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II. Genetics of Mitochondria and Chloroplasts

II. Genetics of Mitochondria and Chloroplasts

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120



H. K . SRIVASTAVA



phosphorus-to-nitrogen ratio was demonstrated by Caspari (1 956) in mouse

mitochondria. The involvement of plasmon in heterosis (Ruebenbauer, 1967)

and other hereditary phenomena (Wilkie, 1964; Gillham, 1978) has also been

documented. Whaley (1952) observed greater efficiency of plasmon in maize

hybrids than in inbreds as judged by the rapidity with which the cytoplasm could

duplicate itself. He also commented that the genetic and physiological studies

concerned with the early phases of growth and development are most likely to

lead to the better understanding of heterosis. The faster germination and growth

exhibited by hybrids as well as the heterotic expression of respiration (Srivastava, 1972) and photosynthesis (Sinha and Khanna, 1975) in many crops

suggested the involvement of organelles in the phenomenon of heterosis. In yeast

Saccharomyces cerevisiae several respiratory-deficient “petite ” mutants have

also been identified with chondriome by a number of criteria (Mounolou el al.,

1966). These observations spurred the rapid development of investigations into

genetics of organelles and their relevance to cell heredity.

A. ORGANELLE

GENOMES



Chloroplasts and mitochondria contain unique genomes located in the unique

DNAs of these cytoplasmic organelles. At the beginning of the 1960s highmolecular-weight double-stranded DNAs were identified in chloroplasts (Sager

and Ishida, 1963) and in mitochondria (Luck and Reich, 1964). The organelle

DNA molecule differed significantly from that of nuclear DNA in size, nucleotide content, and other physicochemical properties. Subsequently, the

mt-DNA was shown to be the camer of the mitochondria1 genome (Michaelis et

al., 1973) and the chloroplast genome of the unicellular green alga

Chlamydomonas reinhardii was associated with ct-DNA (Schlanger and Sager,

1974). Results of many elegantly designed experiments with the yeast and the

green alga by these investigators provide convincing evidence in support of the

following conceptions: ( i ) genes are localized in the chloroplast and mitochondria; ( i i ) organelle genomes can be identified by their non-Mendelian behavior in

reciprocal crosses and also by the high frequency of segregation and recombination in clonal growth of hybrids; (iii) genetic recombination occurs between the

two parental organelle genomes in the progeny of biparental zygotes: biparental

zygotes transmit organelle genes from maternal and paternal parents to the meiotic products; in the progeny of such zygotes organelle genes, unlike Mendelian

genes, continue to segregate during the postmeiotic mitotic division as well as

during the initial meiotic division; ( i v ) the recombinations of nuclear and organelle genes are separated both in time and space: recombination occurs mostly

in vegetative growth of progeny clones, rarely in zygotes where nuclear gene

recombination occurs during meiosis; and ( v ) two types of recombination events



INTERGENOMIC INTERACTION, HETEROSIS, AND CROP YIELD



121



occur, reciprocal and nonreciprocal: alleles segregate 1 : 1 in reciprocal recombination, and 1 : I on the average in nonreciprocal recombination. These findings

have been generalized to other organisms, in part on the basis of experiments and

in part by inference. The recently discovered technique of restriction endonuclease fragment analysis of organelle genomes (Pring and Levings, 1978) from

organisms belonging to geographically distinct areas offers promise in the exploration for plasmon heterogeneity and its relevance to crop improvement.

The results of genetic mapping by two methods, gene-attachment point distances and coconversion frequencies, reveal that the organelle DNA is genetically circular (Sager, 1977). The genomes of mitochondria and chloroplasts

reside in covalently closed circular DNA molecules. Exceptions are the linear

mt-DNA molecules of the ciliated protists Tetruhymena and Paramecium. Each

species has its own unique mt-DNA and ct-DNA molecule; that molecule carries

a complete set of organelle genes. Several important properties of chloroplast and

mitochondrial DNAs are summarized in Table I. Comparative data on bacterial

and blue-green algal DNAs together with eukaryotic nuclear DNA, giving an

idea of the phylogenetic relationship between genetic systems of organelles and

prokaryotic organisms, are also presented in Table I. Both the genomes of

organelles and prokaryotic organisms are endowed with a circular fibrous DNA

organization and an absence of histone. Exceptions are the highly condensed

mt-DNA molecules of the true slime molds, which appear to be complexed with

a basic protein (Kuroiwa et al., 1976), and the mt-DNA of Xenopus oocytes,

which can be isolated in association with protein in the form of structures reminiscent of the nucleosomes of chromatin (Pinon ef al., 1978). The association

of circularity with the non-histone-clad DNA is viewed as a primitive one, while

the histone-clad DNA of eukaryotic organisms is thought to be a derived state

(Uzzel and Spolsky, 1974). The similarities in physicochemical properties shared

by chloroplast, mitochondrial, blue-green algal, and bacterial DNAs provide

evidence that organelles may have originated as endosymbionts within a eukaryotic cell. A highly significant feature of organelle genome, however, is the occurrence of the eukaryotic properties of intercistronic spacer sequences and polyploidy. Many, but not all, genes from nucleated cells carry within themselves

nucleotide sequences (called intervening or spacer sequences) that are not transcribed in the messengers (mRNA) corresponding to these genes. In contrast,

bacterial messengers are direct copies of the genes, without any missing segments (Marx, 1978). Thus, the nucleated cells and organelles seem to possess a

common mechanism, not found in prokaryotic organisms, for producing mRNAs

from which some gene sequences are omitted or deleted. The total amount of

ct-DNA per chloroplast in many crops is (2-10) x lo-'" g. This corresponds to a

molecular weight of (1.2-6.0) X lo9. Using the renaturation kinetic concepts,

the genome size of the ct-DNA in higher plants has been estimated to be ( 1 -2) x

lo* MW (Kolodner and Tewari, 1975). Hence the average content per chloro-



H. K. SRIVASTAVA



122



Table I

Some Properties of Nuclear, Organelle, and Bacterial and Blue-Green Algal DNA"



Parameters

g)

DNA content ( X

Buoyant density (g/cm3)

Dry weight (8)

Guanine-cytosine content (8)

Histones

Repeated DNA sequence

Polyploidy (multiple copies)

Conformation

DNA organization

Satellite band

5-Methylcytosine

Renaturation rate

Recombination (meiosis and mitosis)

DNA condensation (cell division)

Cytoplasmic ribosome (80 S)

Organelle ribosome (70 S )

Intercistronic spacers

Ribosomal RNA

( a ) Large subunit

(6) Small subunit

5 S RNA (most primitive)

Transcription-translation system

(streptomycidchloramphenicaYrifampicin)

Protein synthesis system (cyclohexamide or a-aminitin)

Initiation of protein synthesis



Eukaryotic

nuclear DNA



ct-DNA



mt-DNA



BacteriaVbluegreen algal DNA



4-7

1.725

0.6-1.7

40-65

Present

Present

Present

Linear

30-40 di fibrils

Present

Present

Slowhapid

Yes

Yes

Present

Absent

Present



0.3-0.9

1.697

0.05-0. I5

36-42

Absent

Absent

Present

Circular

25 di fibrils

Absent

Absent

Rapid

Yes

No

Absent

Present

Present



0.1-0.3

1.706

0.02-0.08

36-45

Absent

Absent

Present

Circular

25 di fibrils

Absent

Absent

Rapid

Yes

No

Absent

Present

Present



2-3

1.715

0.4-0.6

38-60

Absent

Present

Absent

Circular

25 di fibrils

Absent

Present

Slow



1.3 X lo6

0.70 x loR

Present

Insensitive



1.28 x 10'

0.75 x lo6

Absent

Sensitive



1.23 X 10'

0.65 x lo6

Absent

Sensitive



1.1 x 106

0.55 X lo6

Present

Sensitive



Sensitive



Resistant



Resistant



Resistant



tRN Arne'



tRNA;""'



tRNA(""



tRNApeL



No

No

Absent

Present

Absent



aAdapted with modifications from Lehninger (1976) and Kung (1977).

which is considered as

bProtein synthesis system in organelle begins with N-formylmethionine (tRNAfmeL),

evidence for a prokaryotic origin of organelles.



plast is about 10-30 times that of genome size, suggesting that the genetic material

is present in about 10-30 copies (like many chromosomes in a nucleus) per

chloroplast. The sequence of organelle genome is therefore amplified manyfold,

representing organelle gene amplification.

Organelle genes appear to be present in multiple copies in every cell. Organelles are thus analogous to highly polyploid nuclei in an organism with a haploid

chromosome number of one. The number of organelle genes is limited by the size

of the DNA molecule. Molecules of mt-DNA range from about 5 p m in length

(about 10 X lo6 MW or 15 x lo3 base pairs) in animals and Chlamydomonas to

about 30 pm(60 x lo6 MW or 90 x lo3 base pairs) in pea plants. Most plants



INTERGENOMIC INTERACTION, HETEROSIS, AND CROP YIELD



123



have ct-DNA molecules close to 45 p m in length, while the liverwort Sphaerocarpus and the protist Chlamydomonas set the known lower and upper limits

with ct-DNA molecules 37 and 62 p m in length, respectively (Birky, 1978). A

single cell may contain many molecules of ct-DNA and/or mt-DNA. These

organelle genomes are packaged into one or many organelles, each of which may

contain many DNA molecules. Finally, there is a third order of packaging; within

an organelle, the DNA is generally observed to be localized in discrete regions

that are most appropriately called “nucleoids” by analogy with similar regions in

prokaryotes (Birky, 1978). The number and packaging of mt-DNA molecules are

even more variable in Saccharomyces. The amount of mt-DNA in these cells

varies with genotype, ploidy, cell volume, and in at least some strains with the

physiological state of the cell (Williamson et al., 1978). These authors conclude

that the average haploid cell has about 50 molecules of mt-DNA, with a range of

8-123. Haploid yeast cells have 10-32 “nucleoids,” and each “nucleoid” is

estimated to contain 4-5 mt-DNA molecules. A distinction, however, must be

made between the repeated DNA sequences and multiple copies of organelle

DNA. The organelle genomes do not seem to possess repeated sequences but are

amplified in multiple (nonredundant) copies distributed at a number of independent DNA-containing areas within the organelle (Gibbs and Prole, 1973), representing organelle genome polyploidy . Gene amplification in terms of ribosomal

RNA genes (rDNA) is of common occurrence in eukaryotes (Tartof, 1975), and

the present observation of multiple gene clusters in organelles suggests that gene

amplification is not restricted to rDNA only, but DNA amplification may perhaps

be found in other extrachromosomal locations. The occurrence and functional

significance of gene clusters in prokaryotes, such as yeast and Escherichia coli,

have become well documented since the classical studies of Demrec (1964) and

Jacob and Monod (1961). In eukaryotes, gene clusters appear to be relatively less

frequent than in prokaryotes, although numerous such clusters have been detected in recent years in mitochondria and chloroplasts (Giles, 1978). The plausibility of the presence of a polyploid genome model and its heterogeneity in a

heteroplasmon within a cell can be considered supportiveto the view of eukaryotic

resemblance of organelles. The eukaryotic nature of modem organelles is further

substantiated by the absence of a 5 S primitive ribosomal RNA (120 nucleotides)

in mitochondria and chloroplast, while such an RNA is of common occurrence in

both bacterial and eukaryotic ribosomes (Schwartz and Dayhoff, 1978). It is

associated with the larger ribosomal subunit and is thought to function in the

nonspecific binding of transfer RNA to the ribosome during protein synthesis. In

most eukaryotic species the 5 S RNA genes (5 S DNA) are of a homogeneous

sequence (Tartof, 1975). The undeniable fact remains that organelles contain

their own genomes and protein synthetic apparatus: both differ substantially from

the corresponding nuclear-cytoplasmic system. The transcription-translation

systems of organelles have a strong resemblance to those of prokaryotes.



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H. K . SRIVASTAVA



B. CODING

CAPACITY

OF ORGANELLES



Both mitochondria and chloroplasts contain distinct genetic systems based on a

covalently closed circular DNA molecule and a 70 S ribosomal proteinsynthesizing machinery. The main question that remains to be resolved is regarding the total content of genetic material present per organelle, and to what extent

the organelle genome is involved in the overall genetic functioning of the cell. It

is intrinsically difficult to make counts of the number of organelles present in a

cell. Rough approximations based on light and electron microscopic views indicate a range from 700-1000 up to an extreme of 200,000 mitochondria in a large

egg of higher plant and animal cells (Grun, 1976). Leaf parenchyma cells of Beta

vulgaris have been studied intensively by Henmann et a f . (1974), who find that

these cells contain 40-50 chloroplasts. Each chloroplast contains 4- 18 nucleoids

(discrete regions inside the organelle packed with DNA molecules), and each

nucleoid is estimated to have 10-100 chloroplast DNA molecules, and an entire

cell possesses a total of about 500-1500 chloroplast genomes. Striking motion

pictures of mitochondria of living plant cells suggest that mitochondria could

fuse with one another and then split, causing changes in the frequency of

mitochondria per cell over time (Honda et a f . , 1971). The numbers of chloroplasts per cell in both palisade and mesophyll tissue in spinach vary from 600 to

6000 depending on the developmental stage and length of leaves (Possingham

and Smith, 1972). Nass (1976) estimates that a mouse L cell in culture contains

about 250 mitochondria each with about 5 mt-DNA molecules, for a total of 1250

molecules per cell. In contrast, Chfamydomonas haploid cells have about 46

mt-DNA molecules (Ryan et a f . , 1978). The actual count of organelles per cell

may not be very important from a genetic point of view. It is imperative to know,

rather, how many organelle DNA gene centers there are per cell, how many

DNA molecules there are per center, and whether mitochondria differ in their

potential genetic capacity within a cell.

Electron microscopic studies in yeast reveal that there are on the average 10

mitochondria per haploid cell (Grimes et a f . , 1974). Knowing the total DNA

per cell (about 13% of the total DNA is mt-DNA), the authors calculated that

there were 2.2 x lo9 MW of mt-DNA per cell; this corresponds to about 44

nucleoids of the yeast mt-DNA, giving about 4 in each mitochondria. Autoradiographic observations coupled with the results of electron microscopic

studies in higher plants indicate that each chloroplast possesses a number of

independent ct-DNA-containing centers (Gibbs et al., 1974). The number of

such centers seems to increase with chloroplast size, varying from one area in

small chloroplasts to a maximum of 40 centers in very large chloroplasts. Since

organelles are known to reproduce by division (Ridley and Leech, 1970), the

ones that contain the greater number of ct-DNA gene centers may be interpreted



INTERGENOMIC INTERACTION, HETEROSIS, AND CROP YIELD



125



as being ready for division. The chloroplast genome from most higher plants has

DNA in the range of (2-10) X

g and the unique genetic message is 40-150

p m long (Tewari and Wildman, 1970). A molecule 40- 150 p m long would have

a molecular weight of (80-300) X 10" since 1 p m has a molecular weight of 2 x

10". If all the assumptions and estimates involved in this evaluation are correct, then

there is room for between 15 and 80 DNA copies of the 40-pm ct-DNA and

between 4 and 20 copies of the 150-pm ct-DNA in each chloroplast.

The definite account of organelle DNA in light of its potential coding capacity

is not yet feasible for want of direct experimental evidence. The mt-DNA may

replicate to produce a mass of intramitochondrial DNA with or without

mitochondria1 division (Grun, 1976). The organelle DNA replication evidently

takes place independently at least on occasion, but the genetic significance of this

replicated mass of DNA is unknown. The hypothesis that organelles possessing

superior DNA replication machinery may be effective in providing superior

organelle activities during plant growth and development is highly speculative,

but is nevertheless worthy of consideration in designing future experiments with

higher plants. Considering the size of mt-DNA in higher plants to be of the order

of 150 X l(r' MW, it could be reasonably assumed that the mitochondrial

genome is large enough to code for both large and small ribosomal RNA species,

a complete set of transfer RNA species, and polypeptide components of enzyme

systems that play key roles in respiratory electron transport and phosphorylation,

e.g., cytochrome oxidase and the F,-F, complex in mitochondria. As regards

chloroplasts, the total genetic capacity of average length 45-pm circular ct-DNA,

assuming asymmetric transcription and allowing for an inverted repeat sequence,

is about 6 X l(r' of polypeptides. A subsequent estimate by Smillie et af. (1973)

reveals chloroplast genome to contain about 2 x 10' nucleotide pairs. If about

1500 base pairs form one functional gene, the chloroplast genome must contain

about (1.3-1.5) x lo3 genes. Taking into account the existence of frequently

repeating homologous chains, intercistron spacers, and regulatory genes, one can

suppose that the chloroplast genome codes for about 150-300 specific proteins

of 50 x 10" MW. It is tempting to conclude that this genetic system synthesizes

most of the entire spectrum of chloroplast polypeptides. Nevertheless, genetic

studies in both algae and higher plants have located the structural genes for

several chloroplast polypeptides in the nuclear genome (Gillham, 1978), and the

results of studies of the biosynthesis of many key enzymes of mitochondria and

chloroplast activities, which are discussed in detail in later sections, indicate that

some of their subunits are synthesized under transcriptional control of organelle

genome, while the other partner polypeptides are made by the nuclear DNA. A

possible interpretation of such observations would be that probably mitochondria

and chloroplasts require the integrated action of both organelle and nuclear

genomes for their structural and catalytic functions.



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H . K . SRIVASTAVA



c. TRANSMISSION

OF O R G A N E L L E S

In the absence of detailed information about the fate of cytoplasmic organelles

before, during, and after syngamy in the sexual offspring, one can only extrapolate from results regarding organelle heredity in terms of the specific source of

the organelle genes. Several studies have shown that the organelles of the male

gametophyte angiosperms are incorporated into the vegetative cell, and the

generative cell and resulting male germ cells receive very few plastids, although,

as in most plants, they contain mitochondria (Mascarenhas, 1975). It is of interest to note that in those cases in which plastids were not found in the generative

cells (e.g., maize-Larson, 1965; petunia-Sassen, 1964; and impatiens-van

Went, 1974) plastid variegation is transmitted only maternally (“status albomaculatus”), whereas in cases in which plastid variegation was found to be

transmitted biparentally (“status paralbomaculatus”), numerous proplastids

have been identified in the male germ cells (e.g., Pefargonium-Lombard0 and

Gerola, 1968; Oneorheru-Walles, 1971 ; Hypericum-Hageman, 1964). Paternal mitochondria and plastids may or may not be excluded during syngamy; they

may even prevail and exclude maternal mitochondria and plastids as indicated by

the exclusive paternal transmission of organelles in some gymnosperms (Giarnordoli, 1974).

In most instances the pattern of non-Mendelian inheritance of organelle traits

follows uniparental transmission. Sager (1954) first reported the occurrence of

streptomycin-resistant chloroplast mutations in Chlamydomonas showing uniparental transmission. In the literature these mutations are referred to by a variety of

terms, including “cytoplasmic,” “chloroplast,” “non-Mendelian, ” “nonchromosomal, ” and “uniparental. ” The occurrence of biparental inheritance,

foreshadowed by the findings of Baur (1909), has been documented in few

higher plants, of which the most thoroughly studied are Oenothera and Pelargonium (Tilney-Bassett, 1973). Tilney-Bassett (1975) cataloged the various genera and species in which non-Mendelian inheritance of chlorophyll-variegated

phenotypes were reported. At present, maternal inheritance for organelle genes

appears to be more common than biparental, but both types are found in the two

major groups of angiosperms: the dicotyledons, with 24 genera maternal and 14

genera with biparental inheritance, and the monocotyledons, with 8 genera maternal and 2 genera with at least a trace of biparental inheritance. Subsequently,

biparental plastid inheritance was reported in Coix lacrymujobi, C. aquatica, and

C . gigantea (Rao, 1975), in Browallia (Semeniuk, 1976), and in Pennisetum

americunum (Rao and Koduru, 1978). In view of the extranuclear location of

mitochondria and chloroplasts, it is not surprising that transmission of the organelle genomes is not governed by the same rules that apply to chromosomal

genes. For fungi (Roodyn and Wilkie, 1968), amphibians (David and Blackler,

1972), and mammals (Hutchinson et al., 1974), there is evidence that the



INTERGENOMIC INTERACTION, HETEROSIS, AND CROP YIELD



127



mitochondria1 genome of an individual is derived solely from the maternal parent. Based on the restriction endonuclease fragmentation analyses of mt-DNA

from normal and Texas maize cytoplasms, Pring and Levings (1 978) have made

a strong claim for an absence of transmission of the paternal mitochondrial

genome; paternal mt-DNA is not expressed at detectable levels in their experiments. However, recent study on ct-DNA distribution in parasexual maize

hybrids, as shown by polypeptide composition of ribulose biphosphatase

carboxylase-oxygenase, indicates that chloroplasts from both parents stay together and are distributed biparentally to daughter cells, giving a preponderance

of one type or the other (Chen et al., 1977). Mammalian eggs are known to be

very rich in mitochondria, and if the concentration of mt-DNA is similar to that

found in the cytoplasm of echinoderm eggs, an equine egg would contain about

1W molecules of mt-DNA (Hutchinson et al., 1974). This would imply that if

paternal and maternal mitochondria replicate at equal rates, then only about

of the mt-DNA in a mammal would be paternally derived. This small quantity

would presumably be below the limit of experimental detection. The possibility

therefore exists that paternal organelle genomes are present in very small numbers in the tissues of higher organisms, and that sufficiently sensitive experiments in the future could detect their presence.

It is of interest to consider the possible evolutionary implications of organelle

genome transmission. An albomaculatus plant that does not have pollen transmission will inherit each generation only organelles of the maternal parent, and

they will all probably be genetically the same. Evolutionary change will occur

only when mutants are produced, and the selection will be restricted to one

mutant at a time. In paralbomaculatus plants a mixture of the different types of

organelles from both parents occurs in the fertilized egg. The heterogenous types

present may compete, mitochondria with mitochondria and chloroplasts with

chloroplasts. In addition, the paralbomaculate system offers possibilities of fusion between unlike types and recombination of their genes to produce new

organelle genotypes. The paralbomaculate hybrid would be more flexible in the

evolution of its organelle genotypes than the albomaculate. The hypothesis that

the fusion of the two chloroplasts, just like nuclear fusion, brings together the

ct-DNA from parental types resulting in an ‘‘interorganellular gene recombination” by physical exchange of pieces of DNA is very attractive (Bastia et al.,

1969; Cavalier-Smith, 1970); however, rigorous experimental testing is required

to fully understand the possible exchange of genetic material during “interorganellular recombination. ” It would be of further advantage to an organism

if the number of organelles in the cytoplasm were reduced before or during

fertilization, so that doubling of such particles did not occur each generation. The

genetic control mechanism to maintain the specific number of organelles in each

species after biparental contribution of contrasting organelles to the zygote is

not known. Considering the fact that meiosis serves this purpose for nuclear



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H . K . SRIVASTAVA



genomes, Jinks (1964) has presented the hypothesis that size differences between

male and female gametes in most species serve the purpose of organelle genome

transmission in the zygote. There is, however, no correlation between egg size

and the number of plastids in Quercus gambelii where the egg and zygote cells

contain negligible or very scarce number of plastids (Morgensen, 1972). Although the information is still minimal, the present examples are a warning that

one should be extremely wary of making assumptions about the paternal and

maternal organelle genome transmission to the zygote.

D. ORIGINS

OF ORGANELLES



It is now established that the replicating chloroplasts of young spinach leaves

actively synthesize DNA, and during their division cycle segregate it to daughter

chloroplasts (Rose et a f . , 1975; Possingham and Rose, 1976). Organelle division

is not necessarily the same as organelle DNA replication, for the DNA might

replicate to produce a mass of intraorganellular DNA but still not produce new

organelles therefrom. Direct evidence that organelles divide following one or the

other mechanism has been elusive for a number of reasons. The organelles are so

small that simply watching them divide in the ordinary microscope is not practical. There are complexities in the interpretations of observations of fixed tissue in

an electron micrograph, but some studies of spinach leaves suggest the possibility that both constriction division by cross-wall or baffle formation occur (Gran

and Possingham, 1972). In some ways, the constriction division of chloroplasts

bears a similarity to cytokinesis in mammalian cells (Robins and Gonata, 1964).

In spinach the daughter chloroplasts formed from a single dividing chloroplast

are usually of equal size, and in this respect chloroplast binary fission differs

from the asymmetric budding that occurs during yeast cell multiplication (Mitcheson, 1971). The constriction process of chloroplasts also differs from division in

bacteria, where distinct cross-wall formation from both sides of the cell usually

precedes daughter cell separation (Higgins and Shockman, 1971; Slater and

Schaechter, 1974). There is evidence that isolated chloroplasts separated from

the environment of the cell can divide using mechanisms that exist within the

plastid itself (Ridley and Leech, 1970; Kameya and Takana, 1971). A doubling of

chloroplast numbers was associated with a doubling of chloroplast silver grains,

which is consistent with a duplication of ct-DNA during the chloroplast division

cycle (Possingham and Rose, 1976; Possingham, 1976). There have been at least

two electron microscopic studies (Marton, 1962; Andre, 1962), on the marine

flagellate alga Chromulina pusilla, and an autoradiographic study involving

growing Neurospora crassa, that seem to show division of mitochondria by

binary fission similar to the constriction division of chloroplasts.

The Chlamydomonas chloroplast is the only case where strong data favor



INTERGENOMIC INTERACTION, HETEROSIS, AND CROP YIELD



129



nonrandom segregation of organelle genomes (Birky, 1978). Because this organism possesses a single large-sized plastid that divides as the cell divides, the

ct-DNA segregation can easily be followed during organelle division, whereas in

yeast, Paramecium, and higher plants the data are compatible with random

segregation of organelles during cell division. It is likely that organelle segregation during cell division has a strong random element, while organelles themselves

have mechanisms to assure nonrandom DNA segregation when they divide

(Birky, 1978). There is in fact substantial ultrastructural and autoradiographic

evidence for numerically equal division of mt-DNA (Kuroiwa et a l . , 1977)

and ct-DNA (Rose et al., 1974) during organelle division. It must be noted that

the term segregation is applied here to the physical movement of organelles or

organelle genomes during cell division. The vegetative segregation of organelle

genes is commonly attributed to a random physical segregation and sorting out of

the organelles containing them during vegetative divisions, since mitochondria

and chloroplasts are usually not closely associated with the mitotic spindle or

other visible apparatus that might assure nonrandom segregation.

DNA replication in isolated chloroplasts was first reported in spinach and

Euglena (Spencer and Whitfield, 1969). Evidence for the semiconservative replication of ct-DNA comes first from the work of Chiang and Sueoka (1967).

These results confirm that ct-DNA is essential as master molecule for chloroplast

division. According to Kolodner and Tewari (1975), ct-DNA replicates by both

the “cairns” and “rolling circle” mechanisms. The “cairns” round of replication is used to initiate the “rolling circle” round of replication of ct-DNA. If this

is true, why are two different modes of DNA replication required by higher

plants? These authors propose that it is probably related to a developmental

aspect of higher plant chloroplast biogenesis. During chloroplast development, a

rather rapid synthesis of a number of copies of ct-DNA molecules is required.

Therefore, the “rolling circle” mechanism might be used for this rapid synthesis, while the “cairns” mechanism may be used for normal replication.

The subject of the evolutionary origin of organelles has aroused much interest

and speculation, but the existing evidence is still far from conclusive. Three main

theories, among many others, explaining the origin of organelles have been

discussed frequently. The first, an endosymbiotic theory, proposes that the cellular organelles of eukaryotes, even including flagella, centrioles, and spindle

fibers, originated from symbiotic prokaryotes like blue-green algae or photosynthetic bacteria that originally existed in the host cells and later became established after some modifications as endosymbionts within eukaryotic cells

(Stainer, 1974). Margulius (1970) has marshaled a great wealth of

morphological, biochemical, and paleontological facts in support of this theory.

It has further been supported and somewhat amplified by Raven (1970), as well

as by Schnepf and Brown (1971) and Taylor (1974), but has been severely

attacked by others, particularly Allsopp (1969), Raff and Mahler (1972), and



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H. K . SRIVASTAVA



Bogorad (1975). The second theory, the origin of organelles by progressive

evolution of prokaryotes, suggests that mitochondria and chloroplasts originated by progressive transformations of the thylakoid membrane systems of

blue-green algae (Allsopp, 1969). Raff and Mahler (1972) postulate that

mitochondria originated from invaginations of the inner cellular membrane,

which first formed respiratory vesicles bound by membranes. These vesicles

converted into mitochondria by the addition of a protein-synthesizing DNA

plasmid, derived from another prokaryote. They believe that the difference in

modem eukaryotes between nuclear-directed and mitochondria-directed protein

synthesis and other metabolic activities is due to divergent selective pressures

that have acted since the acquisition of mitochondria. The many similarities

between organelles and prokaryotic cells (illustrated in Table I) can not be taken

as direct support for the endosymbiont hypothesis because the eukaryotes may

have evolved by gradual transformation of prokaryotes. Bogorad (1975) offers a

third theory, “cluster-clone,’’ for the origin of the eukaryote nucleus and the

DNA contained in chloroplasts and mitochondria. He believes that all of the DNA

found in eukaryote cells originally existed in a nuclear area, perhaps surrounded

by a common nuclear membrane. Later, clusters of genes derived from the

protonucleus became separated and surrounded by their own membranes, thus

forming the plastids and mitochondria. The earlier considerations that some of

the proteins of which these organelles consist are coded by nuclear genes, while

other are coded by genes located in the organelle themselves, could be regarded

as evidence in favor of the “cluster-clone’’ hypothesis or a nucleoplasmic origin

of organelles. It has been reported that there is considerable base sequence

homology between ct-DNA and nuclear DNA in tobacco (Siegel, 1975), broad

bean (Kung and Williams, 1969), and Euglena (Rawson and Haselkorn, 1973).

Furthermore, chloroplast rRNA hybridizes with nuclear DNA in tobacco (Tewari

and Wildman, 1968). These studies appear to favor a direct origin of organelles

rather than an indirect one. Evidence in support of another hypothesis that organelles originated from an invagination of the nuclear membrane has been

presented (Bell, 1970). Whichever theory is finally accepted, evolutionary

modification of both nuclear and organelle DNA during early eukaryote

evolution must be postulated.



111. ORGANELLE INVOLVEMENT IN GENETIC

PHENOMENA

When one considers the diverse effects of cytoplasmic genes on plant growth

and development of the few species of higher plants studied (Stubbe, 1964), the

possibilities of recombination resulting from the biparental transmission of organelle genes (Sager, 1977), and the opportunities for inducing mutations of



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II. Genetics of Mitochondria and Chloroplasts

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