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A unique pollen wall mutation in the family Compositae: ultrastructure and genetics¹

² Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131 USA; ³ Oklahoma Biological Survey and Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, Oklahoma 73019-6131 USA; and ⁴ Samuel Roberts Noble Microscopy Laboratory, University of Oklahoma, Norman, Oklahoma 73019-6131 USA

Received for publication November 9, 1999. Accepted for publication February 10, 2000.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
During a routine screening of pollen fertility in the n = 2 chromosome race of Haplopappus gracilis, a spineless pollen wall mutation was discovered that renders the otherwise functional pollen grains completely unrecognizable as Compositae pollen. Normal Haplopappus pollen is characterized by an outer layer, the ektexine, consisting of large spines supported by a roof (tectum), which in turn is supported by collumellae that are joined basally. A large cavity (cavea) stretches from aperture to aperture and separates columellae bases from the final ektexine unit, the foot layer. The spines, tectum, columellae, and columellae bases are filled with perforations (internal foramina), while the foot layer is without them. Immediately underlying the foot layer is a thickened, lamellate, disrupted, internal foramina-free second exine layer, the endexine. In contrast, the mutant pollen ektexine is a jumble of components with randomly dispersed spines as the only clearly definable unit. The endexine layer is similar to the endexine in normal pollen. The mutation apparently disrupts only the organization of ektexine units, and mutant pollen appears to be without the caveae and foot layer characteristic of normal pollen. In genetic tests, the mutant allele is recessive. There is a simple Mendelian pattern of inheritance of the mutant gene, and its phenotype is under sporophytic control.

Key Words: cavea • Compositae • ektexine • endexine • foot layer • Haplopappus • internal foramina • mutant pollen

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The large family Compositae has several distinct patterns of pollen wall ornamentation such as Helianthoid, Senecoid, Anthemoid, etc. (Skvarla and Turner, 1966 ; Skvarla et al., 1977 ), many of which allow more efficient cross pollination by insects. Although genera and tribes are characterized by distinctive pollen wall architecture, little is known about intraspecific variation and genetic control of the structural differences. In this paper, we describe a pollen wall mutation in the n = 2 chromosome race of Haplopappus gracilis (Nutt.) Gray (Tribe Astereae) that renders the otherwise functional pollen grains completely unrecognizable as Compositae pollen.

As with many other Astereae, normal H. gracilis pollen has a surface of conspicuous densely packed spines, while the mutant surface is spineless, covered instead with randomly dispersed angular and globular fragments. A particular plant produces either normal or mutant pollen but not both, and the distinction between the two types is easily recognized at low power (125x) of a compound microscope. Because of the ease of phenotypic recognition and the small number of chromosomes (Jackson, 1957 ), this mutation will be useful for future genetic analyses designed to locate additional markers on the two linkage groups of the species as described by Jackson (1964) .

Our objectives were to characterize and compare normal and mutant pollen wall ultrastructural differences by scanning and transmission electron microscopy (SEM and TEM) and to determine the inheritance pattern of the mutant trait. Our results should encourage further studies of pollen wall variation within and among populations and lead to a better understanding of pollen wall evolution in the Compositae. One future question to be answered is whether different pollen wall structures within the family are due to single gene or to quantitative effects.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Plant culture
Haplopappus gracilis seeds were collected by R. C. Jackson from a roadside population (No. 8208) in 1989 in central Archuleta County, Colorado (where populations often are transient) and another collection in 1990 from Maricopa County, Arizona (No. 8222), and stored in a refrigerator at 0°C until used. Seeds were germinated in distilled water at room temperature. Seedlings 10–15 mm long were transplanted to hot-water-expanded Jiffy-7 peat pots (Jiffy Products Ltd., Norway) in white plastic dishpans, covered with clear plastic with aeration under light for 3 d, and then grown with small amounts of water without the cover under artificial light until roots were visible. The peat pots were then transferred to 8-inch soil-filled pots and the plants grown in a greenhouse until maturity.

Mutant source
Most plants from population No. 8208 seeds had normal pollen with spines (Fig. 1), but a spineless pollen mutant (Fig. 2) was isolated, propagated by further crosses, and used in the hybridization program.

View larger version (159K): [in this window] [in a new window]   Figs. 1–6. Scanning electron micrographs of normal (Fig. 1 ) and mutant (Figs. 2–6 ) H. gracilis pollen. 1. Polar view showing the characteristic, densely spined pollen surface. Note pillared arrangement of spine bases in the central spines. 2. Polar view showing a smooth surface containing randomly dispersed globular and angular exine fragments mainly aggregated in the interapertural regions (a). The sporopollenin fragments appear to show a faint striate-like surface sculpturing. 3. Equatorial view with aperture region (a) surrounded by coarse exine fragments. 4. Lateral view with similar fragments as noted in polar (Fig. 2 ) and equatorial views (Fig. 3 ). 5, 6. Enlarged globular and angular fragments of material noted in Figs. 2–4 are composed of linear and spongy-appearing sporopollenin. In Fig. 6 at least three spine tips (s) are dispersed among the fragments. All pollen acetolyzed. All scale bars = 1 µm

Monohybrid crosses were made between spineless pollen plants from No. 8208 and spined pollen plants from No. 8222. F1 hybrids were intercrossed to produce 46 F2 seedlings.

Dihybrid crosses were made later between plants with spineless pollen and normal stems and those with normal (spined) pollen and fasciated stems. The fasciated gene is recessive as analyzed by Mangum (1992) and was localized to chromosome A (linkage group 1).

Electron microscopy
Buds from mutant and wild-type plants were divided into two portions. One portion was immersed in 2.5% glutaraldehyde with 0.2 mol/L cacodylate buffer (pH 7.2). Softened anthers were then sliced open, and loose pollen was washed in cacodylate buffer to remove the glutaraldehyde. The other portion was acetolyzed (Erdtman, 1960 ). Both samples were cleaned of detritus by sucrose density gradient sedimentation (Chissoe and Skvarla, 1974 ). For SEM, acetolyzed and nonacetolyzed pollen grains were divided into two groups. Whole pollen grains were examined either by drying with hexamethyldisilazane (HMDS; Chissoe, Vezey, and Skvarla, 1994 ), mounting on specimen stubs and sputter coating with gold; or by drying and made electrically conductive using the osmium-thiocarbohydrazide (OTOTO) method revised by Chissoe, Vezey, and Skvarla (1995) . Other samples for SEM were frozen on an International Cryostat IEC Model CTR microtome as described elsewhere (Vezey et al., 1994 ) and sectioned with a steel knife at a thickness of 20 µm, collected on SEM specimen mounts (JEOL bulk specimen mounts), and sputtered coated with gold. Examination and photography were done with a JEOL-880 SEM equipped with a lanthanum hexaboride gun at 15 kV. For TEM, acetolyzed pollen was stained in cacodylate-buffered 0.5% OsO₄ for 2 h, washed in distilled water, dehydrated through ETOH and embedded in araldite-epon resins (Mollenhauer, 1964 ). Ultrathin sectioning was accomplished with diamond knives, and sections were retrieved on copper grids and stained with uranyl acetate and lead citrate to improve general contrast. Sections were examined with a JEOL-2000 intermediate-voltage TEM.

Pollen terminology
The terminology used to describe Haplopappus pollen is based on Faegri (1956) and Iversen and Troels-Smith (1950) , as adapted to Composite pollen by Skvarla and Larson (1965) and Skvarla et al. (1977) . The terminology recognizes two discrete exine layers after acetolysis treatment (Erdtman, 1960 ): an outer electron dense complex layer termed ektexine, composed of spines, tectum, columellae and foot layer, and an inner less electron dense single layer, termed endexine.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Monohybrid crosses
All 16 F1 hybrids between spineless and spined pollen plants had spined pollen, indicating that the spineless mutant gene is recessive. There were 46 F2 generation plants, but two died before flowering. Of the 44 surviving plants, 33 had spined pollen and 11 were spineless. This fits the expected 3:1 Mendelian proportions for a recessive trait. These data also show that the spineless trait is under sporophytic control (the diploid parental genome instead of the haploid pollen genome).

Dihybrid crosses
Plants homozygous for spineless pollen and normal stems were crossed to those with spined pollen and fasciated stems. The 16 F1 hybrids analyzed had spined pollen and normal stems. Among 88 F2 progeny were 54 spined pollen and normal stems, 17 spined pollen and fasciated stems, 14 spineless pollen and normal stems; three spineless pollen and fasciated stems.

The F2 data give an acceptable fit to a 9:3:3:1 proportion expected for two unlinked traits under simple Mendelian control (²₍₃₎ = 1.94, P = 0.58). For each trait, the F2 progeny also had acceptable fits to monohybrid expectations.

Normal (wild type) pollen
The ektexine in normal H. gracilis pollen has a surface completely ornamented with acute spines 2 µm in height (Fig. 1). Spine tips are supported by basal regions composed of short (<0.1 µm) pillars. The tectum (roof) (<0.25 µm thick) underlying the spines is the distal extension of columellae that are 0.5–1.5 µm in height (Figs. 14, 17). The basal parts of the columellae are united into an irregular layer ranging from 0.25 to 1.0+ µm in thickness (Figs. 7, 8, 14, 17). Columellae bases in each of the three interapertural regions of the pollen wall are separated from the remainder of the exine by a cavity, termed cavea (Blackmore, van Helvoort, and Punt, 1984 ) (Figs. 7, 8, 14). Below each cavea are two exine layers joined as a band (Fig. 14). The upper layer, the foot layer, is part of the ektexine and is the only ektexine layer without internal foramina (Skvarla and Turner, 1966 ). At each aperture margin the foot layer connects with columellae bases to give unity to the pollen wall (Figs. 7, 8). The foot layer is considerably narrower than the lower, less electron-dense endexine layer, which is highly lamellate, disrupted, and lacking internal foramina (Fig. 14).

View larger version (107K): [in this window] [in a new window]   Figs. 14–19. Transmission electron micrographs of normal (Figs. 14, 17 ) and mutant (Figs. 15, 18, 19 ) H. gracilis pollen. 14. Section is in a cavea (ca) region between aperture margins showing spine (s), thin tectum (t), columellae (c), irregularly thickened columellae bases (cb), foot layer (fl), and fragmented endexine (en). Internal foramina are common within exine units above the cavea. 15. Exine region approximately equivalent to Fig. 14 with aperture (a) at lower left. In comparison to Fig. 14 , note complete absence of corresponding spines, cavea, and columellae. Individual exine fragments and the comparatively thick layer overlying the endexine (en) all contain abundant internal foramina. The lower margin of the endexine is fragmented. 16. Section similar to Fig. 15 but in interapertural region. Endexine (en). 17. Internal foramina are dominant in this view of tectum (t), columellae (c) and columellae bases (cb) above cavea region (ca). The cavea, foot layer, and endexine are not shown. 18, 19. Sections for direct comparison (approximately the same magnifications) with some of the ektexine units in normal pollen in Fig. 17 . Note the similarity in abundance and distribution of internal foramina. The prominent ektexine layer directly above the endexine (en) is filled with internal foramina, a marked contrast to the solid foot layer above the endexine in normal pollen (Fig. 14 ). In Fig. 18 a problematic columella unit is indicated (c). Note similarity of fragmented lamellate endexines in both the normal and mutant pollen. All sections acetolyzed. All scale bars = 1 µm

View larger version (158K): [in this window] [in a new window]   Figs. 7–13. Scanning electron micrographs of freeze-fractured sections of normal (Figs. 7,8 ) and mutant (Figs. 9–13 ) H. gracilis pollen. 7. Near-median section showing cytoplasm (CY), three apertures (a), corresponding caveae (ca), and union of columellae bases with foot layers at the aperture margins (arrows). 8. Intercolpal area showing a cavea (ca) surmounted by columellae and thickened spines. At the left and right margins (arrows) the columellae bases join with the foot layer. Exine acetolyzed. 9. Similar view to Fig. 7 (cytoplasm has "fallen out" during sectioning). Note lack of caveae, columellae, and spines. Surface covered with randomly dispersed exine fragments as noted elsewhere (see Figs. 2–4 ). Spine(s). 10. View comparable to Fig. 8 . Globular exine fragments (gf) are common. A bilayering is suggested at the exine margin (arrow) with the upper layer (ektexine) distinguished from the lower layer (endexine) by a slight texturing (see also Fig. 12 ). Cytoplasm (CY). 11. The highly irregular exine fragments include a randomly displaced spine tip (s). Exine acetolyzed. 12. Section shows two exine fragments which appear to contain linear structures and spongy sporopollenin. The exine margin beneath these fragments appears to be delineated into two layers (arrow): an upper, somewhat spongy layer, and a lower, denser layer. Exine acetolyzed. 13. SEM of a rare, isolated exine surface fragment (compare with Fig. 18 for an approximate TEM equivalent) possibly representing a portion of a columellae and tectum unit similar to that in normal H. gracilis pollen. Globular (gf) and angular fragments are common. Exine acetolyzed. All scale bars = 1 µm

Ultrathin sections (Figs. 15, 16, 18, 19) show an endexine layer that is highly lamellate and with considerable disruptions at the lower margin. Directly overlying the endexine is a comparatively thicker and slightly more electron-dense ektexine layer that is perforated with abundant internal foramina, which appear to be several tiers in height. Frozen sections (Figs. 10, 12) further support this bilayering of ektexine and endexine. Loosely attached to the upper part of the ektexine layer are the randomly displaced fragments noted above by SEM. All fragments also contain numerous internal foramina (Figs. 15, 16, 18, 19). Internal foramina in the mutant and wild-type pollen grains appear identical (compare enlarged TEMs in Fig. 17 with Figs. 18, 19).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Mutant pollen grains of H. gracilis bear no resemblance to typical Haplopappus pollen that has been well characterized in light microscopy (e.g., Wodehouse, 1930, 1935 ; Heusser, 1971 ) and SEM (e.g., Martin and Drew, 1970 ; Clark, Brown, and Mayes, 1980 ). (For a complete listing of references for Haplopappus see see the bibliographic volumes of Thanikaimoni, 1972, 1973, 1976, and 1980 .) The highly disorganized nature of the mutant pollen surface does not resemble any member of the Compositae, or pollen of any angiosperm family. While the lamellate and fragmented endexine of mutant pollen is equivalent to the endexine in normal H. gracilis pollen, as well as Compositae exines in general (Skvarla and Larson, 1965 ; Skvarla et al., 1977 ), ektexine correlations are less clear. In mutant pollen, SEM and TEM show isolated spine tips and sporopollenin fragments. The latter we believe contain mostly ektexine units. Because internal foramina are absent in the foot layers of all Compositae exines possessing caveae (Skvarla and Larson, 1965 ; Skvarla and Turner, 1966 ; Skvarla et al., 1977 ), the internal foramina-filled layer directly overlying the endexine in mutant pollen may be equivalent to columellae bases of wild-type Haplopappus. If this interpretation is correct, then mutant Haplopappus pollen grains may be missing caveae and foot layers. While there are groups of Compositae without caveae (viz. Anthemideae, Cardueae, Mutisieae, Lactuceae), there are no Compositae pollen grains without foot layers.

These observations suggest that some organizing factor(s) during pollen wall formation were disrupted by the mutation, interfering with proper assembly of the ektexine. Paxson-Sowders, Owens, and Makaroff (1997) compared pollen wall development in normal and mutant Arabidopsis thaliana pollen in order to follow development of the reticulate pollen wall. They noted that the mutant exine was not differentiated into distinct exine units (see their fig. 18, p. 61), but rather it consisted of a random deposition of unorganized sporopollenin much like that described above for Haplopappus. They suggested that specific stencil or exine receptors to organize the accumulating sporopollenin were absent.

The genetic data show the recessive nature of the spineless mutant gene and indicate a location on chromosome B (linkage group 2). However, this location is not certain because a high recombination frequency approaching 50% between the fasciated and spineless gene on paired homologous chromosomes of linkage group 1 could also yield a good fit to an expected dihybrid ratio.

The spineless mutant that causes such massive disruption of exine development in Haplopappus gracilis indicates its critical importance to pollen wall development, and sequencing its allele could lead to identification of the same gene in related species and other genera. Published data on pollen morphology show numerous fixed taxon differences that undoubtedly are under genetic control, and crosses among interfertile species with such differences could provide useful genetic data. However, population sampling within taxa will more likely provide fertile mutants that could lead to a better understanding of ontogenetic pathways in this critical life cycle stage.

	FOOTNOTES

 
¹ The authors gratefully acknowledge the assistance of Mr. Greg Strout of the Samuel Roberts Noble Electron Microscopy Laboratory of the University of Oklahoma for his expertise in obtaining the pollen transmission electron micrographs.

² Author for correspondence

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Blackmore, S., H. A. M. van Helvoort, and W. Punt. 1984 On the terminology, origins and functions of caveate pollen in Compositae. Review of Palaeobotany and Palynology 43: 293–301.[CrossRef]

Chissoe, W. F., and J. J. Skvarla. 1974 Sucrose density pads for concentration and purification of pollen grains. Stain Technology 49: 123–12.[ISI][Medline]

———, E. L. Vezey, and J. J. Skvarla. 1994 Hexamethyldisilazane (HMDS) as a drying agent in pollen scanning electron microscopy. Biotechnic & Histochemistry 69: 192–198.

———,———, and ———. 1995 The use of osmium-thiocarbohydrazide for structural stabilization and enhancement of secondary electron images in scanning electron microscopy of pollen. Grana 343: 317–324.

Clark, W. D., G. K. Brown, and R. A. Mayes. 1980 Pollen morphology of Haplopappus and related genera (Compositae-Astereae). American Journal of Botany 67: 1391–1393.[CrossRef][ISI]

Erdtman, G. 1960 The acetolysis method, revised description. Svensk Botanisk Tidskrskrift 54: 561–564.

Faegri, K. 1956 Recent trends in palynology. Botanical Review 22: 639–664.

Heusser, C. J. 1971 Pollen and spores of Chile. University of Arizona Press. Tucson, Arizona, USA.

Iversen, J., and J. Troels-smith. 1950 Pollenmorfologiske Definitioner og Typer. Danmarks Geol. Undersögelse 4: Raekke 3, 8.

Jackson, R. C. 1957 New low chromosome number for plants. Science 125: 1115–1116.

———. 1964 Preferential segregation of chromosomes from a trivalent in Haplopappus gracilis. Science 145: 511–513.[Abstract/Free Full Text]

Mangum, P. D. 1992 Localization and characterization of the fasciation mutation in Haplopappus gracilis. Masters thesis, Texas Tech University, Lubbock, USA.

Martin, P. S., and C. H. M. Drew. 1970 Additional scanning electron photo-micrographs of southwestern pollen grains. Journal Arizona Academy Sciences 6: 140–161.

Mollenhauer, H. 1964 Plastic embedding mixtures for use in electron microscopy. Stain Technology 39: 110–114.[ISI][Medline]

Paxson-Sowders, D. M., H. A. Owens, and C. A. Makaroff. 1997 A comparative ultrastructural analysis of exine pattern development in wild-type Arabidopsis and a mutant defective in pattern formation. American Journal of Botany 198: 53–65.

Skvarla, J. J., and D. A. Larson. 1965 An electron microscope study of pollen morphology in the Compositae with special reference to the Ambrosiinae. Grana Palynologica 6: 210–269.

———, and B. L. Turner. 1966 Systematic implications from electron microscopic studies of Compositae—a review. Annals of the Missouri Botanical Garden 53: 220–256.[CrossRef]

———, ———, V. C. Patel, and A. S. Tomb. 1977 Pollen morphology in the Compositae and in morphologically related families. In V. H. Heywood, J. B. Harborne, and B. L. Turner [eds.], The biology and chemistry of the Compositae, 141–248. Academic Press, New York, New York, USA.

Thanikaimoni, G. 1972 Index bibliographique sur la morphologie des pollens d'angiospermes. Institut Francais de Pondichery. Travaux de la Section Scientifique et Technique 12: 1–339.

———. 1973 Index bibliographique sur la morphologie des pollens d'angiospermes. Supplement 1. Institut Francais de Pondichery. Travaux de la Section Scientifique et Technique 12: 1–164.

———. 1976 Index bibliographique sur la morphologie des pollens d'angiospermes. Supplement 2. Institut Francais de Pondichery. Travaux de la Section Scientifique et Technique 13: 1–385.

———. 1980 Quatrieme index bibliographique sur la morphologie des pollens d'angiospermes. Institut Francais de Pondichery. Travaux de la Section Scientifique et Technique 17: 1–336.

Vezey, E. L., L. E. Watson, J. J. Skvarla, and J. R. Estes. 1994 Plesiomorphic and apomorphic pollen structure characteristics of Anthemideae (Asteroideae: Asteraceae). American Journal of Botany 81: 648–657.[CrossRef][ISI]

Wodehouse, R. P. 1930 Pollen grains in the identification and classification of plants V. Haplopappus and other Astereae: the origin of their furrow configuration.Bulletin of the Torrey Botanical Club 57: 21–46.[CrossRef]

———. 1935 Pollen grains. McGraw-Hill. Reprinted in 1959 by HafnerPublishing, New York, New York, USA.

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