A comparison of mitochondrial proteins from diverse cytoplasms of maize by gel electrophoresis

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Title:
A comparison of mitochondrial proteins from diverse cytoplasms of maize by gel electrophoresis
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vi, 61 leaves : ill. ; 28 cm.
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English
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Thornbury, David Walter, 1948-
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Corn -- Cytology   ( lcsh )
Corn -- Disease and pest resistance -- Genetic aspects   ( lcsh )
Mitochondria   ( lcsh )
Hybrid corn   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 56-60).
Statement of Responsibility:
by David Walter Thornbury.
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Typescript.
General Note:
Vita.

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University of Florida
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Full Text









A COMPARISON OF MITOCHONDRIAL PROTEINS FROM
DIVERSE CYTOPLASMS OF MAIZE BY
GEL ELECTROPHORESIS











BY

DAVID WALTER THORNBURY


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY







UNIVERSITY OF FLORIDA


1979

















ACKNOWLEDGEMENTS


The author wishes to express his sincere appreciation to

his major professor Dr. Daryl Pring, for his guidance and patience


throughout this project. The author also wishes to thank the

members of his committee Dr. J. R. Edwardson, Dr. R. J. Mans,

Dr. E. Hiebert, and Dr. D. A. Roberts for their guidance and advice.

A special appreciation goes to Dr. J. R. Laughnan and Dr. S. J. Gabay-

Laughnan for the genetic stocks they provided which were an invaluable

aid to this study.

Finally the author wishes to thank his parents, Dr. and Mrs.

James H. Thornbury, for their faith and encouragement.


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TABLE OF CONTENTS


Page

* ii


ACKNOWLEDGEMENT S


ABSTRACT ..... . ..................... iv

INTRODUCTION .... .. .. . . 1

LITERATURE REVIEW .... . 4

MATERIALS AND METHODS .... .. . 13

Maize Genetic Stocks . .... .. .. 13

Isolation and Purification of Mitochondria ... 13

Fractionation of Mitochondria. . .14

Electrophoresis of Mitochondrial Proteins ... .15

Labeling and Fluorography of Mitochondrial Proteins 18

Isolation of Mitochondrial DNA . ... .19

Agarose Electrophoresis of Mitochondrial DNA ... 20

Restriction Endonuclease Fragment Analysis ... .20

RESULTS . . . 22

Purity of Mitochondrial Membrane Fractions ... 22

Electrophoresis of Mitochondrial Membrane Fractions 22

Electrophoresis of Total Mitochondrial Protein ...... 24

Scms Cytoplasm Instability and the S Protein ... 37

Restriction Endonuclease Analysis of Mitochondrial DNA 44

DISCUSSION . . . 50

LITERATURE CITED. .. . .. .... .56

BIOGRAPHICAL SKETCH . .... . 61

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Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

A Comparison of mitochondrial Proteins From
Diverse Cytoplasms of Maize by
Gel Electrophoresis

By

David Walter Thornbury

June 1979

Chairman: Daryl R. Pring
Major Department: Plant Pathology

Male sterility is a phenomenon of higher plants usually expressed

as a failure to produce functional pollen. Cytoplasmic male sterility

(cms) is transmitted through the female parent only and its inheri-

tance is non-mendelian. In maize (Zea mays, L.), cytoplasmic male

sterility is economically useful in the production of hybrid seed.

By 1970, the Texas male-sterile cytoplasm (Tcms) was predominant

in the production of hybrid maize seed in the United States. The

southern corn leaf blight epidemic of 1970 led to its abandonment,

since Tcms maize lines were highly susceptible to race T of Bipolaris

maydis (Nisikado) Shoemaker. This increased susceptibility resulted

from the production by race T of a host-specific pathotoxin.

The cytoplasm-specific mitochondrial characteristics of

a) differential sensitivity of Normal (N) and Tcms mitochondria to

the pathotoxin from race T of B. maydis, h) differences in the endo-

nuclease restriction fragment patterns between N and cms mitochondrial

DNA, and c) differences among several N versus cms mitochondrial

enzyme levels suggested a dual mitochondrial involvement in the

susceptibility of Tcms lines and in male sterility. The objective

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of this work was to investigate the hypothesis that these differences

may be detected at the protein level. Therefore, mitochondria from

isogenic lines of maize were examined for protein differences assoc-

iated with male sterility and disease susceptibility. Soluable and

membrane mitochondrial proteins were examined by means of sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Two types of protein differences were detected in total mito-

chondrial protein preparations. Differences due to nuclear variation

were seen among mitochondria from four maize lines in the USDA or

S male sterile cytoplasm (Scms). One cytoplasm-specific protein was

found in mitochondria from Scms maize lines. This S protein has an

apparent molecular weight of 133,900 daltons, determined by SDS-PAGE.

Examination of membrane fractions from purified mitochondria revealed

no detectable cytoplasm-specific differences between N and

Tcms maize.

Labeling of the S protein was detected in mitochondria from

shoots germinated in the presence of 3S. Emetine, an inhibitor of

cytoplasmic ribosomes, was used to limit labeling of nuclear-coded

mitochondrial proteins. Inhibition was incomplete, and specific

labeling of motochondrial gene products was not achieved. Analysis

of motochondrial proteins from other maize cytoplasms labeled with 35S

in detached shoots did not reveal protein differences that correlated

with cytoplasmic male sterility or disease susceptibility.

The relationship of the S protein to male sterility was examined

in restorer and non-restorer maize lines and in a line that carries a

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cytoplasmic mutation to fertility. With the exception of the line

carrying the cytoplasmic mutation to male fertility, all Scms lines

tested contained the S protein in their mitochondria. Mitochondria

from Scms maize contain two plasmid-like DNAs. Data from other

workers suggested that these DNAs play a role in male sterility. The

plasmid-like DNAs have also been shown to be absent in lines carrying

this mutation. The presence of fertility restorer genes in the

nucleus had no effect on either the S protein or the plasmid-like

DNAs.

Data from this study indicate that cytoplasmically determined

male sterility and disease susceptibility do not produce gross changes

in the electrophoretic phenotype of maize mitochondria. However, one

unique protein was detected in Scms lines by SDS-PAGE. Although its

function is unknown, the presence of the protein correlates with the

presence of plasmid-like DNAs in Scms maize, suggesting that the S protein

may be a gene product of the plasmid-like DNAs.


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ITi iPTODLICTION


Male sterility in higher plants usually is expressed as.a failure

to produce functional pollen. Male sterility in maize (Zea mays, L.)

can be determined by nuclear genes (Beadle, 1932) or by cytoplasmic

factors (Rhoades, 1933; Duvick, 1965). Cytoplasmic male sterility

(cms) is transmitted only through the female parent and the factors

responsible for its expression are not known. In maize cytoplasmic

male sterility is widely used in the production of hybrid seed.

Cytoplasmic male sterility, used in conjunction with nuclear genes

which restore pollen fertility, eliminates the need for detasseling

in the production of hybrid seed.

Until 1970, the use of cytoplasmic male sterility was the domi-

nant procedure for the production of maize seed in the United States

(Tatum, 1971). Approximately 85 percent of all maize planted in the

United States in that year had one type of male sterile cytoplasm,

called Texas, or Tcms, cytoplasm (Ullstrup, 1972). This situation of

cytoplasmic homogeneity was conducive to the development of the

southern corn leaf blight epidemic of 1970. The epidemic was caused

by a race of the causal agent, Bipolaris maydis (Nisikado) Shoemaker

(formerly Helminthosporium), designated race T, which exhibited highly

specific pathogenicity towards maize hybrids produced using Texas

male sterile cytoplasm (Hooker et al., 1970; Scheifele et al., 1970).

Prior to the epidemic, southern corn leaf blight had been of little

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economic importance in the United States. In 1970, the disease caused

an estimated one billion dollar loss in the maize crop in the United

States (Tatum, 1971).

The specific pathogenicity of race T of B. maydis to Tcms maize

lines raised questions about the nature of cytoplasmic male sterility

and its role in disease susceptibility. Race T produces a host-specific

toxin (Hooker et al., 1970) that inhibits root growth in seedlings

having Tcms cytoplasm but not in those with normal (N) cytoplasm.

Isolated mitochondria from Tcms maize were sensitive to T toxin.

the toxin induced swelling of mitochondria, the uncoupling of

oxidative phosphorylation, and other physiological effects on Tcms

mitochondria (Miller and Koeppe, 1971; Peterson et al., 1975).

Toxin did not have these deleterious effects on the mitochondria

from N cytoplasm maize.

Cytological studies have indicated mitochondrial involvement in

toxin sensitivity and male sterility. Ultrastructural studies by

Aldrich et al. (1977) showed that toxin effects on Tcms mitochondria

were rapid and preceded any other cellular changes. Involvement in

male sterility was indicated by mitochondrial degeneration in Tcms

anthers during microsporogenesis (Warmke and Lee, 1977).

In addition to physiological and cytological evidence for a

mitochondrial role in cytoplasmic inheritance of male sterility and

disease susceptibility, cytoplasm-specific differences have been

found in mitochondrial DNA and proteins. Levings and Pring (1976)

showed cytoplasm-specific differences in restriction endonuclease

digest patterns of mitochondrial DNA from N and Tcms maize.








Mitochondrial differences were also found in other male sterile cyto-

plasms (Pring and Levings, 1978). Chloroplast DNA from N, Tcms, and

Ccms cytoplasms were indistinguishable using these techniques (Pring

and Levings, 1978). Mitochondrial protein differences in N and Tcms

maize were found in carboxymethylated acidic, chloroform/methanol

extracts of submitochondrial particles and of the partially purified

mitochondrial ATPase complex analyzed by isoelectric focusing in

polyacrylamide gels (Barratt and Peterson, 1977). Forde et al.

(1978) found that in vitro amino acid incorporation by isolated maize

mitochondria yielded approximately twenty discrete polypeptides.

They found Tcms maize mitochondria produced one polypeptide that was

different from those of all other cytoplasms. Ccms mitochondria also

produced a polypeptide that was distinct among the mitochondrially

synthesized polypeptides.

Data on differential sensitivity of N and Tcms mitochondria to

pathotoxin from B. maydis, race T and cytoplasm-specific mitochondrial

DNA and protein differences suggested mitochondrial involvement in

cytoplasmic male sterility and in susceptibility in Tcms lines. The

objective of this work was to investigate the possibility of a dual

role of mitochondria in these phenomena by examining mitochondria

from isogenic lines of maize for cytoplasm-specific differences in

protein components. To achieve this objective mitochondrial proteins

were examined by sodium dodecyl sulfate polyacrylamide gel electro-

phoresis (SDS-PAGE) using optical staining techniques as well as

differential labeling with radioisotopes.















LITERATURE REVIEW


Cytoplasmic Male Sterility of Maize


Rhoades (1933) reported male sterility in maize supplied to him

from Peru. He concluded that the male sterility was transmitted by

cytoplasmic factors. Rhoades made no further studies and this source

was lost. In the 1940's, two more sources of male sterility were

found, one in Texas by J. S. Rogers (Rogers and Edwardson, 1952)

termed "Texas" or "T" cytoplasm and the other, called "USDA" or "S"

cytoplasm, by D. F. Jones (Jones et al., 1957).

The use of nuclear genes that restore fertility to cms maize

(Jones et al., 1957) made cytoplasmic male sterility an economically

useful trait. There have been many reported sources of male sterility

in maize (Beckett, 1971), but most of these can be classed as either

a T, S, or C-type according to patterns of fertility restoration. In

the T-type two gene loci, called Rfl and Rf2 for restoration factor,

are involved. A heterozygous condition at both loci results in complete

fertility. Therefore, it can be said that restoration is dependent

upon the genotype of the sporophyte. In S-type a third locus (Rf3)

is responsible for restoration. The heterozygous condition at this

locus (Rf3 rf3) results in semifertile flowers. Half of the pollen

carry rf3 and are sterile; half carry Rf3 and are fertile. The S-type

restoration is thus dependent upon the genotype of the gametophyte

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(Duvick, 1965). Beckett (1971) studied the restoration of thirty

maize lines with male sterile cytoplasms. On the basis of restoration

patterns Beckett was able to distinguish a third cytoplasm group, Ccms,

in addition to T and S. As with Tcms, heterozygous restoration in C

results in 100 percent fertility.

By 1970, the use of Tcms hybrids was the dominant procedure for

the production of maize seed in the United States (Tatum, 1971). The

advantages of complete fertility with heterozygous restoration and the

fact that sterile and partially fertile plants can be distinguished

by visual examination was responsible for this dependence upon one

source. This led to a situation of cytoplasmic homogeneity which

was conducive to the development of the southern corn leaf blight

epidemic of 1970.


Association of Disease Susceptibility with Texas Cytoplasmic Male
Sterility

The appearance of an extremely virulent race of B. maydis

curtailed the use of Tcms hybrids. Race T was found to produce

a host specific toxin (Hooker et al., 1970), and had increased

virulence which is seen in the ability of race T to infect most parts

of the plant. Infection by race O (old race) is restricted mainly to

the leaves. Hooker and his co-workers (1970) found that small amounts

of this pathotoxin caused severe inhibition of root growth in Tcms but

not in normal (N) maize seedlings.

The site of T toxin action in Tcms maize has been investigated

in isolated mitochondria and intact tissues. Miller and Koeppe's

work (1971) indicated mitochondrial involvement in toxin sensitivity.








They observed that the pathotoxin caused immediate and irreversible

swelling of Tcms mitochondria suspended in KC1 medium. The pathotoxin

also induced uncoupling of oxidative phosphorylation and inhibition

of malate oxidation. Mitochondria from N cytoplasm shoots were

unaffected by the pathotoxin. In addition, Peterson et al. (1975)

showed that toxin induced activation of cytochrome oxidase and

succinate-cytochrome c reductase in isolated Tcms mitochondria.

Inhibition of the electron transport chain at a step prior to the

entry of electrons from succinate dehydrogenase was also seen in sus-

ceptible mitochondria. From the inhibition of malate oxidation Flavell

(1975) concluded that a site of toxin action is within the endogenous

NADH dehydrogenase complex or the coupling of malate dehydrogenase to

the complex.

Nine inbred maize lines showed differential sensitivity to toxin

in both uncoupling of oxidative phosphorylation and inhibition of

electron transport (Barratt and Flavell, 1975). It was suggested that

interaction between Tcms mitochondria and nuclear restorer gene

products produced structural changes in the mitochondria, which were

responsible for modifications in mitochondrial response to the patho-

toxin (Barratt and Flavell, 1975; Watrud et al., 1975a). Watrud et

al. (1975b) found that removal of the outer mitochondrial membrane

while having no effect on Tcms mitochondrial response, conferred

sensitivity to normally resistant N mitochondria. They :zu;:. -.ted that

differences in outer membrane permeability to toxin affects the

accessibility of a mitochondrial site to action of the toxin.








Association of Disease Susceptibility and Male Sterility with
Mitochondria


The role of the mitochondrion as the primary site of pathotoxin

action was questioned by Arntzen et al. (1973). They reported no

significant changes in tissue respiration or phosphate uptake and

release in intact roots treated with T toxin at high concentration or

for ten minute exposures at lower concentrations. These results did

not correlate well with toxin effects on isolated mitochondria.

However, Bednarski et al. (1977) found that T toxin treated leaves

and coleoptiles from susceptible plants exhibited up to 60 percent

greater oxygen uptake than untreated controls; resistant plants showed

no effects. An explanation of these conflicting data comes from work

using 2-deoxyglucose to assay mitochondrial function in vivo. Mito-

chondrial contraction in response to addition of 2-deoxyglucose

can be viewed ultrastructurally and indicates the phosphorylative

capacity of mitochondria. Using this approach, Malone et al. (1978)

found that T toxin penetrated root and leaf tissue slowly. However,

once in the cells the toxin had rapid deleterious effect on mitochon-

drial function. In addition leaf studies showed that the toxin effect

was only detectable in cells within 3.0 mm of the fungal lesions.

They concluded that T toxin has in vivo effects similar to those

reported from in vitro experiments, and that mitochondria are a

primary site of toxin sensitivity. Cytological studies by Aldrich

et al. (1977) also indicated that mitochondria are primary sites of

toxin sensitivity. They found ultrastructural changes in root mito-

chondria of susceptible (but not resistant) maize treated in situ with

T toxin. Mitochondrial effects were rapid and preceded any other

detectable cellular changes.





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A role for a plasmalemma target site for T toxin has been

suggested by in vivo studies of Mertz and Arntzen (1977, 1978).

They found selective inhibition of ion uptake and a depolarization

of cell membrane potential in Tcms maize tissues treated with T toxin.

Effects were seen after 10 to 30 minutes exposure to toxin; at

higher toxin concentrations N maize tissues exhibited similar effects

but to a lesser degree.

An association of toxin sensitivity with Tcms mitochondria

indicates a coupling of the susceptibility and sterility phenomena.

Gengenbach and Green (1975) and Gengenbach et al. (1977) used toxin

to select resistant cultures from T callus tissue culture.

Mitochondria isolated from these cultures were unaffected by toxin.

Plants differentiated from the calli were resistant to toxin and male

fertile. Studies by Warmke and Lee (1977) also suggest the mito-

chondrion as the site of determinants conditioning male sterility

in maize. They observed mitochondrial degeneration in the tapetum

and middle layer of T anthers at the tetrad stage of microsporo-

genesis; no plastid changes were detectable until late in anther

development. No mitochondria or plastid alterations were seen in

N anthers.

The association of mitochondria with susceptibility and male

sterility led to the examination of mitochondrial proteins for cyto-

plasm-specific differences. Examination of mitochondrial membranes

using SDS-PAGE suggested quantitative differences between two low

molecular weight species in inner membrane particles and one high

molecular weight species in outer membrane fractions from N and Tcms

maize (Watrud et al., 1975b). Neither Bolen (1975) nor Barratt and








Peterson (1977) were able to detect these differences. Bolen (1975)

was unable to detect cytoplasm-specific protein differences among

five cytoplasmic versions of six inbred lines. Using carboxy-methylated

acidic, chloroform/methanol extracts of submitochondrial particles

and partially purified mitochondrial ATPase complex, Barratt and

Peterson (1977) were able to detect qualitative differences in

mitochondrial protein patterns of both extracts from N and Tcms

versions of the same line. The mitochondrial ATPase complex in yeast

is known to have mitochondrially coded subunits (Schatz and Mason,

1974). Cytoplasm-specific protein differences in translation products

from isolated mitochondria were reported by Forde et al. (1978).

They found that isolated mitochondria from Tcms and Ccms maize each

labeled one polypeptide not present in either N or Scms mitochondria.

The additional Tcms and Ccms polypeptides had apparent molecular

weights of 13,000 and 16,000 daltons respectively.

Levings and Pring (1976) isolated and characterized mito-

chondrial DNA from N and Tcms maize using restriction endonuclease

fragment analysis on agarose gels. Their study revealed cytoplasm-

specific DNA differences between N and Tcms mitochondria. Further

studies of digestion fragment patterns from a number of inbreds and

single crosses in N, Tcms, Scms, and Ccms cytoplasms revealed pattern

differences between the cytoplasms (Levings and Pring, 1978). Pring

et al. (1977) found two unique plasmid-like DNAs in addition to

the high molecular weight mitochondrial DNA from Scms maize lines.

These unique DNAs have molecular weights of 3.45 and 4.1 x 106 daltons,

are not found in other cytoplasms and have not been isolated from

Scms nuclear or chloroplast fractions.





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Scms cytoplasm maize in some lines exhibits a lack of stability

in its male sterility (Singh and Laughnan, 1972). This instability

is seen as reversion to the male fertile condition. The existence of

such cytoplasmic entities as the plasmid-like DNAs and a possible

role for them in the instability of male sterility in Scms were

predicted by Laughnan and Gabay (1975). Conversely, Duvick (1965)

has noted that Tcms cytoplasm is extremely stable. Laughnan and his

co-workers (Singh and Laughnan, 1972; Laughnan and Gabay, 1973) have

found several hundred mutatuons where Scms male steriles have reverted

to fertility. Two kinds of changes were observed; frequent cytoplasmic

mutations from male sterility to male fertility and rare nuclear

mutations giving rise to new fertility restorer genes (Laughnan and

Gabay, 1975). From their studies of the genetics of newly arisen

restorer genes, they proposed the existence of a male-fertility element

which has the characteristics of an episome. They suggest that if

the element is fixed in the cytoplasm the result is a cytoplasmic

change from male sterility to male fertility; conversely, if it is

fixed in the nucleus, it behaves as a new restorer gene. To date

unequivocal evidence to identify the plasmid-like Scms DNAs as

Laughnan's fertility element is not available.

The correlation between mitochondrial sensitivity to T toxin and

susceptibility of Tcms maize lines to race T suggest a mitochondrial

role in susceptibility of Tcms maize to race T, B. maydis. The

interaction between the nuclear and cytoplasmic genomes in restoration

(Duvick, 1965), modification of mitochondrial toxin response (Barratt

and Flavell, 1975; Watrud et al., 1975b), and cytoplasm-specific

differences in mitochondrial DNAs (Levings and Pring, 1976) would




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justify study of mitochondrial gene products in the investigation of

the mechanisms of cytoplasmic male sterility ahd the susceptibility

of Tcms maize to race T of B. maydis.


Origin of Mitochondrial Constituents

Mitochondria are the product of two distinct genetic systems

working cooperatively through separate protein synthesis systems.

From the work on the biosynthesis of mitochondrial proteins in

Ascomycete and animal mitochondria, it has been established that the

majority of mitochondrial proteins are coded for by nuclear genes,

translated on cytoplasmic ribosomes, and then imported into the

mitochondria (Schatz and Mason, 1974). Subunits of F ATPase, cytochrome

b, and coenzyme QH2-cytochrome c reductase are products of mito-

chondrial protein synthesis in fungal and animal systems (Borst, 1977;

Obbink et al., 1976). Evidence of mitochondrial origin for these

proteins comes from studies using differential labeling in the

presence of antibiotics (Schatz and Mason, 1974), cell-free synthesis

of cytochrome-oxidase subunits from mitochondrial RNA (Moorman et al.,

1978) and mapping of genes for mitochondrial proteins (Obbink et al.,

1976). The mitochondrial subunits of cytochrome oxidase have been

shown to be hydrophobic and are postulated to be responsible for the

integration of the cytoplasmic subunits into the inner mitochondrial

membrane.

Studies of planL mitochondrial protein synthesis have been hindered

by technical difficulties in the preparation of coupled, uncontaminated

plant mitochondria. Nevertheless, differences between plant and

other eucaryotic mitochondria have been found, and they cast doubt

on the direct applicability of conclusions drawn from other systems





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to plant mitochondria. Animal mitochondrial DNA has a molecular

weight of 9-12 x 106 daltons (Borst, 1977). Work with Xenopus

(Dawid, 1970) and HeLa cells (Aloni and Attardi, 1971) suggest that

at least 25-30 percent of the mitochondrial genome is involved in

coding for mitochondrial rRNAs and tRNAs. This leaves enough genetic

capacity to code for only about twenty small proteins. The molecular

weight of higher plant mitochondrial DNA, although not unequivocally

established, may be up to 165 x 106 daltons (Levings and Pring, 1978)

Plant mitochondrial DNA could have the capacity to code for as many

as 200 proteins assuming that the "additional" sequences are unique.

Thus the possibility of greater autonomy exists for plant mito-

chondria as compared with that of other eukaryotes.













MATERIALS AND METHODS


Maize Genetic Stocks

Maize stocks used in this study are identified as normal (N)

or male sterile cytoplasm (Tcms, Ccms, Scms, EKcms, Vgcms, CAcms).

The Scms, EKcms, Vgcms, and CAcms cytoplasms are all members of the

S group. Lines were furnished by J. R. Edwardson, University of

Florida; J. R. Laughnan, University of Illinois; and Clyde Black and

Sons, Ames, Iowa. Maize stocks not carrying dominant nuclear restorer

alleles used were: W64A, B37 X NC236, WF9, FR37, Mol7, and N6 X W64A.

Two maize stocks carrying the dominant restorer for Scms used were:

Tr and C103. The line M825L/Oh07 in Vgcms cytoplasm frequently

mutates to male fertility (Laughnan and Gabay, 1975) due to cytoplasmic

mutation, indicated in this study by Vgmf, or a nuclear mutation

to new restorer genes, indicated here as the h, i, and m stocks of

Laughnan (J. R. Laughnan and S. J. Gabay-Laughnan, personal

communication).

Isolation and Purification of Mitochondria

Mitochondria were isolated from 6-day old Zea mays L. seedlings,

germinated in vermiculite at 300 in the dark following the method

of Pring (1974; Pring and Levings, 1978). Mesocotyl and coleoptile

tissues (10-500 g) were harvested and homogenized in medium A, 0.5 M

sucrose, 0.05 M Tris-HCl (pH 7.5), 5 mM Na2EDTA, 0.1 % bovine serum

albumin (Fraction V), and 5 mM 2-mercaptoethanol, in a chilled

Waring blendor for 5 sec at low speed. Batches of less than 50 g

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-14-

were homogenized in a mortar and pestle or a Sorvall Omnimixer for

15 sec at a setting of 5. The homogenate was filtered sucessively

through two layers of cheesecloth and two layers of Miracloth

(Chicopee Mills, Inc., New York). All procedures were done at ice

temperatures, unless otherwise noted. The homogenate was centrifuged

for 15 min at 8,700 g(max) to pellet mitochondria. mitochondria were

resuspended in medium B, 0.3 M sucrose, 0.05 M Tris-HCl (pH 7.5),

5 mM Na2EDTA, and 0.1 % bovine serum albumin, and purified on a

discontinuous sodium diatrizoate gradient (Winthrop Labs) in

medium B with a density of 1.19 g/cc overlayed with an equal volume

of a 1.14 g/cc sodium diatrizoate solution. After centrifugation

for 60 min at 72,000 g(avg), the mitochondria were collected from the

1.14-1.19 g/cc interface and diluted with three volumes of medium C,

0.3 M sucrose, 0.05M Tris-HCL (pHl 7.5). Mitochondria were then

pelleted for 10 min at 17,300 g(max), resuspended in medium C and

repelleted.

Fractionation of Mitochondria

Mitochondrial membrane fractions were prepared by two methods.

The first was a modification by Watrud et al. (1975b) of the Morreau

and Lance (1972) procedure, and uses osmotic shock to rupture the outer

membrane and ATP to contract the inner membrane cores to facilitate

separation on sucrose gradients. Pruified mitochondria were resuspended

to 5 mg/ml mitochondrial protein, estimated by the method of Lowry

et al. (1951), in 1 mM potassium phosphate, pH 7.2, and incubated

for 15 min on ice. The suspension was then made 0.3 M in sucrose,

0.3 mM in MgSO4 and 0.3 mM in ATP and allowed to stand 15 min on ice

with occasional agitation. Treated mitochondria were then layered onto

a discontinuous sucrose gradient of 4 ml 1.18 M and 4 ml 2M sucrose





-15-


in 1 mM KH2PO (pH 7.2) and centrifuged for 90 min at 90,000 _g(avg).

Outer membranes were collected from the 1.18 M sucrose step and inner

membrane cores were caught on the 2 M shelf.

A second method used osmotic shock to rupture the outer membrane

followed by sonication to separate outer and inner membranes.

Membrane separation was assessed by observation of preparations

negatively stained with 1 % potassium phosphotungstate in the electron

microscope. Purified mitochondria were resuspended to 5 mg/ml protein

in 1 mM potassium phosphate at pH 7.2, and incubated on ice for 30 min

to allow osmotic swelling to rupture the outer membranes. The suspension

was then sonicated for 30 sec at 20 % line voltage, using a Biosonic II

sonicator with a microprobe. After sonication the inner membrane

particles were pelleted at 17,300 g(max) for 10 min. The supernatant

and resuspended pellet were then layered on separate 0.7 M, 1.18 M,

2 M sucrose step gradients and centrifuged for 90 min at 90,000 g(avg).

Inner membranes were collected from the 1.18-2 M sucrose interface

and outer membranes at the 0.7-1.18 M sucrose interface.

Membrane fractions from both methods were then diluted three-fold

with 1 mM potassium phosphate (pH 7.2) and pelleted at 250,700 g(max)for

30 min. Pellets were resuspended in appropriate buffers for

electrophoresis and stored at -20.

Electrophoresis of Mitochondrial Proteins

Several electrophoresis systems were used in this study to examine

mitochondrial proteins. Polyacrylamide gels using the Weber and Osborn

(1969) buffer system, 0.1 M sodium phosphate (pH 7.0), were cast in 6 mm

internal diameter X 125 mm long glass tubes coated with 0.5 % Kodak

photoflo-200. A 7 cm, 10 % acrylamide, 0.27 % bisacrylamide,





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0.1 % SDS, separating gel was polymerized with 1.5 ml of fresh

1.5 % ammonium persulfate and 45 pl of TEMED per 30 ml. After

polymerization the water overlay was removed and a 1.5 cm 5 %

acrylamide, 0.135 % bisacrylamide, 0.1 % SDS stacking gel was cast

using 0.05 M sodium phosphate (pH 7.0). The stacking gel was

polymerized with 0.5 ml of fresh 1.5 % ammonium persulfate and 15 pl

of TEMED per 10 ml. Proteins were denatured by heating at 1000 for

5 min in 0.05 M sodium phosphate (pH 7.0), 3 % SDS, 3 % 2-mercapto-

ethanol, and 10 % glycerol. Electrophoresis was conducted at constant

current (8 ma/gel) until the bromphenol blue tracking dye reached the

bottom of the tubes (anode). After electrophoresis, gels were fixed

and stained in 0.1 % Coomassie brilliant blue R250, 50 % methanol,

10 % acetic acid for 2 hr. Gels were destined in 10 % methanol, 7.5 %

acetic acid with frequent changes of solution.

A Tris-sulfate, discontinuous buffer system was used with an

Ortec model 4200 slab gel electrophoresis tank and an Ortec model

4100 pulse power constant power supply. The molarity of this buffer

refers to the molarity of the Tris with sufficient H2SO4 added to

give a pH of 9.0 at 25 A two-step acrylamide gradient gel was cast

using 0.22 M Tris-sulfate, pH 9.0, 0.1 % SDS. The first layer, 3.5 cm

consisting of 12 % acrylamide, and 0.33 % bisacrylamide was overlayed

with a 3.5 cm layer of 10 % acrylamide, 0.27 % bisacrylamide.

Polymerization of each layer was initiated by the addition of 9.6 ml

of 0.105 % ammonium persulfate and 12 Pi TEMED per 20 ml of solution.

The separation gel was overlayed with H20 and allowed to polymerize

for 30 min. After the overlay was removed the unpolymerized

acrylamide was washed off and the surface of the gel blotted with




-17-


filter paper. A 1 cm stacking gel, 5 % acrylamide, 0.135 % bisacryla-

mide, 0.1 % SDS, 0.0375 M Tris-sulfate, pH 9.0, was cast and polymer-

ized with the addition of 4 ml of 0.21 % ammonium persulfate and

0.005 ml of TEMED per 8 ml. After 20 min polymerization time, the

H20 overlay was removed and an 8 % acrylamide, 0.22 % bisacrylamide

well-forming gel was layered and a thirteen-tooth teflon comb inserted

until it made contact with the stacking gel. Polymerization was ini-

tiated with 3.7 ml of 0.21 % ammonium persulfate and 0.005 ml of TEMED

per 8 ml of solution. Buffer concentration was as in the stacking gel.

Proteins were prepared for electrophoresis by heating for 5 min at

1000 in 0.0375 M Tris-sulfate, pH 9.0, 3 % SDS, 3 % 2-mercaptoethanol,

10 % glycerol. Tris-borate electrophoresis buffer (0.065 M Tris,

0.1 % SDS) was prepared by adding 7.86 g Trizma base (Sigma), 0.09 g

H3B03, and 1 g SDS per liter of H20. Electrophoresis was carried out

using pulsed power at a discharge capacitance of 1 pF, at 280 V.

Pulses per second (pps) were increased in 75 pps increments every 5

min until a maximum of 300 pps was reached. Proteins were electro-

phoresed for 3 hr or until the bromphenol blue tracking dye reached

the bottom of the gel.

The Tris-sulfate buffer system was also used to examine proteins

on a 12-16.5 % continuous gradient acrylamide gel in a Studier (1973)

type apparatus. Gel solutions were prepared as described. The 12 %

acrylamide and 16.5 % acrylamide, 0.45 % bisacrylamide solutions were

placed in a gradient maker and poured by gravity feed into a 0.075

x 14.5 x 16.3 cm cell. The volume of the gel was 28 ml, and stir

bars were used in both chambers of the gradient maker to inhibit

polymerization and to mix the solutions. After layering, the gel was





-18-


overlayed with H20, allowed to polymerize for 30 min, the overlay

was poured off, the gel surface rinsed with H20, and then blotted with

filter paper. A 4.5 ml, 5 % stacking gel and an 8 % well-forming

gel were cast as described. Since solubilization of membrane proteins

is incomplete at protein concentrations greater than 10 mg/ml, proteins

were resuspended to less than 10 mg/ml, and electrophoresed for 18-19

hr at 80 V constant voltage with Tris-borate buffers. Gels were

stained with Coomassie brilliant blue R250 and photographed with

Polaroid type 55 P/N film using a Wratten 9 filter.



Labeling and Fluorography of Mitochondrial Proteins


Four-day old etiolated maize seedlings were cut approximately 1

cm above the scutellar node and placed in individual wells of disposa-

ble microtiter plates (Cooke Laboratory Products, Alexandria, VA).

Each well contained 150 pl of 0.01 M sodium phosphate, pH 7.0, with

10 C 35S as carrier free H2SO4 in H20 (New England Nuclear, Boston,

MA). After incubating at 300 for 12 hr, mitochondria were isolated

as described except that samples weighing less than 10 g were homo-

genized in a mortar and pestle. Labeled protein was assayed using

the Mans and Novelli (1961) filter disc procedure and counted with a

Beckman LS-150 liquid scintillation counter, in 6 g/l 2,5-diphenyloxa-

zole (PPO) in Beckman Redisolve I.

Mitochondriial protLeins were electroplorescd in the 'r ri -sul fatc

continuous gradient gel system and the gels prepared for fluorography

according to the method of Bonner and Lasky (1974). Gels were soaked

twice in twenty volumes of dimethyl sulfoxide (DMSO) for 20 min. Gels





-19-


were then impregnated with PPO by soaking for 3 hr in four volumes of

22 % w/v PPO in DMSO. The DMSO was removed by soaking the gel in

twenty volumes of deionized H20 for 1 hr with one change. Gels

were dried onto chromatography paper in a Bio-Rad slab dryer for 1.5 hr

under a vacuum of 68.6 cm of mercury. The dried gels were then

overlaid with Kodak X-Omat RP X-ray film at -700 for varying exposure

times.


Isolation of Mitochondrial DNA

Mitochondrial DNA was isolated following the procedures of Pring

and Levings (1978) Mitochondria from the second cycle of differential

centrifugation were resuspended in medium C and incubated in the

presence of 0.05 g/ml deoxyribonuclease I (Worthington DPFF), 10 mM

MgCl2 for 60 min at room temperature. After incubation the mito-

chondria were diluted with seven volumes of medium B and pelleted for

10 min at 17,300 g(max). Pellets were resuspended in medium C and

pelleted again at 17,300 g(max) for 10 min. Pellets were resuspended

in 0.05 M Tris-HCl, pH 8.0, 0.02 M Na2EDTA, lysed by the addition of

0.5 % SDS, and incubated for 60 min at room temperature in the presence

of 0.2 g/ml Proteinase K (EM Laboratories, Inc., Elmsford, NY).

The mitochondrial DNAs were purified by CsCl/ethidium bromide

preparative ultracentrifugation, as described by Pring and Levings

(1978). Solid CsC1 was added to the mitochondrial preparations to a

density of 1.611 g/cc in the presence of 0.2 g/ml ethidium bromide.

Gradients (10 ml) with the mitochondrial preparation from a maximum

of 300 g of shoots were overlayed with mineral oil and centrifuged at




-20-


200 for 40 hr at 44,000 rpm in a Beckman Ti 75 rotor. Mitochondrial

DNA was visualized using long wave UV-light and the DNA was collected

by piercing the tubes with an 18 ga needle. Ethidium bromide was

removed by three extractions with isopropanol and CsCI removed by

dialysis against 36 mM Tris-HCl, 30 mM NaH2P04, 2.5 mM Na2EDTA (TPE)

at pH 7.8. The DNA preparation was then made 1 M in NaCIO4 and then

deproteinized by three extractions with chloroform/isoamyl alcohol

(95:5, vol/vol). The aqueous layer was then dialysed against the

appropriate buffer for agarose electrophoresis.



Agarose Electrophoresis of Mitochondrial DNA


DNA was electrophoresed at room temperature in 1 % agarose (Sea

Kem, Bausch and Lomb) gels prepared according to the method of

Pring and Levings (1978). Slab gels (0.38 x 14.5 x 14.3 cm) were run

in a Studier type electrophoresis apparatus at 1.9 V/cm for 16 hr with

the TPE buffer system. After electrophoresis gels were soaked in

buffer containing 0.4 jg/ml ethidium bromide for 60 to 90 min. Gels

were photographed over UV light with Polaroid type 55 P/N film using

a Wratten 9 filter.



Restriction Endonuclease Fragment Analysis


Mitochondrial DNA was analysed using HindIII restriction endonu-

clease (Miles Laboratories, Inc.) following the method of Pring and

Levings (1978). Approximately 0.001 g of DNA was digested in sterile

plastic tubes containing enzyme in a 0.05 ml volume of 50 mM NaC1,

6 mM MgC12, 6 mM Tris-HCl, pH 7.5, and 0.005 g bovine serum albumin.





-21-



Reactions were terminated by heating at 600 for 10 min in the

presence of 1 % SDS. Bromphenol blue and glycerol were added to

the digests and the samples electrophoresed on agarose as described

above.
















RESULTS


Purity of Mitochondrial Membrane Fractions


The relative purity of membrane fractions was assayed by the

differential inhibition of NADH-cytochrome C reductase by 0.3 pM

antimycin A (Watrud et al., 1975b). The enzyme activity in the outer

membrane is antimycin A insensitive, while the inner membrane activity

is inhibited by antimycin A. Therefore, the percent inhibition of

enzyme activity in treated membrane fractions over controls can be

used to assess relative purity of the partially resolved fractions.

A comparison of membrane fractions prepared by the osmotic shock

method of Watrud et al. (1975b) and the sonication procedure is shown

in Table 1. These data suggest that the sonication procedure gave

better separation of both membrane fractions. However, neither

procedure completely resolved the two membrane components nor gave

highly reproducible fractions (note wide range among replications).



Electrophoresis of Mitochondrial Membrane Fractions


Mitochondrial membrane fractions from W64A in N, Tcms, and CAcms

cytoplasms were examined for cytoplasm-specific protein differences

on 10 % polyacrylamide cylindrical gels using the Weber and Osborn

(1969) buffer system. Although protein pattern differences were seen


-22-





-23-


Table I. Inhibition of cytochrome c reductase by antimycin A in
outer and inner maize mitochondrial membrane fractions.




Method of Preparation


Osmotic


Sonic


Membrane
Fraction


Outer 17.6 (3.4-30)- 11.3 (0.04-27)

Inner 60.3 (37-79) 65.2 (47-81)



1/
- Numbers in parenthesis indicate the range from nine
replications.




-24-


in individual preparations, there was a lack of reproducibility

among experiments. Therefore, no consistent cytoplasm-specific

protein differences were detected in inner or outer membrane fractions

prepared by the sonication procedure (Figure 1). This conclusion

was based on a minimum of five experiments. However, figures are

only representative of these experiments, and include pattern

variation unique to the individual experiment.

Greater resolution of proteins from these fractions was obtained

using two-step acrylamide slab gels with a Tris-sulfate discontinuous

buffer system. Membrane fractions from W64A in N, Tcms, and CAcms

cytoplasms were examined in this system. No reproducible cytoplasm-

specific protein differences were detected in outer membrane (Figure 2)

or inner membrane (Figure 3) fractions by Coomassie brilliant blue

staining.



Electrophoresis of Total Mitochondrial Protein

Sodium diatrizoate gradient-purified mitochondria were soluablized

as described and 150-200 pg protein samples were electrophoresed on

12 to 16.5 % continuous polyacrylamide gradient gels with the Tris-

sulfate discontinuous buffer system.

The resolution attained with this system resulted in the detec-

tion of line specific protein differences as shown in figure 4. This

figure shows electrophoresis of total mitochondrial proteins from

four lines in Scms group cytoplasms. The sample of Mol7 exhibits one

band at 53 mm not present in lines Wf9, FR37, and N6 X W64A; and is

missing one band at 43 mm which is present in the latter lines.

































SDS-polyacrylamide gel electrophoresis of Inner (A) and
Outer (B) mitochondrial membrane fractions from W64A in
a) Normal, b) Tcms, and c) CAcms cytoplasms run in 10 %
gels with 0.1 M sodium phosphate, pH 7.0, 0.1 % sodium
dodecyl sulfate. Samples were denatured by heating at
1000 for 5 min in 0.05 M NaH2PO4, pH 7.0, 3 % SDS, 3 %
2-mercaptoethanol.


Figure 1.







a b c


I1


-26-


a
UI


b c


A
































SDS-polyacrylamide gel electrophoresis of maize outer
mitochondrial membrane proteins run in 10 % 12 % discon-
tinuous acrylamide gradient gels with 0.22 M Tris-sulfate,
pH 9.0, 0.1 % SDS. Fractions were prepared by the osmotic
(A) and sonication (B) procedures from W64A in a) normal,
b) Tcms, and c) CAcms cytoplasms. Samples were dissociated
by heating at 1000 for 5 min in 0.11 M Tris-sulfate, pH 9.0,
3 % SDS, 3 % 2-mercaptoethanol.


Figure 2.





-28-


a b c a b c




aw-





np
..




a. B"






A p
































SDS-polyacrylamide gel electrophoresis of maize inner
mitochondrial membrane proteins run in 10 % 12 % discon-
tinuous acrylamide gradient gels with 0.22 M Tris-sulfate,
pH 9.0, 0.1 % SDS. Fractions were prepared by the osmotic
(A) and sonication (B) procedures from W64A in a) normal,
b) Tcms, and c) CAcms cytoplasms. Samples were dissociated
by heating at 1000 for 5 min in 0.11 M Tris-sulfate, pH 9.0,
3 % SDS, 3 % 2-mercaptoethanol.


Figure 3.




-30-


a b c
&-me a-;4


a b
$Now II


I VP


I .A
V: .... .......r


9<1
1 L


A

































Figure 4. SDS-polyacrylamide gel electrophoresis of total maize
mitochondrial protein from lines a) WF9, b) Mol7,
c) FR37, and d) N6 X W64A in Scms cytoplasms. Samples
were dissociated by heating at 1000 for 5 min in 0.11 M
Tris-sulfate, pH 9.0, 3 % SDS, 3 % 2-mercaptoethanol and
electrophoresed in 12 to 16.5 % continuous acrylamide
gradient gels with 0.22 M Tris-sulfate, pH 9.0, 0.1 %
SDS.





-32-


abc d




wIl~7-m~


r

.-
L.



h3



W
C~ j


r
o





-33-


A comparison of maize mitochondrial proteins from four cytoplasmic

sources is shown in Figure 5A. No consistent cytoplasm-specific

differences were detected in the mitochondrial proteins from N, Tcms,

or Ccms cytoplasms. The Scms cytoplasm mitochondria contained a

high molecular weight protein band at 12 mm which is distinct from

the other cytoplasms tested. This protein, designated S protein, is

present in all Scms cytoplasm maize lines tested. Figure 5B shows

the presence of this protein in four maize lines and two versions of

the Scms cytoplasm. The molecular weight (mw) of this protein was

estimated by comparing the distance migrated of the S protein with

the distance migrated by standards of known molecular weights on a

5 % acrylamide, 0.136 % bisacrylamide, Tris-sulfate slab gel. Myosin

(mw 200,000 daltons), beta galactosidase (mw 116,394 daltons, Fowler

and Zabin, 1978) and phosphorylase b (mw 94,000 daltons) were used as

molecular weight standards. Linear regression analysis of the rela-

tionship of the log of molecular weight to the distance migrated of

the standards indicated a mean molecular weight of 133,890 635

daltons for the S protein (Table II).

Mitochondria were labeled with 3S in detached seedlings and the

proteins electrophoresed on gradient gels. Proteins were stained

with Coomassie brilliant blue and labeled proteins were visualized

using fluorography. Proteins present in concentrations too low to be

seen using staining can be detected using fluorography; however,

only those proteins synthesized during the labeling period will be

seen. Figure 6A shows a comparison from the same set of stained and

fluorographic protein patterns from N, Tcms and Scms maize mito-

chondria. Although the stained and flourographic patterns were































SDS-polyacrylamide gel electrophoresis of total maize
mitochondrial protein dissociated by heating at 1000 for
5 min in 0.11 M Tris-sulfate, pH 9.0, 3 % SDS, 3 %
2-mercaptoethanol and electrophoresed in 12 to 16.5 %
continuous acrylamide gradient gels with 0.22 M Tris-
sulfate, pH 9.0, 0.1 % SDS.


A. Patterns from a) normal, b) Tcms, c) Ccms, and d) Scms
mitochondria.

B. Patterns from Scms maize in a) WF9, b) Mol7 (EKcms),
c) FR37, and d) N6 X W64A lines.


Figure 5.




-35-


abcd


I --
a











I.


ab















A


abc d

-up"


I -







CI=



t.


z:


.AM l.III .' '


.0 -


r




-36-


Determination of the S protein molecular weight by SDS-
PAGE in 5 % acrylamide with 0.22 M Tris-sulfate, pH 9.0,
0.1 % SDS. Proteins were dissociated by heating at 1000
for 5 min in 0.11 M Tris-sulfate pH 9.0, 3 % SDS, 3 %
2-mercaptoethanol.


Experiment 1 2 3 4 5 6


Distance migrated (cm)

Myosin
(200,000 MW)

Beta Galactosidase
(116,394 MW)

Phosphorylase b
(94,000 MW)


S protein


5.23


9.52


5.1


5.28 5.2


9.35 9.5


11.2 11.4 11.38 11.5 11.6 11.5


8.3


8.5


8.35


Linear regression analysis


Slope


Correlation
coefficient


-0.53 -0.53 -0.53


-0.999 /-0.999


-0.998


-0.53 -0.53 -0.53


-0.999 -0.999 -0.999


133,890

635


All correlations were significant at the 5 % level.


Table II.


Molecular weight of S protein

Mean molecular weight (daltons)

Standard deviation of the mean





-37-


different, no consistent cytoplasm-specific protein differences were

detected among labeled proteins. The S protein seen on stained gels

was not detected under these labeling conditions. Therefore, Scms

mitochondria were labeled using a 0-time labeling procedure. Scms

35
maize seeds were imbibed in the presence of 35S, and after four-day

continuous labeling mitochondria were isolated as before and the

protein patterns examined using Coomassie staining and fluorography

(Figure 6B). Although not readily apparent in this photograph, a

labeled band was detected in the same position (13 mm) as the stained

S protein.

An attempt was made to differentially inhibit cytoplasmic protein

synthesis by enhancing the labeling of mitochondrial gene products.

Emetine, an inhibitor of cytoplasmic protein synthesis (Peska, 1971),

at 300 ig/ml (saturating level) inhibited the incorporation of 35S

into maize mitochondrial protein 85 % (Figure 7). A comparison of

maize mitochondrial proteins labeled with and without emetine is shown

in Figure 8. Consistent cytoplasm-specific protein differences were

not detected under these conditions of incomplete inhibition. Figure

8 shows an example of the inconsistent results obtained using emetine.

All samples were labeled simultaneously; however, in sample b the

antibiotic appears to have stimulated rather than inhibited incor-

poration. This effect was not limited to any one cytoplasm and

was not seen consistently in all experiments.



Scms Cytoplasm Instability and the S Protein


The effects of nuclear restoration and of nuclear and cytoplasmic


























Figure 6. SDS-polyacrylamide gradient gel electrophoresis of total
maize mitochondrial protein.

A. Comparison of the stained and fluorographic patterns of
proteins from maize mitochondria labeled in detached
shoots with 35S. a) Normal cytoplasm mitochondrial
proteins stained with Coomassie brilliant blue, b) flouro-
graph of the same gel; c) stained Tcms mitochondrial
proteins, d) fluorograph of the same gel; e) stained Scms
mitochondrial proteins, f) fluorograph of the same gel.

B. Comparison of the stained and fluorographic patterns of
proteins from Scms mitochondrial proteins labeled by
imbibition of maize seed in the presence of 35S (0-time
labeling), a) Proteins stained with Coomassie brilliant
blue, b) fluorograph of the same gel. S protein is
indicated by arrow.





-39-


a bc de f ab












A B
____ -.














A- B





























35
Figure 7. Inhibition by emetine of incorporation of 3S into mito-
chondrial protein. Four day-old etiolated seedlings were
cut approximately 1 cm above the scutellar node, pretreated
in the presence of emetine for 30 min, and placed in
individual wells of disposable microtiter plates and
allowed to take up 150 ol of 0.01 M sodium phosphate,
pH 7.0 with 10 iC35S as carrier-free H2SO4 with
various concentrations of emetine. After incubation at
300 for 12 hr mitochondria were isolated and incorpora-
tion assayed using the Mans and Novelli (1961) filter
disc procedure. Inhibition was calculated by comparing
incorporation by emetine treated seedlings with that of
untreated controls. The average specific activity of
controls was 2000 cpm/pg mitochondrial protein.





-41-


Concentration of Emetine (ug/ml)






























35
Figure 8. Fluorograph of 3S labeled maize mitochondrial proteins
electrophoresed on SDS-polyacrylamide gradient gels.
Mitochondrial proteins were labeled in detached four day-
old etiolated shoots with 10 PC/shoot 35S as carrier-
free H2SO4. Fluorograph was prepared by exposing Kodak
X-Omat RP X-ray for 24 hrs at -700 to dried gels impreg-
nated with PPO. Normal (a), Tcms (c), and Scms (e)
proteins labeled without antibiotic treatment. Normal
(b), Tcms (d), and Scms (f) labeled in the presence of
emetine.




-43-


a b c de f


4




IJ


1 *





-44-


mutation to male fertility on the presence of the S protein in Vgcms

cytoplasm maize were examined by gradient gel electrophoresis.

Lines Tr and C103 carry the dominant nuclear restorer genes

(Rf3 Rf3) for Scms cytoplasm. A comparison of mitochondrial

proteins from these lines in N, Scms, and Vgcms cytoplasms showed that

nuclear restoration had no detectable effect on the S protein (Figure

9A). This figure is overdeveloped to emphasize the S protein.

Line M825L/Oh07 does not carry the nuclear gene for fertility

restoration. Laughnan and his co-workers have identified several

nuclear mutations to male fertility in the Vgcms version of this line.

These mutations have been characterized as the formation of new

restorer loci. Three of these were examined for the presence of the

S protein. As with lines Tr and C103 the new restorer genes h, i,

and m had no effect on the presence of the S protein (Figure 9B).

Although not readily apparent in Figure 9B, slot a, M825L/Oh07 with

the new restorer h did yield the S protein.

Cytoplasmic mutation to male fertility in the Vgcms version of

line M825L/Oh07 was also examined for the presence of the S protein.

Figure 9B (Slots d, e, f) shows a comparison of this line in N and

Vgcms cytoplasms and in a mutant cytoplasm Vgmf. No S protein was

detectable in the cytoplasmic mutant.



Restriction Endonuclease Analysis of Mitochondrial DNA


The identity of Vgcms cytoplasm as a member of the Scms group

was substantiated by an examination of the mitochondrial DNA from

the h, i, and m versions of M825L/OhO7 in Vgcms. Two criteria were































Figure 9.


SDS-PAGE of total mitochondrial protein solubilized by
heating at 1000 for 5 minutes in the presence of 3 %
SDS, 3 % 2-mercaptoethanol. Photograph is overdeveloped
to emphasize the S protein.


A. Mitochondrial proteins from restorer lines Tr in
a) normal, and b) Scms cytoplasms and C103 in c) normal,
b) Scms, and Vgcms cytoplasms.

B. Proteins from M825L/Oh07 with new restorer genes in
Vg cytoplasm a) h, b) i, c) m, and in d) normal,
e) Vgcms, and f) Vgmf cytoplasmic versions.





-46-


a b c d e a b c d e f







1 4
















B





-47-


used: the presence of the two unique DNAs described by Pring et al.

(1977) and a comparison of restriction endonuclease fragment patterns

of mitochondrial li'!A with that of a known Scms cytoplasm. Figure 10

is a comparison of mitochondrial DNA and HindIII restriction endo-

nuclease digests from W64A in CAcms cytoplasm and h, i, and m in

Vgcms cytoplasm. All four mitochondrial iJA? contain the two plasmid-

like DNAs characteristic of the Scms cytoplasm. DNA digest patterns

from h and i are identical to that of W64A. The digest of mito-

chondrial DNA from m was incomplete which resulted in a smeared

pattern. However, the similarities in this pattern and the patterns

from h and i in addition to the presence of the two plasmid-like

DNAs, provide sufficient data to verify Vgcms as having Scms type

DNAs.

































Figure 10. Agarose electrophoresis of undigested (b, d, f, h) and
HindIII-digested (a, c, e, d) mitochondrial DNAs from
W64A CAcms (a, b) and from M825L/Oh07 m (c, d),
M825L/Oh07 i (e, f), and M825L/Oh07 h (g, h) lines in
Vgcms.




-49-


k r I


f 9 h


4I














DISCUSSION


The objective of this study was to search for aberrant mito-

chondrial gene products from male sterile cytoplasms of maize.

Information on cytoplasm-specific toxin effects on Tcms mitochondria,

and specific mitochondrial DNA differences among the cytoplasms of

maize indicated that such a search may be useful in the elucidation

of the role of nuclear-mitochondrial interaction in Tcms maize suscep-

tibility and in cytoplasmic male sterility.

Three types of protein differences could be expected: line

specific differences, cytoplasm-specific differences associated with

disease susceptibility, and cytoplasm-specific differences associated

with cytoplasmic male sterility. Line specific differences due to

line variation in nuclear genes coding for mitochondrial proteins may

be seen as protein differences from individual lines in the same cyto-

plasm. Susceptibility associated differences may be seen as variation

in protein patterns which distinguish susceptible Tcms mitochondria

from resistant N, Scms, and Ccms mitochondria. Differences associated

with cytoplasmic male sterility may be represented by patterns which

differentiate the male sterile cytoplasms from the normal fertile

cytoplasms. Protein differences which could be associated with line

variation and cytoplasmic male sterility were detected in Scms maize.

However, no mitochondrial protein differences were detected which

could be correlated with disease susceptibility in Tcms maize.

-50-





-51-


No mitochondrial protein difference between N, Tcms, or Scms

maize were detected in mitochondrial fractions prepared using either

the method of Watrud et al. (1975b) or the sonication procedure. The

Watrud procedure was followed exactly; however, mitochondria used in

this study were purified on gradients and have been shown to be free

of microsomal contamination when assayed for the presence of cyto-

plasmic ribosomal RNA (rRNA) (Pring,1974). Membrane fractions

prepared by the sonication procedure exhibited less cross-contamina-

tion, using the cytochrome c reductase assay, than those prepared

by the procedure of Watrud et al. (1975b). Protein differences in

mitochondrial fractions reported by Watrud et al. (1975b) have not

been verified by this work or that of Barratt and Peterson (1977),

who looked at submitochondrial particles prepared by sonication. In

all three cases, mitochondrial fractions were examined on 10 % poly-

acrylamide gels using the Weber and Osborn (1969) SDS system. In

addition, cytoplasm-specific protein differences were not apparent

in Tris-sulfate step gradient gels which gave improved resolution of

protein species.

Mitochondrial protein differences were resolved with continuous

polyacrylamide-gradient gels. Line specific differences were detected

in four nuclear lines in Scms cytoplasm. Bolen (1975), using poly-

acrylamide-gradient gels and isoelectric focusing, also found line

specific protein differences; however, his results are not directly

comparable to those in this study due to differences in the methods

used. These line specific proteins probably represent variants of

nuclear-coded mitochondrial proteins. Contrary to Bolen's work, one




-52-


cytoplasm-specific mitochondrial protein was detected in mitochondria

from Scms maize. This high molecular weight protein, 133,900 daltons,

was present in all Scms lines tested with the exception of the line

with a cytoplasmic mutation to fertility. Absence of detectable

amounts of this protein in the cytoplasmic mutant and other maize

cytoplasms suggests that it is associated with male sterility in Scms

maize. Failure of Bolen to detect this protein is probably due to

differences in mitochondrial preparation or sample solubilization

procedures. The protein was not detected when sample concentrations

exceeded 10 mg/ml mitochondrial protein, estimated by the method of

Lowry et al. (1951). Protein aggregation at higher sample concen-

trations with failure of the aggregated protein to enter the gel may

result in failure to detect the S protein.

In vivo labeling with radioisotopes has been used in animal and

fungal systems to identify mitochondrial gene products. The incon-

sistency and failure of labeling in detached shoots to reveal cyto-

plasm-specific proteins is probably indicative of technical difficul-

ties rather than absence of such proteins. Limited inhibition of

labeling by emetine (85 %) probably results from incomplete

translocation of the antibiotic to all cells. The most successful

examples of this type of experiment have come from studies in yeast.

Therefore, it seems likely that use of cell suspension culture or

protoplast systems would be more efficient for this type of study in

plants.

The results, in this study, of labeling experiments in the

absence of protein synthesis inhibitors revealed that labeling of the

S protein occurred only in shoots germinated in the presence of





-53-


isotopes. Forde et al. (1978) examined protein synthesis in isolated

maize mitochondria. Their results showed that approximately twenty

polypeptides were labeled and detectable by SDS-PAGE. Although mito-

chondria from Tcms and Ccms maize were found to synthesize a single

additional polypeptide; no differences in mitochondrial translation

products were detected between N and Scms mitochondria. The results

from Scms mitochondria in these studies may indicate that the S

protein is made early in the germination process and has a relatively

slow rate of synthesis or degradation.

An important part of this study is the investigation of the

S protein and its relationship to the instability of male sterility

in Scms maize. Genetic studies by Laughnan and his co-workers

(Laughnan and Gabay, 1975) indicated that this instability was due

to an episome-like male fertility element. Fixed in the nucleus, it

behaves as a new restorer gene; if it is fixed in the cytoplasm, the

result is seen as a cytoplasmic mutation to male fertility. The

plasmid-like DNAs described by Pring et al. (1977) are possible

candidates for the role of Laughnan's fertility elements. Pring

et al. (personal communication) have found that the plasmid-like

DNAs are not present in Vgcms lines which have a cytoplasmic

mutation to male fertility.

Agarose gel electrophoresis and restriction endonuclease

fragment analysis of mitochondrial DNA verified the presence of the

plasmid-like DNAs in three nuclear mutants and confirmed that Vgcms

has Scms type DNA. Examination of mutant stock supplied by

Laughnan showed that the presence of the S protein is coincident with

the plasmid-like DNAs (Table III). These data suggest that the S




-54-


protein may be a gene product of these DNAs. This can be proved by

showing that a gene product from one or both of these DNAs is identical

to the S protein found in this study. Identification of the plasmid-

like DNAs as Laughnan and Gabay's (1975) fertility element depends

on evidence which shows the fate and function of these DNAs in the

two types of mutation to fertility.

Data from this study indicate that cytoplasmically determined

male sterility and disease susceptibility do not produce gross changes

in the electrophoretic phenotype of maize mitochondria when examined

on one dimensional gels. The gametophytic type of fertility restora-

tion, mitochondrial DNA content, and the presence of a cytoplasm-

specific protein make Scms distinct among male sterile cytoplasms.

These differences suggest that the mechanism of male sterility may

be different among male sterile cytoplasms. In Scms maize the

association of the plasmid-like DNAs and the S protein with male

sterility provide supportive evidence that the mitochondrial genome

is involved in cytoplasmic male sterility.




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Table III. Relationship of S protein to restoration, male sterility
and the presence of plasmid-like DNAs in Scms maize.



Cytoplasm Male Sterile Plasmid-like S-Protein
DNAs


Restored lines


N
Scms
Vgcms

N
Scms
Vgcms


C 103
C 103
C 103


New restorer genes

M825L/Oh07 h
M825L/Oh07 i
M825L/Oh07 m

Non-restored lines


M825L/Oh07
M825L/Oh07
M825L/Oh07


Vgcms
Vgcms
Vgcms


vgcms
Vgmf















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BIOGRAPHICAL SKETCH


David Walter Thornbury was born in Charleston, West Virginia,

on May 4, 1948. He spent his childhood in Belle, West Virginia, where

he attended Belle Elementary School. He graduated from DuPont High

School in May of 1966. He received his undergraduate training at

West Virginia University, Morgantown, West Virginia, where he was

awarded a Bachelor of Arts in zoology in 1970. He continued his

education at West Virginia University and received a Master of Science

in biology in 1973. In September, 1973, he entered the University of

Florida to commence work towards a Doctor of Philosophy in plant

pathology.

David W. Thornbury is a member of the American Phytopathological

Society. He has accepted a postdoctoral position in virology with

the department of plant pathology at the University of Kentucky in

Lexington.


-61-










I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.





Daryl R. P g, Chairman
Associate Professor of Plant
Pathology



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.




Rusty J.
Pro sor f iochemistry



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.




John R. Edwardson
Professor of Agronomy



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.


I I

Ernest Hiebert
Associate Professor of Plant
Pathology










I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.




Daniel A. Roberts
Professor of Plant Pathology



This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.



June 1979




Dean, lege of Agricultu


Dean, Graduate School





































UNIVERSITY OF FLORIDA
11 II Ii IIIII0 5 1 III
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