The development of in vitro fertilization systems in flowering plants is important for understanding and controlling the mechanisms of fertilization. Here a method is described in which isolated maize gametes fuse. In a medium containing 5 mM calcium chloride, sperm and egg cells adhere for several minutes and then fuse within 10 seconds. The method is specific to male-female gamete pairs and results in 80 percent fusion, whereas fusions with other combinations of gametic and mesophyllic cells are less frequent. Eggs fertilized in vitro do not fuse with additional male gametes, which suggests that a block to polyspermy exists.

Transgenic maize (Zea mays) plants were generated with a construct harboring a maize caffeic acid O-methyltransferase (COMT) cDNA in the antisense (AS) orientation under the control of the maize Adh1 (alcohol dehydrogenase) promoter. Adh1-driven ␤-glucuronidase expression was localized in vascular tissues and lignifying sclerenchyma, indicating its suitability in transgenic experiments aimed at modifying lignin content and composition. One line of AS plants, COMT-AS, displayed a significant reduction in COMT activity (15%-30% residual activity) and barely detectable amounts of COMT protein as determined by western-blot analysis. In this line, transgenes were shown to be stably integrated in the genome and transmitted to the progeny. Biochemical analysis of COMT-AS showed: (a) a strong decrease in Klason lignin content at the flowering stage, (b) a decrease in syringyl units, (c) a lower p-coumaric acid content, and (d) the occurrence of unusual 5-OH guaiacyl units. These results are reminiscent of some characteristics already observed for the maize bm3 (brownmidrib3) mutant, as well as for COMT down-regulated dicots. However, as compared with bm3, COMT down-regulation in the COMT-AS line is less severe in that it is restricted to sclerenchyma cells. To our knowledge, this is the first time that an AS strategy has been applied to modify lignin biosynthesis in a grass species.Lignins are complex phenolic polymers present in all vascular plants. They provide rigidity to conducting xylem elements and fiber cells. Lignins are composed of C 6 C 3 units, principally p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, and are present in various proportions according to botanical, physiological, and cytological criteria (Lewis and Yamamoto, 1990). Throughout the plant kingdom, grass lignins appear to be particularly specialized because they contain not only H, G, and S units, but also additional p-hydroxycinnamic units such as p-coumaric and ferulic acids (Higuchi et al., 1967). Ferulic acid may be ester linked to wall polysaccharides and/or ether linked to G units, thereby forming bridges between lignins and polysaccharides (Jacquet et al., 1995), whereas p-coumaric acid is primarily ester linked to S lignin units in lignified walls (Ralph et al., 1994; Grabber et al., 1996).Lignification in dicotyledons has been extensively studied and most of the known lignin biosynthetic genes have been employed in genetic engineering experiments (for review, see Grima-Pettenati and Goffner, 1999). There are almost no molecular data on lignification in grasses (Collazo et al., 1992;McAlister et al., 1998;Pichon et al., 1998;Selman-Housein et al., 1999;Spangenberg et al., 2001). Although lignification in grass species is likely to share a high degree of similarity to other angiosperms, the aforementioned structural specificity of grass cell walls may also involve a certain degree of grass-specific regulatory mechanisms. An in-depth knowledge of lignification in Graminaeae is of utmost importance because lignins are one of the ...

The characterization of in vitro xylogenic cultures of zinnia (Zinnia elegans) has led to major discoveries in the understanding of xylem formation in plants. We have constructed and characterized a subtractive library from zinnia cultures enriched in genes that are specifically expressed at the onset of secondary wall deposition and tracheary element (TE) programmed cell death. This Late Xylogenesis Library (LXL) consisted of 236 nonredundant cDNAs, 77% of which encoded novel sequences in comparison with the 17,622 expressed sequence tag sequences publicly available. cDNA arrays were constructed to examine dynamic global gene expression during the course of TE formation. As a first step in dissecting auxin and cytokinin signaling during TE differentiation, macroarrays were probed with cDNAs from cells cultured in different hormonal conditions. Fiftyone percent of the LXL genes were induced by either auxin or cytokinin individually, the large majority by auxin. To determine the potential involvement of these categories of genes in TE differentiation, multiplex in situ-reverse transcription-PCR was performed on cells for two genes encoding putative cell wall proteins: Gibberellin stimulated transcript-1, induced by auxin alone, and expansin 5, induced by cytokinin alone. All transcriptionally active TEs expressed both genes, indicating that, although these genes may not be considered as specific markers for TE differentiation per se, they are nevertheless an integral part of TE differentiation program. Among the non-TE population, four different gene expression-based cell types could be distinguished. Together, these results demonstrate the underlying complexity of hormonal perception and the existence of several different cell types in in vitro TE cell cultures.The formation of xylem, or xylogenesis, constitutes one of the most spectacular forms of cell differentiation in plants. Xylem, initiating from meristematic procambial or cambial cells, is a heterogeneous tissue composed of nonconducting cells including parenchyma and fibers, and conducting cells or tracheary elements (TEs; for recent review, see Ye, 2002). In angiosperms, TEs are essential for the transport and storage of water and nutrients in land plants. Although it is clear that TE function is of prime importance in plant development, our knowledge of the cascade of cellular and molecular events from procambium and cambium formation to the initiation of xylem differentiation, cell elongation, secondary wall deposition, and programmed cell death (PCD), is at best fragmentary. Our lack of knowledge is due to the high degree of organizational complexity of vascular tissues throughout the plant and the limited number of cells actually undergoing differentiation at a given time. As a result, the accessibility of vascular cells is limited and makes experimentation difficult in planta.To gain a more in-depth knowledge of xylem formation, two approaches have been largely exploited. The genetic dissection of Arabidopsis (Arabidopsis thaliana) mutants with alt...

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Xylogenic cultures of zinnia (Zinnia elegans) provide a unique opportunity to study signaling pathways of tracheary element (TE) differentiation. In vitro TEs differentiate into either protoxylem (PX)-like TEs characterized by annular/helical secondary wall thickening or metaxylem (MX)-like TEs with reticulate/scalariform/pitted thickening. The factors that determine these different cell fates are largely unknown. We show here that supplementing zinnia cultures with exogenous galactoglucomannan oligosaccharides (GGMOs) derived from spruce (Picea abies) xylem had two major effects: an increase in cell population density and a decrease in the ratio of PX to MX TEs. In an attempt to link these two effects, the consequence of the plane of cell division on PX-MX differentiation was assessed. Although GGMOs did not affect the plane of cell division per se, they significantly increased the proportion of longitudinally divided cells differentiating into MX. To test the biological significance of these findings, we have determined the presence of mannan-containing oligosaccharides in zinnia cultures in vitro. Immunoblot assays indicated that b-1,4-mannosyl epitopes accumulate specifically in TE-inductive media. These epitopes were homogeneously distributed within the thickened secondary walls of TEs when the primary cell wall was weakly labeled. Using polysaccharide analysis carbohydrate gel electrophoresis, glucomannans were specifically detected in cell walls of differentiating zinnia cultures. Finally, zinnia macroarrays probed with cDNAs from cells cultured in the presence or absence of GGMOs indicated that significantly more genes were down-regulated rather than up-regulated by GGMOs. This study constitutes a major step in the elucidation of signaling mechanisms of PX-and MX-specific genetic programs in zinnia.Xylogenesis is one of the most remarkable examples of cell specialization in higher plants. The xylem is the principal water-conducting tissue, transporting water from the root system to the aerial portions of the plant. To ensure this critical function, long files of cells divide and elongate, secondary cell wall material is deposited, the end walls between cells are hydrolyzed, and cell content is destroyed. In angiosperms, the resulting hollow structure, or xylem vessel, is composed of single units called tracheary elements (TEs). TEs are characterized by the different types of secondary wall thickening that are laid down: annular, helical, reticulate, and pitted. Protoxylem (PX) TEs with annular or helical secondary cell wall thickening differentiate while an organ is still expanding, whereas metaxylem (MX) TEs with reticulate or pitted secondary cell wall thickening differentiate after organ expansion has ceased (Esau, 1977). The relative proportions of PX and MX TEs that make up a given vascular bundle vary among plant organs and within a given organ throughout plant growth (Fahn, 1990;Pesquet et al., 2003). Until now, the underlying molecular mechanisms, signaling events, and positional information dict...

For the last 20 years, in vitro xylogenic cultures of Zinnia elegans have been routinely used to study tracheary element (TE) formation. That said, the precise anatomical relationship between in vitro and in planta xylogenesis in Zinnia has been completely ignored. In order to make this comparison, we provide herein a much needed description of xylem tissue of the Zinnia plant. Based on the proportions of secondary wall thickenings, the in vitro TE system most closely resembles hypocotyl vasculature. Moreover, we have shown by confocal microscopy that vessel-like structures of up to five individual TEs are produced in vitro, suggesting that the formation of multicellular structures and cell-cell communication during in vitro TE formation are far more extensive than previously suspected. Finally, as more and more genes become available through genomic approaches of Zinnia TEs, it will be necessary to precisely localize them in planta as a first step in elucidating gene function. Our results provide the histological groundwork for this very purpose.

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