JXB Advance Access originally published online on January 16, 2006
Journal of Experimental Botany 2006 57(3):665-673; doi:10.1093/jxb/erj056
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RESEARCH PAPER |
Drought effects on carbon and nitrogen metabolism of pea nodules can be mimicked by paraquat: evidence for the occurrence of two regulation pathways under oxidative stresses
Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra, Campus de Arrosadía, E-31006 Pamplona, Spain
* To whom correspondence should be addressed. E-mail: cesarai{at}unavarra.es
Received 4 July 2005; Accepted 11 November 2005
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Abstract |
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Biological nitrogen fixation (BNF) is dramatically affected by environmental constraints such as water stress or heavy metals. It has been reported that these stresses induce the over-production of reactive oxygen species (ROS) and, in turn, oxidative stress that may be responsible for the above-mentioned BNF decline at the molecular level. Oxidative stress, occurring under different environmental stresses, has been widely related to physiological damage. However, a direct relationship between oxidative stress and the decline of BNF, independently from any other cellular damage resulting from adverse environmental situations, has yet to be demonstrated. In order to study the likely in vivo relationship between ROS and BNF inhibition in the legume–Rhizobium symbiosis, two paraquat (PQ) doses, 1 (LPQ) and 10 (HPQ) mmol m–3, were applied to pea roots for 96 h in order to exacerbate ROS production. Whole-plant physiology and nodule metabolism parameters were determined every 24 h to monitor the evolution of plant responses to ROS. LPQ provoked BNF decline, which was preceded by a prior decrease in sucrose synthase (SS) activity. However, HPQ gave rise to a faster and more pronounced BNF inhibition, which coincided with a decline in SS and also with a reduction in leghaemoglobin (Lb) content. These results indicate a likely involvement of ROS in the effects of environmental stresses on BNF. Furthermore, these results support the occurrence of two regulation pathways for BNF under oxidative stress, one of these involving carbon shortage and the other involving Lb/oxygen flux.
Key words: Leghaemoglobin, nitrogen fixation, nodule metabolism, oxidative stress, paraquat, Pisum sativum L., ROS signalling, sucrose synthase
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Introduction |
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Biological nitrogen fixation (BNF) carried out by the legume–Rhizobium symbiosis contributes to agricultural sustainability providing the required N input to agroecosystems. Legume–Rhizobium symbiosis is based in the carbon/nitrogen exchange between both partners. Sucrose, the main photoassimilate provided by shoot to nodules, is predominantly hydrolysed by sucrose synthase (SS), leading to fructose and UDP-glucose production. These hexoses enter the glycolytic pathway providing carbon skeletons, predominantly in the form of malate, both for bacteroid respiration and for nitrogenase activity maintenance.
BNF is an extremely complex biological process, which is very sensitive to a wide range of environmental stresses (Sprent et al., 1988
; Zahran, 1999). It has been suggested that regulation of BNF in legume–Rhizobium symbiosis may be dependent on several factors: carbon supply, oxygen availability and N-feedback regulation. Although the supply of assimilates to nodules or current photosynthesis does not regulate BNF under non-stress conditions (Vance and Heichel, 1991), under conditions of mild water stress, nodule SS activity sharply declines (González et al., 1995; Gordon et al., 1997), thus limiting the carbon flux to bacteroid respiration (Gálvez et al., 2005). Oxygen supply appears to be a limiting factor for nodule functioning, and it has also been put forward as a controlling factor for BNF under a wide range of environmental stresses, with reports of an increase in gas diffusion resistance (Minchin, 1997; Denison, 1998). Evidence of phloem N involvement on BNF regulation has also been shown (Neo and Layzell, 1997; Vadez et al., 2000). However, it has not yet been completely clarified which of these: C, O or N, is ultimately responsible, or whether they act in a co-operative way to regulate the legume BNF process (Schulze, 2004). González et al. (2001b) suggested a model with at least two different control pathways within nodules in response to changes in the environment that involve a double carbon/oxygen switch. In this way, moderate and gradual environmental stresses would trigger a down-regulation of SS, whilst abrupt stresses would involve an Lb/oxygen-related control of BNF. Little is known of the signal transduction pathway that links stress perception and the decline of nitrogen fixation and nodule metabolism. The pathway involving SS decline is not sensitive to abscisic acid (ABA), whereas ABA has been shown to trigger a decrease of Lb content in nodules (González et al., 2001a).
Reactive oxygen species (ROS) are continuously produced in plants as by-products of aerobic metabolism (Dalton, 1995). Nitrogen-fixing root nodules exhibit very high respiration rates to maintain the energy and carbon supply that allow the functioning of nitrogenase. The high Lb concentration in the cytosol of infected cells, along with the abundance of catalytic Fe, and the presence of several redox proteins that have the capacity to transfer electrons to O2, together enhance nodule capacity to generate ROS (Becana et al., 2000). Production and removal of ROS must be strictly controlled; as such plants have developed a complex array of non-enzymatic and enzymatic detoxification mechanisms. Nevertheless, the equilibrium between production and scavenging of ROS may be disrupted by a number of environmental constraints such as water stress, pathogens, herbicides, high irradiance or high temperatures leading to oxidative stress situations and thus, cell damage (Apel and Hirt, 2004).
The aim of this work was to analyse the effect of oxidative stress on nodule metabolism and BNF in order to determine a likely role of ROS on BNF regulation under ROS-generating environmental constraints. Plant growth, photosynthesis, nodule antioxidant metabolites and key nodule C/N metabolism parameters were determined in pea plants subjected to two root-applied low concentrations of paraquat.
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Materials and methods |
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Experimental procedures and growth conditions
Pea seeds (Pisum sativum L. cv. Sugar-snap) were surface-sterilized (Labhilili et al., 1995
) and inoculated with Rhizobium leguminosarum biovar. viciae strain NLV8. Single plants were grown in 1 dm3 pots in a 1:1 (v:v) perlite:vermiculite mixture in a controlled environment chamber (22/18 °C day/night temperature, 70% relative humidity, 500 µmol m–2 s–1 (PPF), and 15 h photoperiod), with a N-free nutrient solution (Rigaud and Puppo, 1975). Four-week-old plants were transferred to 1 dm3 hydroponic tanks 4 d prior to the beginning of the treatment under the same environmental conditions.
A screening of paraquat doses supplied to the nutrient solution was performed in order to find out the concentrations that generated an oxidative stress situation in nodule metabolism prior to paraquat being translocated to shoots. Plants were divided randomly into three sets, which were exposed respectively to 0 (controls), 1 (LPQ), or 10 (HPQ) mmol m–3 paraquat (Aldrich Cat.: 85,617-7). The highest concentration used in this study represents one-tenth of the concentrations commonly used for foliar treatments (Iturbe-Ormaetxe et al., 1998) or the lowest concentration used so far in nodules (Dalton, 1992). Physiological parameters were determined at 0, 24, 48, 72, and 96 h after application. Fresh nodule aliquots were immediately frozen in liquid nitrogen and stored at –80 °C for analytical determinations. Plants shoot, roots, and nodules were weighed separately and the dry weights were determined after drying for 48 h at 80 °C.
Gas exchange and chlorophyll content measurements
Net CO2 assimilation rate was measured with a portable IRGA (Li-6200, Li-Cor, Lincoln, NE) in the youngest fully expanded leaf. Chlorophyll content was measured in the same leaf with a Minolta SPAD-502. For apparent nitrogenase activity (ANA) determinations, the plant root systems were sealed and H2 evolution was measured in an open flow-through system under N2:O2 (79:21%) in accordance with Witty and Minchin (1998) using an electrochemical H2 sensor (Qubit System Inc., Canada). The H2 sensor was calibrated with high purity gases (Praxair, Madrid, Spain) using a gas mixer (Air Liquid, Madrid, Spain) flowing at the same rate as the sampling system (500 cm3 min–1).
Extraction and assay of enzymes
Nodules were homogenized in a mortar and pestle with 50 mol m–3 MOPS pH 7, 20% PVPP, 10 mol m–3 DTT, 10 mol m–3 2-mercaptoethanol, 1 mol m–3 EDTA, 20 mol m–3 KCl, 5 mol m–3 MgCl2, at 0–2 °C (5 cm3 g–1 fresh weight). The homogenate was centrifuged for 30 min at 20 000 g, 4 °C.
An aliquot of the supernatant was retained for plant fraction protein (Bradford, 1976) and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31
[EC]
) assay. The rest of the supernatant was desalted by low speed centrifugation (180 g, 1 min) through Bio Gel P6DG columns (Bio-Rad) equilibrated with 50 mol m–3 MOPS pH 7, 20 mol m–3 KCl, and 5 mol m–3 MgCl2. The desalted extract was used to measure leghaemoglobin content (Appleby and Bergersen, 1980) and the following enzyme activities according to González et al. (1998): sucrose synthase (SS; EC 2.4.1.13
[EC]
), alkaline invertase (AI; EC 3.2.1.26
[EC]
), UDP-glucose pyrophosphorylase (UDPGPP; EC 2.7.7.9
[EC]
), malate dehydrogenase (MDH; EC 1.1.1.37
[EC]
), glutamate synthase (GOGAT; EC 1.4.1.14
[EC]
), and aspartate aminotransferase (AAT; EC 2.6.1.1
[EC]
). NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42
[EC]
) activity was assayed according to Ferri et al. (2000).
In order to assess whether a contamination of the nodule plant fraction might occur from bacteroid proteins and to monitor whether PQ may affect the nitrogenase content of bacteroids, bacteroid protein was extracted as described by González et al. (1995). Aliquots of host plant and bacteroid protein extracts equivalent to an equal amount of protein were loaded on to SDS-PAGE and blotted onto nitrocellulose membrane for the inmunodetection of component 1 of nitrogenase. Nitrogenase was not detected in the host plant protein extracts, discounting a contamination of the measured enzyme activities from those of bacterial origin. Furthermore, there were no changes in the level of component 1 at the end of the experiment between controls, LPQ, and HPQ bacteroids.
Extraction and determination of low molecular mass antioxidants
Frozen nodules (200 mg) were crushed with liquid nitrogen to a fine power and subsequently homogenized with 1.5 cm3 of ice-cold 2% metaphosphoric acid, and 1 mol m–3 EDTA (Schützendübel et al., 2002). The homogenate was centrifuged (4400 g, 4 °C, 2 min) and filtered (Millex GV, 0.22 µm).
Antioxidants were analysed by high-performance capillary electrophoresis (CE) in a Beckman Coulter PACE system 5500 (Beckman Instruments, Fullerton, CA, USA) with an associated diode array detector, as described by Herrero-Martínez et al. (2000). The CE instrument was equipped with the P/ACE station software for instrument control and data handling. The background buffer was 60 mol m–3 NaH2PO4 (pH 7) containing 60 mol m–3 NaCl and 0.0001% hexadimethrin bromide. The applied potential was 15 kV, and the capillary tubing (50 µm) was 30/37 cm long. The UV detection wavelength was set at 200 nm and 265 nm. Ascorbic acid (ASC) and reduced glutathione (GSH) were determined directly by injecting an aliquot of the sample extract in the CE as described above. In order to determine dehydroascorbate (DHA) and oxidized glutathione (GSSG), aliquots of nodule extracts were reduced with DTT as described by Davey et al. (2003), then total ASC and total GSH were analysed directly by CE. DHA and GSSG levels were determined as the difference between total ASC and total GSH and the levels of their respective reduced forms. Ascorbate 2-phosphate was used as the internal standard. Recovery of externally added reduced forms of ascorbate and glutathione was always greater than 92%, whilst that of oxidized forms was greater than 98%.
Carbohydrate extraction and determination
Fresh nodules (around 200 mg) were prepared for sucrose extraction in boiling 80% (v/v) ethanol. Ethanol-soluble extracts were dried in a Turbovap LV evaporator (Zymark Corp, Hopkinton, MA, USA) and soluble compounds were redissolved with 4 cm3 of distilled water, mixed, and centrifuged at 20 000 g for 10 min. The supernatant was immediately frozen and stored at –80 °C until its use for sucrose determination. The ethanol-insoluble residue was extracted for starch as in MacRae (1971).
Sucrose of the supernatant fraction and glucose produced from starch hydrolysis were analysed by high-performance capillary electrophoresis as above, with a background buffer of 10 mol m–3 benzoate (pH 12) containing 0.5 mol m–3 myristyltrimethylammonium bromide (MTAB). The applied potential was –15 kV, and the capillary tubing was 50 µm i.d. and 31.4/38.4 cm long. The indirect UV detection wavelength was set at 225 nm.
Statistical analysis
Results were examined by two-way analysis of variance, using Fisher's protected least significant difference (LSD) tests between means. All results discussed in this study were significant at P 
0.05 between treatments at a given day.
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Results |
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Plant growth, expressed as shoot and root dry weight, was not significantly affected after 96 h of both PQ treatments (Table 1). Nodule dry weight experienced nearly a 50% reduction at 96 h following application (Table 1) in HPQ plants, although no differences were found until 72 h (data not shown). Nodule dry weight of LPQ plants was not affected. The photosynthetic rate declined 40% at 48 h of HPQ treatment (Fig. 1A). This reduction was related to stomatal closure leading to a decrease in intercellular CO2 concentration (data not shown). In addition, a 20% chlorophyll content decline occurred at 72–96 h after PQ application (Fig. 1B). Neither CO2 assimilation rate nor chlorophyll content differed from control plants in LPQ plants (Fig. 1). Water status was not affected during the whole experiment in both PQ treatments (data not shown).
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) between 1 mmol m–3 PQ (LPQ) and HPQ.PQ provoked a 50% ASC content decline and an 80% DHA content increase in nodules at 96 h of application (Table 2). ASC+DHA pool size was significantly higher in PQ-treated plants than in the controls (Table 2). ASC content represented 40% of the ASC+DHA pool in controls, whilst in PQ-treated plants it only reached 15% (Table 2). Thus the ASC/(ASC+DHA) ratio declined by 65% in both PQ treatments with respect to the controls (Table 2).
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GSSG increased around 2.5-fold in the HPQ treatment, whereas in LPQ it did not vary (Table 2). GSH content was 25% higher in LPQ treatment, whilst HPQ plants did not differ from control plants. Thus, the GSH+GSSG pool was significantly higher in LPQ, whilst no differences were found in HPQ. The GSH/(GSH+GSSG) ratio only declined in the HPQ treatment due to the great differences in GSSG content (Table 2).
The apparent nitrogenase activity (ANA) was almost completely inhibited after 48 h following HPQ application, whilst LPQ plants only experienced a significant decline at 96 h after PQ application (Fig. 2). This decline of ANA occurred despite the fact that PQ supply did not affect the nitrogenase content of bacteroids (data not shown).
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The leghaemoglobin (Lb) content in LPQ plants only declined significantly at 96 h after application (Fig. 3A), whilst HPQ treatment provoked a decline of Lb content that was significant within the first 24 h after application; this was actually the first monitored parameter that was affected in HPQ plants (Fig. 3A). The nodule plant fraction protein declined 72 h after application in HPQ whereas differences were not observed in LPQ treatment (Fig. 3B).
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Treatment with LPQ provoked a rapid decline of SS activity, significant with respect to control plants 48 h after application, with a further 50% decline 96 h after the onset of treatment (Fig. 4A). However, it must be noted that the other carbon enzyme activities analysed (UDPGPP, PEPC, MDH, and ICDH) remained unaffected (Fig. 4C, D, E, F), with the exception of INV activity (Fig. 4B), which only declined significantly after 96 h. AAT and GOGAT (Fig. 4G, H) activities were also only affected at the end of the treatment (96 h after application). HPQ plants showed a rather general decline in nearly all of the enzymes determined. SS, INV, UDPGPP, MDH, AAT, and GOGAT activities declined significantly after 48 h of PQ application (Fig. 4A, B, C, E, G, H) and PEPC after 72 h (Fig. 4D). On the contrary, ICDH was the only enzyme whose activity increased significantly at 96 h after application (Fig. 4F).
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Sucrose content accumulated significantly in the nodules of both PQ doses at 48 h after application (Fig. 5A). This sucrose accumulation occurred concomitantly with a significant decline in the starch content (Fig. 5B), which may be an alternative source of carbon for bacteroid respiration when glycolysis is limited because of the down-regulation of SS (Fig. 4A). In LPQ plants, this trend of sucrose accumulation continued for the whole period of study. However, in HPQ plants sucrose content returned to control values 48 h after application (Fig. 5A), reflecting a balance between a decreased sucrose flux to nodules as a consequence of photosynthesis inhibition (Fig. 1A) and a decreased carbon use in nodules because of the decline in SS activity (Fig. 4A).
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Discussion |
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Biological nitrogen fixation in legumes (BNF) is known to be dramatically affected by environmental constraints. In a wide range of environmental stresses such as drought (Moran et al., 1994
), low temperatures (Prasad et al., 1994) or heavy metals (Sandalio et al., 2001) ROS are known to be over-produced. Thus, it has been suggested that oxidative stress that may be responsible for the above-mentioned BNF decline at the molecular level. Nevertheless, under these constraints, many other factors affect plant performance independently of ROS action. In the present study, PQ was applied to determine the in vivo effect of oxidative stress on nodule metabolism deprived of complex interactions that natural environmental stresses might create, such as an alteration of long-distance transport or a lack of nutrients.
PQ (methyl viologen or N,N'-dimethyl-4,4'-bipyridylium dichloride) is a compound that exacerbates 
and H2O2 production (Farrington et al., 1973). Its action involves the damage of plant cells by the interference of the electron transport system and the formation of ROS that attack unsaturated membrane fatty acids, rapidly opening and disintegrating cell membranes and tissues. As PQ is applied as a post-emergent herbicide, in laboratory assays it is applied to shoots as its mode of action is enhanced by light during the plant photosynthetic process (Donahue et al., 1997; Iturbe-Ormaetxe et al., 1998). Moreover, it also leads to plant death in plants in the dark and is associated with mitochondrial electron transport (Dodge, 1971). To our knowledge there is only one reference of using PQ applied on soybean nodules (Dalton, 1992). In that work, 0.1–1 mol m–3 PQ was supplied to plants grown in perlite and it provoked antioxidant enzyme activation, ASC content decline, GSH content increase, and nitrogen fixation decline which was measured as acetylene reduction. However, no hypothesis for the ultimate decline of nitrogen fixation was provided, other than the occurrence of general oxidative damage.
Two PQ concentrations were applied to the nutrient solution in order to generate oxidative stress as a consequence of ROS over-production which then affects nodule metabolism prior to being translocated to shoots and, therefore, before any likely photoassimilate depletion for nodule functioning was provoked. These two concentrations (1 and 10 mmol m–3) are significantly lower than those used before, precluding the possibility of rapid and generalized oxidative damage. Thus, the highest concentration used in this study represents one-tenth of the concentrations commonly used for foliar treatments (Iturbe-Ormaetxe et al., 1998) or the lowest concentration used so far in nodules (Dalton, 1992).
Low-molecular mass antioxidants were determined at the end of the treatment (96 h after PQ application) in order to assess whether PQ actually produced an antioxidant response in the nodules. Under both treatments paraquat provoked a nodule redox imbalance, which led to a 65% decline in the ASC/(ASC+DHA) ratio. However, the GSH/(GSH+GSSG) ratio only declined in HPQ nodules due to the highest oxidative stress severity (Table 2). In LPQ nodules, to compensate ROS over-production, both ASC+DHA and GSH+GSSG pool sizes increased (Table 2). There is also a great deal of evidence to support the suggestion that oxidative stress results in increases in enzymatic and non-enzymatic systems associated with the ROS scavenging cycle (Apel and Hirt, 2004; Okpodu et al., 1996). GSH synthesis is enhanced in plants exposed to xenobiotics and environmental stresses, such as in soybean nodules subjected to paraquat (Dalton, 1992) or in Brassica napus plants under salt stress (Ruiz and Blumwald, 2002). The latter attributed the increase of GSH synthesis to an enhancement of S assimilation and cysteine biosynthesis. Although it is likely that a transient response might have occurred earlier within the study period, the analyses carried out at the end of the study period suggested that the severity of HPQ treatment did not allow GSH+GSSG pool size to respond to oxidative stress, whilst ASC+DHA increased as it did in LPQ nodules. Hence, low-molecular mass antioxidant data reveal that although low PQ doses were applied in roots, two different intensities of oxidative stress situations were reached in pea nodules (Table 2).
In the long-term, the effects of plants subjected to HPQ were apparent in shoots, thus provoking photosynthesis rate and chlorophyll content reduction. Thus these parameters declined from 48 h and 72 h of PQ application, respectively (Fig. 1), showing that HPQ concentration was high enough to be translocated to shoots. Nevertheless, photosynthesis decline does not seem to limit the photoassimilate supply necessary for nodule functioning, since sucrose content (Fig. 5A) was significantly higher in HPQ nodules with respect to controls at 48 h after application, due to a decline in sucrose-cleaving enzyme activities (Fig. 4A, B). Thereafter, a decrease in sucrose content of nodules occurred as a consequence of photosynthesis inhibition (Fig. 1), although sucrose content of HPQ nodules was not significantly lower than in control nodules at any time of the study. Therefore, the near complete ANA inhibition occurring 48 h after application (Fig. 2) could not be due to a limited shoot–root carbon-skeleton supply.
Oxygen flux is essential to maintain the energy supply for the functioning of nitrogenase. Lb provides an adequate flux of O2 to the bacteroids at very low concentrations of free dissolved O2 and, as such, Lb changes have been suggested as the main effect on nodule metabolism cause by severe drought in indeterminate nodules (Guerin et al., 1991; Irigoyen et al., 1992) suggesting that O2 limitation would affect bacteroid respiration and energy production. Recently, Ott et al. (2005) abolished symbiotic Lb synthesis in nodules of Lotus japonicus by RNAi, causing the loss of nitrogenase and the absence of BNF. In this work, plants under HPQ treatment showed a decline in Lb content (Fig. 3A) as soon as 24 h following treatment, prior to any detectable change either in ANA (Fig. 2) or in any of the remaining nodule metabolism parameters determined. It is known that H2O2 directly reacts with both the ferrous and ferric forms of Lb and oxidizes them to the inactive ferryl (Fe4+) form (Aviram et al., 1978; Puppo et al., 1993). Thus, the oxidative stress provoked in the present study might be involved in Lb degradation. Indeed, Lb changes have also been described in relation to other environmental stresses, such as salinity (Abd-Alla, 1992), darkness (Gogorcena et al., 1997) or nitrate (Escuredo et al., 1996) and related, at least in part, to the decline in nitrogen fixation. Moreover, in exogenous ABA supply studies, a BNF decline was provoked relating to a reduction in Lb content as well, whilst photosynthesis and other nodule enzymes were not affected (González et al., 2001a).
HPQ plants experienced a rather generalized decline in nearly all measured enzyme activities from 48 h after PQ application (Fig. 4). From 72 h following application, the decline was more pronounced, due in part to a decline in total plant fraction protein content (Fig. 3B). This general effect has been reported previously under situations of severe drought (Gálvez et al., 2005). Only ICDH demonstrated different behaviour, where activity slightly increased at the end of the treatment (Fig. 4F). It has been suggested that this enzyme plays a role that ensures an adequate supply of NADPH for plant defence against oxidative stress (Hodges et al., 2003), or has a function to supply 2-oxoglutarate to the GS-GOGAT cycle in carbon-limiting situations such as water stress (Gálvez et al., 2005). Therefore, in this study ICDH could be activated as a response for the ASC-GSH pathway reducing power demand or as a carbon-skeleton supplier because of the inhibition of nodule glycolytic pathway enzymes (Fig. 4).
LPQ plants experienced BNF inhibition, although the photosynthetic rate was not affected, as has been described in moderate water-stressed symbiotic plants (Durand et al., 1987; Djekoun and Planchon, 1991), thus eliminating the possibility that the BNF decline could be due to a reduction in the shoot carbon supply to nodules. In this treatment, SS activity (Fig. 4A) declined within 48 h after application, prior to any other significant effect being detected in all nodule-metabolism determined parameters. Furthermore, a striking sucrose accumulation (Fig. 5A) is observed due to SS activity decline. Thus, these results support the key role of SS in regulating BNF under moderate oxidative stress, which might be equivalent to mild environmental constraints. It still remains unclear which signal transduction pathway could be responsible for regulating SS under abiotic stresses. This study raises the issue that, in addition to the oxidative damage already described by other authors under environmental constraints that leads to nodule senescence in nodules, a likely SS regulation mechanism mediated by ROS may occur at an earlier stage. The involvement of ROS, mainly hydrogen peroxide (H2O2) having functions as signalling molecules, has been described for other plant systems mediating responses to several stimuli (Neill et al., 2002) such as stomatal closure (Pei et al., 2000), programmed cell death (Beers and McDowell, 2001) or as being involved in plant senescence (Puppo et al., 2005). Since PQ was applied to the nutrient solution of whole plants in the present study, it cannot be precluded that some of the effects shown might be secondary effects derived from PQ effects on roots rather than nodules. However, the mode of action of PQ and the high respiratory rates of nodules strongly suggest a direct action of PQ on nodule performance.
In conclusion, mild oxidative stress generated by root-applied paraquat (1 mmol m–3) provoked a BNF inhibition produced by a shortage of the glycolytic pathway due to a down-regulation of SS. In 10 mmol m–3 PQ treatment, in addition to the carbon flux shortage, BNF inhibition was also related to the striking decline in Lb content that possibly limited O2 availability for bacteroid respiration. The overall results are consistent with the model suggested by González et al. (2001b) for BNF regulation under stress conditions with the occurrence of at least two different control pathways within nodules in response to changes in the environment. The mild and severe oxidative stresses occurring under water stress conditions were mimicked in the present study by low concentrations of paraquat supplied to the nutrient solution, hence providing evidence that ROS could be part of the signal transduction pathway involved in BNF regulation under environmental constraints.
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Acknowledgements |
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The authors thank Gustavo Garijo and Elena Denia for technical assistance, Loli Galvez for western immunoblotting of nitrogenase, and Marta Santos for preliminary work. Daniel Marino is the holder of a grant from the Basque Government. This work was supported by DGI-MEC (Spain) grant AGL2002-02730 and its associated FEDER funding.
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