classification of normal and male-sterile cytoplasms in ... - Europe PMC

CLASSIFICATION OF NORMAL AND MALE-STERILE CYTOPLASMS IN MAIZE. I. ELECTROPHORETIC ANALYSIS OF VARIATION IN MITOCHONDRIALLY SYNTHESIZED PROTEINS B. G. FORDE*, R. J. C. OLIVER

AND

C. J. LEAVER

Department of Botany, The King’s Buildings, University of Edinburgh, Edinburgh EH9 3JH, Scotland AND

R. E. GUNN

AND

R. J. KEMBLEt

Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ, England

Manuscript received November 11, 1979 Revised copy received February 27, 1980 ABSTRACT

Male-sterile cytoplasms of maize have previously been classified into three groups (T, S and C) according to their fertility ratings in various inbred backgrounds. In earlier studies, mitochondria from three male-sterile cytoplasms, representing each of these three groups, have been found to synthesize characteristic variant polypeptides that distinguish them from each other and from those of normal (N) cytoplasm. In order to determine the extent of cytoplasmic variation, we have now analyzed the translation products of mitochondria from 28 additional cytoplasmic sources. The results show that on this basis 18 of the cytoplasms are identical to the USDA (S) cytoplasm, three are identical to the Texas (T) cytoplasm and two are identical to the C cytoplasm. The five remaining cytoplasms are indistinguishable from normal, malefertile (N) cytoplasm. Our classification of the cytoplasms is in general agreement with those based on fertility restoration. However, of three cytoplasms that have previously remained unclassified, two (B and D) have now been assigned to the S group and one (LF) to the N group. No heterogeneity in mitochondrial translation products was detected within the normal or any of the three male-sterile groups. The usefulness of the analysis of mitochondrial translation products as a method for classifying normal and male-sterile cytoplasms is discussed.

HE extensive use of cytoplasmically inherited male sterility in t h e comTmercial production of hybrid maize, first introduced in the early 1 9 5 0 ~ soon ~ led to 80% of the maize (Zea mays L.) grown in the United States sharing the same male-sterile cytoplasm, known as the Texas (T) cytoplasm. This high degree of genetic uniformity is now seen as the major factor responsible for the epiphytotic outbreak in 1970 of southern corn-leaf blight (ULLSTRUP 1972). The cause of the epidemic was a virulent new race of Helminthosporium maydis, * Present address, to which requests for reprints should be sent: Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts. AL5 ZJQ, England. t Present address: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida. Genetics 95: 44-50

June, 1980.

444

B. G. FORDE et

al.

designated race T, that preferentially attacks maize plants carrying the T cytoplasm (HOOKER et al. 1970). As a consequence of the epidemic, there is now an increased interest in obtaining alternative sources of cytoplasmic male sterility. There is also an awareness of the need to introduce into agriculture cytoplasmic variation that is not connected with male sterility. Any increase in cytoplasmic diversity will reduce the probability of an epidemic arising from cytoplasmicallyinherited susceptibilityto disease. In maize, over 80 separate discoveries of cytoplasmic male sterility have been made (DUVICK1965), and many of these have now been classified into one of three groups according to the pattern of restoration of fertility by nuclear genes (DUVICK 1965; BECKETT 1971; GRACEN and GROGAN 1974). This method of classification involves crossing each source of male sterility as female to a series of inbred lines and backcrossing repeatedly. Most inbred lines are found to restore fertility with some sources of cytoplasm, but not others. On the basis of fertility restoration, DUVICK(1965) indicated the existence of two types of cytoplasmic male sterility, the Texas (T) and USDA (S) cytoplasms. A survey of 30 maleand GROGAN sterile cytoplasms by BECKETT (1971) and a similar one by GRACEN (1974), added a third type, designated C cytoplasm. In each of these studies there was, apparently, considerable heterogeneity within each group of cytoplasms, certain inbred lines restoring fertility with some cytoplasms but not with others. This heterogeneity suggested the possibility that cytoplasms that are grouped together on the basis of fertility restoration are not identical. A few of the cytoplasms could not be classified into any of the three groups. The present study was undertaken to assess cytoplasmic heterogeneity within the groups classified as T, S, C (or “normal”) and to provide a possible alternative method for classifying maize cytoplasms, one that does not depend on patterns of fertility restoration. The study was prompted by the initial finding that mitochondria from three male-sterile cytoplasms, one from each of the designated groups (T, C and S ) , could be distinguished from each other and from normal mitochondria by their synthesis of characteristic variant polypeptides (FORDE, OLIVERand LEAVER 1978; FORDE and LEAVER 1980). Mitochondria possess their own genetic system, and evidence is accumulating that they are the carriers of the factors responsible for cytoplasmic male sterility in maize (MILLER and KOEPPE1971; LEVINGS and PRING 1976; PRING and LEVINGS 1978; WARMKE and LEE 1977; FORDE, OLIVER and LEAVER 1978; FORDE and LEAVER 1980). Less than 20% of mitochondrial proteins are translated on mitochondrial ribosomes, the majority being coded for by nuclear DNA and synthesized on cytoplasmic ribosomes. Detection of mitochondrial translation products, however, is made possible by the ability of the mitochondrion to continue protein synthesis after isolation from the cell, allowing the translation products to be labelled with radioactive amino acids to high specific activity. This technique, combined with sodium dodecyl sulphate-polyacrylamide gel electrophoresis, has now been employed to compare the translation products of mitochondria from 32 cytoplasmic sources.

CLASSIFICATION OF MAIZE CYTOPLASMS I

445

MATERIALS A N D METHODS

Maize lines: 31 of the cytoplasms used were in the nuclear background of CO192 XWJ produced by the Eucarpia Northern Maize Committee (GUNN1975) via CO192 cytoplasmic stocks obtained from V. E. GRACEN(Cornel1 University), These cytoplasms were: B, C. CA, D, EK, F, G, H, HA, I, IA, J, L, LF, ME, MY, OY, PS, Q, R, RB, RS, S, SD, SG, T, TA, TC, 381, 234 and the normal maintainer line (nml). The El Salvador cytoplasm was in the nuclear background of W117. Extraction and purification of mitochondria for in vitro lobelling with [35S]-methiom*ne: Maize seeds were surface sterilized with 1% (w/v) sodium hypochlorite, washed overnight in running tap water and rinsed 3 times with sterile distilled water. The seeds were spread evenly on sterile cellulose wadding, saturated with sterile distilled water and germinated in darkness at 30" for 4 days. Subsequent operations were pedormed at 0 to 4", using sterilized and prechilled solutions and glassware. Approximately 30g of etiolated shoots were washed 3 times in sterile distilled water and homogenized for 1 min in 2 ml of grinding medium for each g fresh weight of tissue, using a pestle and mortar. The grinding medium consisted of 0.4 M mannitol, 0.025 M 4-morpholine-propanesulphonicacid (MOPS) pH 7.8, 0.005 M KCl, 0.008 M cysteine, 0.001 M EGTA and 0.1% (w/v) bovine serum albumin. The homogenate was squeezed through 4 layers of muslin and filtered through 2 layers of milk filter (Johnson and Johnson). The filtrate was centrifuged 5 min at 1000 x g, and the supernatant was then centrifuged at 10,000 x g for 15 min. The pelleted mitochondrial fraction was resuspended in 20 ml of wash medium (0.4 M mannitol, 0.005 M MOPS pH 7.5, 0.001 M EGTA and 0.1% bovine serum albumin) using a loose-fitting glass-in-glass Potter homogenizer and given another cycle of low-speed and high-speed centrifugation. The washed mitochondrial pellet was further purified in a continuous 20% (w/v) to 60% (w/v) sucrose gradient, containing 0.001 M EGTA, 0.1% bovine serum albumin and 0.01 M N- [2-hydroxyl-l, I-bis (hydroxymethyl) ethyl] -glycine [Tricine) pH 7.2, by centrifugation at 40,000 x g for 60 min in an M.S.E. 6 x 14 ml swingout rotor. The mitochondria that banded at approximately 42% sucrose were removed and diluted to an osmolarity of 0.5 M before being pelleted at 10,000 x g for 15 min. The final pellet was carefully resuspended in 0.4 M mannitol, 0.001 M EGTA, 0.01 M Tricine pH 7.2 with the aid of a Teflon-in-glass Potter homogenizer. Labelling of mitochondrial translation products with [35S]-methionine: Conditions for incorporation of [35S]-methionine into isolated mitochondria were those described previously (FORDE, OLIVER and LEAVER 1978). Freshly purified mitochondria (2O(r-600 pg protein) were incubated for 90 min at 25", with shaking, in 250 ,ul of medium containing 0.25 M mannitol, 0.09 M KCI, 0.01 M MgCl,, 0.01 M Tricine (pH 7.2), 0.005 M sodium phosphate buffer (pH 7.21, 0.001 M EGTA, 25 pmol of 19 amino acids (omitting methionine), 0.002 M dithiothreitol, 0.001 M GTP and 15 pCi of [S5S]-methicunine (1005 Ci "01-1). In addition, the incubation medium contained 0.01 M sodium succinate and 0.006 M ADP (so that ATP was generated by oxidative phosphorylation) or 0.08 M creatine phosphate, 25 fig creatine phosphokinase and 0.006 M ATP (so that ATP was generated externally) or 0.02 M sodium acetate. The time course of incorporation was routinely followed by removal of 5 p l aliquots onto Whatman 3MM chromatography paper discs. Radioactivity precipitable by hot trichloroacetic acid was determined according to the procedure of MANSand NOVELLI(1961). The amount of radioactivity incorporated into trichloroacetic acid precipitable material in the incubations containing the nonoxidizable substrate sodium acetate is a measure of the extent to which bacterial contamination contributes to the reaction (FORDE, OLIVERand LEAVER 1979). In all cases, the radioactivity incorporated in the presence of acetate was less than 2% of that incorporated in incubations containing succinate. Electrophoretic analysis of mitochondrial translation products: Following the 90 min incubabation period, the incorporation of [35S]-methionine was stopped by the addition of ice-cold buffer containing 0.4 M mannitol, 0.001 M EGTA, 0.01 M Tricine (pH 7.2) and 0.012 M unlabelled methionine. The mitochondria were pelleted at 10,000 x g for 2 min in an Eppendorf 3200 centrifuge, frozen on dry ice and stored at -80" for up to 2 wks. When mitochondria

446

B. G . FORDE

et al.

from all 32 cytoplasms had been isolated and labelled, the pellets were thawed and solubilized i n 65 to 145 $1 of sample buffer [2% (w/v) sodium dcdecyl sulphate, 10% (w/v) sucrose, 5% (v/v) 2-mercaptoethanol and 0 . 0 6 ~Tris-HC1 (pH 6.8)] by incubation at 37" for 3 hr. The solubilized mitochondrial proteins (100-150 pg protein and 500 x IO3 cpm in 25 p l sample buffer) were analyzed by electrophoresis in 15% (w/v) polyacrylamide slab gels containing 0.1% (w/v) sodium dodecyl sulphate (LAEMMLI 1970). The gels were stained for protein with 0.1% Coomassie brilliant blue in 5 % (v/v) acetic acid: 45% (v/v) methanol, destained i n several changes of 5% acetic acid: 46% methanol and dried onto Whatman 3 MM chromatography paper. The radioactively labelled polypeptides were detected by exposing the dried gels for 10 days to Kodak "Blue Brand" X-ray film. Molecular weights were estimated by comparison with standard proteins electrophoresed in parallel tracks. RESULTS

Mitochondria from the T, S and C male-sterile cytoplasms have been found to synthesize variant polypeptides that distinguish them from each other and from normal (N) mitochondria (FORDE, OLIVERand LEAVER 1978; FORDE and LEAVER 1980). Electrophoretic analysis o€ the translation products of mitochondria from a total of 32 cytoplasmic sources has now shown that each can be classified as either N-, T-, S-, or C-type according to which, if any, of the variant polypeptides are synthesized. The four different banding patterns obtained in this survey are shown in Figure 1. Our classification of the 32 cytoplasms on the basis of their mitochondrial translation products is presented in Table 1. The translation products of mitochondria from five cytoplasms were indistinguishable from those of mitochondria from the c c n ~ ~ a (nonsterile) l'7 cytoplasm of C0192; these cytoplasms were therefore classified as N-type. The seven additional, high molecular weight (58,000 to 84,000 daltons) polypeptides that are Synthesized by cms-S mitochondria were also synthesized by mitochondria from 18 other cytoplasms; therefore, these cytoplasms were classified as S-type. The variant 13,000 MW polypeptide that is characteristic of cms-T mitochondria was also synthesized by mitochondria from three other cytoplasms; these were classified as T-type. The 21,000 MW polypeptide that was found in later experiments to be missing from 1980) was not resolved in any of the cms-T mitochondria (FORDE and LEAVER cytoplasms by the electrophoretic procedures used here. Mitochondria from the remaining cytoplasms, like those from the C cytoplasm, synthesized a variant 17,500 M W polypeptide but failed to synthesize a 15,500 MW polypeptide seen in the other cytoplasmic types; therefore, these remaining cytoplasms were classified as C-type. No significant heterogeneity was observed within any of the four groups so that it was possible to classify each cytoplasm unambiguously. Occasionally, however, with extended autoradiographic exposure times, the high molecular weight polypeptides that are characteristic of S-type mitochondria were also detected in mitochondria from some N-type cytoplasms, but always in much lower amounts. This could have been due to contamination from adjacent gel slots canying labelled S-type mitochondria. It is also important to distinguish any labelled high molecular weight polypeptides that are due to bacterial contamination from those of mitochondrial origin (FORDE, OLIVERand LEAVER 1979). This was done

CIASSIFICATION O F MAIZE CYTOPLASMS I

447

- -

- -

70

- - A

60

50

9

9 40 x

30

ds 20 15

1c

---6 N

S

T

C

FIGURE 1.-Polyacrylamide gel rlectrophoresir of 1:olypeptides synthesized by mitochondria from maize plants carrying 4 different types of cytoplasm in the same nuclear background (CO192 x WJ).Mitochondria wcre isolated from 4-day-old etiolated shoots and incubated for 90 min in a medium containing [SYS] -methinnine. The mitochondrial polypeptides were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and the dried gel was outoradiographed. Among thc 32 cytoplasms examined. 4 distinct polypeptide patterns were obtained. The tranclocation products of mitochondria from cytoplasms LF, B, Q and RB have been used here to represent these 4 pattenis, designated N-, S-. T- and C-type, respectively. The arrows indicate those polypeptides that distinguish the four cytoplasmic types.

448

B. G. FORDE

et al.

TABLE 1 Classification of 32 cytoplasms according to their mitochondrial translation products Cytoplasmic grouping (distinguishing feature of nutochondnal translation products*)

Cytoplasm

N-type (“Normal”) C-type (Synthesis of 17,500 MW polypeptide, absence of 15,500 MW polypeptide) T-type (Synthesis of 13,000 MW polypeptide) S-type (Synthesis of high molecular weight polypeptides)

LFt, OY, SG, 181,234, nml

C, RB, El Salvador HA, Q, RS7 T

Bt, CA, D-t7 EK, F7 G, H, J, L, ME, MY, PS, R, S, SD, TA, TC

* See Figure 1.

t Unclassified by fertility restoration (BECKETT1971; GRACEN and GROGAN 1974). by incubating an aliquot of each mitochondrial sample under the normal conditions for incorporating [35S]-methionine except that succinate was replaced by a nonoxidizable substrate such as acetate. Under these conditions, mitochondria are unable to generate the ATP required for protein synthesis, and any radioactivity incorporated into protein may therefore be attributed to bacteria. I n the experiments reported here, the radioactive counts incorporated into trichloroacetic acid-insoluble material in the presence of acetate were always less than 2% of those incorporated in the presence of succinate and were not detected by electrophoresis and autoradiography. The classification in Table 1 is in agreement with those that were based on patterns of fertility restoration (BECKETT 1971; GRACEN and GROGAN 1974). I n the earlier classifications, however, three of the cytoplasms could not be assigned to any of the four groups. We have classified two of these cytoplasms (B and D) as S-type and the third (LF) as N-type (Figure 1 and Table 1). TC cytoplasm has been reported to be an impure stock resulting from a mixture of cms-S and cms-T seed (BECKETT1971). The translation products of mitochondria from the TC cytoplasm were, however, indistinguishable from those of other members of the S group. Synthesis oE the 13,000 M W polypeptide that is characteristic of the T-type mitochondria could not be detected. In agreement with this, we have traced the parentage of the TC stock used in the present study and found it to consist primarily of cms-S seed. DISCUSSION

In these studies, we have been able to classify the 32 cytoplasmic sources into four groups according to their mitochondrial translation products (Table 1). The results are in agreement with previous classifications based on the use of fertility restorer genes (BECKETT1971; GRACEN and GROGAN 1974) and with the conclusions reached in the accompanying paper (KEMBLE,GUNN and FLAVELL 1980). The assay method described here has a number of advantages

CLASSIFICATION O F MAIZE CYTOPLASMS I

449

over the use of restorer genes, including its speed (four-day-old etiolated seedlings are used), its lack of ambiguity and its applicability to cytoplasms that are not male-sterile. Furthermore, although nuclear genes can partially suppress the synthesis of one variant mitochondrial translation product (FORDEand LEAVER 1980), we have found that identification of the four cytoplasmic groups in Table 1 is possible in all nuclear backgrounds studied so far (FORDE, OLIVER and LEAVER 1978; FORDE and LEAVER 1980). Therefore, classification of any additional cytoplasms into one of these groups may be possible regardless of their nuclear backgrounds. The value of the technique has already been underlined by the discovery that, during production of the crosses used in the present study, stocks of two of the S group had inadvertently been interchanged with stocks of two of the C group. We could detect no variation in the mitochondrial translation products within each of the four cytoplasmic groups. This is in contrast to the differences observed in the fertility restoration patterns of members of the same group (BECKETT 1971; GRACEN and GROGAN 1974). However, restriction enzyme analysis of the mitochondrial DNAs of seven S-type cytoplasms also failed to reveal any differand LEVINGS 1978), although the same technique is ences between them (PRING sensitive enough to discriminate between S, T and C cytoplasms and to detect heterogeneity of mitochondrial DNA among normal cytoplasms (LEVINGS and 1977; PRINGand LEVINGS 1978). Therefore, PRING1976; LEVINGS and PRING it is probable that differences in restoration patterns observed between cytoplasms from the same group are usually due to nuclear gene interactions and not to cytoplasmic variation. Nevertheless, heterogeneity within the C group of cytoplasms has been detected by restriction enzyme analysis of the mitochondrial DNAs (PRING, CONDEand LEVINGS 1979). This analysis showed that the C and RB cytoplasms are similar, but not identical, while the El Salvador cytoplasm appeared to be only distantly related to the other C-type cytoplasms. Despite these differences in the mitochondrial DNAs, however, we found no differences between the translation products of mitochondria from these three cytoplasms. This result highlights an important difference between the two techniques for assaying cytoplasmic variation. Analysis of mitochondrial protein synthesis reveals genetic variation that is expressed and therefore likely to be functionally significant. Restriction enzyme analysis is capable of detecting variation at many sites in mitochondrial DNA, but this variation may not always result in altered gene expression. Thus, although restriction endonucleases are sensitive tools for detecting differences between cytoplasmic DNAs, it should be borne in mind that any variation that they reveal may be of no consequence to organelle function and to plant breeding. The finding that there is heterogeneity of mitochondrial DNA within the C group of cytoplasms also shows that it will not always be possible to assign an unknown cytoplasm to a particular group on the basis of restriction enzyme analysis. On the other hand, the correlation that we have found between variant translation products and male-sterile grouping indicates that this analytical technique will provide a reliable method for cytoplasmic classification. The apparent lack of variation in mitochondrial translation products within

450

B. G . FORDE et

al.

each cytoplasmic group suggests that there may be no advantage for plant breeding programs in using one source of male sterility rather than another in the same group (Table 1). Similarly, it seems unlikely that any significant increase in cytoplasmic variation in commercially grown maize will be achieved by introducing a number of cytoplasms from the same group. We thank JANICE FORDE for excellent technical assistance and R. B. FLAVELL for critical reading of the manuscript. This research was supported by a grant from the A.R.C. (Grant No. AG 15 160) to C. J. LEAVER and by a Rank Prize Fund Fellowship held by R. J. KEMBLE. LITERATURE CITED

BECEETT,J. B., 1971 Classification of male-sterile cytoplasms in maize (Zea mays L.). Crop Sci. 11: 724-727. DUVICK,D. N., 1965 Cytoplasmic pollen sterility in corn. Advan. Genet. 13: 1-56. FORDE? B. G. and C. J. LEAVER,1980 Nuclear and cytoplasmic genes controlling synthesis of variant mitochondrial polypeptides in male-sterile maize. P m . Natl. Acad. Sci. U.S. 77: 418-422. FORDE, B. G., R. J. C. OLIVERand C. J. LEAVER,1978 Variation in mitochondrial translation products associated with male-sterile cytoplasms in maize. Proc. Natl. Acad. Sci. U.S. 7 5 : 3841-3845. -, 1979 In vitro study of mitochondrial protein synthesis during mitochondrial biogenesis in excised plant storage tissue. Plant Physiol. 63: 67-73. GRACEN, V. E. and C. 0. GROGAN, 1974 Diversity and suitability for hybrid production of different sources of cytoplasmic male sterility in maize. Agron. J. 65: 654-657. GUNN,R. E., 1975 Report on the activities of the Northern Maize Committee (1974-75). pp. 22-29. Eucarpia, Eighth Meeting of the Mm-ze and Sorghum Section held in ParisVersailles, 15-17 September. HOOKER, A. L., D. R. SMITH,S. M. LINN and J. B. BECEETT,1970 Reaction of corn seedlings with male-sterile cytoplasm to Helminthosporium maydis. Plant Dis. Reptr. 54: 708-712. XEMBLE,R. J., R. E. GUNN and R. B. FLAVELL, 1980 Classification of normal and male-sterile cytoplasms in maize. 11. Electrophoretic analysis of DNA species in mitochondria. Genetics 95: 451-458. LAEMMLI,U. K., 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. 1976 Restriction endonuclease analysis of mitochondrial LEVINGS,C. S., 111 and D. R. PRING, DNA from normal and Texas cytoplasmic male-sterile maize. Science 193: 158-160. -, 1977 Diversity of mitochondrial genomes among normal cytoplasms of maize. J. Hered. 68: 350-354. MANS,R. J. and A. D. NOVELLI,1961 Measurement of the incorporation of radioactive amino acids into protein by filter-paper disk method. Arch. Biochem. Biophys. 94: 48-56. MILLER, R. J. and D. E. KOEPPE,1971 Southern corn leaf blight: susceptible and resistant mitochondria. Science 173 : 67-69. PRING,D. R. and c. s. hvINGs, 111, 1978 Heterogeneity of maize cytoplasmic genomes among male-sterile cytoplasms. Genetics 89: 121-136. PRING, D. R., M. F. GINDE and C. S. LEVINGS,111, 1979 Heterogeneity within the C group of male-sterile cytoplasms. Maize Genet. Coop. News Lett. 53: 42. ULLSTRUP, A. J., 1972 The impacts of the southern corn leaf blight epidemics of 1970-1971. Ann. Rev. Phytopathology 10: 37-50. WARMKE, H. E. and S. L. J. LEE, 1977 Mitochondrial degeneration in T cytoplasmic malesterile corn anthers. J. Hered. 58: 213-222. Corresponding editor: R. L. PHILLIPS