Aspen Tension Wood Fibers Contain β-(1→4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.Contractile cell walls found in plant organs and tissues such as tendrils, contractile roots, and tension wood (TW) have remarkable functions and properties. Their traits have been most intensely studied in TW of hardwoods, where they provide negative gravitropic response capacities to stems with secondary growth, as recently reviewed by Mellerowicz and Gorshkova (2012). These properties are conferred by TW fibers, which in many species contain a so-called gelatinous cell wall layer (G-layer; Norberg and Meier, 1966; Clair et al., 2008). G-layers are formed following the deposition of xylan-type secondary cell wall layer(s) and, thus, can be considered tertiary layers (Wardrop and Dadswell, 1948). They are almost or completely devoid of xylan and lignin and have very high cellulose contents (up to 85%). However, several other polymers appear to be present in TW G-layers, according to recent chemical analyses of isolated G-layers (Nishikubo et al., 2007; Kaku et al., 2009) and immunohistochemical labeling of TW sections (Arend, 2008; Bowling and Vaughn, 2008). Notably, xyloglucan (XG) has been found in G-layers of poplar (Populus spp.) TW (Nishikubo et al., 2007) and at the boundary between secondary cell wall layers (S-layers) and G-layers (Baba et al., 2009; Sandquist et al., 2010). It is also important for tension creation (Baba et al., 2009). However, it is not detectable in mature G-layers by monoclonal antibodies or XG-binding modules (Nishikubo et al., 2007; Baba et al., 2009; Sandquist et al., 2010).Structurally similar G-layers have been also identified in phloem fibers in many fibrous crops, such as flax (Linum usitatissimum), hemp (Cannabis sativa), and ramie (Boehmeria nivea; Gorshkova et al., 2012). These fibers occur in bundles that can be isolated for biochemical analysis. G-layers in fibers from diverse sources have a very similar structure, being largely composed of cellulose (with axial microfibril orientation, high degrees of crystallinity, and large crystallite sizes) lacking xylan and lignin (Mellerowicz et al., 2001; Pilate et al., 2004; Gorshkova et al., 2010, 2012) and having high water contents (Schreiber et al., 2010). In phloem fibers, the G-layers become very prominent, reaching thicknesses up to 15 µm and occupying over 90% of the cell wall’s total cross-sectional areas (Crônier et al., 2005). Pectic β-(1→4)-galactan with complex structures has been shown to be the major matrix polysaccharide of isolated phloem fibers in flax (Gorshkova et al., 2004; Gorshkova and Morvan, 2006; Gurjanov et al., 2007). Some of it is so strongly retained within cellulose that it cannot be extracted by concentrated alkali and can only be obtained after cellulose dissolution (Gurjanov et al., 2008). Such galactan, therefore, is a prime candidate for a polymer entrapped by cellulose microfibrils during crystallization that could substantially contribute to the contractile properties of cellulose in G-layers, according to recently formulated models (Mellerowicz et al., 2008; Mellerowicz and Gorshkova 2012). Furthermore, Roach et al. (2011) have shown that trimming of β-(1→4)-galactan by β-galactosidase is important for final cellulose crystallization, the formation of G-layer structure, and, hence, the stem’s mechanical properties.There is also immunocytochemical evidence for the presence of β-(1→4)-galactan and type II arabinogalactan (AG-II) in G-layers of TW fibers (Arend, 2008; Bowling and Vaughn, 2008). In addition, high-M_r branched galactans have been isolated from TW of Fagus sylvestris (Meier, 1962) and Fagus grandifolia (Kuo and Timell, 1969), with estimated degrees of polymerization (DP) of approximately 300 and complex structure, probably including both β-(1→4) and β-(1→6) linkages, although their exact nature remains unknown. Furthermore, Gal has been identified as one of the major sugars after Glc and Xyl in hydrolysates of isolated Populus spp. G-layers (Furuya et al., 1970; Nishikubo et al., 2007), and the Gal content of cell walls is a proposed indicator of the extent of TW development in beech (Fagus spp.; Ruel and Barnoud, 1978). However, subsequent linkage analyses identified only 2- and 3,6-linked Gal in poplar TW G-layers (Nishikubo et al., 2007), while in flax fibers, 4-linked Gal is the main component (Gorshkova et al., 1996, 2004; Gurjanov et al., 2007, 2008). Thus, the type(s) of galactans present in poplar TW remains unclear, and the galactans have not been shown previously either to have a rhamnogalacturonan-I (RG-I) backbone or to be strongly retained by cellulose microfibrils, as demonstrated for flax gelatinous fibers.To improve our understanding of cell wall properties in TW and their contraction mechanism, in the study presented here, we tested aspects of the recently proposed entrapment model (Mellerowicz et al., 2008; Mellerowicz and Gorshkova, 2012). According to this model, contraction is driven by the formation of larger cellulose structures, sometimes called macrofibrils, via interactions of cellulose microfibrils in the G-layer with each other and forming inclusions containing matrix polymers. This would induce tension within cellulose through the stretching of microfibrils required to surround the inclusions. The model is compatible with available data on the structure and action of gelatinous walls, but the main assumption, that polymers are trapped inside crystalline cellulose, such as that found in flax, has not been tested previously. Therefore, we compared matrix polymers retained by cellulose microfibrils in normal wood (NW) and TW of the model hardwood species hybrid aspen (Populus tremula × Populus tremuloides) that forms TW with gelatinous fibers. For this purpose, we used a combination of sequential cell wall extractions, similar to those used previously to characterize flax gelatinous fibers (Gurjanov et al., 2008), followed by fractionation of polymers by size-exclusion chromatography, immunological analyses, and oligosaccharide profiling by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). The results reveal the main polymers of cellulose-retained fractions and key differences between NW and TW. Comparison of our results and previous findings also indicates that there are both similarities and differences in the constitution of gelatinous fibers in aspen and flax. An updated model of the contractile G-layer of TW fibers based on the data is presented.     

DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.     

Allocation of inner cells to epiblast vs primitive endoderm in the mouse embryo is biased but not determined by the round of asymmetric divisions (8→16- and 16→32-cells)

The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8→16 (primary inner cells) and 16→32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.     

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.     

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a^null blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a^null embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.     

Characterization of polarity development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in Brassica napus L

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical–basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.     

Enhancement of Festuca rubra L. germination and seedling growth by seed treatment with pathogenic Agrobacterium rhizogenes

The study concerned effects of two methods of red fescue seeds treating with wild type Agrobacterium rhizogenes strains (15834 and LBA 1334): soaking and matriconditioning with Micro-Cel E solid carrier. The effects were assessed from germination and seedlings growth rates. The bacterial indole-3-acetic acid (IAA) production and ACC deaminase activity were also determined. Both strains are able to produce the IAA and showed ACC deaminase activity in various amounts. The strains accelerated seeds germination, seedling emergence and development. The beneficial effect on those processes was visible when seeds were soaked for 1 h in bacterial suspension and, especially, when the bacteria were present during Micro-Cel E conditioning. The main effect observed upon inoculation of seeds during priming was increased growth of lateral roots and more complex architecture of the branching root system. Treating seeds with A. rhizogenes simultaneously with Micro-Cel E priming as a carrier is a promising method of enhancing grass germination and seedlings growth, particularly by improving the root system development.     

Antioxidant activity and ROS tolerance in triticale (×Triticosecale Wittm.) anthers affect the efficiency of microspore embryogenesis

To verify the hypothesis that cell redox status regulates the process of microspore embryogenesis (ME), reactive oxygen species (ROS) generation and the activity of enzymatic and non-enzymatic antioxidants were analyzed in eight doubled haploid lines of triticale with significant differences in embryogenic potential. The analyses were performed in anthers excised from freshly cut tillers (control) and from low temperature (LT) pre-treated tillers (3 weeks at 4 °C) in which ME has been initiated. Significant associations between ME effectiveness and the variables studied were found. In control cultures, high superoxide dismutase (SOD) activity appeared crucial for microspore viability. On the other hand, positive though non-linear correlation between ME effectiveness and H₂O₂ generation, and negative correlation with catalase (CAT) activity suggest that some threshold level of H₂O₂ is important for successful ME initiation. LT tillers pre-treatment significantly increased H₂O₂ accumulation, which had a negative effect on ME effectiveness. However, even high level of H₂O₂ did not endanger cell viability as long as the cells exhibited high activity of ROS-decomposing enzymes (SOD, CAT and POX). The ability to sustain antioxidative enzyme activity under cold stress in the dark was another important requirement for high effectiveness of ME, allowing for the generation of the signal initiating microspore reprogramming and simultaneously protecting the cells from the toxic effects of ROS production. The role of antioxidative enzymes cannot be replaced even by high activity of non-enzymatic antioxidants. In conclusion, genetically controlled but environmentally modified cell tolerance to oxidative stress seems to play an important role in triticale ME.     

Characterisation of the wheat phospholipid fraction in the presence of nickel and/or selenium

The influence of nickel (Ni) and/or selenium (Se) on phospholipid composition was studied in shoots and roots of wheat seedlings. Phospholipid differences between samples were analysed using liquid chromatography/electrospray ionization–MS coupled. A total of 39 lipid species were identified. Individual phospholipids were then quantified using a multiple reaction monitoring method. In the roots, Ni toxicity was associated with an elevated level of phosphatidic acid species. In the shoots, the phosphatidylcholine/phosphatidylethanolamine ratio was about fivefold higher than in roots and decreased in Ni-treated samples. Additionally, the concentrations of phospholipid species containing C 18:3 fatty acid were reduced. Lipidome data were then analyzed using principal component analysis, which confirmed the compositional changes in phospholipids in response to Ni and Ni + Se. In contrast, the phospholipid profiles of wheat seedlings exposed to Se alone showed more similarities with the control. Together, our results suggested that the presence of Se, despite a considerable improvement of growth of Ni-treated wheat, did not counterbalance negative effect of Ni on the phospholipid composition in wheat roots and shoots.