fixation by subterranean clover at varying stages of nodule dehydration. II. Efficiency of nitrogenase functioning. Journal of Experimental Botany 41: Diaz del Castillo L, Layzell DB Drought stress, permeability to O diffusion and the respiratory kinetics of soybean root nodules. Plant Physiology 17: Dia del Castillo L, Hunt S, Layzell DB The role of oxygen in the regulation of nitrogenase activity in drought-stressed soybean nodules. Plant Physiology 16: Durand JL, Sheehy JE, Minchin FR Nitrogenase activity, photosynthesis and nodule water potential in soybean plants experiencing water deprivation. Journal of Experimental Botany 38: Fiegenbaum V, Dossantos DSB, Mello VDC, Filho BGD, Tillmann MAA, Dasilva JB Influence of water deficit on the yield component of 3 bean cultivars. Pesquisa Agropecuaria Brasileira 26: Gogorcena Y, Gordon AJ, Ecuredo PR, Minchin FR, Witty JF, Moran JF, Becana M N fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean plants. Plant Physiology 113: Gonza lez EM, Gordon AJ, James CL, Arrese-Igor C The role of sucrose synthase in the response of soybean nodules to drought. Journal of Experimental Botany 46: Gordon AJ Enzyme distribution between the cortex and the infected region of soybean nodules. Journal of Experimental Botany 42: Gordon AJ, Minchin FR, Skøt L, James CL Stress-induced declines in soybean N fixation are related to nodule sucrose synthase activity. Plant Physiology 114: Guerin V, Trichant JC, Rigaud J Nitrogen fixation (C reduction) by broad bean (Vicia faba L.) nodules and bacteroids under water-stress restricted conditions. Plant Physiology 92: Guerin V, Pladys D, Trichant J, Rigaud J Proteolysis and nitrogen fixation in faba bean (Vicia faba) nodules under water stress. Physiologia Plantarum 82: Kirda C, Danso SKA, Zapata F Temporal water-stress effects on nodulation, nitrogen accumulation and growth of soybean. Plant and Soil 12: LaRue TA, Child JJ Sensitive fluorometric assay for leghaemoglobin. Analytical Biochemistry 92: Lowry OH, Roseborough NJ, Farr AL, Randall RJ Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: Minchin FR, Witty JF, Sheehy JE, Muller M Major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions. Journal of Experimental Botany 34: Minchin FR, Iannetta PPM, Fernandez-Pascual M, de Lorenzo C, Witty JF, Sprent JI A new procedure for the calculation of oxygen diffusion resistance in legume nodules from flow-through gas analysis data. Annals of Botany 55: Muller J, Boller T, Wiemken A Pools of non-structural carbohydrates in soybean root nodules during water stress. Physiologia Plantarum 98: Pankhurst CE, Sprent JI Effects of water stress on the respiratory and nitrogen-fixing activity of soybean root nodules. Journal of Experimental Botany 26: Pankhurst CE, Sprent JI Effects of temperature and oxygen tension on the nitrogenase and respiratory activities of turgid and water-stressed soybean and french bean root nodules. Journal of Experimental Botany 27: 1 9. Pimentel C, Jacob-Neto J, Goi SR, Pessanha GG Estresse hidrico em cultivars de Phaseolus ulgaris L. em simbiose com o Rhizobium leguminosarum biovar phaseoli. Turrialba 4: Purcell LC, Sinclair TR Nodule gas-exchange and water potential response to rapid imposition of water-deficit. Plant, Cell and Enironment 18: Ramos MLG Physiological responses of crop legumes to water stress. PhD Thesis. University of Dundee, Dundee, UK. Romanov VI, Gordon AJ, Minchin FR, Witty JF, Skøt L, James CL, Borisov AY, Tikhonovich IA Anatomy, physiology and biochemistry of root nodules of Spring-2 Fix, a symbiotically defective mutant of pea (Pisum satium L.). Journal of Experimental Botany 46: Scholander PF, Hammel HT, Bradstreet ED, Hemmingsen EA Sap pressure in vascular plants. Science 148: Serraj R, Sinclair TR Inhibition of nitrogenase activity and nodule oxygen permeability by water deficit. Journal of Experimental Botany 47: Streeter JG Translocation. A key factor limiting the efficiency of nitrogen fixation in legume nodules. Physiologia Plantarum 87: Witty JF, Minchin FR, Sheehy JE Carbon costs of nitrogenase activity in legume root nodules determined using acetylene and oxygen. Journal of Experimental Botany 34: