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.     

Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.     

Mitochondrial genome diversity in arctic Siberians, with particular reference to the evolutionary history of Beringia and Pleistocenic peopling of the Americas

Through extended survey of mitochondrial DNA (mtDNA) diversity in the Nganasan, Yukaghir, Chuvantsi, Chukchi, Siberian Eskimos, and Commander Aleuts, we filled important gaps in previously unidentified internal sequence variation within haplogroups A, C, and D, three of five (A-D and X) canonical mtDNA lineages that defined Pleistocenic extension from the Old to the New World. Overall, 515 mtDNA samples were analyzed via high-resolution SNP analysis and then complete sequencing of the 84 mtDNAs. A comparison of the data thus obtained with published complete sequences has resulted in the most parsimonious phylogenetic structure of mtDNA evolution in Siberia-Beringia. Our data suggest that although the latest inhabitants of Beringia are well genetically reflected in the Chukchi-, Eskimo-Aleut-, and Na-Dene-speaking Indians, the direct ancestors of the Paleosiberian-speaking Yukaghir are primarily drawn from the southern belt of Siberia when environmental conditions changed, permitting recolonization the high arctic since early Postglacial. This study further confirms that (1) Alaska seems to be the ancestral homeland of haplogroup A2 originating in situ approximately 16.0 thousand years ago (kya), (2) an additional founding lineage for Native American D, termed here D10, arose approximately 17.0 kya in what is now the Russian Far East and eventually spread northward along the North Pacific Rim. The maintenance of two refugial sources, in the Altai-Sayan and mid-lower Amur, during the last glacial maximum appears to be at odds with the interpretation of limited founding mtDNA lineages populating the Americas as a single migration.     

The ancestral carnivore karyotype (2n = 38) lives today in ringtails

Chromosome painting was used to investigate the conservation of high-resolution longitudinal 4',6-diamidino-2-phenylindole (DAPI)/G bands in Carnivore chromosomes. Cat (Felis catus) and raccoon dog (Nyctereutes procyonoides) painting probes were hybridized to the ringtail (Bassaricus astutus), dwarf mongoose (Helogale parvula), and Malagasy civet (Fossa fossa) to identify homologous chromosome elements. The patterns of chromosome segment homology among Carnivore species allowed us to reconstruct and propose the disposition of a high-resolution banded ancestral carnivore karyotype (ACK). Three bi-armed chromosomes consistently found among Caniformia species are represented as 6 homologous acrocentric chromosomes among Feliformia species of Carnivora. However, reexamination of the most basal of Feliformia species, the African palm civet Nandinia, revealed the presence of the 3 heretofore Caniformia bi-armed chromosomes. Because these 3 bi-armed chromosomes are found in both Caniformia and Feliformia lineages, they are presumed ancestral for all Carnivora, suggesting that the ACK chromosome number would be 38, rather than the previously supposed 42. Banded chromosomes of the ACK are used to evaluate the consistency between recently determined molecular phylogenetic relationships and postulated cytogenetic dynamics in the same Carnivore species.     

The role of corpus callosum development in functional connectivity and cognitive processing

The corpus callosum is hypothesized to play a fundamental role in integrating information and mediating complex behaviors. Here, we demonstrate that lack of normal callosal development can lead to deficits in functional connectivity that are related to impairments in specific cognitive domains. We examined resting-state functional connectivity in individuals with agenesis of the corpus callosum (AgCC) and matched controls using magnetoencephalographic imaging (MEG-I) of coherence in the alpha (8-12 Hz), beta (12-30 Hz) and gamma (30-55 Hz) bands. Global connectivity (GC) was defined as synchronization between a region and the rest of the brain. In AgCC individuals, alpha band GC was significantly reduced in the dorsolateral pre-frontal (DLPFC), posterior parietal (PPC) and parieto-occipital cortices (PO). No significant differences in GC were seen in either the beta or gamma bands. We also explored the hypothesis that, in AgCC, this regional reduction in functional connectivity is explained primarily by a specific reduction in interhemispheric connectivity. However, our data suggest that reduced connectivity in these regions is driven by faulty coupling in both inter- and intrahemispheric connectivity. We also assessed whether the degree of connectivity correlated with behavioral performance, focusing on cognitive measures known to be impaired in AgCC individuals. Neuropsychological measures of verbal processing speed were significantly correlated with resting-state functional connectivity of the left medial and superior temporal lobe in AgCC participants. Connectivity of DLPFC correlated strongly with performance on the Tower of London in the AgCC cohort. These findings indicate that the abnormal callosal development produces salient but selective (alpha band only) resting-state functional connectivity disruptions that correlate with cognitive impairment. Understanding the relationship between impoverished functional connectivity and cognition is a key step in identifying the neural mechanisms of language and executive dysfunction in common neurodevelopmental and psychiatric disorders where disruptions of callosal development are consistently identified.     

DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.