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CYTOLOGICAL EVIDENCE FOR
BIPARENTAL TRANSMISSION OF PLASTID DNA IN PISUM SATIVUM.
Corriveau, J. L, A. W. Coleman,
Division of Biology & Medicine
Brown University, Providence, RI USA
and N. 0. Polans
Department of Biological Sciences
Northern Illinois University, DeKalb, IL USA
Genetic evidence is available
describing the mode of plastid inheritance for some 60 angiosperm
genera (8,9). In the majority of these genera (including Pisum)
plastids are inherited maternally. As early as 1930, DeHaan (4) reported
that a chlorophyll deficiency was inherited maternally in Pisum
sativum. Since then, to our knowledge, DeHaan's observations have
been neither corroborated nor challenged.
DNA-fluorochromes are being used
increasingly in pollen biology (2,6). Recently, our lab reported a
DNA-fluorochrome/epifluorescence microscopy protocol which permits
rapid screening for plant species potentially capable of biparental
transmission of plastid DNA (1). When pollen was examined from plant
species known genetically to display biparental plastid transmission, e.g.
Oenothera biennis and Pelargonium zonale (9), plastid DNA
aggregates (plastid nucleoids) were detected in the cytoplasm of the
generative and/or sperm cells. However, in species known genetically
to display strictly maternal
transmission, e.g. Mirabilis jalapa and Nicotiana tabacum
(9), no plastid nucleoids were observed.
The purpose of the present study
is to determine if the cytological evidence for the mode of plastid DNA
transmission in Pisum sativum corroborates the earlier genetic report.
Mature pollen grains obtained
from greenhouse-grown pea plants were subjected to cytologica] analysis as
described by Coleman and Goff (2). Living pollen grains were t irst Incubated at
20-23C for 3 h in depression wells containing 0.5 ml germination medium
(20% sucrose plus 0.01% H3BO3 and 0.02%
CaCl2 in distilled water). Germinated pollen was then fixed in
95% ethanol:glacial acetic acid (3:1) overnight at 4C, before being
transferred to 70% ethanol for storage at 4C. Samples were prepared by
allowing a drop of fixed
pollen to dry on a slide followed by staining with 0.05 mkg/ml
4',6-diamidino-2-phenylindole (DAPI) in McIlvaine's buffer (pH 4). Observations
of DAPI-DNA fluorescence were made using a Zeiss AXI0PH0T epifluorescence microscope equipped
with a 50 W mercury lamp and the Zeiss 48-77-02 combination of excitation
and emission filters. DNase-treated controls served to monitor the
specificity of staining for
DNA. Pea plants were scored as potentially capable of biparental transmission of plastid DNA if plastid nucleoids were
observed in the cytoplasm of the generative cells of germinated pollen.
They were scored as presumably maternal if no plastid nucleoids were
observed. At least 100 pollen grains were examined for each pea
line.
Cytological evidence obtained
from eight pea accessions and cultivar Alaska (Table 1) suggests that
plastid DNA can be transmitted biparentally in P. sativum.
Plastid nucleoids were observed in the generative cells of germinated
pollen grains from each of the pea lines examined. Variability was
observed, however, among these lines with regard to both the
percentage of pollen grains potentially capable of transmitting
plastid DNA (Table 1), and the number of plastid nucleoids found per
generative cell |
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(Figs. 1
and 2). The percentage of pollen grains containing plastid nucleoids in generative cells varied from as
little as 17%, scored for pea accession A1078-234, to over 50% in
accession B78-259 and cultivar Alaska (Table 1). The variability observed in
plastid nucleoid number per generative cell is exemplified by cytological
observations made on pollen from accession A1078-234 and cultivar Alaska.
Among the 17 generative cells which contained plastid nucleoids in
A1078-234, the number of plastid nucleoids per generative cell ranged
from one to five with an average of only two per generative cell (Fig. 1). in
contrast, cultivar Alaska, which had plastid nucleoids in over 50% of the
generative cells scored, displayed between one and ten plastid nucleoids per
generative cell, with an average of 4.5 nucleoids per generative cell (Fig.
2).
Our lab has
developed a DAPI/epifluorescence microscopy protocol which permits the rapid screening of plant
species for the purpose of determining potential mode of plastid DNA
transmission (1). There is a striking correlation found between the
cytological results obtained using our protocol and results
obtained through corresponding genetic studies. Thus far, plastid nucleoids have been
detected in the generative and/or sperm cells of nine plant species known
genetically to display biparental inheritance of plastids, while
the absence of plastid nucleoids has been confirmed in 25 species known to exhibit
maternal inheritance (3).
With these
results in mind, we propose three possible explanations for the conflicting reports of maternal
inheritance of plastids by DeHaan (4) and of biparentalism in this study.
First, although there is ultra-structural evidence for the presence of
proplastids in the male reproductive cells of pea pollen (5),
there is also the possibility that plastid DNA is eventually eliminated. This loss
could occur during sperm cell formation and/or maturation, during
fertilization, or even in the zygote after fertilization (8). If paternal
plastid DNA is eliminated at any of these later stages and, therefore,
subsequent to our present observations, the final result would be strict maternal
inheritance of plastid DNA. Second, there is reason to question the
genetic evidence supporting maternal inheritance of plastids
in pea. Additional data from self-fertilizations and intercrosses, characterized by
both extended generations and larger sample sizes, is necessary to exclude
the possibility that the chlorophyll
deficiency trait reported by DeHaan was not a nuclear controlled plastid deficiency. In fact, chromosomal
irregularities such as the
behavior of chromosome fragments and unpaired alien chromosomes can
mimic results obtained from
authentic plastid DNA mutations (9). To eliminate the possibility of
nuclear-controlled plastid deficiency, the cyto-logical identification of mixed cells in
variegated plants, i.e. cells with both normal green and
chlorophyll-deficient plastids, would have served as a useful control.
Third, there is now genetic evidence that variability in plastid DNA inheritance exists within
species of Oenothera and Pelargonium (10). Similar
genetic variability for plastid inheritance might exist with Pisum as well. If
this is the case, DeHaan may have worked with a pea line which happened to
follow the maternal mode. Such a Finding would resolve the contradictions
between the cytological and genetic evidence. We are currently testing the
possibility of paternal plastid DNA inheritance in Pisum by
analyzing plastid DNA restriction fragment patterns in F1 progeny of crosses between
parents which differ recognizably in their plastid
DNA.
Worthy of
note is the observation that mitochondrial DNA was not
de- |
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Fig. 1. Plastid nucleoid number per
generative cell in germinated pollen from pea accession A1078-234, as
revealed by DAPI/epifluorescence microscopy. |
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Fig. 2.
Plastid nucleoid number per generative cell in germinated pollen from pea cultivar 'Alaska', as
revealed by DAPI/epifluorescence
microscopy. |
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tected
in the generative cells of germinated pea pollen. This cytological observation is in agreement with a recent
report which suggests that the cyanide-resistant pathway (which may be
under the control of the mitochondrial genome) is inherited maternally in pea
(7). |
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1.
Coleman, A W., J. L. Corriveau, and L. J. Goff. 1986. J. Cell Biol. 103:521A.
2. Coleman, A. W. and L. J. Goff. 1985. Stain
Technol. 60:145-154.
3. Corriveau, J. L. and A. W. Coleman. 1987.
Submitted to Amer. J. Bot.
4. DeHaan, H. 1930. Genetica
12:321-440.
5. Hause, G. 1986. Biol Zentralbl.
105:283-288.
6. Hough, T., P. Bernard, R. B. Knox, and.E.
G. Williams. 1985. Stain Technol.
60:155-162.
7. Musgrave, M. E., I. C. Murfet, and J. N.
Siedow. 1986. Plant Cell Envir.
9:153-156.
8. Sears, B. B. 1980. Plasmid
4:233-255.
9. Tilney-Bassett, R. A. E. 1978. The
Plastids. J. T. 0. Kirk and R. A. E. Tilney-Bassett, eds.
Elsevier/north-Holland, Amsterdam,
pp.251-524; 10. Tilney-Bassett, R. A. E. and 0. A. L.
Abdel-Wahab. 1979. Maternal Effects in Development. D. R. Newth and M,
Balls, eds. Cambridge Univ. Press, Cambridge, pp.
29-45.
Seed of the
pea accessions used in this study were supplied by G. A. Marx and seed of Alaska was obtained from
Carolina Biological. Research supported in part by NSF DCB-85-03942 and
USDA 87-CRCR-l-2534 to AWC.
Table 1.
Cytological evidence for potential plastid DNA transmission by the paternal parent in
pea. |
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