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0 Department of Biological Sciences, Box 5640, Northern Arizona University, Flagstaff, Arizona 86011 USA
Received for publication June 22, 1999. Accepted for publication September 30, 1999.
ABSTRACT
Studies using classic genetics as well as restriction fragment length polymorphism analysis have demonstrated that rye, unlike most flowering plants, has biparental inheritance of both plastids and mitochondria. Yet, a previous in-depth ultrastructural study found no plastids in rye sperm cells, and DNA-specific staining revealed no cytoplasmic DNA in the male gametes of this plant. In the present study, we examined serial ultrathin sections of eight rye sperm cells (four pairs) and found unambiguous examples of plastids in all cases. The number of plastids per sperm cell varies from two to 12. The sperm of a pair may vary with regard to plastid number; however, these differences are not consistent among the sperm pairs examined.
Key Words: cytoplasmic inheritance • Poaceae • Secale cereale • sperm cells • sperm dimorphism • three-dimensional reconstruction
Cytoplasmic organelles are inherited in a non-Mendelian fashion in all eukaryotic organisms investigated (reviewed in Kirk and Tilney-Bassett, 1978
; Birky, 1995
; Mogensen, 1996
). Plastids can be inherited strictly maternally, exclusively paternally, or biparentally. Most flowering plants studied have maternal plastid inheritance, but approximately one-third of the genera investigated display biparental plastid inheritance to some degree (Kirk and Tilney-Bassett, 1978
; Sears, 1980
; Whatley, 1982
; Corriveau and Coleman, 1988
; Hagemann and Schroder, 1989
; Smith, 1989
). Due to the scarcity of phenotypic markers, much less is known about mitochondrial inheritance, but so far it appears that strictly maternal transmission of mitochondria is the rule to an even higher degree among flowering plants than is maternal inheritance of plastids (Smith, 1989
; Harrison and Doyle, 1990
; Radetzky, 1990
; Forsthoefel, Bohnert, and Smith, 1992
; Rajora et al., 1992
).
The grasses appear to be typical among the flowering plants in that the vast majority of those studied exhibit maternal organelle inheritance (Fröst, Vaivars, and Carlbom, 1970
; Kirk and Tilney-Bassett, 1978
; Corriveau and Coleman, 1988
; Smith, 1989
). Only two species in the Poaceae, to our knowledge, have been reported to display biparental inheritance of plastids: rye (Secale cereale) and pearl millet (Pennisetum americanum).
In rye, Fröst, Vaivars, and Carlbom (1970)
demonstrated biparental plastid inheritance using a chlorophyll mutant. In the same species, Kirk and Tilney-Bassett (1978)
reported that 33% of the progeny received plastids from the male parent after crosses between green and variegated plants. Soliman, Fedak, and Allard (1987)
used restriction fragment length polymorphism analysis to demonstrate that hybrids resulting from crosses between barley (female parent) and three Secale species (male parents) inherited plastids, as well as mitochondria, biparentally. Only in the case of Triticale, which is the product of wheat (female parent) crossed with rye (male parent), does there appear to be no inheritance of male rye plastids (Vedel et al., 1981
).
In pearl millet, Krishna Rao and Koduru (1978)
studied a variegated phenotype and concluded that the mutant condition exhibited non-Mendelian, biparental inheritance. However, more recent studies (Subrahmanyam et al., 1986
; Reddy and Subrahmanyam, 1988
), also using stripe mutants of Pennisetum americanum, found only maternal plastid inheritance, casting doubt on the occurrence of biparental plastid inheritance in this species. Thus, rye may be the only grass known to exhibit biparental plastid as well as mitochondrial inheritance.
In conflict with the above genetic studies showing biparental inheritance of plastids in rye is the report, based upon a thorough ultrastructural study, that no plastids were found in either the generative or sperm cells of this plant, although mitochondria are abundant in these cells (Cass and Karas, 1975
). Furthermore, in a study using the DNA-specific fluorochrome, DAPI (4',6-diamidino-2-phenylindole), Corriveau and Coleman (1988)
did not detect plastid nucleoids (DNA aggregates) in the generative or sperm cells of Secale cereale. However, Hagemann and Schroder (1985)
have figured a putative proplastid in the generative cell of rye.
We undertook the present study in order to help clarify the apparent discrepancies between the genetic and cytological data. Using serial sections, electron microscopy, and computer-based three-dimensional reconstruction, we demonstrate clear-cut examples of plastids in rye sperm cells.
MATERIALS AND METHODS
Plants of Secale cereale L. cv. Rheidol were grown from seed in the greenhouse at Northern Arizona University. Grains were obtained from the National Small Grains Collection of the United States Department of Agriculture, Agricultural Research Service in Aberdeen, Idaho, USA. Rheidol has been the cultivar of choice for a number of important studies on sexual reproduction in rye (Heslop-Harrison, 1979
; Heslop-Harrison and Heslop-Harrison, 1980, 1981, 1982, 1987
; Shivanna, Heslop-Harrison, and Heslop-Harrison, 1982
).
Anthers were collected shortly before anthesis. They were then cut under moist conditions, transversely into thirds and placed immediately into freshly prepared 4% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 6.8, at room temperature. After 6 h in the fixative, the material was rinsed in the same buffer for 1 h (three changes), then post-fixed in 2% osmium tetroxide (same buffer) for 2 h at room temperature. After a 1-h rinse in distilled water (three changes), the material was dehydrated in a graded ethanol series, then gradually embedded (over a 4-d period) in Spurr's (1969)
low viscosity resin. Polymerization of the resin was carried out at 70°C for 12 h.
Serial ultrathin sections were cut at ~80 nm with a diamond knife and collected on single-slot, formvar-coated, carbon-stabilized grids according to a modification of the technique of Galey and Nilsson (1966)
. The sections were stained in an automatic LKB ultrostainer with uranyl acetate and lead citrate (30 min each).
Three-dimensional reconstruction was carried out using an Indy computer (Silicon Graphics Inc., Mountain View, California, USA) and the IMOD software program (Kremer, Mastronarde, and McIntosh, 1996
).
RESULTS
Eight sperm cells (four pairs) were serially sectioned, and ultrathin sections were observed in detail with the electron microscope. Ultrastructural details of the sperm cells are the same as those reported for rye by Cass and Karas (1975)
, including a double-membrane-bound heterochromatic nucleus, several small vacuoles, polysomes, endoplasmic reticulum, microtubules, dictyosomes, and numerous mitochondria (Figs. 1–3).
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One sperm pair and the vegetative nucleus were reconstructed in three dimensions to show the general organization of the sperm cells, their proximity to each other and to the vegetative nucleus, and the distribution of organelles (Fig. 4). In these examples, one sperm cell contains 12 plastids, while the other contains four plastids. Not all plastids are visible due to masking by the nucleus (Fig. 4).
Another sperm pair (data not shown) had three plastids in one sperm and two in the other. A third sperm pair contained four plastids in one sperm and two in the other. A fourth sperm pair had three plastids present in each of the cells.
As seen in the three-dimensional reconstruction (Fig. 4), the sperm cells are basically similar in shape, except that one has a cytoplasmic extension. The sperms of a pair were connected at one end in all four examples, but some distance always separated the vegetative nucleus from the sperm cells.
DAPI staining of sperm cells, either within whole pollen grains or in semithin sections failed to reveal any cytoplasmic DNA (data not shown).
DISCUSSION
Although it is sometimes difficult to positively identify plastids in their early stages of differentiation, we have confidence in our interpretations of this study. It is well known that the only organelles to possess a double membrane are the nucleus, mitochondria, and plastids, and all three of these organelles are clearly depicted in rye sperm cells. The primary criterion we used for distinguishing between mitochondria and plastids is the lack of cristae in plastids. Moreover, the plastids of this study are essentially similar to those identified in the sperm cells of Plumbago zeylanica (Russell, 1984
), in an enucleated cytoplasmic body of sperm origin in barley (Mogensen, 1988
), and in the generative and sperm cells of alfalfa (Zhu, Mogensen, and Smith, 1990, 1992
).
This study has clarified a discrepancy between genetic and cytological data regarding the presence or absence of plastids in rye sperm cells. Using serial-section electron microscopy, we have shown that all eight sperm cells (four pairs) examined contained at least two plastids, and as many as 12. Based on the results of the present study, most sperm cells of rye seem to contain between two and four plastids each. Thus, it is not surprising that, without the use of a complete series of ultrathin sections, the presence of plastids may be easily missed (Cass and Karas, 1975
).
Our results support the observation of Hagemann and Shroder (1985)
of a proplastid in the generative cell of rye and show that such plastids are retained within the sperm cells.
As was the case with the study by Corriveau and Coleman (1988)
, we were not able to demonstrate cytoplasmic nucleoids within rye sperm cells using DAPI staining on whole pollen mounts, or on semithin sections of embedded material. Other conflicting results between genetic and DNA-staining studies are known. Corriveau and Coleman (1988)
found no cytoplasmic nucleoids in either Phaseolus vulgaris or Nepeta cataria, yet both of these species are reported, on the basis of genetic studies, to exhibit biparental plastid inheritance (Parker, 1934
; Woods and DuBuy, 1951
). Corriveau and Coleman (1988)
suggest that the conflicting evidence may be explained by dubious interpretations from the genetic studies. That is, results from genetic studies may be due to nuclear-controlled plastid deficiencies or viral infection. However, at least in the case of rye, where both ultrastructural (present study) and molecular (Soliman, Fedak, and Allard, 1987
) studies show the existence of plastids in sperm, this explanation seems unlikely. It is possible that the sperm plastid DNA is in a decondensed or other state that renders it undetectable by DAPI staining. It is also possible that the plastid nucleoids stain with DAPI, but are not detected due to their small number as well as their often close proximity to the nucleus, whose own DAPI staining would mask that of the plastid nucleoids. Perhaps the conflicting results between the genetic, ultrastructural, and DNA studies of rye are due to varietal differences in the plants used; however, the fact that Soliman, Fedak, and Allard (1987)
found male plastid and mitochondrial transmission in three species of Secale argues against this.
Although the sperm cells of a pair in rye are not identical, the degree of dimorphism appears to be minimal, especially when compared to that found in some other species (reviewed in Mogensen, 1992
). In Plumbago zeylanica, for example, the smaller sperm of a pair contains an average of 24 plastids, whereas the larger sperm, which has a long cytoplasmic extension, usually contains no plastids (Russell, 1984
).
All of the sperm pairs observed in the present study were connected at one end. Likely, these examples represent a stage just prior to sperm separation, since the sperms are known to be separate at anthesis (Cass and Karas, 1975
; Mogensen and Rusche, unpublished data).
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1 The authors thank Traci Rogers for excellent help with the three-dimensional reconstruction, Brad Blake for professional help with growing the rye plants, and the Organized Research Fund of Northern Arizona University for support. 
LITERATURE CITED
Birky, C. W. Jr. 1995 Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution. Proceedings of the National Academy of Sciences, USA 92: 11331–11338.
Cass, D. D., and I. Karas. 1975 Development of sperm cells in barley. Canadian Journal of Botany 53: 1051–1062.
Corriveau, J. L., and A. W. Coleman. 1988 Rapid screening method to detect potential biparental inheritance of plastid DNA and the results for over 200 angiosperm species. American Journal of Botany 75: 1443–1458[CrossRef][ISI]
Forsthoefel, N. R., H. J. Bohnert, and S. E. Smith. 1992 Discordant inheritance of mitochondrial and plastid DNA in diverse alfalfa genotypes. Journal of Heredity 83: 342–345
Fröst, S., L. Vaivars, and C. Carlbom. 1970 Reciprocal extrachromosomal inheritance in rye (Secale cereale L.). Hereditas 65: 251–260.[ISI]
Galey, F. R., and S. E. G. Nilsson. 1966 A new method for transferring sections from the liquid surface of the trough through solutions to the supporting film of a grid. Journal of Ultrastructural Research 14: 405–410.[CrossRef]
Hagemann, R., and M. B. Schroder. 1985 New results about the presence of plastids in generative and sperm cells of Gramineae. In M. T. M. Willemse and J. L. van Went (compilers), Proceedings of the 8th international symposium on sexual reproduction in seed plants, ferns, and mosses, 20–24 August 1984, Wageningen, The Netherlands, 53–55. Pudoc, Wageningen, The Netherlands.
———, and ———. 1989 The cytological basis of the plastid inheritance in angiosperms. Protoplasma 152: 57–64.[CrossRef][ISI]
Harrison, R. G., and J. J. Doyle. 1990 Redwoods break the rules. Nature 344: 295–296.[CrossRef]
Heslop-Harrison, J. 1979 Aspects of the structure, cytochemistry and germination of the pollen of rye (Secale cereale L.). Annals of Botany 44: Supplement(1): 1–47.
———, and Y. Heslop-Harrison. 1980 The pollen-stigma interaction in the grasses. 1. Fine-structure and cytochemistry of the stigmas of Hordeum and Secale. Acta Botanica Neerlandica 29: 261–276.
———, and ———. 1981 The pollen-stigma interaction in the grasses. 2. Pollen-tube penetration and the stigma response on Secale. Acta Botanica Neerlandica 30: 289–307.
———, and ———. 1982 The growth of the grass pollen tube. 1. Characteristics of the polysaccharide particles ("P-particles") associated with apical growth. Protoplasma 112: 71–80.[CrossRef][ISI]
———, and ———. 1987 An analysis of gamete and organelle movement in the pollen tube of Secale cereale L. Plant Science 51: 203–213.[CrossRef][ISI]
Kirk, J. T. O., and R. A. E. Tilney-Bassett. 1978 The plastids: their chemistry, structure, growth and inheritance. Elsevier/North-Holland Biomedical Press, New York, New York, USA.
Kremer, J. R., D. N. Mastronarde, and J. R. McIntosh. 1996 Computer visualization of three-dimensional image data using IMOD. Journal of Structural Biology 116: 71–76.[CrossRef][ISI][Medline]
Krishna Rao, M., and P. R. K. Koduru. 1978 Biparental plastid inheritance in Pennisetum americanum. Journal of Heredity 69: 327–330.
Mogensen, H. L. 1988 Exclusion of male mitochondria and plastids during syngamy in barley as a basis for maternal inheritance. Proceedings of the National Academy of Sciences, USA 85: 2594–2597.
———. 1992 The male germ unit: concept, composition, and significance. International Review of Cytology 140: 129–147.[CrossRef]
———. 1996 The hows and whys of cytoplasmic inheritance in seed plants. Amererican Journal of Botany 83: 383–404.[CrossRef][ISI]
Parker, M. C. 1934 Inheritance of a leaf variegation in the common bean. Journal of Heredity 25: 165–170.
Radetzky, R. 1990 Analysis of mitochondrial DNA and its inheritance in Populus. Current Genetics 18: 429–434.[CrossRef][ISI]
Rajora, O. P., J. W. Barrett, B. P. Dancik, and C. Strobeck. 1992 Maternal transmission of mitochondrial DNA in interspecific hybrids of Populus. Current Genetics 22: 141–145.[CrossRef][ISI][Medline]
Reddy, M. K., and N. C. Subrahmanyam. 1988 Inheritance and transmission of plastid alterations in a green-white stripe mutant of pearl millet (Pennisetum americanum). Canadian Journal of Botany 66: 179–182.
Russell, S. D. 1984 Ultrastructure of the sperm of Plumbago zeylanica. II. Quantitative cytology and three-dimensional organization. Planta 162: 385–391.
Sears, B. B. 1980 Elimination of plastids during spermatogenesis and fertilization in the plant kingdom. Plasmid 4: 233–255.[CrossRef][ISI][Medline]
Shivanna, K. R., Y. Heslop-Harrison, and J. Heslop-Harrison. 1982 The pollen-stigma interaction in the grasses. 3. Features of the self-incompatibility response. Acta Botanica Neerlandica 31: 307–319.[ISI]
Smith, S. E. 1989 Biparental inheritance of organelles and its implications in crop improvement. Plant Breeding Reviews 6: 361–393.
Soliman, K., G. Fedak, and R. W. Allard. 1987 Inheritance of organelle DNA in barley and Hordeum X Secale intergeneric hybrids. Genome 29: 867–872.
Spurr, A. R. 1969 A new low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructrual Research 26: 31–43.
Subrahmanyam, N. C., M. Satyaprasad, S. A. Rao, and M. H. Mengesha. 1986 Pattern-dependent maternal plastid inheritance in Pennisetum americanum. Canadian Journal of Botany 64: 1081–1085.
Vedel, F., F. Quetier, Y. Cauderon, F. Dosba, and G. Doussinault. 1981 Studies on maternal inheritance in polyploid wheats with cytoplasmic DNAs as genetic markers. Theoretical and Applied Genetics 59: 239–245.[ISI]
Whatley, J. M. 1982 Ultrastructure of plastid inheritance: green algae to angiosperms. Biological Review 57: 527–569.[CrossRef]
Woods, M. W., and H. G. DuBuy. 1951 Heredity and pathogenic nature of mutant mitochondria in Nepeta. Journal of the National Cancer Institute 11: 1105–115.
Zhu, T., H. L. Mogensen, and S. E. Smith. 1990 Generative cell composition and its relation to male plastid inheritance in Medicago sativa. Protoplasma 158: 66–72.
———, ———, and ———. 1992 Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology. European Journal of Cell Biology 59: 211–218.[ISI][Medline]
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