In maize leaves growth at low temperatures causes decreases in maximum catalytic activities of photosynthetic enzymes and reduced amounts of proteins, rather than effects on regulation or co‐ordination of the photosynthetic processes. To test the hypothesis that differential localization of antioxidants between the different types of photosynthetic cell in maize leaves is a major determinant of the extreme sensitivity of maize leaves to chilling damage, oxidative damage to proteins, induced by incubation of maize leaves with paraquat, has been measured and compared with the effects incurred by growth at low temperatures. While the increase in protein carbonyl groups caused by paraquat treatment was much greater than that caused by low temperature growth conditions, most carbonyl groups were detected on bundle sheath proteins in both stress conditions. With one or two exceptions proteins located in the mesophyll tissues were free of protein carbonyl groups in both situations. Paraquat treatment caused a complete loss of the psaA gene products, modified the photosystem II reaction centre polypeptide, D1, and increased the number of peptides arising from breakdown of ribulose 1,5‐bisphosphate carboxylase oxygenase (Rubisco). In contrast, growth at 15 °C increased the abundance (but not number) of Rubisco breakdown products and decreased that of the psaB gene product while the psaA gene product and PEP carboxylase were largely unaffected. Since bundle sheath proteins are more susceptible to oxidative damage than those located in the mesophyll cells, strategies for achieving a more balanced system of antioxidant defence may be effective in improving chilling tolerance in maize.
Maize is one of the most important crops for European agriculture with some 1.3×106 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 expansion of maize growing areas towards more northern climates, amelioration of chilling sensitivity has become a major research target. Optimal growth conditions for maize are between 20 °C and 30 °C. In northern Europe, however, temperatures of between 4–15 °C 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 dramatic damage to young maize seedlings (Fryer et al., 1998). Similarly, a drop in temperature at the beginning of the grain filling 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). Chilling treatment leads to H2O2 accumulation in the leaves of cereals such as maize (Okuda et al., 1991; Kingston‐Smith et al., 1999). Studies on the relationships between CO2 assimilation, photosynthetic electron transport and antioxidant enzyme activities in field‐grown maize suggested that the donation of electrons to oxygen by the photosynthetic electron transport chain was increased by growth at low temperautes (Fryer et al., 1998).
Maize genotypes that are resistant to low temperatures have been reported to have more efficient co‐ordination between photochemistry and carbon assimilation than cold‐sensitive genotypes (Mauro et al., 1997). While many studies have addressed the effects of stress on PSII protein turnover, very little information is available on the turnover of individual proteins associated with the PSI reaction centre. Recently, several reports have indicated preferential sensitivity of PSI to chilling‐induced damage (Terashima et al., 1994; Sonoike and Terashima, 1994; Sonoike et al., 1995; Sonoike, 1995, 1996). The PSI reaction complex is comprised of two subunits which are the products of the psaA and psaB genes. Suppression of the degradation of these subunits under photoinhibitory treatments is observed under anaerobic conditions in which PSI activity is also protected, suggesting the involvement of active oxygen species in photoinhibition of PSI (Sonoike and Terashima, 1994; Sonoike, 1996). Active oxygen species have also been implicated in the mechanism of degradation of other chloroplast proteins such as the D1 protein (Krause, 1994; Miyao et al., 1995), PSI components (Sonoike, 1996) and Rubisco (Garcia‐Ferris and Moreno, 1994; Ishida et al., 1997).
In maize leaves chilling causes an increase in H2O2 concentration, altered gene transcription and activation of proteases resulting in increased protein degradation (Okuda et al., 1991; Prasad et al., 1995; Prasad, 1996). In a previous study (Doulis et al., 1997) it has been demonstrated that the components of the antioxidant defence system are not uniformly distributed between the mesophyll and bundle sheath cells. This led to the hypothesis that the differential distribution of antioxidants was a major determinant of the extreme sensitivity of maize leaves to chilling damage (Doulis et al., 1997). In the present study evidence is provided that this is the case. Carbonyl group formation on proteins from plants grown for several weeks at 15 °C and 18 °C has been compared with those subjected to severe oxidative stress induced by paraquat treatment. Paraquat acts as an alternative electron acceptor from PSI generating superoxide (Dodge, 1994).
Active oxygen species cause protein damage through direct interaction with specific amino acids (lysine, arginine, proline or threonine). This oxidation forms a type of ‘tagging’ making peptide chains more susceptible to protease attack. Ozone‐induced decreases in Rubisco have been shown to be associated with increased carbonyl group formation resulting in increased susceptibility to aggregation and degradation (Landry and Pell, 1993; Eckardt and Pell, 1995). Increased carbonyl formation has been shown in protein extracts from leaves exposed to low temperatures (Prasad, 1996, 1997). Carbonyl formation on individual proteins isolated from the whole tissue, mesophyll and bundle sheath fractions of maize leaves grown under optimal and sub‐optimal growth temperatures or exposed to paraquat has been determined. Specific antibodies were used to identify changes in the relative abundance of individual proteins and their breakdown products and to demonstrate preferential damage to bundle sheath proteins in both stress conditions.
Materials and methods
Maize plants (Zea mays cv. H99) were grown from seed. Seed was germinated in water‐soaked vermiculite for 5 d and then transplanted into 5” pots of John Innes No. 1 compost. Pots were placed in a system of growth chambers forming a temperature gradient tunnel and maintained at 300 μmol m−2 s−1 illumination (12 h photoperiod) at either 20, 18, 15 or 10 °C. In this growth system 20 °C was the maximum temperature obtainable, just below the accepted optimum for maize (22 °C). Plants were well watered and used when the third leaf was expanded (2–3 weeks after germination). Leaves were harvested mid‐way through the photoperiod and frozen in liquid nitrogen until use. Alternatively, oxidative stress was induced by placing excised leaves from plants grown at 20 °C in a solution of 10 mM paraquat for 2 h under strong illumination (1000 μmol m−2 s−1).
Identification of polypeptides
Leaves were ground to a powder in liquid nitrogen before addition of 5 ml of extraction buffer (0.1 M Bicine/NaOH pH 7.8, 5 mM MgCl2, 1 mM EDTA, 0.1% (v/v) Triton X‐100, 5 mM DTT, 1 mM PMSF, 1 mM p‐aminobenzamidine, and 10 μM leupeptin). Once the solution had thawed it was ground further and then clarified by centrifugation (10 000 g at 4 °C for 5 min).
Protein extracts were mixed with an equal volume of double concentration sample loading buffer (Laemmli, 1970) and separated by 15% SDS‐PAGE. Peptides were Western blotted onto nitrocellulose and specific proteins detected by interaction with appropriate antisera. In each case the second antisera was goat‐anti‐rabbit IgG conjugated with alkaline phosphatase.
Alternatively, oxidized proteins in extracts were detected by use of the ‘Oxyblot’ detection system (Oncor, Gaithersburg, MD, USA). Derivatization of oxidized proteins (recognizing carbonyl residues) with 2,4‐dinitrophenylhydrazine (DNPH) to form 2,4‐dinitrophenylhydrazone (DNP‐hydrazone) within the extract was performed according to manufacturers’ instructions before separation by 15% SDS‐PAGE and Western blotting as described previously. The nitrocellulose filters were incubated with primary (anti‐DNP‐hydrazone) and secondary (anti‐rabbit IgG conjugated with horseradish peroxidase) antisera according to manufacturers’ instructions. Cross‐reactions were detected by chemiluminescence (ECL Reagents, Amersham, UK) and recorded by 10–60 s exposure of blue light‐sensitive film (Kodak, UK).
No attempt has been made to quantify differences in band intensity and all results are interpreted on a qualitative basis only. For in‐depth quantitative analysis a much more detailed study of changes in individual bands would be required including accurate identification of polypeptides. This was beyond the scope of the present study.
Source of antibodies
Antiserum raised against Rubisco (holoenzyme) purified from wheat was provided by Dr AJ Keys (IACR, Rothamsted Experimental Station, UK), the anti‐PEP carboxylase antisera used in this study was supplied by Dr J Vidal (INRA, Versailles, France). Antisera cross‐reacting with synthetic peptides corresponding to partial sequence of the subunits of photosystem I were a provided by Professor T Hiyama, Saitama University, Japan) and antisera against the D1 subunit of PSII was donated by Professor H Thomas (IGER, Aberystwyth, UK). The specificity of each antibody is considered in the individual publications of the researchers cited above.
Enzyme activity measurements
The activities of the marker enzymes Rubisco and PEP carboxylase were determined by the incorporation of radiolabelled [14C]CO2 as described (Doulis et al., 1997).
Effect of paraquat on Rubisco and PEPcarboxylase activities
Maximal extractable Rubisco and PEP carboxylase activities were determined in paraquat‐treated leaves and untreated controls (Table 1). When leaves were incubated in the presence of paraquat for 2 h at a light intensity over three times that of the growth irradiance, a large decrease in maximal extractable Rubisco activity was observed. The Rubisco activity extracted from paraquat‐treated leaves was about 50% that of the control, but PEP carboxylase activity was not affected by paraquat treatment (Table 1). Growth at sub‐optimal temperatures caused a large decrease in maximal extractable Rubisco activity at 18 °C and 15 °C, but maximal extractable PEPC activities were increased in plants grown at 18 °C relative to those grown at 20 °C. PEPC activity was severely decreased in plants grown at 15 °C compared to 20 °C (Table 1).
|(μmol min−1 mg−1 chl)|
|20 °C + paraquat||1.37±0.35||3.02±1.05|
|(μmol min−1 mg−1 chl)|
|20 °C + paraquat||1.37±0.35||3.02±1.05|
Oxidative damage to proteins
Leaves from maize plants grown for 2–3 weeks at 20 °C or under sub‐optimal conditions (18 °C or 15 °C) were used to explore the relationships between low temperature‐induced damage to proteins and oxidative damage caused by the pro‐oxidant herbicide paraquat. Soluble protein fractions from leaves exposed to low temperature or treated with paraquat (Fig. 1) were used in western blotting following SDS‐PAGE for either immunodetection or characterization of oxidative damage via a carbonyl detection system (Figs. 1,2, 3, 4). The effect of oxygen free radicals on leaf proteins was investigated by using the ‘Oxyblot’ oxidized protein detection kit. Some differences in the polypeptide patterns between leaves grown at 20 °C, 18 °C and 15 °C were observed as well as differences in polypeptide profiles between leaves grown at 20 °C and then incubated in the presence or absence of paraquat (Fig. 1A). For example, one band (indicated by the arrow in Fig. 1) was clearly present in plants grown at 20 °C, 18 °C and 15 °C, but was absent in leaves following paraquat treatment.
Using paraquat (to exacerbate oxidative stress) it was possible to identify maize leaf proteins which are susceptible to oxidative stress and compare these with proteins which become oxidized during growth at sub‐optimal temperatures (Fig. 1B). For this reason the following figures show comparisons of proteins from paraquat‐treated leaves and extracts from leaves of plants grown at optimal and sub‐optimal temperatures.
Carbonyl formation was evident in several polypeptides in the total soluble leaf protein extracts, even in plants grown at 20 °C (Fig. 1B). Paraquat treatment greatly increased the number of proteins oxidized as shown by the intensity of the signal throughout the lane in comparison to all other samples and treatments (Fig. 1, lane MV). Paraquat‐induced carbonyl formation was much greater than that caused by low temperature growth. Very few proteins showed increased carbonyl formation following growth at low temperatures and in fact labelling in one major band, at about 24–26 kDa, decreased (Fig. 1B). This may be due to changes in protein abundance as well as the number of carbonyl residues. Indeed, a polypeptide at 24–26 kDa was noticeably less oxidized at 15 °C than at higher growth temperatures (Fig. 1).
Mesophyll and bundle sheath extracts were prepared from maize leaves as described previously (Doulis et al., 1997). Relatively few polypeptides in isolated mesophyll extracts contain carbonyl groups (Fig. 2A) relative to bundle sheath proteins (Fig. 2B). Growth at 15 °C increased carbonyl formation in proteins in both mesophyll and bundle sheath compared to 20 °C (Fig. 2). Paraquat treatment enhanced carbonyl formation on bundle sheath proteins (Fig. 2B), but little difference in carbonyl formation was observed in mesophyll proteins (Fig. 2A). The quantitative differences in the amounts of label between Figs 1 and 2 are due to differences in protein loading (see figure legends) between experiments.
Identification of proteins
While it was impossible within the scope of the present study to identify all of the oxidized proteins, it was possible to distinguish the major photosynthetic proteins and their breakdown products by use of specific antibodies (Figs 3, 4). Leaf extracts probed with anti‐PEP carboxylase antiserum showed a dense staining band at a high molecular weight estimated to be between 90 and 135 kDa (Fig. 3A). This band contains at least two polypeptides which were present in all extracts regardless of treatment (Fig. 3A). Lower molecular weight polypeptides were also detected. The abundance of these minor polypeptides was increased at 15 °C relative to higher growth temperatures. Two of the polypeptides identified with this antiserum, at 84 and 25 kDa, which were detected in the leaf extract made from plants grown at 20 °C, were not present when extracts were prepared after paraquat treatment (Fig. 3A).
Anti‐Rubisco antiserum raised against purified wheat Rubisco cross‐reacted with the maize leaf enzyme (Fig. 3B). The large and small subunits are readily visible in Fig. 3B. The intensity of these bands was similar under all growth temperatures and after paraquat treatment. Aggregation products at between 90 and 110 kDa were observed as previously (Desimone et al., 1996). Similarly, breakdown products at 24 and 34 kDa are similar to those described previously (Desimone et al., 1996; Ishida et al., 1997). The abundance of these products increased as growth temperature decreased (Fig. 3B). Paraquat treatment caused little difference in the abundance of these Rubisco breakdown products compared to extracts from 20 °C‐grown plants (Fig. 3B). However, paraquat treatment resulted in the appearance of other polypeptides which cross‐reacted with the Rubisco antiserum at 46, 43 and 38 kDa.
The PSI reaction centres polypeptides derived from the psaA and psaB genes were identified with antisera produced from synthetic peptides (Sonoike, 1996). Four polypeptides of 75 kDa and below were detected with the anti‐psaA antiserum (Fig. 4A). These polypeptides were of equal abundance in leaves grown at all temperatures but they were completely absent from the leaves treated with paraquat (Fig. 4A). One polypeptide of 68 kDa was detected with antisera raised against a synthetic peptide corresponding to the psaB gene product (Fig. 4B). The intensity of this band was greatly reduced in the plants grown at 15 °C which contained approximately 50% of that present in leaves grown at 20 °C. In contrast to the psaA polypeptide, the psaB polypeptide was unaffected by exposure to paraquat (Fig. 4B). The abundance of this protein was similar in 20 °C (control)‐grown plants and those treated with paraquat (Fig. 4B).
The abundance of the PSII, D1, polypeptide decreased slightly with decreasing growth temperature (Fig. 4C). Paraquat treatment not only caused a decrease in the abundance of the polypeptide but also resulted in an increase in its apparent molecular weight (Fig. 4C).
Photosynthetic CO2 assimilation in maize leaves is decreased by low temperatures (Fryer et al., 1998). The D1 reaction centre protein of PSII (a 32 kDa polypeptide), CPI (a heterodimer of approximately 60 kDa monomeric size) and subunit II (a 22 kDa polypeptide of PSI), the cytochrome b6/f subunit IV, cytochrome f, and the b6/f‐subunit of the coupling factor were all substantially decreased by growth at 14 °C compared with 25 °C. Chloroplast‐encoded proteins were much more affected than nuclear‐encoded proteins (Bredenkamp et al., 1992). Chloroplast protein synthesis appeared to continue during growth at sub‐optimal temperatures (Nie and Baker, 1991), but co‐ordinate control of expression of chloroplast‐ and nuclear‐encoded proteins was disrupted (Robertson et al., 1993).
Changes in the composition of the light‐harvesting antenna were observed at low temperatures, for example, a 31 kDa polypeptide, related to CP29, accumulated (Covello et al., 1988; Hayden et al., 1986, 1988). It has been suggested that the accumulation of the 31 kDa polypeptide may lead to inappropriate functioning of the light‐harvesting apparatus (Hayden et al., 1986, 1988). Phosphorylation of CP29 has been shown to occur in conditions of decreased photosynthetic capacity (Bergantino et al., 1995) and this may represent a regulatory mechanism protecting PSII against low temperature‐induced photoinhibition in maize leaves (Mauro et al., 1997). In maize leaves the Rubisco protein is continuously turned over (Simpson, 1978; Simpson et al., 1981; Esquival et al., 1997). Oxidative stress, however, increases turnover and induces partial degradation of the Rubisco large subunit producing several polypeptides (Mehta et al., 1992; Landry and Pell, 1993; Garcia‐Ferris and Moreno, 1994; Desimone et al., 1996). Increased protease activity has previously been demonstrated in maize leaves grown at low temperature (Prasad, 1996, 1997), but proteases responsible for the degradation of abnormal proteins are poorly characterized in plants (Garcia‐Ferris and Moreno, 1994; Desimone et al., 1996; Ishida et al., 1997).
In the present study maize plants were grown at 15 °C, 18 °C or 20 °C and some of the leaves of plants grown at 20 °C were treated with paraquat (Dodge, 1994). Paraquat treatment resulted in a substantial increase in the number of Rubisco breakdown products which were not observed in leaves grown at low temperatures (Fig. 3B). These polypeptides may represent cleavage of short regions from the Rubisco large subunit at either N or C termini. Oxidative cleavage of Rubisco in the presence of paraquat would appear to be different to that occurring in response to low temperatures, since the foliar Rubisco‐derived polypeptide patterns in plants grown at 15 °C and in paraquat‐treated leaves are different.
The bundle sheath chloroplasts of maize leaves are deficient in PSII complexes while PSI is equally distributed between bundle sheath and mesophyll cells (Hatch and Osmond, 1976; Robertson et al., 1993). Hence, direct stress‐induced changes in PSII function will result from phenomena occurring only within the mesophyll chloroplasts. While growth at low temperatures decreased the abundance of D1, similar to that observed many times previously (Nie and Baker, 1991; Bredenkamp et al., 1992), paraquat treatment caused a marked shift in molecular weight, reminiscent of phosphorylation‐dependent changes in mobility observed on PAGE gels (Aro et al., 1993; Zer et al., 1994; Tyystjärvi and Aro, 1996). Oxidation causes a major shift in gene expression (Mayfield and Taylor, 1987; Reiß et al., 1983) and it is highly unlikely that de novo D1 synthesis can continue under these conditions. Differences in the phosphorylation state of other PSII polypeptides associated with a change in the organization of the antenna system have been observed in maize lines varying in temperature‐sensitivity (Mauro et al., 1997).
The PSI reaction centre complex consists of a heterodimer of the psaA and psaB gene products. The abundance of these proteins was different in the paraquat and low temperature treatments. Following exposure to paraquat, the psaA gene product was below the level of detection in leaf extracts. The psaA gene product, therefore, appears to be extremely sensitive to oxidative damage. In contrast, the psaB gene product decreased with decreasing growth temperature but was unaffected by paraquat. PSI subunit II, a polypeptide of 22 kDa, was shown to be absent from maize leaves grown at 14 °C and chilling decreased the abundance of the CPI complex of PSI (Nie and Baker, 1991; Bredenkamp et al., 1992). There is no indication, however, that PSI activity limits photosynthesis in maize plants grown at low temperatures (Kingston‐Smith et al., 1999).
The authors recently proposed that the compartmentation of antioxidants in maize leaves together with the obligate transport of reduced ascorbate and glutathione to the bundle sheath compartment from the mesophyll predisposes maize to low temperature‐induced oxidative damage (Doulis et al., 1997; Fig. 5). Similarly, cysteine, γ‐glutamylcysteine and glutathione were found to be predominantly located in the mesophyll and cysteine was found to be transported between the mesophyll and bundle sheath cells (Burgener et al., 1998). It has been shown that growth at low temperatures increased the abundance of carbonyl groups on proteins (Prasad, 1997). The present study demonstrates that low temperature treatment does not, however, increase carbonyl formation in proteins uniformly in all leaf cell types. There was a marked difference in the abundance of carbonyl groups between proteins originating from the mesophyll or bundle sheath. Very few proteins from the mesophyll contained carbonyl groups even in paraquat‐treated leaves. In contrast, proteins located in the bundle sheath showed extensive carbonyl formation. Similarly, carbonyl groups were present largely in the bundle sheath tissues of plants grown at 15 °C. Hence, bundle sheath proteins are more susceptible to oxidative damage than those residing in the mesophyll cells. The increase in carbonyl groups observed in plants grown at 15 °C or at 20 °C and subjected to paraquat treatment was largely restricted to bundle sheath proteins (Fig. 2B) consistent with a deficit in antioxidant capacity in the bundle sheath cells.
Superoxide dismutase and ascorbate peroxidase are concentrated in the bundle sheath cells, but dehydroascorbate and oxidized glutathione (GSSG) have to be transported to the mesophyll for re‐reduction, since glutathione reductase and dehydroascorbate reductase are localized only in the mesophyll cells (Doulis et al., 1997; Fig. 5). Any constraint on transport between the compartments would adversely affect this cycling. A direct causal relationship between oxidative stress and chilling has not been demonstrated, but it has been shown that the bundle sheath proteins of maize leaves are much more susceptible to oxidative damage than those of the mesophyll. At low temperatures transport of antioxidants between compartments would be impaired, resulting in a net deficit in antioxidant capacity in the bundle sheath and the bundle sheath tissues become effectively cold‐girdled.
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We are extremely grateful to Dr AJ Keys, Dr J Vidal, Professor T Hiyama, and Professor H Thomas for the generous gifts of antibodies. This work was funded by the European Commission (Grant No. AIR1‐CT92–0205, Engineering Stress Tolerance in Maize) and by the BBSRC. The authors thank Janet Williams for growth and maintenance of the maize plants.