Arabinan Metabolism during Seed Development and Germination in Arabidopsis

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan I. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non- cellulosic polysaccharides of embryos. Arabinose levels decline to -15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using 14C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth.     

Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, ¹³C metabolic flux analysis with parallel studies on [1-¹³C^Fru]sucrose, [1-¹³C^Glc]sucrose, and [¹³C₆^Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS^Man or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Xyloglucan Metabolism Differentially Impacts the Cell Wall Characteristics of the Endosperm and Embryo during Arabidopsis Seed Germination

Cell wall remodeling is an essential mechanism for the regulation of plant growth and architecture, and xyloglucans (XyGs), the major hemicellulose, are often considered as spacers of cellulose microfibrils during growth. In the seed, the activity of cell wall enzymes plays a critical role in germination by enabling embryo cell expansion leading to radicle protrusion, as well as endosperm weakening prior to its rupture. A screen for Arabidopsis (Arabidopsis thaliana) mutants affected in the hormonal control of germination identified a mutant, xyl1, able to germinate on paclobutrazol, an inhibitor of gibberellin biosynthesis. This mutant also exhibited reduced dormancy and increased resistance to high temperature. The XYL1 locus encodes an α-xylosidase required for XyG maturation through the trimming of Xyl. The xyl1 mutant phenotypes were associated with modifications to endosperm cell wall composition that likely impact on its resistance, as further demonstrated by the restoration of normal germination characteristics by endosperm-specific XYL1 expression. The absence of phenotypes in mutants defective for other glycosidases, which trim Gal or Fuc, suggests that XYL1 plays the major role in this process. Finally, the decreased XyG abundance in hypocotyl longitudinal cell walls of germinating embryos indicates a potential role in cell wall loosening and anisotropic growth together with pectin de-methylesterification.Seed germination is a complex process that begins with the absorption of water and ends when the radicle breaks through the seed coat (or testa). In Arabidopsis (Arabidopsis thaliana), as in most angiosperms, the embryo is surrounded by the triploid endosperm and the seed coat of maternal origin (Nonogaki et al., 2010; North et al., 2010). The completion of germination requires the growth potential of the embryo to overcome the resistance of the endosperm and testa layers, which is controlled by the hormonal balance between abscisic acid (ABA) and gibberellins (GAs). During seed development, ABA induces embryo growth arrest at the transition from embryogenesis to the phase of reserve accumulation and then induces primary dormancy, thus preventing vivipary and allowing seed dispersal in a dormant state. Dormancy delays germination until environmental conditions become favorable for seedling survival and growth (Finkelstein et al., 2008; Nambara et al., 2010; Graeber et al., 2012). Dormancy depth varies among plant species and between Arabidopsis accessions; however, seed dormancy of the most commonly used accession Columbia-0 (Col-0) is relatively low and can be released by a few weeks of after-ripening (dry storage) or stratification (cold imbibition). Shortly after hydration, ABA is rapidly degraded in both dormant and nondormant seeds, but ABA catabolism is more active in nondormant seeds, leading to lower ABA levels and thus allowing GA activation of germination processes (Millar et al., 2006). GA increases the elasticity of the wall, thereby reducing the resistance of the endosperm while triggering the elongation of the hypocotyl (Nonogaki et al., 2010). Radicle protrusion through the micropylar endosperm is also stimulated by ethylene, which has an antagonist action with ABA on endosperm cap weakening (Linkies and Leubner-Metzger, 2012). Microarray analyses highlighted the importance of cell wall remodeling processes during germination in various species (Penfield et al., 2006; Morris et al., 2011; Endo et al., 2012; Martínez-Andújar et al., 2012; Dekkers et al., 2013). These studies provided compelling evidence that the tissue-specific expression of genes encoding cell wall biosynthesis or modification enzymes, and their differential response to hormonal signals in the endosperm and embryo, influences the rate of germination.Cell walls are constituted of crystalline cellulose microfibrils that are embedded in an amorphous matrix of complex polysaccharides: pectin and hemicelluloses. Xyloglucan (XyG) is the major hemicellulose polymer in the primary cell walls of gymnosperms and most angiosperms, and its binding to cellulose microfibrils by hydrogen bonding contributes to loosening or stiffening of the wall during cell elongation (Cosgrove, 2005). XyG chains can be cleaved and reconnected by endo-transglycosylases/hydrolases (XTH). Other families of proteins also act on XyG chains, such as expansins, which are thought to nonenzymatically modulate XyG interactions with cellulose microfibrils, thereby controlling the distance between the microfibrils. XyG has a backbone of (1→4)-linked β-d-glucopyranosyl residues, which can be substituted with α-d-xylopyranosyl residues at O-6 (Supplemental Fig. S1). The pattern of XyG substitutions is described using a single-letter nomenclature (Fry et al., 1993). The letter G is used for an unsubstituted Glc and X when it is substituted with a Xyl. In Arabidopsis, like in many other dicots, the xylosylation pattern is in general regular, consisting mainly of XXXG-type units. The xylosyl residue can be further substituted at O-2 with a β-galactosyl (L side chain), which in turn can be substituted at O-2 with α-l-fucosyl (F side chain).Many of the biosynthetic enzymes involved in XyG biosynthesis have been identified, including a glucan synthase, xylosyl, galactosyl, and fucosyltransferases (Scheller and Ulvskov, 2010). Among these, two xylosyltransferases, named XXT1 and XXT2, have been shown to be involved in the synthesis of XyG in Arabidopsis, and the double mutant xxt1 xxt2 lacks detectable XyG (Cavalier et al., 2008). Both belong to the GT34 subfamily of glycosyltransferases, and a third enzyme, XXT5 from a separate clade of GT34, may also be involved in XyG synthesis (Zabotina et al., 2008). These glycosyltransferases are Golgi-localized enzymes, which produce substituted XyG precursors that are secreted into the cell wall. Subsequent trimming of XyG chains is performed by apoplastic glycosidases and determines hemicellulose structure and properties in the wall (Scheller and Ulvskov, 2010). A number of genes involved in the XyG metabolism have been identified, including XYL1, BGAL10, and AXY8 encoding, respectively, α-xylosidase, β-galactosidase, and α-fucosidase (Sampedro et al., 2010; Günl et al., 2011; Günl and Pauly, 2011; Sampedro et al., 2012). Loss of function of these glycosidases results in significant alterations in XyG composition. Although XyG has been proposed to be a major player in cell wall extension and plant growth, mutants with altered XyG composition display only minor growth-related phenotypes. The XyG-deficient double mutant xxt1 xxt2 shows no major growth defect except for deformed root hairs (Cavalier et al., 2008). Nevertheless, it was recently reported that the production of Gal-depleted XyG causes dwarfism in the galactosyltransferase mutant mur3 (Kong et al., 2015) in contrast to xyl1 and bgal10, where increased galactosylation results in shorter but wider siliques (Sampedro et al., 2010; Günl and Pauly, 2011; Sampedro et al., 2012). Phenotypes have not been observed from either reduced or increased fucosylation in the fucosyltransferase mutant mur2 and fucosidase mutant axy8 (Vanzin et al., 2002; Günl et al., 2011). AXY8 overexpression does, however, restore hypocotyl elongation in dwarf AUXIN BINDING PROTEIN1 knockdown seedlings. This demonstrates that in muro removal of Fuc residues can modulate cell elongation (Paque et al., 2014).In contrast to the numerous studies on the impact of XyG composition on plant growth, little information is available on the role of XyG in seed development or germination. A recent study highlighted the slower germination rate of xxt1 xxt2 mutant seeds compared to wild type, whereas germination rates of the arabinan-deficient arad1 arad2 and putative pectin methyltransferase qua2 mutants were not affected (Lee et al., 2012). As mentioned above, XyG chain hydrolysis and linkage is catalyzed by XTH activities, one of which, AtXTH31/XTR8, is encoded by an endosperm-specific gene. Loss of function leads to faster germination, suggesting that AtXTH31/XTR8 is involved in the reinforcement of the cell wall of the endosperm during germination (Endo et al., 2012). Here, we report the identification of an additional xyl1 allele from a screen designed to isolate mutants impaired in the hormonal control of germination, based on their ability to germinate on the GA biosynthesis inhibitor paclobutrazol. To investigate the role of XyG metabolism in seed dormancy and germination characteristics, xyl1 seed phenotypes were correlated with spatio-temporal XyG accumulation during seed development and germination. Comparative studies using mutants impaired in two other apoplastic glycosidases, BGAL10 and AXY8, indicate a major role for XYL1 in XyG remodelling processes that affect germination.