Xylem exudation is related to nitrate assimilation

Journal of Experimental Botany, Vol. 47, No. 297, pp. 485-495, April 1996

Journal of Experimental Botany

Xylem exudation is related to nitrate assimilation pathway in detopped maize seedlings: use of nitrate reductase and glutamine synthetase inhibitors as tools Laure Barthes 1 , Eliane Deleens, Agnes Bousser, Jackson Hoarau and Jean-Louis Prioul Laboratoire Structure et Metabolisme des Plantes, CNRS URA 1128, Institut de Biotechnologie des Plantes, Bat 630, Universite de Paris-Sud, Centre d'Orsay, F-91405 Orsay Cedex, France Received 29 September 1995; Accepted 12 January 1996

Introduction

The xylem exudation of detopped 7-d-old seedlings of Zea mays L. doubled when KCI was present in the root medium compared to seedlings maintained on water. It was further enhanced when KCI was replaced by nitrogen compounds such as nitrate, ammonium and glutamine. The role of the nitrate assimilation pathway on the enhancement of xylem exudation rate was investigated using tungstate, an inhibitor of nitrate reductase (NR) activity, and phosphinothricin or methionine sulphoximine, inhibitors of glutamine synthetase (GS) activity. The sap levels of NO3 , NH4+, glutamine, and asparagine was used to ascertain the in vivo inhibition of both enzymes. The tungstate effects were also checked by measuring leaf in vitro NR activity and NR protein content. Xylem exudation rate of detopped seedlings fed with KN0 3 decreased when the nitrate assimilation pathway was blocked either at the NR or at GS sites. This decrease was prevented when urea (acting as NH4+ supply) was given simultaneously with tungstate. KN0 3 does not act directly on exudation, but through the involvement of NH4+. The involvement of glutamine was also shown since GS inhibition resulted in a cancellation of the enhancing effect of KN0 3 on exudation. As change of exudation rate was not linked to change in sap osmolarity, it is assumed that the assimilation chain could modify root water conductance. The role of glutamine was discussed.

The translocation of nitrate from root to shoot is related to water transport. This transport still occurs when water movement is driven by the process named root pressure which causes xylem exudation in detopped plants. Minshall (1964, 1968) reported an enhancement of the xylem exudation rate by nitrate or urea supply in root medium on detopped tomato plants. The nitrate positive effect on exudation could either be due to an increase of osmotic potential difference between root medium and xylem vessels or to an increase of root hydraulic conductance. This work reassessed these relationships on young seedlings of maize (Barthes et al., 1995a) and wheat (Barthes et al., 19956): as the total osmoticum in the sap can not explain the variations in exudation rate, it is assumed that nitrate induced the enhancement of exudation rate via an increase of hydraulic conductance. Whatever the actual causes of water flux enhancement by KNO3, it seems that the entire assimilation pathway would be linked to an increase of xylem flux. This assumption is supported by two facts, (i) urea, considered as an ammonium supplier, had a similar effect to KNO3 (Minshall 1964, 1968; Barthes et al., 1995a) and (ii) the KNO3 effect on exudation was prolonged even after it was removed from the root medium (Barthes et al., 1995a). The hystereris effect of KNO3 could originate from the endogenously stored anions in the root tissue. But, does it act directly or indirectly via reduced forms originating from its assimilation pathway? The similar effect of urea speaks in favour of an indirect effect of nitrate. An attempt to establish such a correlation between exudation and nitrate reduction was reported by Aschroft

Key words: Exudation, maize, nitrate, conductance, NR, GS. 1

To whom correspondence should be addressed. Fax: +33 1 69 33 64 24. Abbreviations: PPT, phosphinothricin; MSO, methionine sulphoximine; NR, nitrate reductase; GS, glutamine synthetase. © Oxford University Press 1996

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Abstract

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inhibitors were compared to concomitant measurements of the total osmolarity of xylem sap. Materials and methods Plant material and growth conditions

Seeds of a maize hybrid (Zea mays L., F7F2) were rehydrated for 24 h in distilled water bubbled with air. One hundred rehydrated seeds were placed on a germinating bed, the scutellum facing down, at a density of 12 seeds dm" 2 in a transparent plastic box (24 x 36 x 13 cm). The germinating bed consisted of a layer of synthetic porous tissue (1 cm thickness) covered with three layers of filter paper. Each box contained 500 ml of 2 mM potassium phosphate (pH 7.0). The boxes were closed and put in a growth chamber maintained at constant temperature (30 °C) and high relative humidity (95-98%). All the boxes were connected to an air-flow inlet which provided water-saturated air at 30 °C. Illumination was provided by a cool white light source (fluorescent tubes Phillips TL 33 at 200;umol photon m" 2 s" 1 ) 16 h d" 1 . Experimental protocol for xylem sap collection

Plants were harvested at 7 d, at the end of the light period, 16 h after the addition of salt and nitrogen compounds in the root medium. All the plants in a box were rapidly detopped at 2 cm above the seed. The first drops were discarded to avoid exudate pollution by damaged cells or phloem sap. Then, the emerging drops of xylem sap were collected with the aid of a peristaltic pump. The xylem sap from each box was pooled at 1,3, 5, and 7.5 h. The rate of exudation for 100 plants was routinely determined and the cumulative volume of exudate calculated. It may be assumed that the water contained in the sap originated only through roots because the contact between seed and tissue and paper bed was negligible. Salt and nitrogen compounds used as exudation enhancers

Inducers were applied in root medium at day 6, 16 h before detopping. Each salt or metabolite treatment was given in a 100 ml concentrated solution added to the root medium. As the initial root medium volume was 500 ml, the solutes were diluted by a factor of about 6 since the root medium volume could be slightly modified after 6d of growth. Each box received 100 ml of a 50 mM salt solution (KC1 or KNO 3 ) providing a concentration of about 8 mM in the rhizosphere. Urea was provided at 25 mM, giving a final N H / concentration of 5 mM. The final concentrations of NO^ and NH44" were specifically determined as described below for xylem sap samples (data not shown). For amino acids, the final concentrations were not checked. Amino acids and urea solutions were supplemented with KC1 in order to reach the same osmolarity (100 mOsmol 1 ~') as in the case of KNO 3 and K G applications. The final osmolarity of the root medium containing inducers was around 16mOsmol I" 1 in all cases. In an early experiment, the effect of KNO 3 , K G and H 2 O on exudation was compared to that of nitrogenous compounds, NH 4 NO 3 , urea [CO(NH 2 ) 2 ] and glutamine. In another experiment, the effects of selected amino acids (asparagine, glycine, proline, and phenylalanine) on exudation were compared to that of H 2 O and glutamine as control assays. Reference assays for KN03 and urea treatment

(1) Time-course of KNO 3 and urea effects on exudation. K N 0 3 and urea were applied 2, 6, 12, 26, and 30 h before detopping.


et al. (1972) who used detopped tobacco plants. Although they failed to observe participation of nitrate reductase (NR, EC 1.6.6.1) in the process, they clearly showed that nitrate-dependent exudation might be increased by the addition of glucose. Touraine et al. (1990) explained the positive correlation between nitrate uptake in root and nitrate reduction in shoot via the role of the carboxylate shuttle. However, their model is not appropriate for xylem transport in detopped plants since the water nutrient recycling from shoot to root is interrupted. One aim of this work was to analyse the concomitant functioning of the nitrate assimilation pathway and the exudation process in an attempt to identify the compounds which are involved in the exudation process. Detopped 7 d seedlings of maize were used as a model. The effects of NO^~ and of other compounds of the assimilation pathway, such as NH^, glutamine or asparagine were compared. The specific effect of exogenous asparagine and glutamine as efficient inducers of exudation could be due either to their nature as end products of the nitrate pathway or to the fact that they could release ammonium through deamination of their amide group. The respective responses of nitrate and ammonium suppliers on exudation enhancement were investigated. Another objective was to check that inhibition of the assimilation pathway really causes a decrease of the exudation process. Specific inhibitors of nitrate reductase and glutamine synthetase (GS, EC 6.3.1.2) were used to limit the assimilation of NO^~ and NH^. Tungstate, a specific inhibitor of nitrate reductase was used when KNO3 was supplied in the medium. As a control, tungstate was also given with urea as the NH^ supplier allowing an inhibition of NR while maintaining a normal nitrogen assimilation. In this case, any restriction in plant growth or activity of the major enzymes involved in malate metabolism and carbon assimilation could be suspected (Deng et al., 1989a). Two inhibitors of the glutamine synthetase activity, phosphinothricin (PPT, Logusch et al., 1991) and methionine sulphoximine (MSO, Fentem et al., 1983; Lee and Ayling, 1993) were used. They were compared in two conditions: (i) when KNO3 was given in the root medium, and (ii) when NH^ was provided externally through urea acting as N H / supply. The in vivo functioning of the assimilation pathway was checked from measurements of the change in sap composition in NO3~, NH4+, and glutamine + asparagine (Minshall, 1964; Ivanko and Ingversen, 1971; Oaks and Hirel, 1985). The variations of solute concentrations confirmed the negative effects of the inhibitors. Finally, the initial hypothesis assuming that nitrate induced enhancement of exudation rate via an increase of hydraulic conductance rather than of the driving force for water transport was checked: the large range of exudation rate of exudation produced by the use of

Xylem exudation and nitrate assimilation pathway (2) KNO 3 and urea as reference assays. These compounds were applied 16 h before detopping. Exudation rate and solute concentrations in the sap were followed for 7.5 h. These reference assays were systematically done with the following set of inhibitor experiments. Inhibitor treatments

Rationale of the method

All the conditions given here was the same as Barthes et al. (1995a) and, more precisely, there was no calcium added in the minimum medium. In the preceding paper, the main concern was the presence of K + in the minimum medium: it modifies slightly the rate of exudation of the treated plants, but, it did not change the enhancement due to KNO 3 compared to K G . In a same way, in separate experiments, it was checked that no marked effects of Ca 2 + on exudation enhancement due to KNO 3 compared to K G assays could be observed (Table 1). Moreover, the further experiments (Barthes et al., 19956) designed for young wheat seedlings were performed with a basic medium supplied with Ca2 + : the time-course and the KNO 3 concentration range which increased exudation rate were very close to those obtained on maize plants without Ca 2 + . So, the enhancement of exudation due to KNO 3 is an easily reproducible phenomenon in various conditions and various plants as described in Barthes (1994). In the following study, each comparative result (KNO 3 /KC1, KNO 3 /urea, inhibited/not) was always obtained in duplicate in a single run of experiments. This run was repeated twice for each case. Osmolarity of the collected sap

Osmolarity was determined on 100/xl sap aliquots with an automatic freezing point depression osmometer (Roebling). The results were expressed in mOsmoll" 1 . Data lower than 4 mOsmol I" 1 were not significant.

Table 1. Exudation rate ofdetopped seedlings growing previously on a minimum medium supplemented with mono-Ca-phosphate (+Ca2+) or not (-Ca2+) Seedlings were detopped after 16 h on water. KC1 or 1CNO3 (lOmM). Seedlings were grown during the 6 d before detopping in a medium containing 1.5 mM K-phosphate. 0.5 mM, pH 7 for the Ca 2+ treatment whereas a medium containing only 2 mM K-phosphate buffer was used for the treatment without Ca2 + . Results are mean + SEM of n replicates. Treatment

+ Ca2 + -Ca

2 +

Exudation rate of seedlings grown on different media (10"- 1 2 m 3 s - ' ) Water

KC1

KNO 3

5.01+0.35 «=15 5.02 + 0.44 «=12

5.76 + 0.44 «=13 5.60 ±0.48

n=\A

n=\\

A7=ll

12.40 + 0.53 13.20 + 0.09

Carbohydrates and amino acid content in xylem sap

Sap was analysed by an HPLC procedure on 25 pi aliquots at 1, 3, 5, and 7.5 h after detopping, as previously described (Barthes et al., 1995a). The total concentration for carbohydrates and for amino acids was obtained by adding up the values of each class of compounds. Results were expressed in mM. The cumulated amounts of metabolites during the total timecourse of sap collection were calculated using the sap exudate volume and were expressed in /umol per 100 plants. Carbohydrates in exudates consisted mainly of glucose and fructose and a small amount of sucrose. The most abundant amino acid was glutamine. Nitrate and ammonium content in xylem sap

Nitrate content was estimated as previously described (Barthes et al., 1995a) by the nitrite colorimetric method (Aslam et al., 1976) after an enzymatic conversion of NO^ into NOJ with a commercial purified nitrate reductase from Aspergillus species (Sigma N 7265). Free ammonium was estimated by the Nessler method (Umbreit et al., 1964). The absorbance was determined on an ELISA microplate on a spectrophotometer (MR 700 Dynatech) at 490 nm. Both results were expressed in mM. Leaf nitrate reductase activity

At detopping, small leaf pieces were picked up from the median part of the second leaf until a 1.2 g fraction fresh weight was collected. Two 1.2 g samples were harvested per box. These samples were fixed in liquid nitrogen and stored at — 80 °C. The protocol used to assay in vitro NR activity was adapted from Aryan et al. (1983) and was previously described (Barthes et al., 1995a). Results are expressed in pkat g" 1 FW. Protein content

Leaf soluble protein content was estimated by the Bradford method (Bradford, 1976) using the Bio-Rad reagent on the supernatant fluid (lOmin at 4000g) of the extract used for NR assay. ELISA test

Leaf NR protein content was estimated by a two-site ELISA test using a mouse monoclonal antibody, anti-NR ZM 96-9-25, as coating reagent (Cherel et al., 1986), and a polyclonal antiserum raised against spinach NR as the second antibody. Sensitive quantification of NR was conducted using alkaline phosphatase labelling the enzyme rabbit antibody as described


NR is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site (Redinbaugh and Campbell, 1985); tungsten can substitute molybdenum in the cofactor structure of the NR, resulting in an inactive enzyme (Heimer et al., 1969). Tungstate was applied at 0.15 mM as in Deng et al. (19896). As tungstate is acting through de novo biosynthesis of an inactive protein, a long-term effect (72 h) was compared to a short-term (16 h) one. It was added simultaneously with K N 0 3 or urea 16 h prior detopping in the short-term experiment (ST) or 72 h before detopping in the long-term experiment (LT). In each case, a control experiment was performed replacing tungstate by molybdate (0.15 mM). GS catalyses the NH^" assimilation into glutamine with glutamate and ATP as other substrates (Oaks and Hirel, 1985). As phosphinothricin (PPT) and methionine sulphoximine (MSO), analogues of glutamate, are acting through competitive inhibition, the inactivation occurred as soon as the inhibitor reached the catalytic site. As an example, root GS activity of barley seedlings was lowered to 20% of the initial value after a 30min incubation with 1 mM MSO (Fentem et al., 1983). Thus, in the present work, the time schedule was shorter than in the tungstate experiment: inhibitors were applied at the onset of detopping for 0 h (initial experiment) or 16 h before detopping for short-term experiments (16 h). MSO (from Sigma) and PPT (gift from INRA, Versailles) were used at a final concentration of 0.25 mM. The purity of the PPT was not checked. Each analogue was applied together with KNO 3 or urea treatments at 6 d, 16 h before detopping (ST) or just at the time of detopping at 7 d on KNO 3 and urea plants (Initial Term, IT).

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by Hoarau et al. (1991). Absorbance values determined at 405 nm in an ELISA spectrophotometer apparatus (MR 700 Dynatech) were linear with extract dilution. A 1/6 dilution was chosen for comparative ELISA quantification of nitrate reductase protein using nitrate-treated plants as control.


rate and can be regarded as exudation enhancers. Two families of efficient exudation N-enhancers may be distinguished. The first is composed of nitrate and metabolites thereof (e.g. ammonium, glutamine) and the second is the exogenous NH^ donors: urea, NH4NO3 and probably glutamine and asparagine. Thus, glutamine and asparagResults and discussion ine are likely to exhibit a dual role, as end-products of the assimilation pathway and as ammonium donors Effects of the A/03~ assimilation pathway functioning on the through the activities of glutamate dehydrogenase or exudation process glutaminase from the plant and its rhizosphere (Peeters N compounds or NH% suppliers: Cumulative amounts of and Van Laere, 1992) and asparaginase (Sieciechowicz exudate increased with time after detopping showing that et al., 1989; Peeters and Van Laere, 1992). KNO3 and maize seedlings were able to maintain the exudation urea were chosen as reference assays for each family process during the 5h experimental period (Fig. 1). because they have a monovalent role, acting either as Compared to water-plants, exudation was doubled by endogenous NH^ donor in the case of KN0 3 or as KC1, but, at the same osmolarity (16mOsmol I" 1 ), mixexogenous NH^ donor in the case of urea. Urea was tures containing KNO3, urea, NH 4 N0 3 or glutamine preferred to an ammonium salt because it is well known induced a stronger enhancement of the exudation (x 2 that NH^1" exhibits some toxicity and generally gives rise compared to KC1, Fig. 1 left). Asparagine and glutamine to plants with a reduced dry matter content (Salsac et al., produced a similar enhancement of exudation whereas 1987). Urea is readily degraded through urease activity other amino acids tested, glycine, phenylalanine and from the plant or companion bacteria, thus delaying proline were less efficient than KC1 (Fig. 1 right). Three NH^ supply and avoiding the accumulation of large compounds involved in the nitrate assimilation pathway, ammonium concentrations. In this way, a decrease of the ", NH.J", glutamine, (asparagine) increased exudation toxic effect of a high ammonium concentration was expected.

Comparative study of KN03 and urea supply: Irrespective of the duration of the treatments before detopping, KN0 3 enhanced exudation more than urea (Fig. 2): exudation appeared earlier with KN0 3 and was rapidly linear after 2 h of incubation, whereas a pronounced lag phase was observed with urea for the first 12 h of incubation. It is likely that the progressive onset of urease activity delayed the accumulation of NH^. The fact that the lag phase decreased after longer incubation is consistent with this hypothesis. A 16 h treatment was chosen for the reference assay for both inducers because it corresponded to the

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Time after detopping (h) Fig. 1. Effect of different root media on cumulative volume of exudate collected after seedling detopping. Media were applied 16 h before detopping. Left, KNO3, urea, NH4NO3, glutamine (GLN) K.C1, and H2O. Right, glutamine, asparagine (ASN), glycine (GLY), phenylalanine (PHE), proline (PRO), H2O as control. Inducers for both the experiments were given as 100 ml of solution at 50 mM for KNO3, KC1 and NH4NO3, 25 mM for urea with 17.5 mM of K.C1, and 50 mM for the different amino acids with 50 mM of KG. As the initial root volume was 500 ml, the final salt and metabolite concentration was lowered by a factor 6.

6

12 18 24 30 Incubation duration (h)

Fig. 2. Time-course of KNO3 and urea assays on cumulative exudate collected after 2, 6, 12, 26, and 30 h.

Xylem exudation and nitrate assimilation pathway

length of the light period. This choice allowed the collection of xylem sap in the dark period and to obtain the maximum NR activity for KNO3 assay (Barthes et al., 1995a). After a 16 h incubation KNO3-treated plants (Fig. 3, left) had an exudation rate around 1.3 ml h" 1 per 100 plants whereas urea-treated plants (Fig. 3, right) had a slightly lower value around 1.0 ml h" 1 per 100 plants. Nitrate is known to be an efficient counterion supporting potassium uptake and deposition in the xylem (Touraine and Grignon, 1981) and a greater value of sap osmolarity could be expected in such conditions, explaining the higher water flux in KNO3-treated plants. However, this interpretation is not supported by the sap osmolarity values (Fig. 4, control plants, open symbols) since the range of osmolarity of the xylem exudate went from 45 to 35 for the KNO3-treated plants and from 65 to 55 for

the urea-treated plants. Urea was supplemented with a KC1 salt in order to maintain the same medium osmotic condition as in the KNO3 experiment. In such conditions, urea-treated plants, exhibited the higher osmolarity and the lower exudation rate. KNO3- and urea-treated plant osmolarity presented a slight dilution with time as previously described (Barthes et al., 1995a). This fact was related to the disappearance of the resistance due to the removal of the vascular system of leaves. The sap of KNO3 plants contained NO^ and NH4+ (Fig. 3, left) and both presented the time-dependent dilution noted for total osmolarity: the initial NO3~ concentration was 10 mM and declined by 38% in 5 h; the initial NH^ concentration was lower, 2 mM, but declined more rapidly, by 55% in 5 h. Exudates of urea plants (Fig. 3, right) contained twice as much NH^" than the sap of KNO3 plants but, as expected, no nitrate. Amino acid concentrations were also higher with urea (3 mM compared to 1 mM). Similar results were obtained by Chaillou et al. (1991). The higher sap amino acid concentrations of urea-supplied plants suggests that a large part of NH^ arising from urea was actually assimilated. Similar results have already been described by Lee and Lewis (1994) using either 15N-nitrate or 15N-ammonium. This interpretation is supported by the fact that carbohydrates decreased in the sap. Carbohydrate concentration was slightly higher in KNO3 assays at the beginning, but it dropped more rapidly than in the urea assays. Between 5 and 7 h, carbohydrates totally disappeared from the sap in both cases, indicating a restriction in their supply. As the carbohydrate pool was mainly represented by glucose (70%) and fructose (25%) and slightly by sucrose (

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