Journal of Experimental Botany, Vol. 52, No. 355, pp. 285-293,
February 2001
© 2001 Oxford University Press
Original Papers |
Abscisic acid induces a decline in nitrogen fixation that involves leghaemoglobin, but is independent of sucrose synthase activity
Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra, Campus de Arrosadía, E-31006 Pamplona, Spain
Received 11 April 2000; Accepted 18 September 2000
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Abstract |
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Sucrose synthase (SS) activity has been suggested to be a key point of regulation in nodule metabolism since this enzyme is down-regulated in response to different stresses which lead to decreased nitrogen fixation. In soybean, a dramatic decline of SS transcripts has been observed within 1 d from the onset of drought. Such a quick response suggests mediation by a signal transduction molecule. Abscisic acid (ABA) is a likely candidate to act as such a molecule as it mediates in a significant number of plant responses to environmental constraints. The hypothesis of ABA controlling nodule metabolism was approached in this work by assessing nodule responses to exogenous ABA supply in pea. Under the experimental conditions, ABA did not affect plant biomass, nodule numbers or dry weight. However, nitrogen fixation rate was reduced by 70% within 5 d and by 80% after 9 d leading to a reduced plant organic nitrogen content. Leghaemoglobin (Lb) content declined in parallel with that of nitrogen fixation. SS activity, however, was not affected by ABA treatment, and neither were the activities of the enzymes aspartate amino transferase, alkaline invertase, malate dehydrogenase, glutamate synthase, uridine diphosphoglucose pyrophosphorylase, isocitrate dehydrogenase, and glutamine synthetase. Nodule bacteroid-soluble protein content was reduced in nodules only after 9 d of ABA treatment. These results do not support the hypothesis that ABA directly regulates SS activity. However, they do suggest the occurrence of at least two different control pathways in nodules under environmental constraints, which include ABA being involved in a Lb/oxygen-related control of nitrogen fixation.
Key words: Abscisic acid, leghaemoglobin, nodule metabolism, Pisum sativum L., sucrose synthase.
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Introduction |
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Nitrogen fixation in legume nodules is severely affected by different environmental constraints, such as drought, salinity, defoliation, darkness, chilling, and nitrate supply. This decline in nitrogen fixation in response to stress has been explained by means of a closure of the oxygen diffusion barrier to reduce oxygen flux into the nodule and avoid nitrogenase damage (Witty et al., 1986
; Hunt and Layzell, 1993), although the mechanism of such a response is not yet determined. Recently, it has been proposed that nodule metabolism may respond to the different stresses regardless of the changes in nodule permeability (Diaz del Castillo et al., 1994; Diaz del Castillo and Layzell, 1995). González et al. found a rapid decline of both sucrose synthase (SS) activity and protein content in soybean plants subjected to a moderate water stress (González et al., 1995). Durand et al. and Djekoun and Planchon showed that nitrogen fixation was more sensitive than photosynthesis to moderate water deprivation (Durand et al., 1987; Djekoun and Planchon, 1991), thus SS decline does not seem to be triggered by photosynthate shortage. Moreover, sucrose is accumulated in nodules subjected to water stress (González et al., 1995). Thus, it has been suggested that the impairment of sucrose metabolism within the nodule could be responsible for the nitrogen fixation decline by limiting the carbon flux for bacteroid respiration (González et al., 1995; Gordon et al., 1997; Arrese-Igor et al., 1999). Gordon et al. found that the decline in SS activity in response to moderate water stress, was related to the down-regulation of the SS gene within 1 d (Gordon et al., 1997). Also, salt and nitrate supply affect SS at the expression level. Altogether these data point to the key role of SS in the regulation of nodule metabolism. However, the signal transduction pathway that links the perception of these environmental stresses and the decline of SS activity remains unknown.
Levels of the plant hormone abscisic acid (ABA) increase as a result of water stress, playing an important role in the plant response to drought, salinity and cold; stresses that involve cellular water stress. Since levels of endogenous ABA increase in tissues subjected to osmotic stress (Henson, 1984, Mohapatra et al., 1988), it appears to mediate physiological processes in response to osmotic stress. Under these conditions, specific genes are expressed that can also be induced in unstressed tissues by the application of exogenous ABA (Gómez et al., 1988; Mundy and Chua, 1988).
It is thought that some of these ABA-responsive genes may encode proteins with osmoregulatory or other protective functions. Guan and Scandalios found that ABA leads to an increase of superoxide dismutase expression (Guan and Scandalios, 1998) which suggests that ABA-mediated metabolic changes lead to increases in the antioxidant defence system. Furthermore, it has been shown that ABA disrupts cortical actin filaments (Eun and Lee, 1997), which may lead to structural regulation of ion-channel activity or cytoskeletal regulation of signalling molecules, such as protein kinases and phosphatases. SS activity was shown to be modulated by a reversible phosphorylation mechanism in maize leaves (Huber et al., 1996) and the same post-transcriptional regulation in soybean nodule SS has also been found (Zhang and Chollet, 1997). Recently, SS regulation has been examined further and it was found that phosphorylation modifies the hydrophobicity of the enzyme, suggesting that regulation could be mediated by partitioning between soluble and membrane-bound enzyme (Zhang et al., 1999).
Although ABA is the major signal transduction operating during drought stress, not all drought-induced- genes are regulated by ABA (see review by Shinozaki and Yamaguchi-Shinozaki, 1997, and references therein). Studies conducted on ABA-deficient or ABA-insensitive mutants have indicated that several drought- and/or cold-induced genes are expressed independently of ABA (Nordin et al., 1991; Gosti et al., 1995). A desiccation responsive gene in Arabidopsis is rapidly induced by water and salt stresses in an ABA-independent manner (Yamaguchi-Shinozaki and Shinozaki, 1993). A transcript of a MAP kinase gene in alfalfa accumulates after drought and cold treatment, but not in response to ABA, indicating that this kinase pathway mediates drought and cold signalling independently of ABA (Jonak et al., 1996). Recently, it has been found that two SS genes of Arabidopsis were modulated by sugar/osmoticum levels in an ABA-independent manner (Déjardin et al., 1999).
The primary nodule response observed following the occurrence of abiotic stresses has been the down-regulation of SS, but as yet there is no direct evidence as to which signal triggers this mechanism. The aim of this study has been to ascertain whether ABA is involved in the signal transduction pathway of down-regulation of SS activity in nodules. Time-course activities of key enzymes of carbohydrate and nitrogen metabolism were examined in relation to nitrogen fixation rates and levels of carbohydrates and amino acids in plants supplied with exogenous ABA.
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Materials and methods |
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Growth conditions and experimental procedures
Pea plants (Pisum sativum L. cv. Sugar snap) were inoculated with Rhizobium leguminosarum biovar viciae strain NLV 8. This strain is Hup-, according to Southern-blot analysis of EcoRI-digested total DNA using a hup-specific DNA probe prepared by dioxigenin labelling of cosmid pAL618 containing the entire hup gene cluster from Rhizobium leguminosarum bv. viciae strain UPM791 (Matamoros et al., 1999
). Plants were grown in 1 dm3 pots, with a 2:1 (v:v) mixture of vermiculite:perlite as the substrate, with a nutrient solution lacking nitrogen (Rigaud and Puppo, 1975) 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).
When plants were 4-weeks-old, a set was watered daily with 50 ml of a nutrient solution containing 100 mmol m-3 ABA and a control set was watered daily with the same volume of nutrient solution. This represents twice the daily transpiration rate observed in these plants. ABA was supplied in the form of ±ABA (Sigma Chemical Co). This concentration has been shown to be adequate to simulate the regulation of the plant water status in legumes (Pardossi et al., 1992). Data were collected in three independent experiments. Data in Table 1 reflects the mean of the three experiments, whilst the remaining data were collected in two out of the three experiments performed. Physiological determinations were carried out at days 0, 1, 5, and 9. Leaf and nodule samples for protein extraction and analytical determination were frozen in liquid nitrogen and stored at -80 °C. Nodules, roots and shoots were separated and dried for 48 h at 70 °C for dry weight determinations.
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Water relations and gas exchange measurements
Leaf water potential was measured 2 h after the beginning of the photoperiod using a pressure chamber (Soil Moisture Equipment, Santa Barbara, CA) as described previously (Scholander et al., 1965), assuming that total leaf 
is in equilibrium with petiole . Osmotic potential was determined on the same leaf with a vapour pressure osmometer (Wescor Inc. 5500, Logan, UT), after samples were frozen and thawed (Frechilla et al., 1999). The turgor potential was calculated by the difference between water and osmotic potential.
Net CO2 assimilation rate, leaf conductance and intercellular CO2 were measured with a portable IRGA (LI-6200, Li-Cor, Lincoln, NE). Transpiration rate per plant was calculated gravimetrically each day of the experiment; a substrate-filled pot containing no plants was used to estimate evaporation losses.
For nitrogen fixation determinations, H2 evolution of the intact plants, whose root systems were sealed into the growth pots and housed in the growth chamber, was measured in an open flow-through system (according to Witty and Minchin, 1998) using an electrochemical H2 sensor (Qubit System Inc., Canada). The detector 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 (100 ml min-1). Apparent nitrogenase activity (ANA) was determined under N2:O2 (79%:21%). After reaching steady-state conditions total nitrogenase activity (TNA) was determined under Ar:O2 (79%:21%). Nitrogen fixation rate (NFR) was calculated as (TNA-ANA)/3. Electron allocation coefficient to N2 (EAC) was calculated as a percentage of (1-(ANA/TNA)).
Extraction and assay of enzymes
Nodules were homogenized in a mortar and pestle with 50 mol m-3 MOPS, 10% PVPP, 10 mol m-3 DTT, 1 mol m-3 EDTA, 20 mol m-3 KCl, 5 mol m-3 MgCl2, pH 7 at 0–2 °C (5 cm3 g-1 fresh weight). The homogenate was centrifuged for 30 min at 20 000 g, 2 °C.
Samples (50 mm3) of the supernatant were retained for the phosphoenol pyruvate carboxylase (EC 4.1.1.31) assay (González et al., 1995) and for protein (Bradford, 1976) and leghaemoglobin (Lb) determinations (Appleby and Bergersen, 1980). One cm3 aliquots were desalted by low speed centrifugation (180 g, 1 min) through 5 cm3 columns of Bio Gel P6DG (BioRad) equilibrated with the extraction buffer (see above) without PVPP. The desalted extract was used to determine the following enzyme activities: alkaline invertase (EC 3.2.1.26), aspartate amino transferase (EC 2.6.1.1), glutamate synthase (EC 1.4.1.14), glutamine synthetase (EC 6.3.1.2), malate dehydrogenase (EC 1.1.1.37), sucrose synthase (EC 2.4.1.13) and uridine diphosphoglucose pyrophosphorylase (EC 2.7.7.9) (according to González et al., 1998) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) (according to Chen et al., 1988).
The pellet remaining after removing the host plant soluble proteins by centrifugation (see above) was washed (1 cm3 extraction buffer) and centrifuged three times for 10 min at 14 000 g, on each occasion discarding the washings. The pellet was then resuspended in extraction buffer without PVPP (5 cm3 g-1 original nodule fresh weight) and the bacteroids broken by sonication (10x30 s, 2 °C). The supernatant after centrifugation (20 000 g, 2 °C 30 min) was used for bacteroid protein determination (according to Bradford, 1976).
Analytical determinations
Fresh material was exhaustively extracted in boiling 80% (v/v) ethanol. Ethanol soluble extracts were dried in a Turbovap LV evaporator (Zymark Corp, Hopkinton, MA) and soluble compounds were redissolved with 4 cm3 of distilled water, mixed and centrifuged at 20 000 g for 10 min. Total soluble sugars were measured in the supernatant by the anthrone method (Spiro, 1966) and sucrose (according to González et al., 1995). Free amino acids were assayed using the acid ninhydrin method of Yemm and Cocking (Yemm and Cocking, 1955). Malate was also analysed in this supernatant by ion chromatography in a DX-500 system (Dionex, Salt Lake City, UT) by gradient separation with a Dionex IonPac AS11 column (2.5 mol m-3 NaOH/18% methanol to 45 mol m-3 NaOH/18% methanol in 13 min).
The ethanol-insoluble residue, remaining after the extraction of soluble compounds, was extracted for starch (as in MacRae, 1971), and the glucose produced was analysed enzymatically (as described by González et al., 1995).
Organic nitrogen content was determined by Kjeldahl analysis (AOAC, 1990).
Statistical analysis
Results were examined by one-way analysis of variance. All effects discussed in this study were significant at P
0.05 in Fisher's (protected) least significant difference (LSD) tests between means.
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Results |
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The effect of ABA on plant growth was monitored throughout the 9 d of the experimental period. The effect of the daily supply of 50 ml of nutrient solution containing ABA was monitored in relation to controls, where the same volume of nutrient solution without ABA was given. Table 1
summarizes growth parameters at the end of the experimental period. ABA supply did not affect plant dry weight, shoot to root ratio, nodule numbers or nodule dry weight (Table 1). Conversely, organic nitrogen content declined significantly in shoots as a consequence of ABA treatment, although organic nitrogen content of roots was less affected (Table 1).
Transpiration rate, calculated gravimetrically, of ABA- treated plants declined gradually from the onset of ABA supply to day 5 and afterwards it remained at values that represented c. 20% of the initial controls (Fig. 1B). This was in agreement with the decline of stomatal conductance in ABA-treated plants within 1 d of ABA supply to 65% of controls, with a further decline thereafter (Fig. 1A). Stomatal closure led to a slight decrease in intercellular CO2 concentration in ABA-treated plants that also progressed with time (Fig. 1D). Despite the rapid stomatal closure and the decreased intercellular CO2 concentration, photosynthesis was maintained at rates c. 90% of control values (Fig. 1C). The photosynthetic rate is likely to be only slightly affected under these conditions because the decreased intercellular CO2 concentration following stomatal closure allowed the maintenance of CO2 flux into the leaves. Under these conditions, the relatively low rates of carbon assimilation (Fig. 1C) are likely to be still CO2-saturated, possibly associated with a moderate photosynthetic photon flux in the growth chamber, and it remains to be determined whether this moderate effect on photosynthesis also occurs under saturating light intensity.
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The water potential of ABA-treated plants was slightly higher than that of control plants throughout the study (Fig. 2A
). Plants experienced a rapid stomatal closure in the presence of ABA (Fig. 1A) whilst plants did not experience water shortage. Thus, both osmotic (Fig. 2B) and turgor potential (Fig. 2C) showed a transient variation after 1 d of ABA treatment, possibly reflecting the balance between full availability of water and stomatal closure, but came back to control values during the study period.
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Apparent and total nitrogenase activities were not affected within 1 d after ABA supply, but they declined significantly after 5 d (Fig. 3A
, B). Nitrogen fixation rate, calculated as the difference between TNA and ANA and the theoretical relationship between H2 and N2 reduction, showed a similar pattern to the above parameters (Fig. 3C). However, EAC was stable within the first 5 d and showed a significant decline at day 9 (Fig. 3D).
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The Lb content of ABA-treated plants showed a significant decline after 5 d (Fig. 4B
). At that time, nodule plant fraction and bacteroid soluble protein contents did not show any significant effect by ABA, whilst the latter only declined by day 9 (Fig. 4C, D). Conversely to Lb, SS activity was virtually unchanged throughout the experiment (Fig. 4A). Measured SS activity was completely abolished by the glycoside arbutin (data not shown), showing that the lack of changes in SS activity was not due to artefactual, side reactions. Other enzyme activities involved in nitrogen and carbon metabolism in nodules of ABA-treated plants (alkaline invertase (0.17), UDP-glucose pyrophosphorylase (1.22), phosphoenol pyruvate carboxylase (0.21), malate dehydrogenase (15.7), isocitrate dehydrogenase (0.32), glutamine synthetase (0.14), glutamate synthase (0.018), and aspartate amino transferase (0.61, all in µmol product mg-1 protein min-1)) did not show any significant variation during the studied period.
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ABA supply led to a transient total soluble sugar accumulation in the leaf (Fig. 5A
). However, as control plants also showed a trend to accumulate total soluble sugars with age, control and treated plants did not show any difference at the end of the experiment. Likewise, starch content increased in leaves in response to ABA (Fig. 5C). Conversely, leaf total free amino acids of ABA-treated plants was significantly lower than controls at the end of the study period, in agreement with values of shoot organic nitrogen content (Table 1), as a consequence of the sharp decline in nitrogen fixation experienced by the ABA-treated plants after 5 d of treatment (Fig. 3).
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No significant changes were found in nodule carbohydrates (Fig. 5B
) and starch (Fig. 5D). Despite the reduced nitrogen fixation rate found in ABA-treated plants, amino acid content was not affected in nodules, although a transient increase could be detected after 1 d of ABA supply (Fig. 5F), possibly reflecting a decreased xylem flux, as a consequence of stomatal closure (Fig. 1A). Nodule sucrose content was not affected by ABA treatment, with values almost constant c. 3 mg g-1 nodule fresh weight, suggesting that nodules did not experience photosynthate shortage. Moreover, the concentration of malate, the organic acid that fuels bacteroid metabolism, was also unaffected by the ABA treatment, with values c. 1.4 mg g-1 nodule fresh weight.
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Discussion |
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Roots in drying soil produce ABA (Cornish and Zeevaart, 1985
), which is transported to leaves (Zhang and Davies, 1989). The increased concentration of ABA stimulates stomatal closure and reduces transpirational water loss from leaves, therefore preventing plant's drying. Thus, plants integrate the opposite demands of maximizing CO2 uptake and minimizing transpiration water loss (Cowan, 1982; Socias et al., 1997). It has been shown that nodulated legumes showed a better gas exchange response to drought than non-nodulated plants, a fact that is related to the hormone balance (Goicoechea et al., 1997) and to complex interactions with photorespiratory flux and stomatal conductance (Frechilla et al., 2000). In this study, transpiration rate and conductance were significantly reduced by ABA supply, showing that ABA was efficiently translocated through the xylem, although photosynthesis rate was only slightly affected throughout the study (Fig. 1). Under these experimental conditions, total soluble sugar accumulated in the leaf within 1 d from the onset of ABA supply (Fig. 5A) and leaf starch content was also higher than control values throughout the study period (Fig. 5C), which confirms that CO2 fixation was not markedly affected by this treatment. In addition, plant growth was not affected by ABA (Table 1), which is consistent with an uninhibited photosynthetic process. Since the aim of this study was to examine ABA effects on nodule metabolism, this experimental situation where photosynthesis was unaffected was an important requisite in order to avoid interference between the ABA effect and a possible limitation of photosynthate supply to nodules. As expected, the stomatal closure triggered by ABA (Fig. 1) in an environment where plants do not actually experience water shortage, leads to an increase in leaf water potential (Fig. 2A). This is the opposite of the situation found in water-stressed plants and, therefore, the conclusions drawn from this study should be understood in the context of the likely involvement or not of ABA in the signal transduction pathway that links drought perception and a decreased SS activity which, in turn, leads to a decline in nodule nitrogen fixation. However, at the whole-plant level, it cannot be expected that exogenous ABA supply would mimic every physiological plant response to drought.
A significant reduction both in ANA and TNA was described in a conventional closed standard system of ABA-treated plants of Faba vulgaris, without any marked effect on growth (Bano and Hillman, 1986). However, the interpretation of such results is complicated in view of the problems associated with the standard assay (Minchin et al., 1983). Indeed, they suggested a lack of photosynthate supply to nodules as the main cause of nitrogen fixation decline, since lower leaves of ABA-treated plants showed an early senescence. However, the ABA treatment performed in this study did not affect nodule mass (in agreement with the results of Bano and Hillman,1986), but, in addition, no photosynthate shortage to nodule metabolism is evident in view of the concentrations of total soluble sugars (Fig. 5B), sucrose and malate.
It was found that SS may be regulated in soybean nodules by reversible phosphorylation (Zhang and Chollet, 1997) and, moreover, at least two different sites were found to be susceptible to phosphorylation by a calcium-dependent protein kinase (Zhang et al., 1999). ABA activation of protein kinases has been described in different systems (Esser et al., 1997; Burnett et al., 2000; Hong et al., 1997). However, SS activity was not affected by ABA when nitrogen fixation was inhibited by 80%, ruling out the hypothesis of an ABA-mediated SS response to environmental stresses. Déjardin et al. found expression of two SS genes in Arabidopsis differentially expressed in relation to water/osmoticum stresses and both were independent of ABA signals (Déjardin et al., 1999), Sus1 expression was related to the perception of osmotic potential decreases and Sus2 was independent of sugar and osmoticum signal. In other plant systems, SS is known to be induced by changes in carbohydrate availability (Koch et al., 1992; Heim et al., 1993; Koch, 1996) and by low O2 environments (Taliercio and Chourey, 1989; Ricard et al., 1991; Xue et al., 1991). However, both regulation mechanisms seems to have a limited role in nodules (Arrese-Igor et al., 1999).
Lb changes have been suggested as the main effect on nodule metabolism caused by severe drought in indeterminate nodules (Guerin et al., 1991; Irigoyen et al., 1992), suggesting that the decline in nitrogen fixation was due to a reduction in Lb content which would limit oxygen supply to bacteroids and affect respiration and energy production. Indeed, under those conditions an increase in nodule malate content was reported (Irigoyen et al., 1992). This would be consistent with a decreased oxygen supply and the induction of fermentative pathways (De Vries et al., 1980). Lb changes have been also described following 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, with the decline in nitrogen fixation. However, such effects are not evident when these stresses occur in a moderate and gradual situation (González et al., 1995, 1998; Gordon et al., 1997). Moreover, preliminary data suggest that the down-regulation of SS during drought is followed by a depletion of organic acids in nodules under mild drought (L Gálvez, EM González, C Arrese-Igor, unpublished results). Lb declined in nodules of ABA-treated plants (Fig. 4B) in parallel to nitrogen fixation (Fig. 3) after 5 d of ABA treatment prior to any detectable change in the protein content of the plant and bacteroid fractions of nodules (Fig. 4C, D). The pathway of Lb degradation in vivo is largely unknown. Most studies suggest that Lb degradation is due to a particularly rapid activation or decompartmentation of proteases located in the infected cells that have a high affinity for Lb at the acidic intracellular pH that may occur under different stresses (Pladys et al., 1991). Substantial decline of the bacteroid fraction protein was observed after 9 d treatment, suggesting irreversible damage caused by ABA (Fig. 4D). This is further supported by a marked decline in the EAC (Fig. 3D). A marked reduction in the functional bacteroid tissue was also described after preliminary examination of nodule anatomy of plants treated with ABA for 14 d (Bano and Hillman, 1986). An unexpected feature was the low EAC shown by this symbiosis (Fig. 3D). Although the theoretical value for EAC is 0.75, in vivo losses of H2 from legumes are often much greater than 25% of the nitrogenase electron flux. Moreover, EAC seems to respond to a variety of factors, such as temperature and ATP and reducing power availability. It has also been shown that the same bacterial strain may display different relative efficiencies when nodulating different hosts (López et al., 1982). Indeed, when this strain nodulates another pea variety (Frilene) EAC was shown to be 0.67. Moreover, changes in carbon availability lead to a decrease in EAC to 0.52 (PM Cabrerizo, EM González, PM Aparicio-Tejo, C, Arrese-Igor, unpublished results). The basis for this effect is not understood yet.
It has been shown that a moderate and progressive water deficit stress, triggers the SS response in nodules (González et al., 1995, 1998; Gordon et al., 1997). In these studies, SS activity and expression were down-regulated, whilst Lb was not affected. Thus, exogenous ABA supply seems to simulate a situation of abrupt stress. A severe drought might also lead to Lb reduction (Guerin et al., 1991; Irigoyen et al., 1992) mediated by a rapid rise in ABA levels. Therefore, the stress intensity may determine nodule responses. It remains to be determined whether the decline in nitrogen fixation is solely caused by the decreased Lb levels. Indeed, at day 5, when nitrogen fixation declined by 70% (Fig. 3C), Lb concentration only declined by 50% (Fig. 4B). Thus, it cannot be discarded that a second factor, such as direct effects of ABA on nodule O2 diffusion may also be involved in the decline of nitrogen fixation. In conclusion, the occurrence of at least two different control pathways in nodules under environmental constraints is put forward, with ABA being involved in a Lb/oxygen-related control of nitrogen fixation under abrupt or severe stresses, whilst mild or gradually developed stresses would be mediated by an ABA-independent SS pathway.
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Acknowledgments |
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Authors thank Anthony J Gordon, Pedro M Aparicio-Tejo and Mercedes Royuela for their critical reading of the ms, Monica Ayerra for technical assistance, Leszek Kleczkowski for providing results prior publication, Tomás Ruiz-Argüeso for valuable advice on relative efficiency and the unknown referees for their constructive comments. Seeds were kindly supplied by Bonduelle (Milagro, Spain). Loli Gálvez was the holder of a grant from the Spanish Ministry of Education (Plan FPU). This work was supported by DGESIC (PB98-0545) and CICYT (AGF97-0458).
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Notes |
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1 To whom correspondence should be addressed. Fax: +34 948 168930. E-mail: cesarai{at}unavarra.es
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