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IS THE PLASTID AN
ENDOSYMBIONT?
William P. Hall
(1966: Biology Dept., Southern Illinois University, Edwardsville, Ill.
62025/USA)
2010: Engineering Learning Unit, University of Melbourne, Vic.
3010/Australia
Cover Notes
SUMMARY (1979)
Hans Ris proposed in 1961 that chloroplasts might be highly derived endosymbiotic
microorganisms, originally related to blue-green algae. Evidence from 1966 and
earlier is reviewed to test this proposal. Elegant experiments using the unique genetic
system offered by Oenothera (the evening primroses) clearly show that plastids carry
heritable characters not under nuclear control. Two or even three distinctive kinds of
plastids may coexist and retain their identities in a single line of heteroplastidic cells.
The distinctive characters of a line of plastids were even maintained in contact with a
foreign nuclear genome for more than 10 generations of reproduction of the host
plant. Studies in Epilobium, corn, tobacco, and other plants further demonstrate the
heritability and mutability of an independent plastid genome. Time-lapse micro-
cinematography, electron microscopy, histochemistry, cell fractionation, tracer and
biochemical studies, and DNA hybridization all show that plastids are reproduced
only from pre-existing plastids, that they contain DNA differing in many traits from
nuclear DNA, that they contain their unique ribosomes, and that even when isolated in
vitro or in enucleated cells they still synthesize their own DNA, transcribe at least
some RNA, and synthesize some protein. In all of these characters plastids more
closely resemble complete blue-green algea than they do other parts of the eukaryote
cell.
What do these findings imply for ideas about the origins and early evolution of cells?
There are two major anomalies in the previously accepted dogma that all eukaryotes
trace from phytoflagellates, which are supposed in turn to derive from blue-green
algae:
1. the diversity of very simply organized free-living sarcodina is incompatible
with their derivation from the vastly more complex and highly organized
phytoflagellates.
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2. There is an unbridgeable structural gap between blue-green algea and the
simplest phyto-flagellates. There is a close relationship of cell structures of a
blue-green algae and a single chloroplast. Nothing is left over to serve as
evolutionary anlagen for the remaining structures of the eukaryote cell.
These anomalies vanish if chloroplasts evolved independently, and only secondarily
united in a symbiosis with eukaryote cells. It follows that each type evolved
independently from the primordial organic soup. Chloroplasts derive from a line of
"producers" which evolved stereochemically complex systems of coupled electron
transfer reactions to cope with a decline in the quality of the soup. Cytoplasmic
motility would be incompatible with coupling the systems stereochemically.
Eukaryotes trace from early "consumers" which evolved simple cytoplasmic motility
to sop up adsorbed building blocks, and then graduated to phagocytosing "producers."
The cytoplasmic shearing forces provided strong selection pressures for the evolution
of nucleoprotein chromosomes and nuclear membranes. More complexly specialized
motility apparatuses trace easily from a generalized cytoplasmic motility based
initially on only a few different kinds of molecules.
Origins of the paper [1979]
The manuscript was first submitted in Hampton L. Carson's Genetics and Evolution
course at Washington University, St. Louis., May 3, 1966. It was revised Summer,
1966, in hopes of finding a sponsor for its publication. It was shown at the cell
biology meetings in Ames, Iowa, with no result, and since then I have had no time to
update the presentation. The typescript includes 26 pages of text, and 66 references.
Although old, and certainly not current with the literature, I have decided to try
submitting the MS as is to Evolutionary Theory. Many of the ideas are still fresh and
deserve further development.
Further comments [2005]
The 1979 abstract and comment was included on a version of the paper distributed
with job applications for biology positions in 1979-80. As things transpired, I failed to
find the kind of academic position that allowed me to continue my career in biology.
The version here was scanned, OCRed and converted to HTML in 2004 from a
photocopy of the summer 1966 version included in 1979-80 job applicatoins. The
only changes made from the raw OCRed text were to correct OCR conversion and
spelling errors and to add HTML markup and links.
It is interesting to compare my 1966 MS with Lynn Sagan/Margulies' 1967 article, On
the Origin of Mitosing Cells", J. Theoretical Biology, p. 225 and 1970 book, Origin of
Eukaryotic Cells, Yale University Press. To me, the most significant indicator that
chloroplasts had an independent genetic system was the phenotypic evidence for the
inheritance of plastid features in ways that could not be explained by inheritance via
genetic systems based on nuclear chromosomes. The other area where our analyses
differed significantly was in the origin of the flagellar motility apparatus. My theory is
that flagellar locomotion of eukaroyotic cells is a directly evolved extension of an
already motile cytoplasm, where Sagan/Margules hypothesized that the flagella
evolved through the endosymbiosis of spirochaete bacteria. There is now
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overwhelming evidence that endosymbiosis of chloroplasts and mitochondria
certainly occurred, and in fact, may have occurred more than once.
Had I succeeded in finding a sponsor for the paper at the 1966 cell biology
conference, I have no doubt that it would have become a classic paper in cell and
evolutionary biology. However, given that I had no perceptible qualifications in cell
biology, that I was only a masters degree student in a university that had no accredited
masters degree program, and that the study was a distraction my research program on
chromosomal evolution and speciation in lizards, I did not persevere in trying to
publish it following the lack of interest at the cell biology conference. It took all of
my intellectual effort to move from the non-existent graduate program at Southern
Illinois University, Edwardsville to Harvard University's PhD program.
IS THE PLASTID AN
ENDOSYMBIONT?
William P. Hall, III
Introduction
In 1961 Hans Ris proposed that chloroplasts may be highly evolved and modified
derivatives of ancient endosymbiotic microorganisms related to the photosynthetic
monera (the blue-green algae and bacteria). The hypothesis revived ideas expressed
by Altmann, (l890), Mereschkowsky, (1905), and Famintzin, (1907). In this and
another paper (Ris and Plaut, 1962) Ris cited genetic evidence for partial plastid
autonomy (Rhoades, 1955; Granick, 1955), cytochemical and cytological work from
his lab (Ris and Plaut, 1962), and comparative studies on blue-green algae (Ris and
Singh, 196l) as new support for the hypothesis.
Following Ris's papers, there has been an almost exponential increase in the
publication of information about the autonomous nature of the chloroplast; yet, to my
knowledge, only one writer (Swift, 1965) and his co-workers (Kislev, et. al., 1965)
supported, or even mentioned, the possibility that chloroplasts may be derived from
endosymbionts. Yet, many investigators have cited the Ris papers for other reasons.
No one has publicly discussed the important and far reaching implications of the Ris
hypothesis.
My review will show that the Ris hypothesis certainly provides a valid explanation for
a large mass of data concerning the genetics, chemistry, and physiology of the plastid,
which cannot be readily explained in other ways. After this evidence is presented,
some of the more obvious implications of the hypothesis will be discussed. An
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examination of these implications will also expose several independent avenues of
approach calling for the same conclusion.
Evidence derived from plastid studies, indicating that these bodies arose as
independent organisms, is particularly strong in five categories:
1. Experiments demonstrate that at least part of the plastid's inheritance is
independent of nuclear control.
2. Several cytological studies show that plastids are derived by division from
previously existing plastids and not from any other cellular source.
3. There is strong evidence that the partially autonomous genetic system
contained within the plastid is based on its own unique species of DNA.
4. This unique DNA is synthesized within the plastid, and this synthesis is
independent of nuclear control.
5. The plastid uses this unique DNA to control the synthesis of proteins by a
plastid specific ribosomal system.
In this paper I will pay particular attention to the first category of evidence, since it
has the longest history and shows most clearly the independent nature of the
chloroplast. The genetic experiments are supported by other evidence showing that
the plastid does, in fact, possess the physical and biochemical pre-requisites expected
of a genetically unique, self-reproducing organism.
THE GENETIC EVIDENCE
A large mass of genetic evidence has been published showing that some plastid traits
are not controlled by nuclear genes. This information was reviewed by several authors
(Caspari, 1948; Weier and Stocking, 1952; Rhoades, 1955; Granick, 1955; and von
Wettstein, 1961). The elegant studies of plastid inheritance within the genus
Oenothera, and some of the more recently published work will be discussed here as
examples of the available evidence.
To understand the studies of Oenothera plastid inheritance, it is necessary to
understand the unique nuclear inheritance system found in these relatives of the
evening primrose. This genetic system was reviewed by Cleland (1962). Many
Oenothera races have two distinct haploid sets of chromosomes. A specific haploid
chromosome set is called a Renner complex which has a specific haploid genotype
called a genome. The complex and its associated genome is usually given a Latinized
name. One Renner complex differs from others by a series of reciprocal, whole-arm
translocations, arranged so that all of the chromosomes of two different complexes
pair in meiosis to form a single complete circle. Meiotic disjunction is not random;
each Renner complex segregates as an intact unit into a meiospore. Most complexes
possess one or more balanced gametophytic or zygotic lethals that effectively prevent
the formation of individuals homozygous for a specific complex. Two different
Renner complexes form a single linkage group of two genomes which are generally
heterozygous for most loci. Because of the balanced lethals, this is the only
propogatable genotype in many lines. Therefore, the line is "true-breeding," even
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though heterozygous. The genetic system of Oenothera is particularly useful for the
study of plastid inheritance, because single Renner complexes enter given gametes.
This allows the production of hybrids with accurately known and reproducible
combinations of nuclear genomes.
The comments on Oenothera plastid genetics are primarily based on Cleland's review
(1962). Added information was derived from the other reviews previously cited.
In 1913, De Vries observed anomalous inheritance of plastid traits in some Oenothera
hybrid crosses. Reciprocal crosses of Oe. hookeri (homozygous for the hhookeri
Renner complex) and Oe. lamarkiana (a balanced heterozygote with the gaudans and
velans Renner complexes) produced an un-explainable distribution of progeny. When
hhookeri was used for the female parent and lamarkiana provided the pollen, all
classes of progeny had normal chloroplasts. When the reciprocal cross was made,
with lamarkiana as the female, the progeny of the gaudans and hhookeri complexes
were normal, while the velans.hhookeri progeny had defective yellow plastids.
However, according to the Oenothera genetic system, the hhookeri.velans progeny
from the first cross and the velans.hhookeri from the second should have had exactly
the sane phenotypes. The two crosses were identical except that different species were
used for the female parent.
Later, Renner (1924) reported that about 15% of the yellow F1 hybrids from similar
crosses of lamarkiana and hookeri had green flecks or sectors. Self fertilized flowers
from green sectors of the hybrid produced green plants, while selfed flowers from the
yellow areas produced only yellow plants. A re-examination of the reverse cross
showed the reciprocal pattern. Similar observations were made in hybridization
experiments with many other Oenothera species. Renner offered the following
hypothesis to explain this strange situation. He proposed that plastids derived from
one race or species could differ in genetic quality from plastids derived from another
race.1 Renner further suggested that Oenothera plastids were generally inherited along
with the egg cytoplasm although on occasion a few male plastids might be introduced
with the sperm nuclei. Once in the egg, the male plastids would segregate randomly
with the female plastids during embryogeny which would lead to the formation of
sectoral chimeras or localized areas where the plastids differed in functional ability.
The evidence for this process of segregation will be discussed in detail below.
In the specific example under consideration, it was proposed that a few hookeri
plastids entered the lamarkiana egg with the sperm nuclei. Once present in the egg
cytoplasm, the hookeri plastids segregated randomly into meristamatic cells during
embryogeny. These cells eventually formed the green areas in the adult plant. The
hookeri plastids could become green, since they were able to function properly in
association with the velans.hhookeri nucleus; although the lamarkiana plastids were
defective in this same association.
Renner (1936) further concluded that differences between plastid races must be
caused by genetic differences within the plastids themselves, and not by any other
factors in the cytoplasm. Many different crosses showed that plastids derived from
either pollen or egg, from species A, were defective in combination with specific
hybrid genomes formed with species B. This was irrespective of which species
provided the egg cytoplasm to the hybrid zygote. It was proposed that these genetic
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differences between plastid types must have come about by mutation. As evidence,
Renner reported finding six separate instances of naturally occurring plastid
mutations. These mutants were incapable of becoming green in any genetic
environment to which they could be transferred. These were definitely plastid
mutations rather than nuclear gene mutations since each was shown to be inherited
exactly as were the plastid differences previously discussed. Renner estimated that
these mutations were found in .0005 of the examined plants.
Stubbe (1957) provided further evidence that Oenothera plastids differ genetically
from one another in the same cellular environment. He made cytological studies of the
early developmental stages of hybrid plants, when mixtures of plastid types could still
be found within single cells. The two plastid types could be easily distinguished when
they occurred in the same cell, or after segregation into different cells. In one
experiment he was able to produce a hybrid possessing three distinct plastid types. In
this plant Stubbe found areas where all three plastid types could be seen in the same
cell. The existence of several plastid classes in the same cell, under the identical
cytoplasmic conditions after many cell divisions, certainly indicates that different
genetic determiners reside within individual plastids.
The long term genetic stability of Oenothera plastids was elegantly proved by
Schwemle et al. (1938). The experiment was begun by crossing a particular plastid
type into association with a foreign genome where the plastids became defective. This
produced an obviously weak plant. These weak hybrid plants were selfed and
propagated sexually for up to l4 generations of the host plant. After a few generations
the hybrid lines gradually became fully green and recovered vigor. One might have
assumed that the defective plastids adapted to the new genome, but suitable
outcrosses showed that it was actually the hybrid genome which adapted to the
plastids! Plastids transferred from the adapted line into the same, but not adapted,
genome produced a phenotype identical to the the original weak hybrid. Plastids
carried for several generations with the hybrid genome also immediately recovered
their normal appearance when crossed back into their normal nuclear association, thus
indicating that the plastid's genome was not changed after thousands of duplications
during long association with a foreign nuclear genome.
The independent nature of the Oenothera plastids has also been used as a taxonomic
tool for the classification of plastid classes and for the determination of nuclear
genome relationships. Schotz (1954) used a fortuitous plastid mutation which was
incapable of becomming green in any nuclear combination as a standard for
comparing division rates of naturally occurring plastid classes. In some tests two
different plastid classes could be compared to the mutant type in association with
identical nuclear genotypes. Different characteristic rates of division were found for
different classes of plastid in this study.
Stubbe (1959) recognized five distinct classes of plastid after studying more than 500
genome combinations involving l4 distinct Oenothera races. These races were
grouped into "superspecies" based on their characteristic plastids He also found that
the genomes of the Renner complexes could be placed in three classes according to
the effects combinations of them had on the five plastid classes.
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I believe the material just presented is entirely adequate to prove that plastids of the
genus Oenothera have inheritable factors which are clearly independent of the nuclear
genome. This inheritance must also depend on genetic information found within each
of the individual plastids. However, before this conclusion is entirely accepted, can
any alternative explanations be found, or can the work be invalidated?
One might question the reliability or precision of the investigators, or the care with
which the genetic experiments were performed. Although I am not able to criticize the
original work, since most is in German, I don't think this would be reasonable. I think
it would be difficult for several independent investigators to reach the same
conclusion or to make the same mistakes. Erroneous conclusions are particularly
doubtful in the light of the many control experiments reported.
Some experiments were specifically designed to rule out any cytoplasmic factors that
might influence inheritance of the plastid phenotype. Also, it does not seem likely that
any possible external or internal influence could cause phenotypically distinct plastids
to be inherited, particularly when these types could be found in the same cell.
It is remotely possible that phenotypic effects like those observed could be caused by
viruses. However, the reported results would seem to require every plastid in all
Oenothera lines to be infected by virus in order to explain the taxonomic distribution
of plastid phenotype classes. If present, these hypothetical viruses would necessarily
seem to be temperate in nature since there is no indication that viruses of one cell ever
infect plastids in another cell, or even other plastids in the sane cell.2 Finally, if
temperate viruses were actually present in all of the chloroplasts, they would seem to
require the presence of an endogenous genetic system for transmission. Since none of
these alternatives provide reasonable explanations for the data, there is no alternative
but to assume that Oenothera plastids are genetically distinct entities.
If plastids are really genetically distinct, they should be mutable, and the mutant
plastids and their progeny should segregate at each cell division and not just in
meiosis as do nuclear genes. Some of the Oenothera experiments seemed to indicate
mutation and mitotic segregation, but much better evidence is available.
Recently, Michaelis (1959) reviewed some of his studies of the inheritance and
segregation of cytoplasnic mutations in Epilobium, a genus related to Oenothera. The
inheritable nature of Epilobium plastids had already been demonstrated (Michaelis,
1949; 1958b). In the 1959 paper, Michaelis discussed statistically tested cytological
observations which were made on the distribution and segregation of mutant plastids
during the growth of individual plants. Since the natural mutation rate for plastids in
this species was low (.0008 in 68,699 cultivated plants), plastid mutations were
artificially induced by exposing experimental plants to ionizing radiation which was
enough to raise the plastid mutation rate by a factor of 10 (Michaelis, 1958c).
Plastid mutations were studied in 172 plants where the radiation had induced a
chlorophyll variegation. Wherever the abnormal plastid condition reached the flowers
of the variegated plant, the inheritance of the trait was tested. In all of these cases, the
mutation was inherited only maternally, indicating that it was non-nuclear. A
statistically tested examination of the distribution of leaf variegation in 128 of the
studied plants showed that areas of mutated chloroplasts were randomly distributed in
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all but seven cases. This was taken to indicate that specific developmental processes
were not involved.
Michaelis was unable to distinguish cytological differences between mutant and
normal plastids until they began to become green in the maturing leaf. Cytological
study of mature leaves from all of the 172 variegated plants showed 50 where
individual, cells contained both plastid types, 92 where plastid differences were
cytologically indistinguishable, and 30 plants where no mixed cells could be found. In
this latter case all plastids in a given cell had either the mutant or normal phenotype.
Assuming random assortment of plastid types at each mitotic division, statistical
models predicted that sister cells should have similar ratios of mutant to normal
plastids, and that deviations from this ratio should follow certain distributions.
Cytological examination . of leaves containing mixed cells showed that the plastid
types were distributed as expected. Thus, implying that genetically distinct plastids
were segregating at random.
Starting with different numbers of segregating units, statistical predictions were made
of the distribution of homoplastidic normal, heteroplastidic, and homoplastidic mutant
cells to be expected after various numbers of mitotic divisions. For low numbers of
segregating units, the theory predicted that the cell divisions of one individual plant
should be adequate to produce fixation of plastid types in most cells. In theory, these
predictions would allow the discrimination of effects caused by chloroplast mutations
from effects possibly caused by different segregating factors in the cytoplasm.
However, for the test to be useful, the point of mutation and the number of subsequent
cell divisions must be known. Naturally, practical application of the theory is difficult,
since it is usually impossible to localize the point of a mutation.
However, Michaelis found a particularly fortunate event. A back mutation was
observed in a leaf growing from a purely mutant sector of a variegated plant. It was
determined that the mutation event must have occurred during one of the leaf
primordium's first cell divisions. It was also known, within an order of magnitude,
how many cell divisions were required for the formation of the mature leaf. Cell
families were studied to determine how many cell divisions were required to sort out
the progeny of the mutant plastid. The cytological observations were then compared
with the statistical tables. The comparison indicated that more than 10 and less than
20 segregating units must have been assorted. The average number of plastids in
meristematic cells of this species was observed to be 12, with a range of 5 to 20;
which was certainly within the limits of the statistical prediction. Plastids were the
only cytoplasmic units present in so few numbers. Mitochondria were present in the
next lowest numbers, but were so common that segregation of a mitochondrial mutant
would have taken much longer. Michaelis concluded that segregation of a mutant
plastid could be the only cause of the observed varigation.
After this test Michaelis examined the thirty cases of plastid variegation where no
mixed cells were found. He proposed that mutant and non-mutant plastids were still
segregating in early mitotic divisions. But, in these instances, the plastids within the
sane cell might interact through the exchange of diffusible substances. He suggested
two possible ways the plastids could influence one-another. First, the mutant plastid
might manufacture a substance that damaged the normal plastids during the
maturation process. In the other type of interaction, the normal plastid might
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manufacture a substance that would allow the mutant form to develop normally.
Theory predicted that the two types could be distinguished clearly by an examination
of the distribution of mutant and normal cells. Michaelis found clear examples of both
types of mutation in his material.
Burk, et. al. (1964) reported a similar study of the somatic segregation and
histogenesis of a plastid controlled variegation in tobacco. These authors made a
detailed study of the propagation and genetics of a naturally occurring plastid
mutation. They found that the heteroplastidic condition seemed to be preferred in
meristematic tissue--i.e. few cells tended to become homoplastidic. Also the mutant
condition dominated the normal plastid during the maturation process and suppressed
the normal plastid's phenotype if the mutants were present in excess.
The Burk article is also important because they thoroughly studied the distribution
and fixation of the mutation in the various histogenic layers of the plant. Burk et. al.
noted that Michaelis did not consider this particular aspect in his 1958 study. Also
many other studies of variegation in a wide variety of plants were reviewed. They
concluded that many variegations could be explained best by the early fixation of
plastid mutations in one or more histogenic layers. This is why so few examples of the
mutations have been confirmed by the finding of heteroplastidic cells. They remarked
that it would be unlikely for many mutants to be favored in the heteroplastic
condition.3
It should also be mentioned scattered evidence exists for plastids of the lower
eukaryote plants (those plants that have nuclear membranes and divide by mitosis)
also show genetic continuity. Granick (1955) reviewed most of the important breeding
and cytological studies concerning the lower plants. More recent plastid studies in
these plants have generally been aimed towards finding the physical and chemical
basis for plastid inheritance.
A summation of the plastid inheritance studies provides a reasonably clear picture of
the functional nature of the plastid associated genetic system.
1. This genetic system is very stable-being propagated for thousands of
replications without observable change.
2. At a minimum, the genetic system regulates certain chemical and
morphological aspects of chloroplast development.
3. The genetic system is subject to occasional mutations, which may affect
developmental or growth processes in diverse fashions.
4. The genetic system is discrete and located within each of the plastids.
5. The plastid traits seem to show clonal inheritance and segregation within the
cellular environment.
6. The plastid's genetic system is completely isolated from the nuclear genome.4
7. The plastid genomes can, and do, undergo evolutionary changes, like the
formation of species, apparently as an adaptive response to an evolving
cytoplasm.
This report has presented good experimental verification for all of these aspects of the
plastid specific genetic systems. However, it is realized that the experiments reviewed
here certainly do not represent all of the available work, and it is also realized that
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much of the published work is ambiguous or misleading. Finally, it is known that
some of the papers contradict the statements just made. Yet the evidence presented
still seems to be valid, and being valid, still needs an explanation.
Other Evidence
Many investigators, particularly before good cytological techniques were developed
for the electron microscope, have claimed that chloroplasts are derived from the cell
nucleus. Or, they have claimed that chloroplasts are the specialized progeny of
mitochondria, which were derived from the nucleus. Other authors claim a de novo
origin. Weier (1963) and Granick (1963) refute these ideas. In the first place, the
genetic evidence indicates that the plastid is inherited independently. In the second
place, good cytological studies (where proplastids and mitochondria were
distinguished) never show any merging of nucleus and plastid or of mitochondria and
plastid. Recent cytological studies have shown that proplastids and promitochondria
are always distinctly different objects in good electron microscope preparations.
Jensen (1965) clearly showed distinct proplastids and mitochondria in the plant egg
cell. Other papers show plastids in the pollen tube (Hanson, 1965 [missing
reference]). Many published electron micrographs show plastids in the process of
division. Green (1964) took clear timelapse micro photographs of the division of
Nitella plastids covering more than one replication cycle. In short, there is abundant
cytological evidence that the plastids are in fact derived from previously existing
plastids by fission, and not from any other source.
Since the genetic stability and low mutation rate which are characteristic of the plastid
genomes are also characteristic of the genetic systems of free-living organisms, one
would expect both of these systems to have the same chemical foundation. Since all.
known organisms carry their hereditary information on DNA, one would expect to
find DNA within the isolated plastid if this unit is actually a functioning organism.
This has been demonstrated many times with a great variety of techniques. Swift
(1965) and Gibor (1965) reviewed earlier experiments. More recent studies
demonstrating plastid DNA were done by Hotta, et. al. (1965) and Shipp, et. al.
(1965).
The earliest experiments attempting to demonstrate the presence of DNA in isolated
chloroplasts were generally poorly controlled and inconclusive. Recent works have
been very precise and carefully controlled. For instance, control experiments are
frequently conducted to rule out the possibility of bacterial or mitochondrial
contamination, which generally involve the addition and subsequent separation of
known contaminants. Studies have been made on plastids taken from organisms
ranging from the phytoflagellates to spinach and tobacco. All of the recent
experiments have conclusively shown that the plastid DNA is, in fact, DNA; that it
generally differs from the nuclear DNA in buoyant density and base pair ratio x, and
that it does not hybridize with the nuclear DNA. This last experimental technique
proves there are few areas where the base sequences (or the genetic code) are similar
(Shipp, et. al. 1965).
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In addition to the studies of isolated chloroplasts, there are also several electron
microscope cytochemical studies (Ris and Plaut, 1962; Kislev, et. al, 1965). These
show the physical presence of DNA strands within plastids taken from a variety of
sources.
Studies have shown that chloroplasts can synthesize their own DNA in intact cells, in
anucleate cells, and also when the plastids are isolated in vitro. The incorporation of
various radioactive or isotopically tagged compounds into DNA within the plastid has
been demonstrated. DNA synthesis has been observed while various antibiotics and
other chemical inhibitors were used to insure that no information could be transferred
to the plastid through the cytoplasm. Kislev, et. al. (1965), Hotta, et. al. (1965),
Janowski (1965), and Shephard (1965) all performed experiments of this nature.
Endogenous DNA synthesis was also demonstrated in the following, more complex
experiments.
The endogenous synthesis of DNA, RNA, and proteins within a variety of plastids has
been shown by several investigators (Goffeau and Brachet, 1965; Schwartz, et. al.
1965; Sissakian, et. al. 1965; and Sheppard, 1965). The line of information transfer
from the plastids endogenous DNA to the production of specific proteins has been
traced. No part of this line depends on information transferred from the cell's nucleus.
Plastid ribosomes have been isolated from the cell's chloroplast faction (they
generally have a different sedimentation rate or buoyant density than the cytoplasmic
ribosomes) and used for the transcription of specific RNA messages into specific
proteins. Interestingly enough, in one such experiment (Schwartz, et. al. 1965) the
plastid ribosomes could accurately transcribe the message carried by a coliphage virus
RNA, while they only synthesized nonsense when programmed by a tobacco mosaic
virus RNA. This may be a very interesting result, since it is my understanding that
TMV RNA generally operates with cytoplasmic ribosomes of a eukaryote cell, while
the colliphage RNA would be adapted to operate with the ribosomes of a moneran
cell.
Discussion
The papers I have reviewed show that plastids do in fact have many attributes of
independent organisms. This is compatible with the idea that chloroplasts are
endosymbiotic descendants of primitive free-living photo-synthetic monerans, now
living in the cytoplasm of eukaryote plants. However, it is practically certain that
plastids are incapable of an extracellular existance, whatever their evolutionary origin
nay have been.
If plastids are desendents of free-living monerans, would one expect to find the close
metabolic relationships between plastids and eukaryote cells that are seen today?
The initial plastid-eukaryote association must have taken place in the Cambrian
period or earlier, since the earliest multicellular green algae and vascular plant fossils
are known from the late Cambrian. This would allow at least 5x108 years for
refinement of the symbiotic association. The presymbiotic plastid ancestors must have
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been competent to synthesize most, if not all, compounds needed for their structural
development and growth. After entering eukaryote cells, the plastid ancestors would
have a readily available and concentrated source of structurally and metabolically
important compounds surrounding then. The eukaryote cell would probably depend
on the plastid for energy fixation, and possibly for some complex synthetic activities.
Under these circumstances natural selection would probably favor increased
specialization of the partners. Mutations that increased the energy fixing abilities of
the primitive plastids would probably be most favored by selection, while there would
seem to be little selective value for the retention of metabolic pathways for the
synthesis of compounds also manufactured by the eukaryote cytoplasm. In fact,
photosynthetic efficiency could probably be increased most easily by the selective
elimination of "useless" metabolic machinery with the concurrent expansion of the
photosynthetic apparatus. In this reduction, the plastid would be likely to retain only
the genetic information needed to specify and control its specialized functions. This
could lead to an associated reduction in the amount of plastid DNA.
The elimination of "useless" plastid functions night give selective advantages to the
host cell which must be responsive to the external environment. If the plastids
incorporated compounds manufactured by the host cytoplasm, then the host nucleus
would have genetic control over these compounds. Therefore, environmental natural
selection operating on the nuclear genome, could directly affect plastid morphology
and function. Since most eukaryote organisms have sexual processes allowing rapid
evolution, which are apparently lacking in the plastids5 and the hypothetical plastid
ancestors (cyanophycae) transfer of genetic control to the host nucleus could have
considerable selective value.
Considering the selective factors just discussed, and assuming a moneran origin, a
highly evolved plastid should have little or no endogenous genetic system, and it
should contain, at most, only a few structures not directly active in its primary
functions. If no plastid genetic system remained, there would be no way to prove that
plastids ever had endogenous genetic systems. However, the papers previously cited
have shown that even the plastids of modem plants have enough of an hereditary
mechanism to be observed in genetic experiments and by cytochemical techniques.
The smallest estimate of the amount of chloroplast DNA (Gibor and Izawa, 1963) was
1x10-16 gm per plastid from Acctabularia. Other studies cited by Gibor and Granick
(1964) provide estimates of 1x10-16 to 10x10-16 gram DNA per higher plant plastid.
Gibor and Granick using data from the literature, calculated that Euglena plastids each
had 40x10-16 grams of DNA. Edelman, et. al. (1964), using their own data, calculated
that Englena chloroplasts each had a minimum of 12x10-16 grams of DNA. Gibor and
Granick observed that a DNA content of 1x10-16 was characteristic of some of the
more complex viruses. Edelman, et. al. remarked that an E. coli cell carried around
16x10-16 grams of DNA. This data suggests that the chloroplasts of the most primitive
flagellates probably carry almost as much genetic information as do some of the
plastid's free living relatives. The more highly evolved organisms, which probably
posses more highly evolved plastids, show a considerable reduction in the amount of
DNA that they carry. This is precisely the picture that would be expected if the
chloroplasts were derived from a free-living moneran ancestor.
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Of course the acceptance of this hypothesis would cause some unique taxonomic
problems. Dillon, (1962), Lwoff (1951), Hutner and Provasoli (1951), Goodwin
(1964), as well as many others, propose that phytoflagellates and therefore higher
plants also, are derived as a whole (they didn't think of the other possibility) from the
blue-green algae [monerans lacking nuclear membranes], because of many chemical
and structural similarities between their photosynthetic systems. Since no other
properly photosynthetic forms are known, almost no one has considered any
alternative possibilities. Obviously, the idea that only the photosynthetic part of the
eukaryote cell is derived from the photosynthetic moneran is incompatible with the
idea that the whole eukaryote cell is derived from that source. Since there is so much
evidence supporting the endosymbiotic nature of the plastid, perhaps there is
something wrong with those taxonomical assumptions.
Acceptance of the moneran origin of the plastid would also cause difficulties for the
protozoan taxonomist. The viewpoint of many protozoologists (Kudo, 1954, Hall,
1953, Pitelka, 1963, Lwoff, 1951, Hutner and Provasoli, 1955, etc.) is that eukaryote
heterotrophic protozoans were initially derived from the photosynthetic eukaryote
cells. This viewpoint, of course, leaves the eukaryote cell without any point of origin.
Therefore, if the Ris hypothesis is accepted for consideration, it must be assumed that
the protozoans represent an independent line of evolution, separate from the
photosynthetic monerans. Then it would seem reasonable that this line provided the
host cells colonized by the presumptive plastids.
Where and how might this second line have started? (Don't ask the modern
protozoologists though. Apparently they haven't seriously considered the problem
since it was settled during the 30's and 40's (Lwoff, 1951).) Before 1931,
protozoologists used a simple rule-of-thumb to decide which of two organisms had
the most primitive characteristics:6 The simpler of the two organisms was considered
to be the closest to the primitive condition. As presently understood, the laws of
thermodynamics and information theory would probably provide theoretical support
for this viewpoint (Margalef, 1963).
Before the 1930's almost any protozoologist would agree that the amoeba-like
protozoans, because of their simplicity, must have been closer to the ancestral animal
condition than any other organism which could be examined with the light
microscope. In addition to the visible simplicity there are many other valid reasons,
for accepting this hypothesis.
As already noted, members of the order Amoebina have the simplest structure, on
both visible and ultrastructural levels. (Pitelka, 1963) Virtually all protozoan groups
can be derived by one, or at the most a few, steps of increasing specialization and
complexity from an ameboid ancestor. Testaceans are more complicated because they
have tests (Kudo, 1956). Actinopods are more complicated because randomly oriented
motility molecules are rearranged into orderly polymers (Kitching, 1964). Some of
the amoebina tend towards the development of polymerized, motile, pseudopodia
which are not flagellar in nature (Bovee, 1964). Some past amoeba probably found a
particularly successful spatial, arrangement of the polymer fibers leading to the
famous 9+2 pattern of the flagellum. Interestingly enough, in the sarcodinan groups
there are some other patterns reminiscent of flagellar structure in complexity
(Kitching, 1964, Roth, 1964).
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One very good clue to primitivencss is the absence of evidence hat any of the free-
living, non-testate Amoebina reproduce sexually;7 although sexual mechanisms are
found, in one form or another, in most of the other protozoan types. This distribution
would be expected if meiosis is a specialized and advanced type of mitosis.8 As one
further example, the flagellate mitotic and chromosomal apparatus seems to be much
more closely related to that of the higher plants and animals, than it is to the genetic
apparatus of some of the amoeba (Kudo, 1954, Cleveland, many papers, Saito, 1961,
McClellan, 1959). The Sarcodina seem to have the greatest variety of unique mitotic
apparatuses. One would expect to find just this experimentation with many forms in
the most primitive group. The more advanced groups would show specializations of
the more successful mitotic mechanisms.
In short, I think a little reflection will show that reasonably direct lines of
advancement and evolution nay be drawn to the modern protozoan groups, if the
amoeba is taken as the ancestral form. On the other hand, if the flagellates are
assumed to be ancestral; complex series of advancements and regressions must be
assumed in order to develop any reasonable protozoan phylogenies. The principle of
William of Occam, sometimes known as parsimony, tells one to choose the simplest
of the available alternatives.
For the sake of completeness, the large gap of structural complexity between the most
advanced living monerans, and the simplest photosynthetic eukaryotes should be
pointed out. Why are the lines of structural evolution so clear both above and below
this gap? If the eukaryote organism [as a whole] is derived directly from the
monerans, where are all of the intermediate structural forms? Dillon (1962) remarked
on this absence, and mentioned the wide nature of the gap.
Taking stock--it now appears that there were at least two independent lines of
evolution early in the phylogeny of life. The first line leading to the development of
non-motile producers reached its culmination with the development of the complex
synthetic apparatus characteristic of the blue-green algae. The second stock must have
had many characteristics of the amoebas. However, the presence of motility
compounds [i.e., macromolecules] in this line provided fertile substrates for the
evolution of complex kinetic structures, such as filaments, mitotic spindles,
microtubules, flagella and eventually muscle fibers.
Under many circumstances, the combination of advanced synthetic and kinetic
abilities could have great selective value. A symbiotic association would have many
obvious advantages over both the pure moneran or eukaryotc cell types.9 It is also
reasonable that this association, being so adaptive would have taken place many
times. In fact, it still happens with fair frequency, as is seen by the number of
symbionts known in metazoan animals (Barnes, 1963; Trager, 1960; Lederberg,
1952). Lederberg noted several recent cyanophyte endosymbioses in protozoans,
although he rejected the Famintzin, Moreschowski idea. The diatoms may also be an
independent line derived from an early sarcodinian offshoot, since its centriole is
totally unlike that found in most eucells (Drumm and Pankratz, 1963).
To this point we have been looking at the evolution of the chloroplast from a
viewpoint in the present. How would the situation appear if it were examined from a
viewpoint preceding the origin of life?
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Oparin has described the stage setting for this drama rather well. The atmosphere of
the abiotic world was a reducing one, probably high in simple hydrocarbons and
ammonia, with a liquid phase of water (Berkner and Marshall, 1965). This
atmosphere was bombarded with high energy radiation, which activated the simple
carbon and nitrogen compounds present. These activated compounds would then react
to lose the excess potential energy they carried. This process would lead naturally and
probably to the formation of large reservoirs of high potential, information rich,
organic compounds. These would be reasonably stabile, since there would be
practically no free oxygen available to attack them. It might be noted that the famous
experiments of Calvin, Miller, Fox, etc. confirm the probability of this process. It is
also interesting to note that Barghoorn (Barghoorn and Schopf, 1966) believes that he
may have an electronmicrograph of the fossilized evidence of these organic
compounds.
I think that almost anyone familiar with the Oparin hypothesis (1938) would agree
that the first "living" organisms were necessarily very simple heterotrophs which fed
on the geochemically produced biomolecules. Under these circumstances, the
heterotrophs would eventually consume their abiotic food supply, which would favor
the evolution of synthetic ability. If this path was actually followed, there may be
several living remainders. The chemosynthetic and photosynthetic bacteria seem to be
more primitive than the blue-green algae in their synthetic abilities.
It is interesting to note that none of these primitive producers have developed large
size or a successful system of motility. Why?
It is suggested that complex arrays of specifically organized organic molecules,
arranged in precise stearic configurations, are necessary for efficient photosynthesis.
Any system of motility that might disturb this stearic configuration would obviously
be maladaptive for an organism that depended on said configuration for its dinner.
Apparently the bacterial flagellum and the bending motion of Oscillatoria have been
the most successful attempts towards motility. Nowhere in these motile structures is
there anything that could reasonably give rise to either a cytoplasmic ameboid notion,
or to the highly complex and specific structural arrangement of the eukaryote
flagellum. It also seems that cytoplasmic motility night be a prerequisite for the
attainment of large cell size. The moneran cell size is probably limited by the internal
diffusion rate of important compounds. Any cytoplasmic motility for internal
circulation would probably disturb the stearic configuration of the synthetic
machinery and therefore be maladaptive. Again, the moneran cannot reasonably be
the ancestor of the [entire] eukaryote cell.
This leaves only one avenue to consider--one which has been completely forgotten
about or ignored in the literature. What kind of metabolism did the first "living"
organisms have?
These were heterotrophs, and probably had a fementative metabolism. These [first]
organisms must have made their living by making small alterations on abiotic
macromolecules in order to fit them to their needs. Initially this metabolism probably
required only a few enzymes, since the organism was a direct product of the abiotic
chemicals initially available. As the food supply became harder to obtain, two classes
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of adaptive alternatives would be available. The first alternative; development of
increasing enzymic specialization for synthesis has already been considered.
The second alternative would require only the development of a simple motility. I
think this is the easiest and most probable line of evolution from a proto-heterotrophic
organism. The only adaptive requirement needed for motility would be the
development of a somewhat specialized membrane and a large number of a few kinds
of [macro]molecules which could change shape when stimulated to do so by a change
in, say, ionic concentration. Even a motility sufficient to allow the proto-heterotroph
to roll around on the immediate substrate would have adaptive value, since this would
allow biomolecules to be sopped up from a large substrate surface, rather than
requiring diffusion to supply the food substances. This type of adaptation would
certainly not require as much information (or consequently as many genetic
mutations) as would be required for the development of the structural complexity of
the autotrophic producer. As long as some organisms specialized for autotrophic
production, the evolution of a motile heterotrophic line would seem to be a logical
necessity. Once this second line started, easy modifications would lead to
phagotrophy and the development of specialized kinetic structures, such as flagella
and the spindle apparatus. Involvement of the cytoplasm in motility would put an
immediate selective premium on the development of a system of internal membranes
to protect the genetic structures, an arrangement having little obvious value for the
reasonably akinetic monerans. A constantly changing and wearing membranous
system would place a selective value on the development of areas of membrane
manufacture, such as the dichtysomes or golgi bodies. In short, the evolutionary
origin and development of a proto-heterotrophic line of organisms would be expected
to lead to the evolution of just those organelles characteristic of the eukaryote cell.
The subsequent combination of these two lines to form the autotrophic eukaryote
would also be probable. Cytoplasmic motility and complex biosynthesis were shown
to be directly antagonistic functions. However, a combination of the two [within one
cell] would have some obvious advantages over either of the original states. For
instance, the combination of motility and photosynthetic ability must have opened the
great surface areas of the primordial oceans to autotrophic organisms (Berkner and
Marshall, 1965, Fischer, 1965). Before symbiosis, the autotrophic monerans, and
hence the heterotrophic organisms feeding on them, were probably limited to
substrate areas within the range of visual light and below the penetration of
ultraviolet. With the symbiotic combination of biosynthesis and motility, pelagic
communities night become possible or likely. This is a morphologically feasible
combination, since the moneran's external membrane would protect the stearic
configuration of its synthetic machinery from the kinetic sources of the eukaryote's
cytoplasm.
Summary
I have just barely scratched the surface of the ideas released by the tentative
acceptance of Hans Ris's hypothesis that the chloroplasts of higher plants are really
not part of the plant at all, but are, in fact, endosymbiotic microorganisms. Even if the
idea is totally wrong--which I doubt--it should be seriously considered for the new
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and untried experimental approaches and viewpoints that it can so abundantly
generate.
Perhaps a serious and considered study of the facts and understanding created by the
acceptance of this hypothesis would lead to some reasonable ideas concerning the
origin and evolution of the mitochondria, which also show some signs of genetic
independence.
Notes
1 It would seem that plastids from a given race might be able to function properly with
the chemical substrates elaborated under the direction of some genome combinations,
but become defective when placed in association with other combinations. This same
explanation could apply equally well to inherited plastid defects under Mendelian
control.
2 Someone reported unsuccessful attempts to transfer the hypothetical virus, but I can't
find the source at this writing.
3 This is, of course, the idea of natural selection. When two species compete for the
same habitat (in this case, within a single cell) one or the other will be eliminated.
Both types would be expected to remain in the same cell only if they were mutually
beneficial.
4 Rhoades (1955) says that specific gene loci may induce plastid mutations, but once
induced they are permanent. The iojap locus in corn provides an example. Regarding
the iojap locus, one should remember that specific nuclear gene loci are known which
increase the mutation rate at other nuclear loci.
5But see Gillham (1965). Gillham presented no evidence that these genes were
located in the chloroplast. Assortment of the mutant traits suggested some 40
equivalent units were involved; perhaps suggesting that the genes night be located in
mitochondria.
6 This consideration necessarily excluded forms specialized for parasitism.
7 With the possible exception of the coprozoic genus Sappina where part of the life
cycle seems very reminiscent of the primitive sexual process described for the
Bascidomyceete fungi (Darlington, 1958).
8 This does not rule out the possibility that other quasi-sexual mechanisms may allow
some genetic recombination to take place. There are several suggestions of possible
mechanisms that may be discussed in a further paper.
9 Fischer, (1965) discusses in detail the adaptive significances of this symbiotic
arrangement. In his article he only saw this value in terns of a zoochlorellar or
zooanthellar association, without considering what would happen to the endosymbiont
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over the course of a proposed 2.7 billion years. In short, his ecological analysis was
probably quite accurate, but its generality was missed.
Bibliography
Altmann, R. 1890. Die Elementarorganismen und ihre Beziehungen zu den Zellen.
Veit und Comp. 145 p.
Barghoorn, E.S. and W.J. Schopf. 1966. Microorganisms three billion years old from
the precambrium of South Africa. Science 152, 758-763
Barnes, Robert D. 1963. Invertebrate Paleontology. W.B. Saunders, Philadelphia. 632.
Berkner, L.V. and L.C. Marshall. 1965. On the origins and rise of oxygen in the
earth's atmosphere. Journal of Atmospheric Science, 22:225-261.
Bovee, R.C. 1964 (1963). Morphological differences among pseudopodia of various
small Amebas and their functional significance. in Allen, R.D. and N. Kamiya, Eds.
Primitive Motil Systems in Cell Biology. Academic Press, N.Y. 189-219.
Burk, L.G., R.N. Stewart and H. Dorman. 1964. Histogenesis and genetics of a
plastid-controlled chlorophyll variegation in tobacco. A.J. Bot. 51:713-724.
Caspari, R. 1948. Cytoplasmic inheritance. Adv. Genet. 2:1-66.
Cleland, R.E. 1962. Cytogenetics of Oenothera. Adv. Genet. 11:147-237.
Corliss, J.O. 1960. Comments on the phylogeny and systematics of the protozoa.
Systematic Zool. 8:169-190.
Darlington, C.D. 1958. The Evolution of Genetic Systems. Basic Books, Inc., New
York. 1958. 265 p.
Dillon, L.S. 1962. Comparative cytology and the evolution of life. Evolution 16:102-
117.
Drum, R.W. and H.S. Pankratz. 1963. Fine structure of a diatom centrosome. Science
142:61-63.
Edelmen, N., C.A. Cowan, H.T. Epstein, and J.A. Schiff. 1964. Studies on chloroplast
development in Euglena. VIII: Chloroplast associated DNA. Proc. N.A.S. 52:1214-
1219.
Famintzin, A. 1907, Die Symbiose als Mittel der Synthese von Organismen.
Biologisches Centralblatt 27:353-364.
Hall [1966] - Unpublished MS
http://tinyurl.com/29mlkh7
18
Fischer, A.G. 1965. Fossils, early life, and atmospheric history. Proc. N.A.S. 53:1205-
1215.
Geitler, L. 1959. ----------------- . Vol. 11:530-
Gibor, A. 1965. Chloroplast heredity and nucleic acids. Amer. Nat. 99:229-239.
Gibor, A. and S. Granick. 1964. Plastids and mitochondria: inheritable systems.
Science 145:890-897.
Gibor, A. and M. Izawa. 1963. The DNA content of the chloroplasts of Acetabularia.
Proc. N.A.S. 50:1164-1169.
Gillham, K.H. 1965. Linkage and recombination between nonchromosome mutations
in Chlamydomonas reinhardi. Proc. N.A.S. 1560-1567
Goffeau, A. and J. Brachet. 1965. Deoxyribonucleic acid-dependent incorporation of
amino acids into the proteins of chloroplasts isolated from anucleate Acetabularia
fragments. Biochim. Biophys. Acta 95:302-313.
Goodwin, T.W. 1964. The plastid system of flagellates. in Hutner, S.H. ed.
Biochemistry and Physiology of Protozoa III. Academic Press, N.Y., 319-339.
Granick, S. 1955. Plastid structure, development and inheritance. in Rhuland, W., ed.
Handbuch der Pflanzenphysiologie I. Springer-verlag, Berlin, pp. 507-564
Granick, S. 1963. The Plastids: their morphological and chemical differentiation. in
Cytodifferentiation and Macromolecular Synthesis. Academic Press, New York, pp
144-
Green, P.B. 1964. Cinematic observations on the growth and division of chloroplasts
in Nitella. A.J. Bot. 51:334-342.
Hall, R.P. 1953. Protozoology. Prentice-Hall, New York. 682 p.
Hotta, Y., A Bassel, and H. Stern. 1965. Nuclear DNA and cytoplasmic DNA from
tissues of higher plants J. Cell Biol. 27: 451-457.
Hutner, S.H. and L. Provasoli. 1951. The phytoflagellates. in Hutner, S.H. and A.
Lwoff ed. Biochemistry and Physiology of Protozoa, I. Academic Press, New York,
pp. 27-128.
Janowski, M. 1965. Synthese chloroplastique d'acides nucleiques chez Acetabularia
mediterrania. Biochim. Biophys. Acta 103:399-408.
Jensen, W.A. 1965. The ultrastructure and composition of the egg and central cell of
cotton. A.J. Bot. 52:781-791.
Kislev, N. H. Swift, and L. Oogorad. 1965. Nucleic acids of chloroplasts and
mitochondria in Swiss chard. J. Cell Biol. 25:327-344.
Hall [1966] - Unpublished MS
http://tinyurl.com/29mlkh7
19
Kitching, J.A. 1964. The axopods of the sun animalcule Actinophrys sol (Heliozoa).
in Allen, R.D. and N. Kamiya eds. Primitive Motile Systems in Cell Biology.
Academic Press, N.Y. pp. 445-455.
Kudo, R.P. 1954. Protozoology. Charles C. Thomas, Springfield, Ill. 966 p.
Larson, D.A. 1965. Fine-structural changes in the cytoplasm of germinating pollen.
A.J. Bot. 52:139-154.
Lederberg, J. 1952. Cell genetics and hereditary symbiosis. Physiol. Rev. 32:403-430.
Lwoff, A. 1951. Introduction to biochemistry of protozoa. in Hutner, S.H., and A.
Lwoff eds. Biochemistry of protozoa I. Academic Press, N.Y. pp. 1-26.
McClellan, J.F. 1959. Nuclear division in Pelomyxa illinoisensis Kudo. J. Protozool.
6:322-331.
Margalef, R. 1963. On certain unifying principles in ecology. Amer. Nat. 97:357-374.
Mereschkowsky, C. 1905. Ueber Natur und Ursprung der Cromatophoren im
Pflanzenreiche. Biol. Centralbl. 25:593-604.
Michaelis, P. 1949. Ueber Abanderungen des plasmatischen Erbgutes. ZZschr. indukt.
Abstamm. 83:36-85.
Michaelis, P. 1958b. Ueber die Bedeutung des plasmatischen Erbgutes fur die
Mannigfaltigkeit. Biol. Zbl. 77:165-196.
Michaelis, P. 1958c. Untersughungen zur Mutation plasmatischer Erbtrager,
besonders de Plastiden. Planta 51:600-634, 722-756.
Michaelis, P. 1959. Cytoplasmic inheritance and the segregation of plasmagenes. X.
International Congr. Genet. I. 375-385.
Oparin, A.F. 1938. The Origin of Life. Dover Publications, N.Y. 270 p.
Pitelka, D.R. 1963. Electron-Microscope Structure of Protozoa. MacMillan Co., N.Y.
269 p.
Renner, O. 1924. Die Schakung der Oenotherenbastarde. Botan. Zentr. 44:309-336.
Renner, O. 1936. Zur Kenntnis der nichtmendelinden Bunheit der Laubblatter. Flora
30:218-290.
Rhoades, M.M. 1955. Interaction of genetic and non-genetic hereditary units and the
physiology of non-genic funcions. in Ruhland, W., ed., Encyclopedia of Plant
Physiology I. Springer-Verlag, Berlin. pp. 19-57.
Ris, H. 1961. Ultrastructure and molecular organization of genetic systems. Can. J.
Genet. and Cytol. 3:95-120.
Hall [1966] - Unpublished MS
http://tinyurl.com/29mlkh7
20
Ris, H. and W. Plaut. 1962. Ultrastructure of DNA containing areas in the chloroplast
of Chlamydomonas. J. Cell Biol. 13:383-391.
Ris, H. and R.N. Singh. 1961. Electron microscope studies on blue-green algea. J.
Biophys. Biochem. Cytol. 9:63-80.
Roth, L.F. 1964. Motile systems with continuous filaments. in Allen, R.D. and N.
Kamiya. eds., Primitive Motile Systems in Cell Biology. Academic Press, N.Y. pp.
527-546.
Saito, M. 1961. Studies in the mitosis of Euglena I. On the chromosome cycle of
Euglena viridis Ehrbg. J. Protozool. 8:300-307.
Schotz, F. 1954. Ueber Plastidenkonkurrenz bei Oenothera. Planta 47:182-240.
Schwartz, J.H., J.M. Eisenstadt, G. Brawerman, and N.D. Zinder. 1965. Biosynthesis
of the coat protein of coliphage f2 by extracts of Euglena gracilis. Proc. N.A.S. 53:
195-200.
Schwemele, J., E. Haustein, J. Sturm and M. Binder. 1938. Genetische und
Zytologische Untersuchungen an Eu-Oenotheran. Teil I bis VI. Z. Induktive
Abstammungs. u. Verbungslehre 75:358-800.
Shephard, D.C. 1965. An autoradiographic comparison of the effects of enucleation
and actinomycin D on the incorporation of nucleic acid and protein precursors by
Acetabularia chloroplasts. Biochem. Biophys. Acta 108:635-643.
Shipp, W.S., F.J. Kieras and R. Haselkorn. 1965. DNA associated with tobacco
chloroplasts. Proc. N.A.S. 54:207-213.
Sissakian, N.M., L.I. Filippovitch, E.N. Svetailo and K.A. Aliyev. 1965. On the
protein-synthesizing system of chloroplasts. Biochim. Biophys. Acta. 95:474-485.
Stubbe, W. 1959. Dreifarbenpansschierung bei Oenotheren I. Entwischung von drei in
der Zygote vereinigten Plastidomen. Ber. Deut. botan. Ges. 70:221-226.
Swift, H. 1965. Nucleic acids of mitochondria and chloroplasts. Amer. Nat. 99:201-
227.
Trager, W. 1960. Intracellular parasitism and symbiosis. In Brachet, J. and A.E.
Mirsky eds., The Cell. Academic Press. N.Y. pp. 151-213.
de Vreis, H. 1913. Gruppenweise Artbildung, unter specieller Beruksichtigung der
Gattung Oenothera. Borntrager, Berlin. 365 p.
Weier, T.E. 1963. Changes in the fine structure of chloroplasts and mitochondria
during phylogenetic and ontogenetic development. A.J. Bot. 50:604-611.
Weier, T.E. and C.R. Stocking. 1952. The chloroplast: structure, inheritance and
enzymology II. Bot. Rev. 18:14-75.
Hall [1966] - Unpublished MS
http://tinyurl.com/29mlkh7
21
von Wettstein, D. 1961. Nuclear and cytoplasmic factors in development of
chloroplast structure and function. Can. J. Botany 39:1537-1545.
Hall [1966] - Unpublished MS
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