The influence of nitrogen supply on antioxidant enzymes in plant roots

© CSIRO 2004 10.1071/FP03130 1445-4408/04/010001

Functional Plant Biology, 2004, 31, 1–9www.publish.csiro.au/journals/fpb

CSIRO PUBLISHING

The influence of nitrogen supply on antioxidant enzymes in plant roots

Leonardo Oliveira MediciA, Ricardo Antunes AzevedoB, Richard John SmithC and Peter John LeaC,D

ADepartamento de Ciências Fisiológicas, Universidade Federal Rural do Rio de Janeiro, Seropédica CEP 23890-000, R.J., Brazil.

BDepartamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba CEP 13418-900, S.P., Brazil.

CDepartment of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK.DCorresponding author; email: [email protected]

Abstract. Plants of Zea mays L., Hordeum vulgare L. and Arabidopsis thaliana (L.) Heynh. were grown atdifferent concentrations of nitrogen, as NH4NO3, and the antioxidant enzyme activities and quantities in the roots,were studied. Maize plants were grown at 1 and 10 mM N for 32 d after germination and their roots were analysedby native PAGE for the isoenzymic profiles of glutathione reductase (GR; EC 1.6.4.2), catalase (CAT; EC 1.11.1.6)and superoxide dismutase (SOD; EC 1.15.1.1) and by spectrophotometric assays for total activity of CAT and GR.Barley and A. thaliana plants were grown at five N concentrations (0.25, 0.5, 1, 10 and 20 mM) for 27 and 26 d,respectively, and the isoenzymic profile of the three enzymes were analysed in their roots. The number of GRisoforms in the three plant species was increased at high N supply compared with low N. In addition, the CAT andGR activities detected in the roots were increased in plants grown at higher N. The increase in activity and numberof bands of GR at high N in the roots of all three plants studied was not accompanied by a change in activity or thenumber of SOD isoforms. The possibility that the application of high N promotes the formation of reactive oxygenspecies by stimulating an increased rate of growth is discussed.

Keywords: Arabidopsis thaliana, barley, catalase, glutathione reductase, maize, nitrogen, roots, superoxidedismutase.

Introduction

A deficiency of nitrogen is the major limitation of maizeproductivity, since it is an essential plant nutrient (Lea andMorot-Gaudry 2001) and 60% of world agriculture ischaracterised by low inputs (Thomason et al. 2002). How-ever, in other sections of agriculture, economic, environ-mental and public health concerns exist owing to theapplication of excess N (Matson et al. 1998; Britto andKronzucker 2002). Therefore a better understanding of howplants respond to N supply is required for several reasons(Gray et al. 2002; Carswell et al. 2003). Several traits havebeen studied to improve this understanding, for examplechlorophyll concentration, plant height, timing of leafsenescence, anthesis–silking interval, kernel number, activi-ties of enzymes controlling the assimilation of N andquantitative trait loci (Reggiani et al. 1999; Bertin andGallais 2000; Majerowicz et al. 2002; Fageria and Baligar

2003; Ray et al. 2003). A new quantitative genetic approachof associating metabolic activity and agronomic traits withDNA markers has indicated that leaf nitrate accumulationand the reactions catalysed by nitrate reductase andglutamine synthetase represent key elements for controllingnitrogen-use efficiency in maize (Hirel et al. 2001).

The enhancement of metabolism and growth by N mayrequire the presence of additional defence mechanismsagainst reactive oxygen species (ROS) (Mullineaux andCreissen 1997). In order to prevent or alleviate the damagethat ROS may cause in plant tissues, plants utilise a range ofantioxidant enzymes that are able to metabolise ROS to lesstoxic compounds; these include superoxide dismutase,ascorbate peroxidase, glutathione reductase and catalase(Polle 2001; Noctor et al. 2002). One aspect, still littlestudied, is the regulation of the activity of these antioxidantenzymes, by the supply of nitrogen to the plant.

Abbreviations used: CAT, catalase; DPIP, 2,6-dichloro-N-(4-hydroxyphenyl)-1,4-benzoquinoneimine sodium salt; GR, glutathione reductase;GSH, GSSG, reduced and oxidized forms of glutathione, respectively; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide;SOD, superoxide dismutase; ROS, reactive oxygen species.

2 Functional Plant Biology L. O. Medici et al.

Superoxide dismutase (SOD; EC 1.15.1.1), which detox-ifies superoxide by converting it into H2O2 and O2, has beenclassified into three distinct groups according to the type ofmetal co-factor (Fe, Cu/Zn and Mn) (Scandalios 1997;Alscher et al. 2002; Fornazier et al. 2002a). Fe–SOD has notbeen found in all studies (Ferreira et al. 2002), but whenobserved in plant tissues, has been shown to be located inthe chloroplasts (Alscher et al. 2002). The abundantCu/Zn–SODs are found in the cytoplasm, peroxisomes andchloroplasts (Alscher et al. 2002; Del Rio et al. 2002). Thelocation of Cu/Zn–SOD has been shown by immunogoldlabelling to include the nucleus and apoplast (Alscher et al.2002). Mn–SOD has been shown to be located in themitochondria and also possibly in the peroxisomes(Scandalios 1997). Seven genes have been isolated fromArabidopsis thaliana, three encoding Cu/Zn, three encodingFe and one Mn–SOD (Kliebenstein et al. 1998), while inmaize five genes encoding Cu/Zn and four encodingMn–SOD have been detected (Fink and Scandalios 2002).Limited information on two SOD genes from barley iscurrently available in the NCBI database (Thomas 2003),although six Cu/Zn and two Mn–SOD isoenzymes fromlight grown leaves could be separated by native PAGE(Azevedo et al. 1998).

Glutathione reductase (GR; EC 1.6.4.2) is a flavoproteinthat catalyses the NADPH-dependent reduction of theoxidized form of glutathione (GSSG) to reduced glutathione(GSH) (Mullineaux and Creissen 1997). The functions ofGSH within the plant cell are wide ranging and include:(i) a store of reduced sulfur, (ii) a substrate for glutathioneS-transferases, which are involved in the removal of toxiccompounds and other key metabolic reactions, (iii) partici-pation in the ascorbate–glutathione cycle, which is a majorroute of peroxide removal in cells under both normal andstressful conditions, and (iv) maintenance of the sulfhydrylgroups of cysteine in a reduced form, which is implicated inthe regulation of enzyme activity (Noctor et al. 2002).Although the majority of the GR activity has been shown tobe located in the chloroplasts or plastids, significant activityhas also been detected in the cytosol, mitochondria andperoxisomes (Noctor et al. 2002). Two GR genes have beenisolated from pea leaves, one that encodes a protein that istargeted equally to both chloroplasts and mitochondria(Mullineaux and Creissen 1997; Chew and Whelan 2003)and one that is located in the cytoplasm (Stevens et al.2000). In higher plants, the implication of GR in the defenceagainst oxidative stress has been studied in several stressfulconditions such as drought (Del Longo et al. 1993), salinity(Rios-Gonzalez et al. 2002), ozone (Azevedo et al. 1998),heavy metals (Vitória et al. 2001; Pereira et al. 2002) orchilling (Kornyeyev et al. 2003).

Catalase (CAT; EC 1.11.1.6) decomposes H2O2 into H2Oand O2 (Fornazier et al. 2002b). Three CAT genes have beencharacterised in maize (Scandalios et al. 1997), and

A. thaliana (Frugoli et al. 1996), while only two have beenidentified in barley (Skadsen et al. 1995). Extensive studiesby Scandalios and colleagues have shown that native PAGEcan readily separate three isoenzymes and that hetero-tetrameric forms can also be detected (Scandalios et al.1997; Guan and Scandalios 2002). The enzyme is abundantin the glyoxisomes of lipid-storing tissues in germinatingbarley, where it decomposes H2O2 formed during theβ-oxidation of fatty acids (Holtman et al. 1994), and in theperoxisomes of the leaves of C3 plants, where it removesH2O2 produced during photorespiration by the conversion ofglycolate into glyoxylate (Kendall et al. 1983; Willekenset al. 1997). A third class of CAT is located in vasculartissues and may be involved in protection against environ-mental stress (Willekens et al. 1994).

The present study was conducted to examine the effectsof a range of concentrations of N supply that were eitherlimiting or in excess, on the activities of the antioxidantenzymes GR, CAT and SOD, in the roots of maize, barleyand A. thaliana.

Materials and methods

Plant and experimental conditions

Three S5 maize (Zea mays L.) lines, numbered as 2, 4 and 6, and theirthree hybrids, numbered as 24 (2×4), 26 (2×6) and 46 (4×6), were used.Lines 2 and 4 originated from the commercial hybrid AG311(Agroceres, Brazil), whereas line 6 originated from the commercialhybrid AG302 (Agroceres, Brazil). These lines and hybrids seeds wereplanted and grown in a glasshouse under natural daylight, in Januaryand February 2001 in Piracicaba, Brazil, with temperatures in therange 23–33°C. Plants were grown in 1.2-L pots containing sand and1 or 10 mM nitrogen (NH4NO3), to which was added a modifiedHoagland’s solution (Hoagland and Arnon 1950), in which the calciumnitrate and potassium nitrate were substituted by calcium chloride andpotassium sulfate. A volume of 1.2 L of nutrient solution per pot wasreapplied every 4 d. Water was continuously supplied by capillarity.The plants were harvested 32 d after germination and samples 10–12cm from the root apex were collected for biochemical analysis. Theexperimental design was a randomised complete block. Four replicateswere used for shoot and root dry weight determinations, threereplicates were used for CAT and GR activities and two replicates wereused for native PAGE.

Barley (Hordeum vulgare L. cv. Maris Mink) seeds weregerminated in moistened paper for 5 d and grown in a glasshouse undernatural daylight during July 2002 in Lancaster, UK with temperaturesin the range 12–31°C. The plants were grown in 1-L pots (three plantsper pot) containing sand and 0.25, 0.5, 1, 10 or 20 mM N (NH4NO3) inthe modified Hoagland’s solution described above. A volume of 1 L ofnutrient solution per pot was reapplied every 4 d. Water wascontinuously supplied by capillarity and after 27 d of treatment,samples 10–12 cm from the root apex were collected for biochemicalanalysis. Five replicates were used for shoot and root dry weightdeterminations and two replicates were used for native PAGE.

Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) plants wereplanted and grown in 20-mL pots containing sand (20 plants per pot) ina growth chamber at 25°C, with a 14-h photoperiod under fluorescentlamps providing a photosynthetically active irradiance of90 µmol photons m–2 s–1. The same five concentrations of N in themodified Hoagland’s solution used for barley were also used forA. thaliana. The nutrient solution was reapplied every 7 d, by the

Nitrogen and antioxidant enzymes in plant roots Functional Plant Biology 3

addition of 20 mL solution per pot. Water was continuously suppliedby capillarity. Each N concentration was applied to 10 pots and allroots were collected after 26 d of N treatment. Two replicates were usedfor shoot and root fresh weight determinations and for native PAGE.

All plant samples collected were frozen in liquid nitrogen andstored at –80°C for further analysis.

Enzyme extraction

Enzyme extraction and preparation were carried out at 4°C. The roottissue was reduced to powder in a pestle and mortar with liquidnitrogen and then homogenised (2:1 buffer volume:fresh weight) with100 mM Tris buffer (pH 7.8) containing 1 mM dithiothreitol, 10% (v/v)glycerol and 0.2% (w/v) insoluble polyvinylpyrrolidone. Thehomogenate was centrifuged at 15000 g for 30 min and the supernatantwas stored in separate aliquots at –80°C to be used for GR, CAT andSOD analyses. Barley and A. thaliana extracts were concentrated withcentrifuge filters (VIVASPIN, Sartorius Ltd, Surrey, England).

Enzyme assays

Glutathione reductase activity was determined as described byAzevedo et al. (1998). GR activity was assayed spectrophotometricallyat 30°C in a mixture consisting of 1 mL 100 mM potassium phosphatebuffer (pH 7.5) containing 1 mM 5,5′-dithio-bis(2-nitrobenzoic acid),1 mM GSSG and 0.1 mM NADPH. The reaction was started by theaddition of 25–50 µL of plant extract. The rate of reduction of oxidizedglutathione was followed by monitoring the change in absorbance at412 nm for 1 min against a GSSG-free blank.

CAT activity was determined as described by Azevedo et al.(1998). CAT activity was assayed spectrophotometrically at 25°C in areaction mixture containing 1 mL 100 mM potassium phosphate buffer(pH 7.5) containing 2.5 µL H2O2 (30% solution) prepared immediatelybefore use. The reaction was initiated by the addition of 15 µL of plantextract and the activity determined by following the decomposition ofH2O2 by the change in absorbance at 240 nm for 1 min against aH2O2-free blank.

Protein determination

The concentration of protein in the extracts for a comparison ofenzyme activity and to ensure equal loading on the native gels wasdetermined spectrophotometrically at 595 nm as described by Bradford(1976) using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad,Hercules, CA), with bovine serum albumin as a standard.

Native PAGE

Polyacrylamide gel electrophoresis was carried out under non-denaturing conditions in gels containing 9% polyacrylamide with a 4%stacking gel. A constant current of 50 mA per gel was applied and thetemperature set to 4°C. Electrophoresis buffers and gels were preparedas described by Laemmli (1970) except that SDS was excluded. Equalamounts of protein (enzyme extracts) of all samples were loaded onthe gels.

GR activity staining

Glutathione reductase activity following native PAGE was determinedas described by Lee and Lee (2000). Protein (23, 16 and 64 µg) wasapplied to each lane for maize, barley and A. thaliana samples,respectively. Gels were incubated in the dark for 30 min at roomtemperature in 50 mL of a reaction mixture containing 250 mM Trisbuffer (pH 7.5), 3.4 mM GSSG, 0.5 mM NADPH, 50 mg of3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT) and 10 mg of 2,6-dichloro-N-(4-hydroxyphenyl)-1,4-benzo-quinoneimine sodium salt (DPIP). Blank gels were stained withsolution without GSSG, to serve as a negative control for GR activity.For barley and A. thaliana a standard sample of GR from

Saccharomyces cerevisiae (Sigma Chemical Co., St Louis, MO) wasused to serve as a positive control for GR activity.

CAT activity staining

Catalase activity following native PAGE was determined as describedby Vitória et al. (2001). Protein (50, 190 and 64 µg) was applied toeach lane for maize, barley and A. thaliana samples, respectively. Gelswere incubated in 0.003% H2O2 for 10 min and then developed in a1% (w/v) FeCl3 and 1% K3Fe(CN)6 (w/v) solution for 10 min. Oneunit of bovine liver CAT (Sigma Chemical Co.) was applied to all gelsto serve as a positive control for CAT activity.

SOD activity staining

Superoxide dismutase activity following native PAGE was determinedas described by Azevedo et al. (1998). Protein (23, 16 and 9 µg) wasapplied to each gel lane for maize, barley and A. thaliana samples,respectively. The native PAGE gel was rinsed in distilled–deionizedwater and incubated in the dark for 30 min at room temperature in areaction mixture containing 50 mM potassium phosphate buffer(pH 7.8), 1 mM EDTA, 0.05 mM riboflavin, 0.1 mM nitrobluetetrazolium, and 0.3% N,N,N′,N′-tetramethylethylenediamine. At theend of this period, the reaction mixture was poured off, the gel rinsedwith distilled–deionized water and then illuminated in water until thedevelopment of colourless bands of SOD activity on the purple-stainedgel. A standard sample of bovine SOD (Sigma Chemical Co.) wasapplied to all gels to be used as a positive control for SOD activity.

Statistical analysis

The F-test was performed to compare the effects of the two Nconcentrations on each inbred line or hybrid of maize, separately. TheTukey average test was performed to compare the effects of thedifferent N concentrations on the means of the data obtained for maize,barley and A. thaliana.

Results

The addition of N at 10 mM caused a significant increase inthe dry weight of the roots of all maize plants tested, withgreater increases being detected with the hybrids (Table 1).For barley, the dry weight of the roots of plants grown at10 mM N was significantly higher than those of plants grownat the other N concentrations (Table 2).

Nitrogen at 20 mM caused a reduction in barley rootgrowth compared with 10 mM N, although the root weight ofplants grown at 20 mM was similar to that at 1 mM N andhigher than that obtained from plants grown at 0.5 and0.25 mM N. There was a similar response by the A. thalianaroots; the fresh weight of the roots of plants grown at 10 mM

was significantly higher than those of plants grown at 1, 0.5and 0.25 mM N. The fresh weight of the roots of plantsgrown at 20 mM N was not different from those grown at theother concentrations, although they appeared shorter andthicker. (Table 2; Fig. 1). Flowering occurred first in theA. thaliana plants grown at 1 mM N, and had not occurred atthe time of harvest in the plants grown at 20 mM N (Fig. 1).

GR activity in the maize roots was increased dramaticallyin all maize genotypes following the application of high N,by factors ranging from 4- to 15-fold (Table 1). Followingstaining for GR activity on the native gels, at least one moreelectropositive isoenzyme was detected in all the roots of the


maize lines that had received the high N treatment (Fig. 2A).These results agree with those of total GR activity, althoughlarger differences between N treatments were detected bythe spectrophotometric assay (Table 1). Similarly in barleyroots, only very weak GR bands were visible on the nativegels in the low N treated roots, but the stained bandsrepresenting the two most electropositive enzymes increaseddramatically in intensity in the high N treatments, while theintensity of the most electronegative band was reduced(Fig. 3A). For A. thaliana, the effect of increased N on thestaining of GR activity on the native gels was less obvious,but there was still evidence of increased activity of the moreelectropositive isoenzymes (Fig. 4A).

The high-N treatment caused increases in total CATactivity in the majority of the roots of maize, with line 4 andhybrids 24 and 26 exhibiting significant increases (Table 1).Following native PAGE staining, it can be seen that in maize,only the most electronegative CAT isoenzyme was stimu-lated by high N, while at low N, the electropositive forms

were more predominant (Fig. 2B). In barley roots, threemajor bands of CAT activity were detected, the intensity ofwhich was greatest in the lowest and highest N treatments(Fig. 3B). For A. thaliana there was little evidence of anyeffect of N treatment on the activity of the CAT isoenzymes,with perhaps a slightly reduced activity at the lowest Ntreatment (Fig. 4B).

The patterns of SOD isoenzymes visualised after PAGEshowed a remarkable consistency in the roots of all threeplants species subjected to the different N treatments. It wasdifficult to identify any differences in any of the maize lines(Fig. 2C), whereas in barley there was slightly lower activityof the more electropositive isoenzymes in the higher Ntreatments (Fig. 3C), a result that was also observed inA. thaliana (Fig. 4C).

Discussion

For maize, the higher level of N (10 mM) stimulated rootgrowth (Table 1), and there was evidence that the hybridlines performed better than the original lines, which agreeswith the observations of Rahman et al. (1994), who foundhybrid vigour for root growth. For both barley andA. thaliana, 10 mM N induced the optimum root growth(Fig. 1; Table 2). Thus, it would appear that under theexperimental conditions used, 20 mM N is in excess for bothbarley and A. thaliana and may cause the induction of astress response.

Although the effects of increased N supply on primarymetabolism have been studied extensively, the effects on theinduction of stress and in particular the antioxidativeresponse, have not been investigated. In this study, theapplication of high N to the roots of maize plants inducedlarge increases in total GR activity ranging from 4- to15-fold (Table 1). Following staining on native gels, addi-tional isoenzymic bands of GR activity were detected in the

Table 1. Effect of N concentration on shoot and root dry weight and antioxidant enzyme activities of maizeF-test for the effect of increased N on each line (2, 4 and 6) and hybrid (24, 26 and 46) of maize, for means of shoot dry weight(g plant–1), root dry weight (g plant–1), CAT activity (µmol min–1 mg–1) and GR activity (µmol min–1 mg–1) at high (10 mM) andlow (1 mM) nitrogen (N) concentrations. *, **, Significant at 5 and 1% levels, respectively; NS, not significant. Means followed

by the same letter within each row do not differ by the Tukey test at the 5% level

Trait 2 4 6 24 26 46

Shoot dry weight F ** ** ** ** ** **High N 6.0b 5.7b 6.2b 10.0a 7.6b 10.2a

Low N 1.5ab 1.4ab 1.3b 1.9a 1.7ab 1.7ab

Root dry weight F ** ** ** ** ** **High N 1.7d 2.7cd 3.0bc 4.0ab 3.2bc 5.0a

Low N 0.5b 0.8ab 0.6b 0.9a 0.8ab 1.0a

CAT activity F NS * NS ** * NSHigh N 33.3a 28.0a 31.5a 46.7a 42.4a 23.7a

Low N 23.2ab 9.7b 22.4a 22.2ab 26.3ab 19.9ab

GR activity F ** ** ** ** ** **High N 1543a 903a 1457a 799a 1430a 1512a

Low N 238a 208a 141a 205a 98a 210a

Table 2. The effect of nitrogen supply on the fresh and dryweights of barley and A. thaliana shoot and roots

Means of dry and fresh weights (mg plant–1) of roots and shoots ofbarley and A. thaliana plants grown at five nitrogen concentrations.Means followed by the same letter within each row do not differ by the

Tukey test at the 5% level

N concentration (mM)0.25 0.5 1 10 20

BarleyShoot dry weight 83.6b 101.1b 157.4b 729.4a 664.1a

Root dry weight 57.5c 74.1c 133.4bc 304.6a 203.9b

Arabidopsis thalianaShoot fresh weight 1.7b 2.7b 5.8b 24.8a 28.0a

Root fresh weight 0.9b 1.1b 1.2b 2.0a 1.5ab


0.25 mM 0.5 mM 1 mM 10 mM 20 mM

Fig. 1. A. thaliana plants grown at five increasing N concentrations for 26 d. One plant for eachtreatment is shown.

A

B

C

2H 2L 4H 4L 6H 6L 24H 24L 26H 26L 46H 46LFig. 2. Isoenzymic profile following native PAGE of (A) GR, (B) CAT and (C) SOD activity,isolated from the roots of maize lines (2, 4 and 6) and hybrids (24, 26 and 46), grown under high,10 mM (H) and low, 1 mM (L) N supply.


roots of the high-N-treated plants, although the intensity ofthe isoenzyme profile suggested a smaller increase in GRactivity (Fig. 2A). The increases observed could be related tothe new additional bands induced by the high level of Napplied to the maize plants. Furthermore, the extra bandswere not only observed in the maize lines tested, but also inthe hybrids produced between them. Discrepancies betweentotal GR activity and the intensity of staining followingnative PAGE have been discussed by Mullineaux andCreissen (1997), who indicated that changes in the amountof a particular isoenzyme can be more important thanalterations in the total GR activity. In the mesocotyl of maizeseedlings exposed to chilling, large changes in the GRisoenzyme banding pattern were observed by native PAGE,

A

B

C

0.25 mM 0.5 mM 1 mM 10 mM 20 mM

Fig. 3. Isoenzymic profile following native PAGE of (A) GR,(B) CAT and (C) SOD activity, isolated from the roots of barley grownat five N concentrations.

A

B

C

0.25 mM 0.5 mM 1 mM 10 mM 20 mM

Fig. 4. Isoenzymic profile following native PAGE of (A) GR,(B) CAT and (C) SOD activity, isolated from the roots of A. thalianagrown at five N concentrations.


while only minor changes in total activity were detected(Anderson et al. 1995).

The staining profiles of GR activity following PAGE ofthe roots of maize, barley and A. thaliana, clearly indicatedthe presence of at least one additional more electropositiveGR isoenzyme in the high-N-treated plants (Figs 2A, 3A,4A). Interestingly, the GR banding pattern observed for themaize roots in 2H and 6H (Fig. 2A) corresponds very wellwith that shown by Anderson et al. (1995) in the cyto-plasmic fraction of maize mesocotyls and suggests that thenew isoenzymic forms of GR may be located in thecytoplasm.

The large increase in GR activity and the appearance ofthe additional isoenzymic bands caused by high N, could bedue to changes in gene transcription and de novo proteinsynthesis. Two genes encoding GR have been characterisedin pea (Mullineaux and Creissen 1997; Stevens et al. 2000)and A. thaliana (Xiang and Oliver 1998). In pea GOR1isparticularly unusual in that the encoded peptide is targetedto both the mitochondria and the plastid (Chew and Whelan2003). The product of the GOR2 gene contains no targetingsequences and the electropositive isoenzyme activity, ispresumably located in the cytoplasm (Stevens et al. 2000).However, there are other possible posttranslational mecha-nisms that could give rise to this increase, such as glyco-sylation, phosphorylation, different oxidation states,carbamylation and C-terminal proteolysis (Mullineaux andCreissen 1997).

In Anabaena PCC 7120 growth on ammonium caused anincrease in the activity of GR, in comparison with growth onnitrate or from the fixation of N2 (Jiang et al. 1995). Theinvolvement of GR with the metabolism of the N was notclear and Jiang et al. (1995) suggested ammonium toxicityas a possible explanation. The recent report of Rios-Gonzalez et al. (2002) has produced conflicting dataconcerning the effect of N on the activity of GR in roots.Both maize and sunflower had a higher growth rate in thepresence of nitrate rather than ammonium. Higher GRactivity was detected in the roots of the nitrate-treated maizebut higher GR activity was observed in the roots ofammonium-treated sunflower. Rios-Gonzalez et al. (2002)observed that the youngest parts of the nitrate-treated rootscontained a higher GR activity than the mature parts.However, the effect of N on the isoenzymes of GR was notdetermined in either sunflower or maize.

There was clear evidence of increased CAT activityinduced by high N in maize roots. According to Andersonet al. (1995), only one form of catalase (CAT1) is detectablein maize roots, however Scandalios et al. (1997) demon-strated that the genes encoding both CAT 1 and CAT3 wereexpressed in roots. According to their positions on the PAGEgel (Fig. 2B), it is the CAT3 isoenzyme that shows the largeincrease in activity. Acevedo and Scandalios (1996) demon-strated that the CAT3 isoenzyme protein was particularly

stable during the senescence of maize silks followingpollination. The data for barley was less clear (Fig. 3B). Inbarley, there was a higher intensity of CAT activity in theroots of plants grown at the lowest N concentration, whichcould be related to senescence of the roots in these plants, asthey exhibited a yellowing of the leaves. However, inA. thaliana there was evidence of a slight increase in themost electronegative form of CAT activity (Fig. 4B) follow-ing the application of increased N, which could correlatewith the peroxisomal form proposed in A. thaliana byFrugoli et al. (1996).

In our own hands and that of several workers, the nativePAGE system has been shown to readily separate themitochondrial Mn–SOD, cytoplasmic Cu/Zn and plastidicCu/Zn–SOD isoenzymes (Scandalios et al. 1997; Azevedoet al. 1998; Lee and Lee 2000; Vitória et al. 2001; Pereiraet al. 2002). Despite the existence of a large number ofdifferent genes controlling the activity of the Fe, Mn andCu/Zn forms of SOD (see Introduction), there was littleevidence of any of the SOD enzyme isoenzyme activities,being regulated by N supply in any of the three plants tested(Figs 2C, 3C, 4C). Thus, it would appear that increased Nnutrition does not have a regulatory control of all of theenzymes involved in the detoxification of ROS, in particularthe large number of different forms of SOD.

Mullineaux and Creissen (1997), proposed an expla-nation for the increased GR activity detected in ammonium-grown Anabaena (Jiang et al. 1995), as being due toincreases in growth rate, photosynthesis and metabolicactivity, which would require an enhancement of thescavenging of ROS. The data presented in this paperdemonstrating the dramatic increases in GR activity follow-ing the application of increased concentrations of ammo-nium nitrate, as well as some of that presented by Rios-Gonzalez et al. (2002), support the hypothesis ofMullineaux and Creissen (1997), that an increase in growthrate causes a demand for GSH and, consequently an increasein GR activity. GR also plays an important role in maintain-ing high concentrations of reduced glutathione for use innumerous other redox reactions in the cell including, forexample, those involved with protein and DNA synthesisand amino acid transport (Noctor et al. 2002), for all ofwhich, there would be a greater demand with increasedgrowth.

Sánchez-Fernández et al. (1997) presented three lines ofevidence for the participation of GSH in the regulation ofcell division in the primary meristem of A. thaliana roots.(i) Artificially increased endogenous GSH concentrationsstimulated cell division, (ii) artificially decreased GSHconcentrations reduced cell division, and (iii) high concen-trations of endogenous GSH were associated with activelydividing initial cells, but not the slowly cycling cells of thequiescent centre. Noctor et al. (2002) have argued that thereare two key factors concerned with the availability of GSH


in plant cells, the concentration and the GSH/GSSG ratio.The latter ratio is normally in the order of 9:1 and isregulated by GR. Although attempts have been made tocalculate the rate of turnover of GSH and GSSG, this hasonly been achieved in actively photosynthesising leaves(Polle 2001; Noctor et al. 2002).

It has been recently proposed by Rodriguez et al. (2002)that leaf expansion in maize requires the formation of ROS;a similar reaction may thus also occur in roots. Thegeneration of ROS in the apoplast and subsequent actionleads to the formation of hydrogen peroxide (Rodriguezet al. 2002). Thus, the expansion process could induceconcurrent increases in the activity of some of the enzymesinvolved in the metabolism of ROS, in particular hydrogenperoxide, in order to prevent their toxic action within thecell. This could explain the N-induced increases detected ofGR and to a lesser extent CAT, but not of SOD.

Acknowledgments

The authors gratefully acknowledge the financial support ofthis work provided by Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP, Grant no. 98/12461-0 toRAA) and the British Council (to RAA, RJS and PJL).LOM and RAA received PhD and research fellowships,respectively, from the Conselho Nacional de Desenvolvi-mento Científico e Tecnológico (CNPq-Brazil).

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Manuscript received 14 July 2003, received in revised form 9 October2003, accepted 3 November 2003

The influence of nitrogen supply on antioxidant enzymes in plant roots

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