PHYSIOLOGIA PLANTARUM 89: 847-853. 1993Printed in Denmark - all rights reserved

Copyright © Physiologia Plantarum 1993tSSN 0031-9317

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Time-course of the phosphinothricin effect on gas exchange andnitrate reduction in Medicago sativa

M. Lacuesta, B. Gonzalez-Moro, C. Gonzalez-Murua and A. Muiioz-Rueda

Lacuesta, M., Gonzalez-Moro, B., Gonzalez-Murua, C. and Munoz-Rueda, A. 1993.Time-course of the phosphinothricin effect on gas exchange and nitrate reduction in

. - Physiol. Plant. 89: 847-853.

The long-time effect of phosphinothricin (PPT) on gas exchange and nitrate meta-bolism in intact plants of lucerne {Medicago sativa L. cv. Aragon) was investigated.Photosynthetic CO: uptake, stomatal conductance, and transpiration were measuredwith an Infra-Red Gas Analyzer (IRGA). Under photorespiratory conditions, CO2uptake continuously decreased after PPT treatment. The decrease of photosynthesis ledto an increase in the intemal CO2 concentration, which in turn caused stomatal closureand a reduction of transpiration rate. Nitrate reduction from plants sprayed with PPTwas assayed both in vitro and in vivo. In vivo nitrate reductase was measured with andwithout nitrate in the infiltration medium. Both types of nitrate reductase assaysindicated that the enzyme was inhibited in plants treated with PPT; however, theenzyme appeared more affected when the in vivo assay was used than when the one invitro was applied. The nitrate reduction was pronouncedly affected after 24 h of PPTtreatment, when glutamine synthetase (GS, EC 6.3.1.2.) activity and gas exchangewere inhibited by more than 60%. The data suggest that the inhibition of GS leads toinhibition of photosynthesis, which, in turn, means lack of NADPH and nitrate, thesubstrates for nitrate reductase. The inhibition of GS also leads to a high ammonialevel, which will produce a secondary inhibition of nitrate reductase activity.

Key words - Glufosinate, glutamine synthetase activity, internal CO2 concentration,lucerne, Medicago sativa, nitrate reduction, photosynthesis, quantum yield, stomatalconductance, transpiration.

M. Lacuesta, B. Gonzalez-Moro, C. Gonzdlez-Murua and A. Muiioz-Rueda (corre-sponding author), Depto de Biologia Vegetal y Ecologia, Facultad de Ciencias, Univ.del Pais Vasco/EHU, Apartado 644, E-48086 Bilbao, Spain.

Introduction

Phosphinothricin (PPT) is a non selective herbicide(Schwerdtle et al. 1981), which has received special at-tention because it can be used as a tool for the study ofnitrogen metabolism. It is an analogue of glutamate andbinds to the active site of glutamine synthetase (GS) incompetition with glutamate (Lea and Ridley 1989).

The inhibitory effect of PPT on GS seems to be ir-reversible (Manderscheid and Wild 1986). Wild andManderscheid (1984) pointed out that the inhibition ofGS was the primary site of action of phosphinothricin andruled out any effect on the in vitro activity of otherenzymes of nitrogen metabolism. However, in vitro ni-

trate reductase activity from plants treated in vivo withPPT was also diminished, although the inhibition was along time effect and not evident until 24 h after treatment(Lacuesta et al. 1990). Because glutamine synthetaseplays a central role in the assimilation of ammonia, theinhibition of that comer-stone enzyme completely dis-turbs the nitrogen metabolism (Lacuesta et al. 1989,1990, Wendler et al. 1990, Shelp et al. 1992).

PPT also inhibits photosynthesis in C3 plants (Wendlerand Wild 1990, Lacuesta et al. 1992) as well as in C4plants (Wendler et al. 1990, Shelp et al. 1992, Gonzalez-Moro et al. 1993). Initially, it was proposed that the buildup of toxic ammonia, was the cause of the photosyntheticinhibition. However, in view of the lack of correlation

Received 14 April, 1993; revised 28 July, 1993

55 Physiol. Plant. 89, 1993 847

between ammonia accumulation and inhibition of photo-synthesis (Wild et al. 1987, Lacuesta 1992), other mecha-nisms have been proposed. For instance, the decrease ofamino donors that are needed to complete the glyoxylatepathway, could lead to an accumulation of glycolateand/or glyoxylate (Gonzalez-Moro et al. 1993), which inturn should inhibit the CO2 assimilation as a consequenceof inhibition of ribulose bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39; Wendler et al. 1992).The decrease of amino donors also restricts the return ofglycerate to the Calvin cycle (Blackwell et al. 1988),which may contribute to the inhibition of photosynthesis.

Practically all studies concerning the effect of PPT onphotosynthesis have been carried out in short-time ex-periments, using excised leaves fed with PPT solutionsthrough the petiole. Long-time experiments with excisedleaves involve the risk of impairment of the transpirationstream, after which the resultant stomata closure leads toinhibition of photosynthesis. In order to avoid confusingresults, it is necessary to discard leaves in which thestomatal resistance has increased. This difficulty isavoided when intact plants are treated with PPT. More-over, whole plant treatments make it possible to study thedynamics of the PPT effects, and the sequence of eventsproduced in nitrogen metabolism as well as in gas ex-change. In the present investigation we have analysed thetime-course of gas exchange processes in lucerne plantssprayed with several concentrations of PPT. The resultsare discussed in relation to the inhibition of GS, the maintarget for PPT. We have also studied the behaviour of invivo nitrate reductase activity, and the results are put inrelation to the effects of PPT on photosynthesis, on tran-spiration, and on nitrate uptake.

Abbreviations - C,, internal CO2 concentration; GDH, gluta-mate dehydrogenase (EC 1.4.1.3.); y-GHM, gamma glutamylhydroxamate; GOGAT, glutamate synthase (EC 1.4.7.1.); GS,glutamine synthetase (EC 6.3.1.2.); IRGA, infra red gas ana-lyzer; NR, nitrate reductase (EC 1.6.6.1.); PPT, phosphino-thricin; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxy-genase (EC 4.1.1.39).

The plants used for phosphinothricin experiments were4 weeks old from sowing, at the vegetative stage. Groupsof 8 plants were sprayed with 10 ml of an aqueoussolution of 0, 250, 500 or 1 000 |JLM of a commercialpreparation of glufosinate (phosphinothricin, HoechstIberica S.A., Barcelona, Spain). Measurements weremade 2, 4, 8, 24 and 48 h after PPT application. Theexperiments were repeated in 5 independent series.

Preparation of leaf homogenates

Leaf homogenates were prepared according to Lacuestaet al. (1989). Fresh leaf material (2 g) was washedwith deionized water before grinding in an Omnimixer atmaximal speed for 1 min with 8 ml of ice-cold grindingmedium, containing 0.5 mM EDTA (disodium salt),1 mM DTT, 2 mM MgCL, 20% (v/v) glycerol and 100mM imidazole-acetate buffer, pH 7.8. The homogenatewas filtered through 4 layers of cheese cloth and cen-trifuged at 35 000 g for 20 min. The supematant was usedfor enzyme assays. All determinations were performedwithin 2-A h after extraction, and samples were main-tained in an ice bath until used.

Enzyme assays, protein content and dry weights

GS activity was measured spectrophotometrically at 540nm by the formation of y-glutamyl hydroxamate in thesynthetase reaction as described by Lacuesta et al. (1989).

The nitrate reductase activity was measured both invivo and in vitro. The NR activity was measured in vivo,with or without 50 mM KNO3, and expressed as nmolnitrite mg"' protein h"' (Munoz-Rueda et al. 1986a,b).Propanol (1%, v/v) was used instead of Triton X-100. Invitro nitrate reductase activity was assayed in extractsobtained as described by Wallace (1986), using 0.5%BSA instead of casein. The enzyme activity was assayedessentially as described by Lillo (1983), except that theNO3 concentration in the reaction medium was 20 mM.

Soluble protein was determined according to Bensa-

Materials and methods

Plant material and growth conditions

Seeds of Medicago sativa L. (cv. Aragon) were placed ina Perlite-containing seed-bed, covered with plastic andwatered with Hoagland's solution (Arnon and Hoagland1940). After emergence, the plants were transferred to 2-1plastic pots containing Perlite (Cafinpa S.A,, Barcelona,Spain), covered with quartz sand and watered every twodays with Hoagland's solution and once a week withdeionized water in order to avoid salt accumulation.The plants were grown in a growth chamber (25/20°C,day/night temperature, 50/70% relative humidity, and aphotoperiod of 14 h at 330 ixmol m-^ s"' PAR). Illu-mination was provided by fiuorescent lamps (SylvaniaF48T12SHO/VHO) and light bulbs.

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Time (h)

Eig. 1. Time-course of the effect of PPT on GS activity inleaves of lucerne plants sprayed with H2O ( • ) , 250 |xM PPT( ^ ) , 500 |JLM PPT ( • and 1 000 ^x.M PPT (Bg . Data given arethe mean (± S E ) of 5 independent preparat ions with 3 replica-tions in each. .• l"i': .;,;,.-••.?"' ..̂ ;-;<-•.••:.>; •\W ' :\y^:- -'. :."; i'......-. r.;

848 Physiol. Plant. 89, 1993

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20 40 60 80 100

GS activity (% of control)

Eig. 2. Relation between photosynthesis (% of control) and theinhibition of GS activity (% of control) in sprayed lucerneplants. Values for the control plants were 8.2 ±0.4 fxmol CO2m"̂ s"' for photosynthetic rate and 7.6 ±0.5 |xmol y-GHM mgprotein"' h"' for GS activity. Data represent the values obtainedduring 48 h after treatments with PPT.

doun and Weinstein (1978), using crystalline bovine se-rum albumin (Sigma fraction V) as the standard.

Dry weights of leaves were obtained after drying in anoven at 85°C for 48 h.

Gas analysis system

Net photosynthesis, transpiration and stomatal con-ductance were determined as described by Becerril et al.(1989) using an IRGA type LCA-2 (Analytical Develop-ment Company Ltd, Hertforshire, UK). The rate of CO2fixation, stomatal conductance, transpiration rate, internalCO2 concentration and water use efficiency were deter-mined by the standard equations for an open gas ex-change system (Long and Haligren 1985). Gas exchangewas determined on 5 plants per treatment, and each treat-ment was assayed 3 times.

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Stomatal conductance (% of control)

Eig. 4. Relation of net photosynthesis (D) and intemal CO2concentration (Ci; ^ ) with stomatal conductance in lucerneplants sprayed with PPT. Data represent the values obtainedduring 48 h after treatments with PPT.

Results

GS activity was reduced soon after treatment with phos-phinothricin (Fig. 1). Approximately 60% inhibition wasproduced by 1 000 |JLM PPT already about 2 h afterapplication. At the end of the experiment, GS activity wasreduced by 50% with 250 |JLM and by 95% with thehighest PPT concentration.

Under photorespiratory conditions, GS inhibition isaccompanied by a fall of net photosynthesis (Wild et al.1987, Lacuesta et al. 1992). The relationship between theinhibiton of GS activity and the decrease of photosyn-thesis is given in Fig. 2. Photosynthesis did not changeuntil GS was inhibited by about 60%, but then an almostlinear relation between the inhibition of GS activity andthe decrease in photosynthesis could be observed.

Inhibition of photosynthesis increased with increasingPPT concentration and with time after treatment (Fig. 3 A),and was almost complete (>90% inhibition) at the end ofthe experiment and the highest concentration of PPT.

In PPT-treated leaves both stomatal conductance (Fig.3B) and transpiration (Fig. 3C) continuously decreased,showing a pattem of inhibition similar to that of net

Eig. 3. Time-course of theeffect of PPT on (A) netphotosynthesis, (B) stomatalconductance, (C)transpiration and (D) internalCOT concentration (C,). ( • )250 |xM PPT, ( • ) 500 \xMPPT and (A) 1000 JJLM

PPT. Data points relate tothe control values at eachtime. The control valueswere practically unchangedwith time, the mean of thembeing: 8.2 ±0.4 iJimol CO2m"- s~' for photosynthesis;148 ± 10 mmol air m"- s"'for stomatal conductance;4.9 ±0.3 mmol H2O m"- s"'for transpiration, and 227 ± 6ixmol CO2 mol"' air for C;.Each data point is the meanof 10-15 measurements.

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55* Physiol. Plant. 89, 1993 849

Tab. 1. Time-course of the effect of PPT on water use efficiency in luceme plants. Values are expressed as mmol CO2 mol ' H2O.Data given are the mean (± SE) of 5 independent experiments with 3 replications in each. Numbers within a column followed by the.same letter or without a letter do not differ significantly (Tukey's r-test, ^ = 0.05).

PPT (|xA/)

- 0 ''"'''250500

1 000

2

' " 1.70±0.081.75±0.091.81 ±0.09 ,1.85±().13

4

'" 2.11 ±0.111.98±0.092.23±0.112.02±0.14

Time (h)

8

2.44±0.202.09±0.102.14±0.222.04±0.19

24

1.65±0.08a1.55±O.IO b0.91 ±0.09 c0.51±0.12 d

48

1.60±0.12 a1.10±0.11 b0.80±0.11 b1.20±0.24 a

photosyntheis (Fig. 3A). However, the decrease in watervapour exchange was always smaller and later than theinhibition of photosynthesis, indicating that PPT treat-ment led first to a reduction of CO2 assimilation and onlylater to a decrease in water vapour exchange. With 1 mMPPT, both stomatal conductance and transpiration werereduced by more than 80% at the end of the study.

Internal CO2 concentration supplies information aboutthe balance between CO2 diffusion into the mesophylland the capacity of Rubisco for CO2 fixation. Despite thestomatal closure, the internal CO2 concentration did notdrop (Figs 3D and 4), but actually increased until 24 hafter treatment. This rise coincided with the diminution ofnet photosynthesis. At the end of the experiment, theintemal CO2 concentration fell again, perhaps indicatinga disturbance of stomatal function; but it never com-pletely reached as low as the initial control values.

The relationship between net photosynthesis and sto-matal aperture was linear, at the same time as stomatanever became completely closed (Fig. 4).

The water use efficiency, that is the ratio between CO2assimilation and water vapour given off, decreased 24 hafter starting the PPT-treatment, although a slight in-crease could be noted at the end of the series with thehighest PPT-concentration assay (Tab, 1). Spraying lu-cerne plants with PPT resulted in a decrease in quantumyield (Tab. 2). The inhibition grew with time and in-creasing PPT concentrations.

Unlike GS, which was reduced by PPT not only in vivobut also in vitro (Lacuesta et al. 1989, 1990), NR activitywas not affected when 1 000 [xM PPT was added to theplant extract (in vitro effect; data no shown). When NR

Tab. 2. Time-course of the effect of PPT on quantum yield.Values are expressed as percentage of the control value at eachtime, the overall mean being 40±3 nmol CO2 (ixmol photon)"'.Data given are the mean of 5 independent experiments with 3replications in each.

PPT Time (h)

02505001000

. . - • - % .

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was assayed in vivo in PPT-treated leaves without (Fig.5A) and with (Fig. 5B) NO3 in the infiltration medium,we observed a decrease of in vivo enzyme activity at 48and 24 h after PPT-treatment, respectively. Though bothtypes of assay showed similar inhibition pattems, the PPTeffect was stronger when nitrate was present in the infil-tration medium.

A close correlation became apparent between on theone hand inhibition of photosynthesis and decrease intranspiration rate and, on the other hand, nitrate reductaseactivity (all expressed as percentages of control values;Fig. 6). Although both net photosynthesis and transpira-tion rate seemed to control NR activity, the decrease oftranspiration affected the enzymatic activity more thandid reduction of photosynthesis.

Discussion

Although it is clear that the primary effect of phosphino-thricin is through the inhibition of glutamine synthetase,other additional sites of action seem to be involved, some

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Eig. 5. Time-course of the effect of PPT on NR activity assayedin vivo without (A) and with (B) NOj in the infiltration medium.Plants were sprayed with H:0 ( • ) , 250 jxM PPT (HB), 500\LM PPT (iD and 1 000 |xM PPT (^). Data given are the mean(± SE) of 5 independent preparations with 3 replications in each.

850 Physiol. Plant. 89, 1993

Eig. 6. Relation of NRactivity with CO2assimilation (open symbols)and transpiration (filledsymbols) in plants treatedwith PPT. Data points relateto the control values at eachtime. In vitro NR activity (Aand B), and NR activityassayed in vivo without (Cand D) and with (E and E)NO3 in the infiltrationmedium. Values are obtainedfrom Eigs 3 and 5.

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CO2 assimilation (% of control)

20 40 60 80 100

Transpiration (% of control)

as a consequence of that inhibition and others as con-comitant changes induced by PPT. These observationsinduced us to study the dynamics of PPT effects onnitrogen metabolism as well as gas exchange on thewhole plant level.

PPT inhibits GS activity already after 2 h of treatmentat 1 000 (xM (Lacuesta et al. 1990; present data. Fig, 1).In air, PPT also gives a quick inhibition of photosynthesisin leaf sections (Sauer et al. 1987, Wild et al. 1987,Lacuesta et al. 1992). Conversely, under non-photo-respiratory conditions (1% O2 or 0.7% CO2) PPT did notaffect photosynthesis to a noticeable extent (Wild et al.1987, Lacuesta et al. 1992), suggesting that the inhibitionof photosynthetic CO2 assimilation is arised through in-hibition of photorespiration.

The reduction of retum of carbon from the photo-respiratory pathway as a consequence of the inhibition ofGS by PPT, should interrupt the carbon recycling to thereductive pentose phosphate pathway (Walker et al.1984). Moreover, as a consequence of the accumulationof glyoxylate and/or glycolate produced (Sauer et al.1987, Wendler et al. 1990, 1992, Gonzalez-Moro et al.1993) ribulose bisphosphate carboxylase/oxygenase ac-tivity will be inhibited (Ziegler and Wild 1989; our data,not shown). Ultimately, the decrease of CO2 fixation (Fig.3A) would be produced.

A continuous decrease in transpiration rate and a re-

duction of stomatal conductance were registered (Fig. 3Band C). The decrease of stomatal conductance observedcannot be the cause of the decrease of photosynthesis,since decreases in stomatal aperture and transpirationwere not perceptible until 24 h after the plants hadbeen treated with 250 |xM and 500 |xM PPT, whereas thesame PPT-treatments reduced net photosynthesis alreadyafter 8 h.

In spite of the decrease in stomatal aperture, the in-temal CO2 concentration did not diminish; actually, a risewas observed during the first 24 h (Fig. 3D and Fig. 4),This indicates that the inhibition of photosynthesis leadsto an increase of intemal CO2 concentration, which intum causes a reduction of transpiration rate. However, adirect effect of PPT on stomata behaviour cannot beexcluded since Ullrich et al. (1990) reported that PPTcaused membrane depolarization and a disturbed level ofK+ within cells. The loss of K"̂ from guard cells wouldlead to a reduction of stomatal aperture (Raschke 1987)and could explain the initial stomatal closure observed atthe highest PPT concentration (Fig. 3B) as well as therecovery of intemal CO2 concentration (Fig. 3D) ob-tained at the end of the experimental time, when thecorrect performance of stomatal movement may be dis-turbed. Taken all in all, we can, however, assume anon-stomatal inhibition of photosynthesis. The effect of

Physiol. Plant. 89, 1993 851

PPT on water vapour exchange is a consequence, and notthe cause, of the inhibition of photosynthesis.

In vitro, PPT does not affect the activity of enzymes ofnitrogen metabolism others than GS. Wild and Mander-scheid (1984) and Troigsch et al. (1989) like ourselves(data not shown), observed that in vitro NR activity wasnot affected by PPT. On the contrary, the in vivo assayshowed inhibition of NR activity by PPT (Fig. 5). Thisfact provides a more clear picture of the in situ NOjassimilation in the intact plant. Without exogenous sub-strate, the in vivo assay refiects not only the enzymecapacity but also the availability of both intemal nitrateand energy (Fig, 5A), whereas, with nitrate in the me-dium, it basically depends on the supply of reductiveenergy (Fig, 5B).

PPT treatment also caused a high accumulation ofammonia (Lacuesta et al. 1989, 1990), and this ammoniacould inhibit NR activity (Srivastava 1980), However,other mechanisms may also have to be taken into accountfor the inhibition of NR activity observed after treatmentswith PPT.

The in vitro inhibition of NR activity from plantssprayed with phosphinothricin (Lacuesta et al. 1990) sug-gests that PPT affects the enzyme level. Moreover, NRactivity is controlled by the fiux of nitrate via the tran-spiration stream to the leaves (Shaner and Boyer 1976,Muiioz-Rueda et al. 1986a,b); so that the inhibition ofnitrate uptake (Troigsch et al. 1989), and the reduction oftranspiration rate in PPT-treated plants (Fig. 3C) couldcause the reduction of NR activity (Fig. 5). In addition,the greater decrease in NR activity when NO3 is presentin the infiltration medium (compare Fig. 5A and B) alsosuggests a lack of reductive energy necessary for nitratereduction. The inhibition of photosynthesis reduces theenergy availability and provokes the decrease of the tran-spiration rate. Also, as a consequence of the inhibition ofphotosynthesis, the fiux of nitrate to the leaves is dimin-ished and, consequently, a lesser NR activity is registeredin PPT-treated plants compared with control plants.

The results obtained in this work show that the reduc-tion of NR activity is more related with the disturbance inthe gas exchange than with GS inhibition itself. Thisagrees with the hypothesis proposed by Kaiser andBrendle-Behnish (1991), who pointed out that CO2 assi-milation determines NO3 assimilation through modula-tion of NR.

We conclude that the chain of events provoked by theaction of PPT on the whole plant is basically as follows:the inhibition of GS produces a disturbance of the photo-respiratory cycle, preventing the retum of carbon to theCalvin cycle. The inhibition of photosynthesis leads to anincrease of intemal CO2 concentration, which in tumreduces stomatal conductance, so that a reduction of thetranspiration rate is achieved. The effect caused on otheraspects of nitrogen metabolism would basically be aconsequence of this change in gas exchange. Thus, the invivo inhibition of nitrate reductase may be attributed notonly to the high levels of ammonium but also to the

decrease of transpiration, the reduction of nitrate uptake,and the lack of reductive energy, as consequence of theinhibition of photosynthesis.

Acknowledgements - This work was partially supported bygrants UPV 118.310-EO16/90 and UPV91 118-E099/91 fromthe Universidad del Pais Vasco. M.L. held a fellowship from theMinisterio de Educacion y Ciencia (PEPI, Programa Nacional).

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Physiol. Plant. 89, 1993 853