Electrophoretic Comparison of Mitochondrial Polypeptides from Maize Lines Susceptible and Resistant to Helminthosporium maydis Race T

The mitochondrial polypeptides from maize lines susceptible and resistant to Helminthosporium maydis were studied by singleand two-dimensional gel electrophoresis. Approximately 120 polypeptides were detected by two-dimensional gel electrophoresis. However, it was not possible to detect qualitative differences between the mitochondrial polypeptides of the two inbreds. These observations are discussed with reference to the putative mechanism of action of the pathotoxin. Maize (Zea mays) inbred lines with Texas (T) cytoplasm are susceptible to toxin produced by race T of Helminthosporium maydis, whereas lines with normal (N) cytoplasm are resistant. Adding the pathotoxin in vitro to susceptible mitochondria from T cytoplasm causes changes in respiration rate and oxidative phosphorylation, but the toxin does not affect maize mitochondria isolated from the N cytoplasm type (9). Plant mitochondria have their own distinct protein synthesis system (12). When [35S]methionine is incorporated in vitro by mitochondria from N and T cytoplasm, there are some differences between the translation products (2). By contrast, incorporation of [35S]methionine in vivo, during sprouting, shows no differences between translation products localized in the mitochondria (4). A comparison between mitochondrial proteins of normal and susceptible inbreds (F7N and F7T) by singleor two-dimensional gel electrophoresis is reported in this paper. MATERIALS AND METHODS Seeds from F7N (male fertile and toxin resistant) and F7T (male sterile and toxin susceptible) inbreds were provided by 'Societe Francaise de Mais. ' Maize seeds were surface-sterilized with NaOCl, soaked in tap water overnight, and germinated in darkness at 28? C for up to 4 d. F7T seeds germinated less quickly than did F7N seeds. Mesocotyls were separated and homogenized in a medium containing 0.3 M mannitol (2 ml/g), 0.15% mercaptobenzothiazol, 5 mm EGTA,2 5 mm EDTA, and 0.1% BSA. The grinding was done without sand in a chilled mortar (2-40C). The homogenate was squeezed through two layers of Miracloth, and the pH was then adjusted to 7.0, if necessary. The homogenate was centrifuged at l0,OOOg for 30 min in a MSE HS18. Mitochondria were purified from the pellet on a discontinuous sucrose gradient containing 10 mM succinate, 10 iM Mops, and 0.1% BSA by centrifugation at 50,000g for 90 min in a Beckman SW25 rotor. The densities of the layers were 1.22, 1.20, 1.18, 1.15, and 1.12 g ml-'. The purified mitochondria which banded between the 1.18 and 1.15 layers were resuspended in a medium containing 0.3 M mannitol and 10 mm Mops, and they were pelleted at lO,OOOg for 15 min in a MSE HS 18 centrifuge. Protein concentration was determined according to Gornall (3). After solubilization of mitochondrial proteins, single-dimensional electrophoresis was carried out using 10% polyacrylamide gels containing 0.1% SDS, according to Laemmli (5). Two-dimensional electrophoresis was performed according O'Farrell (10, 11). Ampholines, with a pH range of 3 to 10, were purchased from LKB Instruments S.A. (France). At the end of the first-dimension electrophoresis, gels were immersed in 10% glycerol, 5% 2-mercaptoethanol, 0.3% SDS, and 60 mm Tris-HCl. Gels were stored at -27? C in the same medium before running the second dimension of electrophoresis or eventually staining with Coomassie blue. The second-dimension slabs were 7 to 22% polyacrylamide gradients. RESULTS The mitochondrial preparations can be shown to have the capacity to oxidize malate and succinate with good respiratory controls and P/O ratios (results not shown). As has been reported elsewhere, the N and T mitochondria are not significantly different in this respect. Two-dimensional gels of three kinds were run. IEF gels with small amounts of protein provided the greatest degree of resolution (Figs. 1 and 2). Proteins with low mol wt were not detected in these gels, where small tubes with 50 or 100 ,ug of protein provided the first dimension. However, low mol wt proteins were known to be present, since samples of the first-dimensional gels contained at least two clear peaks (Fig. 3). To detect these peaks on twodimensional gels, larger tubes, with 250 ,tg of protein, were run using the IEF method. Despite the generally lower resolution on these gels, it was possible to identify several proteins of low mol wt that were undetectable in the other IEF gels (Fig. 4). NEPHGE gels are expected to resolve basic proteins better than IEF gels (13). Since these gels require a short period of electrophoresis in which equilibrium is not reached, they require careful control to facilitate comparisons between experiments. Although the overall resolution is not as good as it is with IEF, several low mol wt basic proteins were revealed by this method but not by the others (Figs. 5 and 6). Possible quantitative differences were seen which ' Supported by the Action Thematique Programmee No. 4122 from Institut National de la Recherche Agronomique and the Centre National de la Recherche Scientifique. 2 Abbreviations: EGTA, ethyleneglycol-bis-(,8-amino-ethyl ether) N,N'tetraacetic acid; Mops, morpholinopropane sulfonic acid; HMT, Helminthosporium maydis toxin; IEF, isoelectric focusing; NEPHGE, nonequilbrium pH gradient electrophoresis. 1217 This content downloaded from 157.55. 39.253 on Sat, 11 Jun 2016 05:45:22 UTC All use subject to http://about.jstor. org/terms 1218 DIANO Plant Physiol. Vol. 69, 1982