Overexpression of Mn-superoxide dismutase in maize leaves leads to ...

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Journal of Experimental Botany, Vol. 51, No. 352, pp. 1867±1877, November 2000

Overexpression of Mn-superoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities Alison H. Kingston-Smith1 and Christine H. Foyer 2,3 1

Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, Wales, UK 2 IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK Received 26 June 2000; Accepted 4 July 2000

Abstract The effect of increased Mn-superoxide dismutase (SOD) on antioxidant enzymes and metabolites was studied using transformed maize, TG1q and TG2q. The progeny of the backcross of each of the primary transformants with the parental line generated two populations denoted M6884 and M6885. These were grown at optimal (25 8C) and sub-optimal (18, 14 and 10 8C) temperatures to assess the impact of elevated SOD activity on cold tolerance and the antioxidant defences in maize. The plants of the M6885 population had similar foliar SOD activities to the untransformed maize plants. Within the segregating M6884 population 50% of the plants had elevated SOD activity (up to four times the activity of the untransformed controls) and 50% of the plants contained the product of the transgene. In untransformed plants grown at 25 8C and 18 8C, SOD activity was not detectable in mesophyll extracts. Similarly, increased foliar SOD activity in the M6884 transformed maize did not lead to detectable mesophyll SOD activity. Increased foliar KCN-insensitive SOD activities were accompanied by enhancement of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities; enzymes which are localized exclusively in the leaf mesophyll tissues. Increased foliar SOD activity had no effect on the hydrogen peroxide, glutathione or ascorbate 3

contents of the leaves. This suggests that increased recycling of reduced ascorbate was required to compensate for enhanced hydrogen peroxide production in transformed plants. Key words: Chilling, glutathione reductase, maize, oxidative stress, superoxide dismutase.

Introduction Maize is one of the most important crops for European agriculture with some 1.3 3 106 hectares of maize grown in northern Europe alone. In addition to its importance in the human diet, the energetic value and high nutritional quality make silage maize an important option for animal feeding. Since maize originates from tropical regions it is not surprising that it is particularly sensitive to low temperature stress. Following the expansion of maizegrowing areas towards more northern climates, acclimation to chilling conditions has become a major research target. Optimal growth conditions for maize are between 20 8C and 30 8C. In northern Europe, however, temperatures of between 4±15 8C are frequently encountered in the early growing season. Moreover, the combination of high light intensities and low temperatures, such as those experienced on cold but sunny mornings in spring, can cause irreversible damage to young maize seedlings

To whom correspondence should be addressed. Fax: q44 1582 763010. E-mail: [email protected] Abbreviations: APX, ascorbate peroxidase; ASC, ascorbate; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, glutathione (reduced form); GSSG, glutathione (oxidized form); MDHAR, monodehydroascorbate reductase; MnSOD, manganese associated superoxide dismutase; PEPc, phosphoenolpyruvate carboxylase; Rubisco, ribulose 1,5 bisphosphate carboxylaseuoxygenase; SOD, superoxide dismutase.

ß Society for Experimental Biology 2000

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(Fryer et al., 1998). Similarly, water de®cits or a drop in temperature at the beginning of the grain-®lling period can cause a substantial decrease in yield (Prioul, 1996). Stress tolerance has, therefore, become a major selection criterion in current maize breeding programmes. The damage caused to mature and developing leaves by low temperature stress occurs primarily in the chloroplasts, leading to inhibition of photosynthesis and premature senescence (Nie and Baker, 1991; Nie et al., 1992, 1995). Chilling treatment leads to H2O2 accumulation in the leaves of cereals such as maize (Prasad et al., 1995; Okuda et al., 1991; Kingston-Smith et al., 1999). Studies on the relationships between CO2 assimilation, photosynthetic electron transport and antioxidant enzyme activities in ®eld-grown maize suggested that the donation of electrons to oxygen by the photosynthetic electron transport chain was increased by growth at low temperatures (Fryer et al., 1998). It was found that while the H2O2 content of maize leaves was increased by growth at sub-optimal temperatures, the chilling-induced increase in H2O2 was independent of irradiance (Kingston-Smith et al., 1999). This suggests that photosynthesis is not directly responsible for the low temperature-induced increase in foliar H2O2. Accumulation of H2O2 is potentially harmful since it can lead to oxidative damage and loss of structure and function. It may, however, also have a regulatory role in signal transduction during low temperature stress (Okuda et al., 1991; Prasad et al., 1995). H2O2 is scavenged by the ascorbate peroxidases and catalases of plant cells (Asada, 1994; Willekens et al., 1997). Ascorbate and glutathione are important foliar antioxidants which are maintained in their reduced active forms by the enzymes of the ascorbate-glutathione cycle (Foyer et al., 1994; Noctor and Foyer, 1998). The enzymes of this cycle, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, have been considered to be important in limiting the degree of photodamage experienced by maize leaves upon exposure to chilling temperatures (Jahnke et al., 1991; Massacci et al., 1995; Hodges et al., 1997; Fryer et al., 1998). Maize leaves may be particularly sensitive to low temperature-induced oxidative damage because of the differential partitioning of antioxidants between the different types of photosynthetic cells (Doulis et al., 1997; Burgener et al., 1998) and the sensitivity of proteins in the bundle sheath cells to oxidative damage (KingstonSmith and Foyer, 2000). While Z. mays is inherently low temperature-sensitive, some degree of acclimation to suboptimal growth temperatures has been observed (Leipner et al., 1997). Acclimation caused increased accumulation of the antioxidants glutathione and a-tocopherol allowing greater stability of thylakoid membrane reactions. Similarly, phosphorylation of CP29 within the PSII core

of resistant maize lines has been shown to aid dissipation of excess light energy at low temperatures and hence protect PSII from photoinhibition (Mauro et al., 1997). Overexpression of a mitochondrial manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) in the chloroplasts of Z. mays var. H99 resulted in effects on foliar tolerance to chilling (Van Breusegem et al., 1999a, b; Van Breusegem, 1997). Leaves from the transformed maize plants showed enhanced tolerance to the oxidative stress induced by incubation of leaf discs in the pro-oxidant herbicide, methyl viologen. Van Breusegem et al. concluded that overproduction of MnSOD in the chloroplasts increases the antioxidant capacity of the maize leaves (Van Breusegem et al., 1999a, b). In the present study, the effect of increased MnSOD on the antioxidant system and cold tolerance was examined in a maize population derived from a backcross between the untransformed line H99 and the primary transformants produced (Van Breusegem et al., 1999a). The effects of the transformation on the total foliar activities of antioxidant enzymes (APX, MDHAR, DHAR, GR), as well as SOD, were investigated. Transformed and untransformed maize lines were grown at optimal and sub-optimal growth temperatures. Mesophyll extracts were prepared from maize leaves grown at different temperatures as described previously (Doulis et al., 1997) to determine the intercellular distribution of SOD activity. At all growth temperatures no SOD activity was detectable in the mesophyll fractions from untransformed and transformed maize leaves.

Materials and methods Plant material Primary transformants of maize plants overexpressing MnSOD from tobacco were produced in the Department of Genetics at the University of Gent (Van Breusegem et al., 1999a). These were denoted TG1q and TG2q (Van Breusegem et al., 1999a). The transformed plants were produced by a backcross of the primary transformants from two individual transformation events with the parental line (H99), to form two heterozygous populations; M6884 and M6885 (Co-op de Pau, France). Thus, these populations were analogous to the R1 populations derived (Van Breusegem et al., 1999a) for which the segregation of the transgene has previously been confirmed by PCR (Van Breusegem et al., 1999a, b). Therefore, from the results of Van Breusegem et al., 50% of the plants in each of the transformed populations could be expected to contain the transgene in heterozygous combination (Van Breusegem et al., 1999a). Thus, transgene-containing plants would be expected to exhibit enhanced SOD activities compared with control values. This protocol also provided transformed controls (not expressing the transgene) within the population in addition to untransformed controls which were studied at the same time. Thus the relationship between SOD activity and that of other antioxidant enzymes could be compared within a near-isogenic population.

Antioxidants in transformed maize Maize seeds were germinated in darkened trays of damp vermiculite at 22u25 8C (nightuday). On emergence of the coleoptile each seedling was transplanted into a 599 pot of compost (John Innes No. 1). The young plants were grown at optimal conditions 22u25 8C (nightuday) with a 16 h photoperiod (750±1000 mmol m 2 s 1). Experiments were performed on the leaves from plants that were 2 weeks old (second leaf fully expanded) or 3 weeks old (third leaf fully expanded). Alternatively, plants were grown on at sub-optimal temperatures (18, 14 or 10 8C) at an irradiance of 250±350 mmol m 2 s 1 during a 16 h photoperiod. Plants grown at 18 8C or 14 8C were used when they were at an equivalent growth stage to those grown at optimal conditions (4±5 weeks old). Plants grown at 10 8C were used when the second leaf had emerged (5±6 weeks old). Mesophyll extraction Mesophyll extracts were prepared from maize leaves by an extrusion technique (devised by Leegood, 1985) as described by Doulis et al. (Doulis et al., 1997). The tip and base of the leaf were removed (retaining 75% of the leaf length) and the leaf was divided in half by excision of the midrib. One half was immediately frozen in liquid nitrogen and stored at 80 8C until needed for determinations of whole leaf enzyme activities. The second half was placed on the pre-cooled (4 8C) aluminium block (Leegood, 1985; Furbank et al., 1985; Doulis et al., 1997) together with 0.2 ml ice-cold extraction buffer (50 mM Bicine, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 0.1% Triton X-100, and 10 mM dithiothreitol). The roller (cooled to 4 8C) was placed gently on the leaf and gently rolled so that the mesophyll sap was extruded from the cut end of the leaf and collected under vacuum into a 1.5 ml microfuge tube containing 0.1 ml buffer at 4 8C. This sample was then immediately frozen in liquid nitrogen and stored at 80 8C until analysis. Mesophyll extracts have a relative low protein content, hence samples from leaves 4 and 5 of each transformed plant were pooled to provide enough protein for full evaluation. Enzyme activity determinations All enzyme activities were measured at 20 8C. Assays of ribulose 1,5 bisphosphate carboxylaseuoxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPcase) were performed on mesophyll and whole leaf extracts in order to determine the extent of contamination of the mesophyll fraction by bundle sheath material.

Rubisco: Maximal Rubisco activity was determined by incorporation of [14C]-CO2 into acid stable product (according to Parry et al., 1988). The Rubisco contained in extracts was activated by pre-incubation with 10 mM NaH12CO3 for 10 min. The 1 ml reaction mixture contained assay buffer (100 mM BicineuKOH, pH 8.0), 20 mM MgCl2, 50 ml activated extract, 10 mM NaH14CO3 (at 0.5 mCi mmol 1), 0.33 mM ribulose bisphosphate (RuBP). After 1 min HCOOH was added to a final concentration of 5 M. The solution was dried at 80 8C overnight. The residue was resuspended in 0.5 ml water and 4.5 ml Scintran `Cocktail T' was added. The radioactivity contained in the acid stable residue was then determined by scintillation counting. PEPcase: PEPcase activity was determined as described previously (Murchie et al., 2000). SOD: Total extractable SOD activity was measured as described previously (McCord and Fridovitch, 1969). The inhibition of the colour formation (measured at 560 nm) was

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determined by addition of 0±50 ml of the extract to a reaction mixture containing 50 mM HEPESuKOH buffer (pH 7.8), 0.05 units xanthine oxidase, 0.5 mM nitroblue tetrazolium, and 4 mM xanthine. One unit of SOD activity is equivalent to the volume of extract needed to cause 50% inhibition of the colour reaction. The cyanide-insensitive SOD activity was determined by pre-incubation of the extract with 20 mM KCN (final concentration) for 30 min at 4 8C, after which the extractuKCN solution was used in assays as described above. Hereafter the activity remaining in the presence of KCN is considered to be MnSOD activity. Ascorbate peroxidase: Ascorbate peroxidase (APX) activity was determined by a modification of a method described earlier (Nakano and Asada, 1987). The oxidation of ascorbate to dehydroascorbate (DHA) was followed at 265 nm in a 1 ml reaction mixture containing 50 mM HEPESuKOH (pH 7.6), 0.1 mM EDTA, 0.05 mM ascorbate, 10 ml extract, and 0.1 mM H2O2. Dehydroascorbate reductase: Dehydroascorbate reductase (DHAR) was assayed by following the increase in absorbance at 265 m of a 1 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 2.5 mM reduced glutathione (GSH), 0.2 mM DHA, and 10 ml extract (as described in Doulis et al., 1997). Monodehydroascorbate reductase: Monodehydroascorbate reductase (MDHAR) was determined as described previously (Miyake and Asada, 1992). The decrease in absorbance at 340 nm was monitored in a 1 ml reaction mixture containing 50 mM HEPESuKOH (pH 7.6), 0.1 mM NADPH, 2.5 mM ascorbate, 50 ml extract, and 0.4 units ascorbate oxidase. Glutathione reductase: Glutathione reductase (GR) activity was determined as described previously (Foyer and Halliwell, 1976). The oxidized glutathione (GSSG)-dependent oxidation of NADPH was followed at 340 nm in a 1 ml reaction mixture containing 100 mM sodium phosphate buffer (pH 7.8), 0.5 mM GSSG, 50 ml extract, and 0.1 mM NADPH. Chlorophyll: Enzyme activities were related to the chlorophyll content of each extract. Chlorophyll was determined by the method of Arnon in which aliquots of extract were diluted in 80% acetone and the absorbances at 645 and 663 nm were measured (Arnon, 1949). Ascorbate, H2O2, glutathione: Ascorbate, dehydroascorbate, reduced glutathione, glutathione disulphide, and hydrogen peroxide were extracted and measured according to the method of Doulis et al. and Kingston-Smith et al. (Doulis et al., 1997; Kingston-Smith et al., 1999). Isoelectric focusing on polyacrylamide gels The second leaves from 3-week-old maize plants were ground to a fine powder in liquid nitrogen. One ml of extraction buffer (10 mM TrisuHCl pH 7.2, 10 mM EDTA, 5 mM b-mercaptoethanol, and 0.1% (vuv) Triton X-100) was added and the mixture allowed to thaw. The SOD activity of the samples was determined as described above and a sample volume equivalent to 2 units of activity was loaded onto ampholine-PAG plates, pH gradient 3.5±9.5 (Pharmacia Ltd) according to manufacturers instructions. Proteins were separated by electrophoresis for 1.5 h at constant power of 30 W at 4 8C. The gel was incubated in the dark for 30 min in a solution of 0.2% (wuv) nitroblue-tetrazolium in water. This was replaced by a solution of 37.5 mM phosphate buffer (pH 7.8) containing 15 mg

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riboflavin and 416 ml TEMED. The gel was incubated in this solution for 15 min in the dark. The gel was then rinsed in 10 mM phosphate buffer (pH 7.8) and illuminated. Bands of SOD activity were detected from the appearance of clear bands on a dark blue background. Isoforms revealed on stained gels were quantified by scanning densitometry (GS-710 Calibrated Imaging Densitometer fitted with Quantity One version 4 image analysis software: BioRad, UK). SDS PAGE and Western blotting

Denatured proteins extracted from maize leaves were separated by SDS PAGE and blotted according to manufacturers instructions (Mini Blot, BioRad, UK). Blots were probed with an antisera specfic for MnSOD from tobacco and which did not cross-react with the native maize enzyme (Van Breusegem et al, 1999a). Statistical analysis Correlations between SOD activity and enzyme activities or metabolite content were examined by fitting a linear equation to the data set (FigP software, Biosoft, UK) Calculation of 95% confidence limits assumed a t-distribution such that confidence p limits ˆ x"tsu n (Bailey, 1976).

Results SOD activities were compared in the leaves of two populations of the tranformed maize M6884, M6885 and in the untransformed control line, H99 (Table 1). The foliar SOD activities of the M6885 population were not statistically different from those of the untransformed controls at optimal growth temperatures (22u25 8C; Table 1), but the SOD activities of the leaves of the M6884 population were signi®cantly higher. The SOD activities of the leaves were similar, however, when the lines were grown at sub-optimal temperatures (18 8C; Table 1). The second leaves to emerge on the stem had lower SOD activities than later emerging leaves (Fig. 1A; Table 1). Data on the SOD activities of leaves of individual plants of the M664 population are shown Fig. 1. These data are placed according to their total SOD activity (Fig. 1A) and cyanide-insensitive (Fig. 1B) and cyanidesensitive SOD activity (Fig. 1C). Leaf extracts were subjected to isoelectric focusing, the activities of the various SOD isoforms was detected on the gels and then quanti®ed by scanning densitometry (Fig. 2). The presence of the product of the transgene in individual M664 plants was con®rmed by cross-reaction with speci®c antiserum after Western blotting with replicate samples from each leaf extract (data not shown). In the absence of inhibitors, six major bands of SOD activity with pI values of 4.4, 4.8, 4.95, 5.05, 5.2, and 5.6 were observed in all plants. The MnSOD (KCNinsensitive) isoform, detected at pI 5.6, was of low abundance in M665 plants and in untransformed plants

Table 1. Foliar SOD activities in the third and fourth leaves of transformed and untransformed maize Total SOD KCN-insensitive SOD (units mg 1 chlorophyll) (units mg 1 chlorophyll) 22u25 8C Untransformed M6884 M6885

196.8"19.0 (16) 387.4"82.8 (59) 244.1"81.1 (18)

144.9"27.9 (3) 198.3"23.4 (41) 100.8" 24.4 (18)

18u18 8C Untransformed M6884

202.4"52.5 (5) 139.9"17.9 (26)

± 94.1"18.1 (10)

(Fig. 2). About half of the M664 plants showed signi®cantly higher leaf SOD activities than the untransformed control line, consistent with the 50 : 50 segregation predicted by PCR analysis (Van Breusegem et al., 1999a). SOD activity was measured in the leaves of 25 individuals from the segregating population of M6884 plants grown either at optimal (22u25 8C nightuday) or sub-optimal temperatures and the SOD activities of leaves of individual plants were then ranked in ascending order (Fig. 3). Leaves of plants grown below 18 8C have less chlorophyll than those grown at higher temperatures (Kingston-Smith et al., 1999a, b), the following data are, therefore, presented on a surface area basis. In plants grown with the 22u25 8C nightuday regime, half of the segregating population had similar total foliar SOD activities to those measured in untransformed controls (Fig. 3A) and one half had higher SOD activities. In maize grown with an 18u18 8C nightuday regime leaf SOD activity was greatly increased on a surface area basis compared to plants grown at 22u25 8C. Only 15% of the maize seedlings grown at 10 8C survived (Kingston-Smith and Foyer, 2000) and hence fewer samples were available for analysis at this growth temperature. Leaf SOD activity in the surviving maize grown at 10 8C or 14 8C was higher than at 22u25 8C (Fig. 3). The presence of the product of the transgene in the M6885 population, as detected by speci®c MnSOD antisera, varied with growth temperature. The Mn SOD protein was not as abundant in maize leaves grown at low temperatures as those grown at higher temperatures. The amount of MnSOD protein was quanti®ed by the relative intensity of the cross-reaction when equal amounts of leaf protein were loaded on Western blots (Fig. 4). In comparison with the result when plants were grown at optimal temperatures, the cross-reaction observed for extracts from leaves grown at the lowest temperatures was poor but just visible. The ratio of transgene negative: positive plants within the population was 1 : 1.9 at 22u25 8C, 1 : 1.3 at 18 8C, 1 : 1 at 14 8C, and 1 : 0.3 at 10 8C (Fig. 4). The survival of maize plants grown at suboptimal temperatures was, therefore, not increased by the presence of the transgene.

Antioxidants in transformed maize

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Antioxidant enzymes and metabolites

The activities of the antioxidant enzymes APX, MDHAR, DHAR, and GR were measured in whole leaf extracts from transformed lines M6884, M6885 and untransformed plants (Table 2). The average values obtained for these populations showed that line M6885 had a slight increase in average leaf MDHAR activity and nearly a 3-fold decrease in DHAR activity, whereas APX and GR were largely unchanged (Table 2). However, line M6884, in which SOD activities were greatly increased relative to the untransformed controls, showed a slight

Fig. 2. Typical densitometer scans after isoelectric focusing electrophoresis and activity staining showing peaks (bands) of activity present in untransformed (A) and transformed plants (B; transgene positive, C; transgene negative) when two units of SOD activity were loaded per track. The peaks corresponding to intrinsic or introduced MnSOD are indicated by an arrow.

Fig. 1. SOD activity in transformed M6884 plants (a±o) and untransformed maize plants (p, q). (A) Total SOD activity measured spectrophotometrically, (B) relative band densities of the MnSOD isoform, resolved by isoelectric focusing electrophoresis and densitometry, (C) relative band densities of the CuZn SOD isoform, resolved by isoelectric focusing electrophoresis and densitometry. Plants transgene positive for tobacco MnSOD are indicated with an asterisk. Dotted lines indicate the upper 95% confidence limit for mean values obtained for untransformed plants.

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Fig. 3. The effect of growth temperature on total SOD activity in the second leaves of transformed maize plants (line M6884) expressed on a surface area basis. Twenty-five plants were grown at (A) 22u25 8C, (B) 18u18 8C, (C) 14u14 8C and the survivors analysed. The mean and (SE) for total SOD activity for the second leaf of each of three untransformed plants is shown by the closed circle on (A). Plants heterozygous for tobacco MnSOD are indicated with an asterisk. The dotted line indicates the upper 95% confidence limit for mean values obtained for untransformed plants.

Fig. 4. Semi-quantitative, antigenic detection of tobacco MnSOD in transformed maize plants grown at 22u25 8C, 18 8C, 14 8C or 10 8C. Molecular weight markers are 35.0, 28.4 and 20.0 kDa.

Table 2. Comparison of mean enzyme activities in leaves of maize grown at optimal conditions (22u25 8C, 750 mmol m 2s

1

light)

Mean values"SE of (n) observations are shown. Enzyme activity (mmol min 1mg

Untransformed M6884 M6885

1

chlorophyll)

APX

MDHAR

DHAR

GR

3.57"0.63 (5) 2.03"0.28 (24) 3.21"0.60 (13)

0.27"0.03 (5) 0.69"0.06 (24) 0.37"0.04 (13)

5.76"0.53 (5)1 24.58"3.32 (24) 1.41"0.66 (13)

0.65"0.08 (5) 1.14"0.08 (24) 0.55"0.04 (13)

Antioxidants in transformed maize

decrease in APX activity and substantial increases in the average values for MDHAR, DHAR and GR activities. When, however, the relative activities from individual plants were plotted against the endogenous KCNinsensitive SOD activity (Fig. 5) more consistent trends

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were observed. A weak correlation was observed between KCN-insensitive and total SOD activity in the leaves (Fig. 5A). A stronger correlation was observed between KCN-insensitive SOD and both MDHAR and GR (Fig. 5C, E). There was no evidence for an increase in

Fig. 5. The relationships between total SOD activity and the activities of Mn-SOD and the antioxidant enzymes APX, MDHAR, DHAR, and GR measured in whole leaf extracts from leaves of the transformed plants. Closed circles, line M6884; open circles, line M6885. The coefficient for the fit of a linear equation is shown in each case.

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APX activity in response to increasing leaf SOD activity (Fig. 5B). A positive correlation between DHAR activity and KCN-insensitive SOD was also observed (Fig. 5D). The amounts of reduced ascorbate and glutathione were similar in the leaves of plants showing very different SOD activities (Fig. 6A, B). Similarly, leaf hydrogen peroxide contents were not signi®cantly changed by increased SOD activities (Fig. 6C). Since Rubisco is a very high abundance, soluble protein, localized in the stroma of the bundle sheath chloroplasts, its presence in mesophyll extracts provides a very sensitive indicator of the degree of contamination by bundle sheath material (Leegood, 1985; Doulis et al., 1997). Only mesophyll extracts which had low bundle sheath contamination (Table 3) as judged by Rubisco activities were used to study the compartmentation of SOD in maize leaves (Tables 4, 5). Total and KCNinsensitive SOD activities were measured in whole leaves and leaf mesophyll extracts from untransformed plants (Doulis et al., 1997) and the transformed line M6884 (Table 4). As in untransformed plants, SOD activity was below the limits of detection in the mesophyll extracts from transformed maize (Table 4). Similar results were obtained when plants were grown at 18 8C (Table 5). Hence growth of transformed maize at sub-optimal temperatures did not cause a marked induction of SOD activity in the mesophyll compartment even in plants Table 3. Estimation of contamination of mesophyll extracts by bundle sheath components by analysis of Rubisco activity in the third and fourth leaves of transformed and untransformed maize Mean"SE for (n) measurements are shown. Rubisco activity in whole leaf % of Activity in mesophyll extract (mmol min 1 mg 1 chl) Transformed plants 2.71"0.60 (18) 2.58"0.13 (6) Untransformed plantsa a

12.9"3.44 (5) 22.6 (6)

Data from Doulis et al. (1997).

Table 4. Compartmentation of SOD between mesophyll and bundle sheath of third and fourth leaves from transformed maize line M6884 grown at 22u25 8C Plant Fig. 6. The relationships between total SOD activity and the foliar antioxidant (ascorbate and glutathione) and hydrogen peroxide content measured in acid extracts from leaves of the transformed plants. Closed symbols, line M6884; open symbols, line M6885. In untransformed plants values were: reduced ascorbate 27.6"6.5, total ascorbate 38.0"3.2, GSH 3.1"0.5, GSSG 1.3"0.2, H2O2 3.0"1.4 (mean and SE of five determinations).

1 2 3 4 5 6 7 8

Activity in whole leaf (units mg 1 chlorophyll)

% of Activity in mesophyll extract

Total SOD

MnSOD

Total SOD

MnSOD

130.1 221.3 246.0 351.9 446.1 455.6 461.5 510.6

96.9 202.0 83.5 316.7 223.0 116.6 70.5 142.4

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

Antioxidants in transformed maize Table 5. Compartmentation of SOD between mesophyll and bundle sheath of third and fourth leaves from transformed maize line M6884 grown at 18u18 8C Plant

1 2 3 4 5 6 7 8 9 10 11

Activity in whole leaf (units mg 1 chlorophyll)

% of Activity in mesophyll extract

Total SOD

MnSOD

Total SOD

MnSOD

35.2 67.5 72.3 90.6 100.1 111.1 137.4 171.4 177.8 184.1 327.0

35.2 67.5 ± 76.3 73.1 98.0 45.4 31.9 149.5 178.6 184.5

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

with three times the foliar SOD activity of untransformed controls.

Discussion Maize species, which differ in sensitivity to low temperatures, have different antioxidant capacities. In the lowtemperature-tolerant Z. diploperennis the ascorbate pool was twice that of the temperature-sensitive Z. mays (Hull et al., 1997) and the ascorbate peroxidase appeared to show a better acclimatory response to low temperatures in the former than in the latter. Similarly, decreased catalase, ascorbate peroxidase (APX), and monodehydroascorbate reductase (MDHAR) activities were found to contribute to chilling sensitivity at the early stages of maize development of four inbred lines of Z. mays (Hodges et al., 1997). Conversely, increased catalase, glutathione reductase (GR) and guaiacol peroxidase activities were related to chilling tolerance in pre-emergent Z. mays seedlings (Prasad, 1997). More recently, a strong relationship between glutathione and chilling tolerance has been demonstrated in maize (Kocsy et al., 2000). A range of total and MnSOD activities were measured for both populations of transformed maize although the enhancement of activity above control values was far more pronounced in line M6884. Overexpression of MnSOD was shown to increase the stress tolerance of the two maize lines used in the present study (Van Breusegem et al., 1999a). Segregation of the co-transferred herbicide resistance gene in backcross populations was 1 : 1 as was the presence of transgenic protein and mRNA (Van Breusegem et al., 1999a). This leads to the assumption that enhanced MnSOD activity also segregates equally (Van Breusegem et al., 1999a). In the studies described here the presence of the product of the transgene in individuals was con®rmed by cross-reaction with

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speci®c antiserum. Although 60% of individuals studied from line M6884 expressed the protein product of the transgene, MnSOD activity was signi®cantly enhanced in only 25% of the population (Figs 1, 3). Isoelectric focusing revealed that increased KCN-insensitive SOD activity was accompanied by enhancement of the MnSOD speci®c activity band. However, the presence of the transgenic protein did not automatically confer elevated MnSOD activity and some individuals contained both the transgene and SOD activities similar to untransformed plants. The presence of the product of the transgene did not result in the appearance of detectable SOD activity in maize mesophyll extracts. SOD activity was localized almost exclusively in the bundle sheath tissues in both transformed and untransformed plants despite the fact that expression of the tobacco MnSOD was under the control of the CaMV35S promotor. This is consistent with previous observations on untransformed maize leaves (Doulis et al., 1997; Pastori et al., 2000a) and with in situ localization studies conducted on maize primary transformants (TG1q and TG2q) grown at optimum temperatures (Van Breusegem et al., 1998). In addition, SOD was not detectable in the mesophyll extracts of leaves of plants grown at either optimal or sub-optimal temperatures. The data show that the increase in SOD activity at sub-optimal growth temperatures is due entirely to bundle sheath located isoforms. There is no evidence from these plants to support increased protection of maize mesosphyll against superoxide generation by growth at low temperatures and thus mesophyll proteins are as susceptible to oxidative damage as mesophyll cells of untransformed plants (Kingston-Smith and Foyer, 2000). This suggests that the 35S-promoter, which is constitutively expressed in tissues of dicotyledonous plants, was not able to confer SOD activity to the mesophyll of maize leaves (a monocotyledonous plant) due either to transcript instability or promotor inhibition (as discussed in Van Breusegem et al., 1998). Alternatively, MnSOD transcripts may simply not be translated in mesophyll cells as is the case for glutathione reductase transcripts in bundle sheath cells (Pastori et al., 2000b). Increased foliar SOD activity was accompanied by corresponding increases in MDHAR, DHAR and GR activities, but no SOD-dependent changes in APX activity, ascorbate or glutathione were observed. Increased MDHAR, DHAR and GR activities may be required to maintain the foliar ascorbate pool in the transformed plants with higher SOD activities and, hence, maintain APX-mediated conversion of H2O2 to H2O. The observation that foliar APX activity was not increased in response to increasing SOD activity suggests that foliar ascorbate and APX activity were suf®cient to destroy H2O2 produced in the transformed plants. This would also explain why the H2O2 content of the transformed

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plants with increased SOD activity was similar to that of the untransformed controls. The increased activities of DHAR, MDHAR and GR suggest that ¯ux through the regenerative phase of the antioxidant defence cycle is increased in plants with increased MnSOD, even under optimal growth conditions. GR, DHAR and MDHAR are localized almost exclusively in the mesophyll cells (Doulis et al., 1997). Even when maize plants were grown at suboptimal growth temperatures, no GR activity was detected in the bundle sheath cells (Pastori et al., 2000a, b). It is logical to assume, therefore, that increases in the activities of GR, DHAR and MDHAR arising from enhanced SOD activity occur only in the mesophyll cells. This must confer an advantage since transformed maize overproducing MnSOD predominantly in the chloroplasts of the bundle sheath cells had enhanced oxidative stress tolerance (Van Breusegem et al., 1999). It is tempting to suggest that, should this apparently obligate partitioning of antioxidant enzyme activity be overcome by appropriate transformation technology, even greater bene®t could be obtained. Acknowledgements This work was supported by the European Commission (AIRCT92±0205, Engineering Stress Tolerance in Maize) and the BBSRC. The authors are grateful to Mrs Janet Williams for providing technical support.

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