Distinct Cell Wall Architectures in Seed Endosperms in Representatives of the Brassicaceae and Solanaceae

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.     

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.