Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.Plant cell walls are multifunctional viscoelastic networks mainly composed of polysaccharides. Many of these polysaccharides, including xylans, (gluco)mannans, xyloglucans (XyGs), and pectins, have various degrees and patterns of acetyl esterification (Gille and Pauly, 2012; Pawar et al., 2013). The biological role of cell wall acetylation is not well understood, but it is believed to be important for pathogen resistance and plant development, and the acetylation of pectin also impacts upon the mechanical properties of cell walls (Manabe et al., 2011; Orfila et al., 2012; Pogorelko et al., 2013). In vitro, acetyl groups influence susceptibility to enzymatic degradation of pectin and xylan (Selig et al., 2009; Chen et al., 2012; Gou et al., 2012; Orfila et al., 2012; Pogorelko et al., 2013), and therefore acetylation may constitute a barrier to cell wall deconstruction. Alkali treatment of wall materials, which hydrolyzes the ester bonds, is broadly used to make polysaccharides more extractable. The treatment does not only facilitate the degradation of xylan and pectins, but also improves the deconstruction of cellulose, as the depolymerization of noncellulosic polymers results in a better accessibility to cellulose by degrading enzymes (Selig et al., 2009). Low levels of acetylated polysaccharides in plant feedstocks would be desirable for downstream processing in biorefineries, firstly, because the cell wall material of plant feedstocks with low level of acetylation is expected to be more easily extracted and, secondly, because less acetate, which is highly toxic to microorganisms such as yeast (Saccharomyces cerevisiae), would be released during extraction (Manabe et al., 2011; Gille and Pauly, 2012; Pawar et al., 2013). However, although reducing the O-acetylation level of xylan by approximately 60%, as observed in the walls of the Arabidopsis (Arabidopsis thaliana) eskimo1 mutant, enhances enzymatic degradation of isolated xylan (Yuan et al., 2013), enzymatic hydrolysis yields of whole wall materials have been reported to actually be decreased (Xiong et al., 2013). This presumably results from a tighter association between these now lowly substituted xylan polymers and cellulose (Xiong et al., 2013).Recently, we reported REDUCED WALL ACETYLATION2 (RWA2), the first protein to be involved in cell wall acetylation in planta (Manabe et al., 2011). RWA2 is a member of a small family consisting of four proteins in Arabidopsis, and its loss-of-function mutants display 20% reduction of acetylation in a range of polysaccharides that include XyG and pectins. We have hypothesized, based on phylogenetic analysis, expression pattern, moderate reduction in acetylation, and the absence of morphological phenotype, that RWA proteins have redundant functions in a biochemical reaction that occurs prior to the actual acetylation of specific polysaccharides. Independently to our research, a quadruple mutant of RWA has been reported to display reduction in xylan acetylation, secondary cell wall thickness, and mechanical strength of the stem (Lee et al., 2011). Meanwhile, Gille et al. (2011) have discovered a new family of proteins involved in the acetylation of specific polysaccharides: the plant-specific DOMAIN OF UNKNOWN FUNCTION (DUF) 231 family (also known as TRICHOME BIREFRINGENCE-LIKE [TBL] family). The loss-of-function mutants altered xyloglucan4 (axy4)/tbl27 and axy4L/tbl22 lack O-acetylation specifically of XyG in certain tissues, while eskimo1/tbl29 mutants contain reduced O-acetylation of xylan (Xiong et al., 2013; Yuan et al., 2013). The TBL/DUF231 family proteins and the RWA proteins have sequence similarity to the N-terminal and C-terminal regions of the fungal protein Cas1p, respectively (Anantharaman and Aravind, 2010). This could suggest that the TBL and RWA proteins function in protein complexes where the determinants of substrate specificity reside in the TBL partner (Manabe et al., 2011). However, because there are many more TBL proteins than RWA proteins (e.g. 46 TBL proteins versus four RWA proteins in the genome of Arabidopsis), it is likely that they do not form discrete and invariable complexes. Crossing of rwa2-3 and a leaky allele of axy4, axy4-1, resulted in a double mutant with partially additive phenotype (Gille et al., 2011). Its XyG acetylation is lower compared with either single mutant. From this analysis, RWA2 and AXY4 have been hypothesized to work in synergy, although the function of RWA2 might be substituted by other RWAs (Gille et al., 2011). Here, we have generated all the combinations of double, triple, and quadruple mutants of all four members of RWA family to further investigate the functional diversity and redundancy and to explore the function of cell wall acetylation and the role of RWAs in the network of acetylation-related enzymes. The triple and quadruple mutants we have obtained displayed severe and distinct phenotypes such as extreme dwarfism. This contrasts with the very mild phenotypes reported by Lee et al. (2011). Taken together, RWAs have partially redundant functions in the process of cell wall acetylation and show distinct impacts upon different cell wall polysaccharides.     

Une abeille afrotropicale spécialisée dans la récolte du pollen de Graminées (Poaceae): Lipotriches notabilis (Schletterer 1891) (Hymenoptera Apoidea Halictidae)

Dans les zones de savanes de l’Afrique, un genre d’abeille, Lipotriches Gerstaecker 1858, s’est spécialisé dans la collecte du pollen de graminées. Un site de nidification et l’aire de butinage de Lipotriches notabilis ont été suivis pendant trois années dans la région de Ngaoundéré au Cameroun. Le régime alimentaire pollinique est composé presque exclusivement du pollen de graminées, notamment Brachiaria ruziziensis abondant dans cette région. Le maïs est aussi visité et l’abeille contribue indirectement à la pollinisation par la mise en suspension dans l’air du pollen. Comme les graminées n’offrent pas de ressource sucrée, les femelles comme les mâles de L. notabilis butinent de temps en temps les Asteraceae pour la collecte de nectar. La consommation du pollen de graminées par les femelles a été aussi observée. Lespèce niche en bourgades dont la taille varie d’une dizaine à une centaine de nids. Le nid creusé dans un sol horizontal est du type progressif. Il comprend un tumulus, une cheminée verticale, un conduit principal vertical pouvant atteindre 65 cm de profondeur et en moyenne 3 conduits latéraux obliques de 4 à 16 cm aboutissant dans une ou plusieurs cellules successives. Les cellules des conduits latéraux sont approvisionnées simultanément et fermées de manière régressive. On compte un maximum de 10 cellules par nid. Généralement un nid est habité par une seule femelle, mais certains nids sont habités par deux ou trois. Lactivité de butinage de cette espèce est limitée dans la matinée. Dès 7 heures, elle s’envole pour le site de butinage. Après 11 heures, il n’y a généralement plus de nids ouverts. Les mâles n’ont pas été aperçus au niveau du site de nidification et l’accouplement a lieu sur le site de butinage. La période d’activité commence avec la saison des pluies en avril et se termine au début de la saison sèche en décembre, avec la fanaison des graminées.     

Contribution à la connaissance des Hymenoptera Apoidea de Nouvelle-Calédonie et de leurs relations avec la flore butinée

Seulement 21 espèces d’Apoidea sont répertoriées de Nouvelle-Calédonie. La pauvreté de cette faune est paradoxale si on la compare à la richesse et l’endémicité de la flore de l’île, mais s’expliquerait par le fait qu’elle a été isolée avant ou peu après l’apparition des Apoidea vers – 130 millions d’années. Les auteurs comparent aussi les données de plusieurs îles des océans Pacifique et Indien. Deux nouvelles espèces de Halictidae sont décrites: Homalictus cocos et Lasioglossum (Chilalictus) delobeli. Une nouvelle synonymie est établie: Homalictus risbeci (Cockerell, 1929) = Homalictus crotalariae (Cockerell, 1929). Deux taxons, Chalicodoma umbripenne et Megachile laticeps, sont signalés pour la première fois en Nouvelle-Calédonie. Les relations entre les Apoidea et les 22 espèces végétales, sur lesquelles ces insectes ont été capturés, sont présentées et commentées.     

Protected and threatened components of fish biodiversity in the Mediterranean sea

The Mediterranean Sea (0.82% of the global oceanic surface) holds 4%-18% of all known marine species (~17,000), with a high proportion of endemism [1, 2]. This exceptional biodiversity is under severe threats [1] but benefits from a system of 100 marine protected areas (MPAs). Surprisingly, the spatial congruence of fish biodiversity hot spots with this MPA system and the areas of high fishing pressure has not been assessed. Moreover, evolutionary and functional breadth of species assemblages [3] has been largely overlooked in marine systems. Here we adopted a multifaceted approach to biodiversity by considering the species richness of total, endemic, and threatened coastal fish assemblages as well as their functional and phylogenetic diversity. We show that these fish biodiversity components are spatially mismatched. The MPA system covers a small surface of the Mediterranean (0.4%) and is spatially congruent with the hot spots of all taxonomic components of fish diversity. However, it misses hot spots of functional and phylogenetic diversity. In addition, hot spots of endemic species richness and phylogenetic diversity are spatially congruent with hot spots of fishery impact. Our results highlight that future conservation strategies and assessment efficiency of current reserve systems will need to be revisited after deconstructing the different components of biodiversity.     

Conservation genetics of an island-endemic lizard: low Ne and the critical role of intermediate temperatures for genetic connectivity

Island populations are at higher risk of extinction than mainland populations. Therefore, understanding the factors that facilitate connectivity is particularly pressing for the conservation of island taxa. Sceloporus occidentalis becki, the Island Fence Lizard, is an endemic taxon restricted to the Northern Channel Islands, part of a nearshore archipelago in Southern California, USA. Since the Last Glacial Maximum, fence lizard habitat on the Northern Channel Islands has decreased with rising sea levels and increasing temperatures that have reduced the availability of woody vegetation. More recently, the introduction (and subsequent removal) of invasive ungulates over the last 170 years and recovery of vegetation has resulted in further dramatic habitat modification. Given the potential for genetic bottlenecks, the history of habitat alteration, and topographic and landscape complexity, we used landscape and population genetic approaches to characterize patterns of genetic diversity and structure of Island Fence Lizards on Santa Cruz Island, the largest of the Northern Channel Islands. Our analyses revealed shallow population structure across the island, low effective population size (N_e?=??~?200), and evidence for a recent genetic bottleneck. Landscape genetic analyses showed that connectivity is facilitated by tree canopy cover and shrubland, as well as by intermediate temperatures, emphasizing the importance of woody vegetation and habitats with variable thermal regimes as the climate warms. Combined, these population and landscape genetic analyses suggest that the Island Fence Lizard is of greater conservation concern than currently appreciated, and increased conservation management focus is warranted for this island endemic.

     

Interaction of Age and Mechanical Stability on Bone Defect Healing: An Early Transcriptional Analysis of Fracture Hematoma in Rat

Among other stressors, age and mechanical constraints significantly influence regeneration cascades in bone healing. Here, our aim was to identify genes and, through their functional annotation, related biological processes that are influenced by an interaction between the effects of mechanical fixation stability and age. Therefore, at day three post-osteotomy, chip-based whole-genome gene expression analyses of fracture hematoma tissue were performed for four groups of Sprague-Dawley rats with a 1.5-mm osteotomy gap in the femora with varying age (12 vs. 52 weeks - biologically challenging) and external fixator stiffness (mechanically challenging). From 31099 analysed genes, 1103 genes were differentially expressed between the six possible combinations of the four groups and from those 144 genes were identified as statistically significantly influenced by the interaction between age and fixation stability. Functional annotation of these differentially expressed genes revealed an association with extracellular space, cell migration or vasculature development. The chip-based whole-genome gene expression data was validated by q-RT-PCR at days three and seven post-osteotomy for MMP-9 and MMP-13, members of the mechanosensitive matrix metalloproteinase family and key players in cell migration and angiogenesis. Furthermore, we observed an interaction of age and mechanical stimuli in vitro on cell migration of mesenchymal stromal cells. These cells are a subpopulation of the fracture hematoma and are known to be key players in bone regeneration. In summary, these data correspond to and might explain our previously described biomechanical healing outcome after six weeks in response to fixation stiffness variation. In conclusion, our data highlight the importance of analysing the influence of risk factors of fracture healing (e.g. advanced age, suboptimal fixator stability) in combination rather than alone.