| Abstract: | In some species, a crucial role has been demonstrated for the seed endosperm during germination. The endosperm has been shown to integrate environmental cues with hormonal networks that underpin dormancy and seed germination, a process that involves the action of cell wall remodeling enzymes (CWREs). Here, we examine the cell wall architectures of the endosperms of two related Brassicaceae, Arabidopsis (Arabidopsis thaliana) and the close relative Lepidium (Lepidium sativum), and that of the Solanaceous species, tobacco (Nicotiana tabacum). The Brassicaceae species have a similar cell wall architecture that is rich in pectic homogalacturonan, arabinan, and xyloglucan. Distinctive features of the tobacco endosperm that are absent in the Brassicaceae representatives are major tissue asymmetries in cell wall structural components that reflect the future site of radicle emergence and abundant heteromannan. Cell wall architecture of the micropylar endosperm of tobacco seeds has structural components similar to those seen in Arabidopsis and Lepidium endosperms. In situ and biomechanical analyses were used to study changes in endosperms during seed germination and suggest a role for mannan degradation in tobacco. In the case of the Brassicaceae representatives, the structurally homogeneous cell walls of the endosperm can be acted on by spatially regulated CWRE expression. Genetic manipulations of cell wall components present in the Arabidopsis seed endosperm demonstrate the impact of cell wall architectural changes on germination kinetics.Angiosperms are a diverse group of seed plants that reproduce by a double fertilization event; the first produces a zygote and the second a specialized nutritive tissue known as the endosperm. The endosperm and the maternally derived testa (seed coat) evolved to protect the embryo until conditions are favorable for germination and establishment of the next generation (Rajjou and Debeaujon, 2008; Linkies et al., 2010). Endosperm from cereals/grasses, such as maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aestivum), is vital for human and animal nutrition and is therefore of global economic importance (Olsen, 2007). In many seeds, such as some representatives of the Brassicaceae, the endosperm is entirely absent at seed maturity, the storage reserves having been absorbed by the cotyledons during embryo development. Arabidopsis (Arabidopsis thaliana) and Lepidium (Lepidium sativum) are notable exceptions in that they have retained a thin layer of endosperm tissue in the mature seed (Müller et al., 2006; Linkies and Leubner-Metzger, 2012).Some seeds exhibit primary dormancy at maturity that has been induced by abscisic acid (ABA; Hilhorst, 1995; Kucera et al., 2005). In its simplest sense, dormancy can be thought of as a block to germination of an intact viable seed under favorable conditions (Hilhorst, 1995; Bewley, 1997). A more sophisticated definition was proposed by Baskin and Baskin (2004), who state that a dormant seed does not have the capacity to germinate in a specified period of time under any combination of normal physical environmental factors that are otherwise favorable for its germination. Seed dormancy can be imposed by the embryo, the seed coat (including the endosperm), or a combination of both depending on the plant species (Bewley, 1997).The endosperm has been shown to be an important regulator of germination potential in several systems, including tomato (Solanum lycopersicum; Groot et al., 1988; Toorop et al., 2000), tobacco (Nicotiana tabacum; Leubner-Metzger et al., 1995; Petruzzelli et al., 2003), Arabidopsis (Bethke et al., 2007), and Lepidium (Müller et al., 2006; Linkies et al., 2009; Voegele et al., 2011). Arabidopsis continues to be an important model for elucidating the hormonal and genetic networks that regulate dormancy and germination (Kucera et al., 2005; Holdsworth et al., 2008), and new bioinformatic methods are providing insights into the evolutionary conservation of such networks in angiosperms (Bassel et al., 2011). Research using the close relative Lepidium, whose larger size makes it amenable to biomechanical techniques, has given insight into the hormonal control of endosperm weakening during germination and established that the mechanism of control is conserved between Arabidopsis, Lepidium, and tobacco (Müller et al., 2006; Linkies et al., 2009; Voegele et al., 2011). It has been reported that ABA is a key regulator of germination in tobacco, Arabidopsis, and Lepidium, controlling the process of endosperm rupture but not testa rupture (Leubner-Metzger et al., 1995; Petruzzelli et al., 2003; Müller et al., 2006). Microarray analyses of ABA-treated Arabidopsis and Lepidium seeds revealed that many cell wall remodeling enzyme (CWRE) genes are down-regulated upon exogenous application of ABA (Penfield et al., 2006; Linkies et al., 2009). Therefore, it follows that ABA impacts cell wall remodeling, which influences germination kinetics. The endosperm is therefore an important control tissue for seed germination and represents a useful model to investigate cell wall architectures and their remodeling.Cell walls are robust, multifunctional structures that not only protect cells from biotic and abiotic stresses, but also regulate growth, physiology and development (Albersheim et al., 2010). Cell walls are fibrous composites in which cellulose microfibrils are coextensive with/cross-linked by noncellulosic polysaccharides. In dicotyledonous plants, xyloglucan (XG) is a major polymer that can cross-link cellulose (Cosgrove, 2000). Load-bearing fibrous networks impart tensile strength to cell walls and are embedded in more soluble, gel-like matrices of pectic polysaccharides, glycoproteins, proteins, ions, and water. The constituent pectic polymers are currently classified as homogalacturonan (HG), rhamnogalacturonan I RG-I; also comprising arabinans and type 1 (arabino)galactans as side branches] and rhamnogalacturonan II, and xylogalacturonan (XGA) (Willats et al., 2001; Caffall and Mohnen, 2009). Pectins are involved in a diverse range of processes, including the regulation of intercellular adhesion/cell separation at the middle lamella, regulating the ionic status, and the porosity of cell walls that influences the access of CWREs to substrates (Willats et al., 2001). Noncellulosic polysaccharides exhibit numerous structural elaborations and differ in their glycan, methyl, and acetyl substitution (Caffall and Mohnen, 2009; Burton et al., 2010). Such modifications have the potential to impact their functionality, including their ability to interact with other wall components and their susceptibility to degradation and modification by CWREs.Studies using Arabidopsis (Iglesias-Fernández et al., 2011), Lepidium (Morris et al., 2011), and tomato (Groot et al., 1988) have highlighted a role for endo-β-mannanases (EBMs), enzymes that degrade heteromannan polysaccharides, during seed germination. In hard seeds with heteromannan-rich endosperms, such as carob (Ceratonia siliqua), date (Phoenix dactylifera), Chinese senna (Senna obtusifolia), and fenugreek (Trigonella foenum-graecum), however, it has been proposed that thinner walls in the micropylar endosperm (ME) and not EBM activity are responsible for allowing radicle protrusion during germination (Gong et al., 2005). Therefore, enzymatic cell wall remodeling and native cell wall architectural asymmetries both have the potential to impact on germination.Although studies on the molecular networks controlling germination have indicated a role for several classes of CWREs in endosperm remodeling and the promotion of germination (Penfield et al., 2006; Kanai et al., 2010; Morris et al., 2011), there is a paucity of information relating to the characterization of such changes at the cell wall level and, indeed, cell wall structures themselves. This study focuses on the targets of CWRE genes currently thought to be involved in seed germination (i.e. cellulose, XG, heteromannan, and pectic polysaccharides). We show that all three seeds possess a similar core cell wall architecture containing unesterified HG, arabinan, and XG. In tobacco, the core cell wall architecture is restricted to the ME, whereas in Arabidopsis and Lepidium, this architecture is observed throughout the endosperm. A further unique feature of the tobacco endosperm is abundant heteromannan. We also outline, using Arabidopsis, to what extent cell wall components contribute to the regulation of seed germination. |
