Christian Raquin

CORRELATED VARIATION IN MICROTUBULE DISTRIBUTION, CALLOSE DEPOSITION DURING MALE POST-MEIOTIC CYTOKINESIS, AND POLLEN APERTURE NUMBER ACROSS NICOTIANA SPECIES (SOLANACEAE)'

In most flowering plants, a single cytokinesis follows the two meiotic divisions during pollen-grain ontogeny. Aperture pattern (i.e., aperture number and distribution on pollen surface) ontogeny could be linked to the processes ensuring the apportionment of the cytoplasm to the four microspores.This apportionment is achieved by radial arrays of microtubules organized around the nuclei. The cleavage planes are defined in the overlapping regions of opposing arrays extending from different nuclei. We followed the establishment of these arrays in two different lines of plants belonging to the genus Nicotiana that produce pollen grains with different aperture numbers. Different distributions of the microtubules have been observed, which can be interpreted as resulting from variation in the interactions between nuclei; these distributions appear to be correlated with aperture number.As a consequence, we propose that simultaneous cytokinesis allows the formation of multiple pollen morphologies. This mechanism is consistent with aperture number distribution within angiosperms and provides clues to help our understanding of the evolution of aperture number.

Males outcompete hermaphrodites for seed siring success in controlled crosses in the polygamous Fraxinus excelsior (Oleaceae).

Polygamy (including trioecy and subdioecy), the co-occurrence of males, hermaphrodites, and females in natural populations, is a rare and poorly studied breeding system expressed in Fraxinus excelsior L. (Oleaceae), a wind-pollinated tree. Here we investigate siring ability of pollen from male vs. hermaphrodite individuals to better understand this sex polymorphism. We conducted single-donor and two-donor pollination experiments and compared both fruit set and seed siring success, assessed with polymorphic microsatellite markers, of male and hermaphrodite individuals. Single pollen donor crosses allowed us to verify the male function of hermaphrodites. However, pollen from hermaphrodites was much less proficient than male pollen, with males siring 10 times as many fruits in single donor pollination treatments. This result was strengthened by the surprisingly low reproductive success of hermaphrodites in pollen competition conditions: of the 110 seedlings analyzed three were selfed and only one was sired by the hermaphrodite donor. The remaining 106 were sired by the male pollen donor. These results raise the question of the maintenance of male fertility in hermaphrodites in Fraxinus excelsior. Male function of hermaphrodites in this species now needs to be assessed under field conditions.

Evolution of oil-producing trichomes in Sisyrinchium (Iridaceae): insights from the first comprehensive phylogenetic analysis of the genus

BACKGROUND AND AIMS Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined. METHODS Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species. KEY RESULTS AND CONCLUSIONS Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.

Maternal inheritance of chloroplasts in the horsetail Equisetum variegatum (Schleich.)

Abstract Reliable data concerning the transmission of chloroplasts in the Pteridophyta are needed both for phylogenies based on chloroplast DNA (cpDNA) sequences and in order to study the evolution of this trait in conjunction with the evolution of the life cycle and the sexual reproduction of land plants. For the first time, this paper describes organelle transmission in the division Sphenophyta, represented by the extant genus Equisetum. By following the fate of polymorphic cpDNA during three intraspecific reciprocal crosses we found no trace of paternal transmission in Equisetum variegatum. The seemingly strict maternal transmission of cpDNA in this species suggests that uniparental chloroplast inheritance preceded the evolution of heterospory in the seed-plant lineage.

Postmeiotic cytokinesis and pollen aperture number determination in eudicots: effect of the cleavage wall number

Summary. In eudicot postmeiotic tetrads, apertures are usually joined in pairs in highly conserved areas. These appear to be located at the last points of contact persisting at the end of cytokinesis between the cytoplasm of the future microspores. In order to investigate the relationship between cytokinesis and aperture formation, aperture distribution within postmeiotic tetrads and the progression of meiosis were studied in Nicotiana tabacum cv. Ambalema. This variety (inbred line) produces about 85% tricolporate pollen and 15% tetracolporate pollen grains. In addition, about 7% of tetrads are composed of four equal-sized microspores and a supernumerary pseudomicrospore of small size and an equal proportion of tetrads exhibit unpaired apertures (these apertures are not joined in pairs within tetrads). Observation of cytokinesis indicates that both unpaired apertures and pseudomicrospores could result from the persistence of late communications between microsporocytes. Observations of tetrads indicate that an increase in the number of elements that are separated during cytokinesis is correlated with an increase in microspore aperture number. All data converge to support the hypothesis that aperture site determination is partly controlled by the number of walls formed to separate the different elements of the tetrad.

Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia

Restriction fragment length polymorphism, polymerase chain reaction–restriction fragment length polymorphism and simple sequence repeat (SSR or microsatellites) analyses were performed to detect chloroplast DNA polymorphisms between two ash species, Fraxinus excelsior and F.angustifolia. Only one SSR locus was found to be polymorphic, confirming the very close relatedness of these species. Inheritance of this marker was studied in hybrids obtained from controlled crosses between the two tree species. Results indicated, for the first time in Oleaceae, that chloroplasts are maternally inherited. This chloroplast SSR marker is now used concomitantly with nuclear markers to analyse ash populations in sympatric areas.

Molecular and Morphological Characterization of Reciprocal F1 Hybrid Ash (Fraxinus excelsior × Fraxinus angustifolia, Oleaceae) and Parental Species Reveals Asymmetric Character Inheritance

Hybridization between Fraxinus excelsior and Fraxinus angustifolia is common. However, identifying hybrids in natural populations is difficult because the closely related parental species share many morphological characters and the inheritance pattern of these characters in hybrids is unknown. We evaluated how morphological characters are inherited and whether morphological and molecular markers can efficiently discriminate artificial first-generation hybrids. Reciprocal F1 hybrids of F. excelsior with F. angustifolia were examined using six microsatellite DNA marker loci and 14 morphological characters. Plants were divided into four groups (F. angustifolia, F. excelsior, the F1 hybrid with F. angustifolia as the maternal parent, and the F1 hybrid with F. excelsior as the maternal parent). The F1 hybrids showed intermediate morphology in most characters, and the range of variation overlapped with the parental species. Canonical discriminant analysis using only the morphological traits separated the four groups without any overlap between the two parental species. F1 hybrids from different maternal parent species could therefore be distinguished. A further analysis that combined molecular and morphological traits allowed clear separation of the four groups and strongly confirmed the a priori defined groups. Our results suggest that intermediate characters can be expected in F1 hybrids of ash but differences may be observed due to maternal/paternal effects.

Successive microsporogenesis affects pollen aperture pattern in the tam mutant of Arabidopsis thaliana

BACKGROUND AND AIMS The tam (tardy asynchronous meiosis) mutant of Arabidopsis thaliana, which exhibits a modified cytokinesis with a switch from simultaneous to successive cytokinesis, was used to perform a direct test of the implication of cytokinesis in aperture-pattern ontogeny of angiosperm pollen grains. The aperture pattern corresponds to the number and arrangement of apertures (areas of the pollen wall permitting pollen tube germination) on the surface of the pollen grain. METHODS A comparative analysis of meiosis and aperture distribution was performed in two mutant strains of arabidopsis: quartet and quartet-tam. KEY RESULTS While the number of apertures is not affected in the quartet-tam mutant, the arrangement of the three apertures is modified compared with the quartet, resulting in a different aperture pattern. CONCLUSIONS These results directly demonstrate the relationship between the type of sporocytic cytokinesis and pollen aperture-pattern ontogeny.

Formation and function of a new pollen aperture pattern in angiosperms: The proximal sulcus of Tillandsia leiboldiana (Bromeliaceae)

Pollen grains are generally surrounded by an extremely resistant wall interrupted in places by apertures that play a key role in reproduction; pollen tube growth is initiated at these sites. The shift from a proximal to distal aperture location is a striking innovation in seed plant reproduction. Reversals to proximal aperture position have only very rarely been described in angiosperms. The genus Tillandsia belongs to the Bromeliaceae family, and its aperture pattern has been described as distal monosulcate, the most widespread aperture patterns recorded in monocots and basal angiosperms. Here we report developmental and functional elements to demonstrate that the sulcate aperture in Tillandsia leiboldiana is not distal as previously described but proximal. Postmeitotic tetrad observation indicates unambiguously the proximal position of the sulcus, and in vitro germination of pollen grains confirms that the aperture is functional. This is the first report of a sulcate proximal aperture with proximal germination. The observation of microsporogenesis reveals specific features in the patterns of callose thickenings in postmeiotic tetrads.

ENVIRONMENTAL SEX DETERMINATION IN THE GENUS EQUISETUM: SUGARS INDUCE MALE SEX EXPRESSION IN CULTURED GAMETOPHYTES

Horsetails (Equisetum, Sphenophyta) are homosporous, and sexual differentiation of Equisetum gametophytes is under the influence of environmental conditions. Still, the environmental cues responsible for sex determination of Equisetum gametophytes in vitro and in the wild have remained elusive. Here, we show that significantly different sex ratios are obtained when gametophytes are grown on media with or without sugar. In our experimental conditions, male gametophytes outnumber females in the presence of 60–120 mM sucrose and 120 mM glucose, whereas in the absence of sugar, most gametophytes differentiate as female. A similar effect is also observed on already differentiated female gametophytes, which become hermaphroditic sooner when cultured in the presence of sucrose in vitro. Interestingly, these results are reproducible within and across species representative of the two subgenera Equisetum and Hippochaete, indicating that the entire genus may share an identical pattern of environmental sex determination.