HELIA.22. Nr. 3O. o.p. 155-162. (1999) UDC 631.524.8:633.854.78

CHARACÎERIZATION OF ELECTROPHORETICVARIAIIITS FOR Cw/ZnSOD IN SUNFLOWER:

RESPONSE TO OXIDATTVD STRESS

M. Fambrinil, v.D. Rossir, L. sebastiani2, p. Vernieri3 and c. pugliesil-

;Diparttmento di Biologia delle piante Agrarie, sezione d.i Genetica, piso., Italg' Scuola Superiore di Studi flniuersitari e d.i perfezionamento',S. Anna,,.Piso.. ItaIAa Dipartimento di Biologia delle ptante Agrarie, Sezione di Orticolturae Floricoltura, Pisa, Italg

Receiued: Nouember 1 7, I 997Accepted: March I S, I 999

SUMMARY

Superoxide dismutases (SODs) and abscisic acid (ABA) are implicated inthe response to environmental stresses. We have evaluated the susceptibilif ofu-'--l ' a mutant with ABA detciency and a variant isoform of chloroplàsticCu/ZnSOD, to oxidative stress. The results indicate that u.r-l leaves were Iessdamaged by paraquat (MV) and H2o2 treatments in respect to the control,while no statistical differences were detected when water

"tra"" *r" imoosedby leafdehydration.

Spectrophotometric assays did not evidence a higher level of SOD activityin Lrr-l extracts than in the control, conlîrming that ABA concentration does notmodiÛ/ SOD actvity. The better response of ro--l leaves to MV treatment couldbe attributed to a higher efficiency ofthe chloroplâstic isoform variant of SODto induce the dismutation of superoxide anion radicals to hydrogen peroxideand oxygen, under oxidative stress.

Key words: Heriolnthus.rnnuus L., ABA-deficient mutant, electrophoreticlsoz5me variant, oddative stress, superoxide dismutese

INTRODUCTION

Environmental stresses (chilling, drought, salinity, air pollurants, etc.) actdirectly or indirectly through the formation of activated oxygen species. plants haveevolved different enzymatic and non-enzymatic mechanisms that can reduce oxida-tive stress by detoxi$ring harmful oxygen species (scandalios, l99g). superoxidedismutases (soDs; Ec 1.15. l.l ) are enzymes strongly implicated in the response tooxidative stress. SODs catalyze the disproportion of superoxide radical anions tohydrogen peroxide and molecular oxygen (Bowler et at., lgg2). These enzvmes. that

I Corresponding author, fax: + 39 SO b76 ZbO, e-mail: cpugliesi@agr.unipi.it

156 HELIA,22, Nr.30, p.p. 155-162, (1999)

have been found in all aerobic organisms, cooperate with other enzymes involved in

hydrogen peroxide-detoxification systems and play an essential role in protecting

liying cells against the indirect deleterious effects ofsuperoxide free radicals (Scan-

dalios, 1993; Bowler et al., 1994). In plants, based upon metal ions present in the

active centre, three forms of the enzyme exist: copper/zinc (Cu,/ZnSOD), manganese

(MnSOD) and iron (FeSOD), which are localized in different cellular compartments.

The FeSOD is a dimeric enzyrne present in prokaryotes and within the chloroplast

of several plant species. The MnSOD appears to consist of identical subunits in

either dimeric or tetrameric arrangement. The enzyrne is widely distributed arnong

prokaryotic and eukaryotic organism. In plants, MnSOD has been found in the

mitochondrial matrix (Arron et al. , Ig76: Jackson et aI. ' 1978; Baum and Scandal-

ios, l98l; Scandalios, 1993; Bowler et al., 1994) and in glyoxisomes and peroxi-

somes of some species (Sandalio and del R'o, lg87; Sandalio et aL' 1987; Corpas

et aI., 1994). Plant Cu/ZnSOD is a dimeric enzyme, always present in the cytosol,

consisting of two identical subunits. In addition to the cytosolic Cu/ZnSOD"r.i' a

more anodic isoform is located in the chloroplast stroma (Asada et al., 1973;

Bowler et aL., 1994). Cu/ZnSOD activity has also been reported in the matrix spaces

of mltochondria (Arron et aI., 1976; Jackson et aI., 1978; Sandalio et aI.' 1987)

and in $lyoxysomes of watermelon, cotton, cucumber and sunflower (Sandalio and

del R'o, 1987; Corpas et aI., 1994).

Recently an ABA-deficient mutant (ro-l ) of sunflower (Helianthus annuus L.),

characterized by abnormal stomatal behaviour (Pugliesi et al., 1994; Fambrini ef

at., 1994) and several inbred lines and varieties were electrophoretically analyzed

for SODs isozymes (Fambrini et al., 1996). A banding pattern (zymogram) was

identified in u-I in which chloroplastic Cu/ZnSOD"6 has decreased mobility differ-

ing from all other tested genotypes (Fambrini et al., 1996); the variant was coded

by a nuclear gene with two codominant alleles (Fambrini et o,I., 1997). A descrip-

tion of response to oxidative stress of this electrophoretic va-riant, also in relation to

the leaf ABA content, is presented in this paper. Moreover, since the isoform of Cu/

ZnSOD variant is located in the chloroplast, we induced in this organelle an oxida-

tive stress by means of ttre herbicide methyl viologen (MV, a redox-compound that

transfers electrons from photosystem I to oxygen, forming the superoxide anions) to

testitsfunctionality.Theeffects of H2O2andwaterstressonleaf disksof thetr-l-I

mutant and control line were also analyzed: the extent of cellular damage was quan-

tified by solute leakage, as a measure of membrane disruption. The relationships

between stresses and the anti-oxidative enz\rynes activity in the different genotypes

are discussed.

HELIA,22, Nr.30, p.p. 155-162, (1999) r57

MATERIALS AND METHODS

Plant materlals

The inbred lines and varieties of sunflower (Helianthus o,nnuus L.)used in thisstudy were obtained from the Agricultural Plant Biologl Department, Pisa (Italy).Achenes were germinated in Petri dishes, on filter papers moistened with distilledwater, in growth chamber at25-+ l"C in the dark. After 3-4 days, germinated seedswere transferred to 8 cm diameter pots containing a mixture of soil and sand plusan initial dose of complete fertilizer (Osmocote 14-14-14, Sierra UK, trngland).Plants were grown in a growth chamber at zl-rl'C, 1.O-l,l kPa WD. A l6h pho-toperiod was provided by fluorescent lighting (Sylvania Day Light F36W56) withlight intensity of l80 pmol quanta m-2 s-I. Flower buds of Cl (normal type) andu.r-.I (mobility variant) were hand emasculated and crossed; F2 populations wereobtained by selfing F1 plants. Experiments were started with 3-week-old plants atthe four-leafstage.

ABA extraction and determlnation

Quantitative analysis of ABA was performed on crude aqueous extracts by asolid-phase radioimmunoassay (RIA) based on the use of a monoclonal antibody(DBPAI) raised against free (S)-ABA, as previously described (Fambrini et al.,1994). Leaf disks (20-30 mg FIÀ/) were weighed, immediately frozen in liquid nitro-gen and extracted with distilled water (water:tissue ratio 50: l, v/w) for l6h at 4"C inthe dark. Experiments to validate RLA results with sunflower crude extracts havepreviously been reported (Pugliesi et at., 1994).

Itl[\I, H2O2 and dehydratlon treatments: solute leakage meaaurement

MV (methyl viologen: l,l'-dimethyl-4,4'-bipyridtnium dichloride) damage wasanalyzed as described by Bowler ef al. (1991) \Mith modifications. Leaf disks(Q= 1.3+ cm) collected from u-.1 mutant and Cl control line, were incubated in Petridishes containing distilled water or MV solution at 2l"c in the dark. After I 6h, theywere illuminated for 3h (35 pmol quanta m-2s-r) and transferred to the darkness foradditional l6h. For H2o2 treatments, leaf disks for both genotypes were incubatedat2l"c for 4h under fluorescent lamps (35 pmol m-2s-r). Leaf dehydration was con-ducted as previously described (Fambrini et al., lg94). The samples were thenplaced in flasks ( 15O ml) \Mith 8 ml distilled water and after 24h the conductivity ofthe solutions obtained was measured; a second measurement was performed afterautoclaving (121'c for 15 min) to release all solutes. Finaly, according to senGupta et aI., (1993), the relative leakage ratio was determined by dividing the firstvalue by the conductivity of the sample after autoclal'ing ( looo/o of leakage).

15a HELIA,22, Nr. 30, p.p. 155-162, (1999)

Superoxlde dlsmutase activlty assay

The SOD activity assay, based on the method of Beuchamp and Fridovich( 1971) as modited by Dhindsa and Matowe (1981), is based on measuring the inhi-bttton by SOD of the photochemical reduction of nitroblue tetrazolium. The reac-tion mixture contained 50 mM phosphate buffer pH 7.8, O.l mM EDTA, 13 mMmethionine, 75 pM nitroblue tetrazolium, 2 pM riboflavin, and the enzyme extract.Riboflavin was added last and the reaction was initiated by placing the tubes underfluorescent lamps. The reactlon was terminated after 1O min. by switching off theItght. Unilluminated samples served as blanks. The tubes were stirred and the bluecolor was measured at 560 nm. The volume of enzyme extract corresponding to5O7o lnhibition of the reaction was considered as one enzvme unit.

Stattstical analysis

Statistical significance between mean values was assessed using a Student's t-test. The data in Table 2 were analyzed by multifactorial analysis of variance, anddifferences among means were determined by LSD, at P:O.OI.

RESULTS AND DISCUSSION

The inbred lines and varieties of sunflower showed two distinct superoxide dis-mutase isoz5rmes resolved by polyacrylamide gel electrophoresis, conduced in non-denaturating conditions on proteins extracted from cotyledons (Figure l).

COTYLEDONS CI{LOROPLASTS

Cl Cl x w-1 w-l Cl w-LAlAl ArAz Azlrz

Cu/ZnSOD"o,

+

Flgure 7: Cu/ZnSOD tsozgme patterns oj protein extractsJrom cotgledons or isolated, chlo-roplasts oJ su4flotoer homozggotrs pa.rental tApes AjAl ftnbred tines: C1), A2A2(ABA-deJtctent mutant: u-1) and Ft hAbrids A jA2 (Cl x to-l). The more anod.iclsozgmes are the chloroplast-located Cu/Zn superoxide dismutase(CutZnSODchù.(Schematic zlmogram redralDn:from Fambrtni et al., j996; jgg7).

Samples of leaf chloroplast extract.s revealed that the more anodic isozyme isthe chloroplast-associated superoxide dismutase (Cur/ZnSODcn), while the cathodicone detected in cotyledons could be a c5rtosolic or glyo<ysomal cu/zn superoxide

HELIA,22, Nr.30, p.p. 155-162, (1999) 159

dismutase (Cu/ZnSOD"*) (Fambrini et aI., 1996; 1997). The ur-l is an ABA-defi-cient mutant with altered stomatal behaviour (Pugliesi et al., 1994; Fambrini et al.,1994), and it showed a different electrophoretic pattern in which the Cu/ZnSOD"irlhas decreased mobility (Figure 1). The Cu/ZnSOD"r1 showed in u-I the samemobility displayed by the normal tlpe (Fambrini et al., 1997). The genetic analysissuggests that the mobility variant for Cu/ZnSOD"11 is coded by a single locus withcodominant alleles (Fambrini et al., 1997 ). Moreover, the results of heterozygousplants that exhibit the heterodimeric isozyme with electrophoretic mobility interme-diate between those of their respective homodimeric forms also indicate that theCu/ZnSOD"lt exists as a functional dimer (Figure I ).

To evaluate the association between the electrophoretic variant of CùZnSOD"1displayed by the u-l genotype and its ABA-deficiency, an experiment of water stresswas performed. Detached leaves of a small F2 population were allowed to dehydrateand leaf ABA contents were measured after a 4O-5Oo/" fresh weight was lost. Prelim-inary data (Table l) indicate a minor increase in ABA level in A2A2 genotype withrespect to the A1A1 and A1A2 genotypes. This result could suggest the associationbetween the two traits, but analysis of larger F2 progenies is required.

Table l: ABA content in detached leaves of the F2 population (AlAl, Al42 and A2A2genotypes)placed on a bench aT2l"C, under ligfrt (l8O pmol qua-nta m-'s-'), with the abaxialsurfaces uppermost, and allowed to dehydrate (4O-5Oo/o FW loss).

GenotypeABA (ng g initial FW)

Non stressed StressedA1A1 and A1A2

AzAz

42j a

42.3 a

411 a

180 bValues in the same column, followed by different letters, are significantly different (P< 0.0 1 ) accordingto Student's f-test.

SODs and ABA have been reported to be involved in tolerance to active oxygenspecies (AOS) produced in plants during common environmental stresses. Here, weevaluate the susceptibility of w-I, a mutant with ABA deliciency and a variant iso-form of chloroplastic Cu/ZnSOD, to oxidative stress. The results (Table 2) indicatethat ru-I leaves are less damaged by paraquat (MV) and H2O2 treatments withrespect to the control, while no statistical differences were detected when waterstress was imposed by leaf dehydration.

Table 2: The oxidative stress on leaf disks of two sunïlower genotypes (u-r--l and Cl): effect ofmethyl viologen (MV), H2O2 and leaf dehydraton on relative leakage ratio.

Relative leakage ratio

Treatment

êâh^+,,^^ MV solution (M) H2O2 solution (M) Leaf dehydration (o/")J-, ,vrr rV

w-l (AzAz) 9.4a 11.9a 62,2a 10.9a 18.4a 34.4a 13.1 a 15.8a 16.9aC1 (AjAl) 11.2a 40.8b 64.2a 13.4a 24.9b B.6b 12.4a 12.9a j7.7a

Values in the same column, followed by different letters, are significanily difierent (p< o.o1)according to LSD test.

160 HELIA,22, Nr.30, p.p. 155-r62, (1999)

Resistance to MV has been found in genotypes with constitutively enhancedSOD activity. In our case, spectrophotometric assâys do not evidence higher level ofSOD activity in ur-l extracts than in the control (also after herbicide treatment: datanot shown). We could speculate that better response of u.r-l leaves to MV treatmentcan be attributed to a higher efficiency of the chloroplastic isoform variant of SODto induce the dismutation of superoxide anion radicals to hydrogen peroxide andoxygen. Additional experiments are necessary to test this hypothesis, because themutant showed also a tolerance to H2O2 which strongly reduces the activity of Cu/ZnSOD (Casano et aI., 19971.

CONCLUSIONS

Many unresolved questions remain about the regulation of the expression ofsod genes and a role of hormones. It has been postulated that abscisic acid isinvolved in a differential transcriptional activation of some of the SOD genes (Zhuand Scandalios, 1994; Sakamoto et al., 1995; Kurepa et al., I99Z); however, in allcases studied total enzymatc activity remained constant (zl'n and scandalios,I 994; Kurep a et al. , 19971. Our results confirm that ABA concentration in plant tis-sue does not modiff soD activity. In fact, endogenous ABA level in ro-l is substan-tially lower than in the control but no statistical differences in SOD activity wereobserved.

ACKNOWLEDGEMENTS

The technical assùstance oJ D. Bertini in conducting this studg ts grate-JUIIg acknouledged.

REFERENCES

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Asada, K., Urano, M. and Takahashi, M., 1973. Subcellular location of superoxide dismutasein spinach leaves and preparation and properties of crystalline ipinach superoxidedismutase. Eur. J. Biochem. 36:252-266.

Baum' J.A., and Scandalios, J.G., lg8l. Isolation and characterizaton of the cytosolic and_ -mitochondrial superoxide dismutases of maize. Arch. Biochem. Bioph. 206: 249-264.Beauchamp, c.o., and Fridovich, I., 1971. superoxide dismutase: imprôved assays and an

assay applicable to acrylamide gel. Anal. Biochem. 44:276-2g7.Bowler, C., Slooten, L., Vandenbranden, S., De Rycke, R., Botterman, J., Sybesma, C., Van

Montagu, M., and Inzé, D., 1991. Manganesé superoxide dismutase can reduce cellulardalnage mediated by oxygen radicals in transgenic plants. EMBo J. lo: lz23-1782.

Bowler, C., Van Montagu, M., and ltzé, D., 1g92. Superoxide dismutase and stress tolerance.Ann. Rev. Plant Physiol. 43: 83-l 16.

Bowler,,C., Van Camp, W., Val Montagu, M., and Inzé,D., 1994. Superoxide dismutase inplants. Crit. Rev. Plant Sci. 13: 199-218.

Casano, L.M., G6mez, L.D., Lascano, H.R., Gonzales, C.A. and Trippi, V.S., 1997. Inactivationand degradation ofCuZn-SOD by acûve oxygen species in wliéat chloroplasts exposed tophotooxidative stress. Plant Cell phvsiol. SB-: 435-440.

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corpas, F.J., Bunkelmann, J.,-Jiang, L. and rrelease, R.N., 1994. Identification of superoxidedismutase isoforms in glyoxyÀomes of oilseeds. plant physiol. lo5: s-146.

Dhindsa, R.S. and Matowe, w., 1981. Drought tolerance in two mosses: correlated withenz]matic defence against lipid peroxidation. J. Exp. Bot. 32: 79_91Fambrini, M.,Pugliesi, c., vernieri, p., pardossi, A. arld Baroncelli, s., 1994. characterizationof a- wilty sunllower (Hetianthus annuus L.) mutant. II. Water relations, stomata_lconductalce. abscisic acid content in leaves and xylem sap ofplants subjected to waterdeficiency. J. Exp. Bot.46: t8O9-l8lS.Fambrini, M.' sebastiani, L., Rossi, y.p., prrgriesi, c. and cavallini, A., 1996. cu,rZn SoD

isozJmres in an Al}A-deficient mutant of sunflower. In: From Molecular Mechanism to thefJant:_3n Int-egrated Approach. loth FEspp_cgngreg, september 9-i3, Frorence, Italy.Plant Physiol. Biochem. Special Issue; Sl8_O22,7. %1.

Fambrini, M., Sebastiani, L., Rossi, V.D., cavallini, A. and pugtiesi, c., 1997. cenetic analysisof an electrophoretic variant for the chloroplast-assoclated form of Cû/Zn "rrp..oiid.dismutase in sunflower (Helianthus an uuê L.). J. Exp. Bot. 48: 1143-l 146.

Jackson, C., Dench, J., Moore,4.L., Halliwell, 8., Foyer, C.H. and Hall, D.O., 1978. Subcellularlocalisation and iclentilcauon of superoxide dismutase in the leaves of higher plants. Eur.J. Biochem. 91: 339-344.Knrepa, J., Hérourt, D., van Monta€Ir, M. and rryzé, D.,1g97. Differential expression of crzn-and Fe-superoxide dismutase gerres of tobacco during develop-..rt, oJduurr. stress, andhormonal treatments. plant CéI physiol. Ba: 468_42-0.Pu$iesi, C., Fambrini, M., Vernieri, P. and Baroncelli S., 19g4. Characterization of a wiltysunflower (Helianthus annuus L.)mutant. I. Abs_cisic acid content, tigfrt-aart ctrrnge* Éthe stomatal conductance and genetic analysis. J. Exp. e"t. as, sèâise.Sakamoto, 4., Okumura, T., Kaminaka, H., Sumi, K. and Tanaka, K., 1995. Structure anddifferential response to abscisic acid of two promoters for the c5rtosolic copper/zinc-superoxide dismutase genes, soDCc,t alld soôcc2, in rice protoplists. FEBS Lett. 358:62-66.sandalio, L.M. and del R'o, L.4., 1982. Localization of superoxide dismutase in gfyoxysomes

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sanda-lio, L.M., palma, J.M. and del Rio, L.A., rgaz. Locarization of manganese superoxide, dismutase in peroxisomes isolated from pisum so,tilum L. plant sci. sl: l_g.scandalios' J.G" rgg3. oxygen stress and superodde dismutases. plant phvsiol. ror: 7-r2.Sen Gupta, A., Heinen, J.L., Holaday, A.S., Burke, J.J. and a:r.", n.o., rggg. Increasedresistarce to oxidative stress in transgenic ptzrnts that orr...*pi.""

"hloroptastic cJznsuperoxide dismutase. proc. Natl. Acaa. Sci. USA 90: 1629_lô33.zhrt' D' and scandalios, J.G., r9g4. Differential accumulation of manganese-superoxide1i"-:Fq.^gutlcripts in maize in response t" au"cisi" à"id^""à

-lrifrr"o"-oticum. plant

Physiol. tO6: t73-t28.

CARACTERIZACION DE VARIAN'TES ELDCTROFORETICASPARA CùZnSOD EN EL GIRASOL: RDACCION AL ESTRESOXIDANTE

RESUMEN

La dismutacion de superoxido (soD) y el acido abscisico (ABA) son incru-idos en las reacciones vegetales aI estres dél medro ambiente. Hemos evaluadola sensibilidad de r-u-I, mudante careciente der acido abscisico y de una vari-ante isoforme cloroplastica Cu/Zn de la dismutacion de superoxido, al estresoxidante' Los resultados han mostrado que las hoJas fueron menos daiadaspor paracuato y los tratamientos con H202 al respecto del control, mientras lasdiferencias estadisticas no se manifestaron

",tatrào el estres fue causado por ra

dehidratacion de hoias.

L6.2 HELLA,22, Nr.30, p.p. r55-162, (1999)

El analisis espectrofotometrico no mostro mas a-lto nivel de la actividad

de la dismutacion de superoxido en los extractos del mudante u-l que ese en

el control. Eso confirma que la concentracion del acido abscisico no provoca

las modifïcaciones de la actividad de la dismutacion de superoxido. Mejor

reaccion de hojas uJ--l al tratamiento por paracuato puede atribuirse a la mas

grande capacidad de la variante isoforme cloroplastica de la dismutacion de

superoxido de provocar la dismutacion de los radicales de superoxido arrion al

peroxido hidrogeno y oxigeno, durante el estres oxidante.

CARACTÉRISATION DE VARIANTESÉr,BctnopHoRÉJIguES ouR LE cdZnsoD DAIïs LETOURNESOL, RÉACtION AU STRESS OXYDAI\IT

RÉSUMÉ

Les dismutations superoxydes (SOD) et I'acide abscissique (ABA) sont

impliqués dans les réactions au stress environnemental. Nous avons évalué la

sensibilité de ru-I, un mutant ayant une carence en ABA et des variantes iso-

morphes de cbloroplastique cu"/ZnsoD à un stress oxydant. Les résultatsindiquent que les feuilles u-J avaient été moins endommagées par le paraquat(MV) et par les traitements H2O2 en comparaison avec le groupe contrôle' alorsqu'aucune différence statistique n'avait été détectée quand Ie stress dû au

manque d'eau avait été imposé par déshydradation des feuilles.

L'analyse spectrophotométrique n'a pas montré un niveau plus éIevé de

I'activité soD dans les extraits ru-l que dans le ÊFoupe de contrôle, confirmantque la concentration ABA ne modi{îe pas I'activité soD. La meilleure réactiondes feuilles ru-.I au traltement MV peut être attribuée à une plus grande capac-

ité de la variante soD isomorphe chloroplastique de provoquer une dismuta-tion de radical anion superoxyde au peroxyde d'hydrogène et à I'oxygène dans

des situations de stress oxvdant.