First insights into the transcriptome and development of new genomic tools of a widespread circum-Mediterranean tree species, Pinus halepensis Mill.

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next-generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA-Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene-based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48,629 contigs and covered about 54.6 Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein-coding sequences resulted in the detection of 34,014 SNPs across species, with a Ka /Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione-s-transferase, a cellulose synthase and a cobra-like protein. A large number of new markers (3334 amplifiable SSRs and 28,236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384-SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next-generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.

Materials nanocomposits a partir de whiskers de cel•lulosa i matriu polimèrica de cautxú

De acuerdo con los objetivos generales del proyecto y plan de trabajo previsto, para esta anualidad, se obtuvieron fibras y microfibras de celulosa a partir de dos fuentes: celulosa vegetal de pino y eucalipto y celulosa bacterial. Las microfibrillas han sido utilizadas como material de refuerzo para la fabricación de materiales compuestos a partir de caucho natural, policaprolactona y polivinil alcohol. Las muestras se fabricaron mediante la técnica de "casting" en medio acuoso y temperatura ambiente. Las muestras fueron caracterizados en sus propiedades mecánicas, físicas y térmicas. Se observó que, en general, la adición de las microfibrillas de celulosa en las matrices poliméricas provoca una mejora sustancial en las propiedades mecánicas del material en comparación con el polímero sin reforzar. Los resultados pueden resumirse de la siguiente manera: 1.Fabricación de materiales compuestos a base de caucho natural y fibras de celulosa. Se obtuvieron fibras y nanofibras de celulosa que fueron modificadas químicamente y usadas como refuerzo en matriz de caucho. Los resultados mostraron mejora de propiedades mecánicas del material, principalmente en los materiales compuestos reforzados con nanofibras. 2. Obtención de whiskers de celulosa y su utilización como material de refuerzo en una matriz de policaprolactona. Se obtuvieron whiskers de celulosa a partir de pasta blanqueada. La adición en una matriz de policaprolactona produjo materiales compuestos con propiedades mecánicas superiores a la matriz, con buena dispersión de los whiskers. 3. Obtención de fibras de celulosa bacterial y nanofibras de celulosa, aislamiento y utilización sobre una matriz de polivinil alcohol. Se obtuvo celulosa bacterial a partir de la bacteria Gluconacetobacter xylinum. Además se fabricaron nanofibras de celulosa a partir eucalipto blanqueado. La celulosa bacterial como material de refuerzo no produjo importantes mejoras en las propiedades mecánicas de la matriz; en cambio se observaron mejoras destacables con la nanofibra como refuerzo.

Estudi del comportament de materials nanoestructurats en el tractament d'aigües contaminades amb arsènic

El SPION (Super Paramagnetic Iron Oxide Nanoparticles) ha estat estudiat com un nou adsorbent per eliminar l’arsènic d’aigües contaminades. Les condicions òptimes de treball es van assolir per un pH de 3,6 i per concentracions inferiors als 100ppm. No es van trobar interferències significatives produïdes pels cations Cu, Ni i Zn en l’adsorció de l’As, sent el fosfat l’anió que més interfereix. Una esponja de cel·lulosa s’ha utilitzat com a suport del SPION, per disminuir les agregacions de les nanopartícules en suspensió i per proporcionar una material adequat per l’adsorció en continu, experiment amb columnes. Així, es va obtenir un augment de la capacitat d’adsorció del SPION per l’As(V), mentre que per l’As(III) continua sent baixa, per tant s’augmenta la selectivitat per l’As(V). Les interferències aniòniques afecten d’igual manera a l’adsorció de l’As(III) i l’As(V) quan l’adsorció és en continu o en discontinu. Els cations metàl·lics no interfereixen en l’adsorció de l’arsènic, a excepció del coure que és adsorbit i porta a la disminució de l’adsorció d’arsènic.

The suitability of steam exploded vitis vinifera and alkaline lignin for the manufacture of fiberboard

The main objective of this study was to explore the suitability of Vitis vinifera as a raw material and alkaline lignin as a natural binder for fiberboard manufacturing. In the first step, Vitis vinifera was steam- exploded through a thermo-mechanical vapor process in a batch reactor, and the obtained pulp was dried, ground, and pressed to produce the boards. The effects of pretreatment factors and pressing conditions on the chemical composition of the fibers and the physico-mechanical properties of binderless fiberboards were evaluated, and the conditions that optimize these properties were found. A response surface method based on a central composite design and multiple-response optimization was used. The variables studied and their respective variation ranges were: pretreatment temperature (Tr: 190-210ºC), pretreatment time (tr: 5-10 min), pressing temperature (Tp: 190-210ºC), pressing pressure (Pp: 8-16MPa), and pressing time (tp: 3-7min). The results of the optimization step show that binderless fiberboards have good water resistance and weaker mechanical properties. In the second step, fiberboards based on alkaline lignin and Vitis vinifera pulp produced at the optimal conditions determined for binderless fiberboards were prepared and their physico-mechanical properties were tested. Our results show that the addition of about 15% alkaline lignin leads to the production of fiberboards that fully meet the requirements of the relevant standard specifications

Biopolymers

Plants naturally synthesize a variety of polymers that have been used by mankind as a source of useful biomaterials. For example, cellulose, the main constituent of plant cell wall and the most abundant polymer on earth, has been used for several thousand years as a source of fibers for various fabrics. Similarly, rubber extracted from the bark of the tree Hevea brasiliensis, has been a major source of elastomers until the development of similar synthetic polymers. In the last century, the usefulness of plant polymers as biomaterials has been expanded through the chemical modification of the natural polymers. For example, a number of plastics have been made by substituting the hydroxyl groups present on the glucose moiety of cellulose with larger groups, such as nitrate or acetate, giving rise to materials such as cellulose acetate, a clear plastic used in consumer products such as toothbrush handles and combs. Similarly, starch has been used in the manufacture of plastics by either using it in blends with synthetic polymers or as the main constituent in biodegradable plastics. The advent of transformation and expres- sion of foreign genes in plants has created the possibility of expanding the usefulness of plants to include the synthesis of a range of biomolecules. In view of the capacity of certain crops to produce a large quantity of organic raw material at low cost, such as oils and starch, it is of interest to explore the possibility of using transgenic plants as efficient vectors for the synthesis of biopolymers. Such plant based biopolymers could replace, in part, the synthetic plastics and elastomers produced from petroleum, offering the advantage of renewability and sustainability. Furthermore, being natural pro- ducts, biopolymers are usually biodegradable and can thus contribute to alleviate problems associated with the management of plastic waste. In this article, the emphasis will be on the use of transgenic plants for the synthesis of two novel classes of industrially useful polymers, namely protein based polymers made from natural or artificial genes, and polyhydroxyalkanoates, a family of bacterial poly- esters having the properties of biodegradable plastics and elastomers.

Poly-N-Acetyl Glucosamine Fibers Induce Angiogenesis in ADP Inhibitor-Treated Diabetic Mice

Background: It has been previously demonstrated that short-fiber poly-N-acetyl-glucosamine (sNAG) nanofibers specifically interact with platelets, are hemostatic, and stimulate diabetic wound healing by activating angiogenesis, cell proliferation, and reepithelialization. Platelets play a significant physiologic role in wound healing. The influence of altered platelet function by treatment with the ADP inhibitor Clopidogrel (CL) on wound healing and the ability of sNAG to repair wounds in diabetic mice treated with CL were studied.Methods: Dorsal 1 cm2 skin wounds were excised on genetically diabetic 8-week to 12-week-old, Lep/r-db/db male mice, and wound healing kinetics were determined. Microscopic analysis was performed for angiogenesis (PECAM-1) and cell proliferation (Ki67). Mice were either treated with CL (P2Y12 ADP receptor antagonist, CL) or saline solution (NT). CL wounds were also treated with either a single application of topical sNAG (CL-sNAG) or were left untreated (CL-NT).Results: CL treatment did not alter wound healing kinetics, while sNAG induced faster wound closure in CL-treated mice compared with controls. CL treatment of diabetic mice caused an augmentation of cell proliferation and reduced angiogenesis compared with nontreated wounds. However, sNAG reversed the effects of CL on angiogenesis and partially reversed the effect on cell proliferation in the wound beds. The sNAG-treated wounds in CL-treated mice showed higher levels of cell proliferation and not did inhibit angiogenesis.Conclusions: CL treatment of diabetic mice decreased angiogenesis and increased cell proliferation in wounds but did not influence macroscopic wound healing kinetics. sNAG treatment did not inhibit angiogenesis in CL-treated mice and induced faster wound closure; sNAG technology is a promising strategy to facilitate the healing of complex bleeding wounds in CL-treated diabetic patients.

Membrane and walls: who is master, who is servant?

Specialised plant cell types often locally modify their cell walls as part of a developmental program, as do cells that are challenged by particular environmental conditions. Modifications can include deposition of secondary cellulose, callose, cutin, suberin or lignin. Although the biosyntheses of cell wall components are more and more understood, little is known about the mechanisms that control localised deposition of wall materials. During metaxylem vessel differentiation, site-specific cell wall deposition is locally prevented by the microtubule depolymerising protein MIDD1, which disassembles the cytoskeleton and precludes the cellulose synthase complex from depositing cellulose. As a result, metaxylem vessel secondary cell wall appears pitted. How MIDD1 is tethered at the plasma membrane and how other cell wall polymers are locally deposited remain elusive. Casparian strips in the root endodermis represent a further example of local cell wall deposition. The recent discovery of the Casparian Strip membrane domain Proteins (CASPs), which are located at the plasma membrane and are important for the site-specific deposition of lignin during Casparian strip development, establishes the root endodermis as an attractive model system to study the mechanisms of localised cell wall modifications. How secondary modifications are modulated and monitored during development or in response to environmental changes is another question that still misses a complete picture.

Transmission Fourier transform infrared microspectroscopy allows simultaneous assessment of cutin and cell-wall polysaccharides of Arabidopsis petals.

A procedure for the simultaneous analysis of cell-wall polysaccharides, amides and aliphatic polyesters by transmission Fourier transform infrared microspectroscopy (FTIR) has been established for Arabidopsis petals. The combination of FTIR imaging with spectra derivatization revealed that petals, in contrast to other organs, have a characteristic chemical zoning with high amount of aliphatic compounds and esters in the lamina and of polysaccharides in the stalk of the petal. The hinge region of petals was particular rich in amides as well as in vibrations potentially associated with hemicellulose. In addition, a number of other distribution patterns have been identified. Analyses of mutants in cutin deposition confirmed that vibrations of aliphatic compounds and esters present in the lamina were largely associated with the cuticular polyester. Calculation of spectrotypes, including the standard deviation of intensities, allowed detailed comparison of the spectral features of various mutants. The spectrotypes not only revealed differences in the amount of polyesters in cutin mutants, but also changes in other compound classes. For example, in addition to the expected strong deficiencies in polyester content, the long-chain acyl CoA synthase 2 mutant showed increased intensities of vibrations in a wavelength range that is typical for polysaccharides. Identical spectral features were observed in quasimodo2, a cell-wall mutant of Arabidopsis with a defect in pectin formation that exhibits increased cellulose synthase activity. FTIR thus proved to be a convenient method for the identification and characterization of mutants affected in the deposition of cutin in petals.

Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

Mutations in LACERATA (LCR), FIDDLEHEAD (FDH), and BODYGUARD (BDG) cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis), of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE), which encodes a protein of RNA-processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

Genetic and hormonal regulation of cambial development.

The stems and roots of most dicot plants increase in diameter by radial growth, due to the activity of secondary meristems. Two types of meristems function in secondary plant body formation: the vascular cambium, which gives rise to secondary xylem and phloem, and the cork cambium, which produces a bark layer that replaces the epidermis and protects the plant stem from mechanical damage and pathogens. Cambial development, the initiation and activity of the vascular cambium, leads to an accumulation of wood, the secondary xylem tissue. The thick, cellulose-rich cell walls of wood provide a source of cellulose and have the potential to be used as a raw material for sustainable and renewable energy production. In this review, we will discuss what is known about the mechanisms regulating the cambium and secondary tissue development.

Evidence for an organic origin of pedogenic calcitic nanofibres

Calcium carbonate nanofibres are found in numerous terrestrial environments, often associated with needle fibre calcite. This study attempts to mimic the natural system and generate comparable crystalline structures. A comparison of natural and synthesized nanofibre structures, using HRTEM as well as electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI), has demonstrated that this type of nanocrystal can result from precipitation on organic templates, most likely cellulose nanofibres. This study emphasizes the fundamental role of organic templates in the precipitation of calcium carbonate in vadose environments, even at the nanoscale.

Poly-N-acetyl glucosamine fibers are synergistic with vacuum-assisted closure in augmenting the healing response of diabetic mice.

BACKGROUND: Vacuum-assisted closure (VAC) has become the preferred modality to treat many complex wounds but could be further improved by methods that minimize bleeding and facilitate wound epithelialization. Short fiber poly-N-acetyl glucosamine nanofibers (sNAG) are effective hemostatic agents that activate platelets and facilitate wound epithelialization. We hypothesized that sNAG used in combination with the VAC device could be synergistic in promoting wound healing while minimizing the risk of bleeding. METHODS: Membranes consisting entirely of sNAG nanofibers were applied immediately to dorsal excisional wounds of db/db mice followed by application of the VAC device. Wound healing kinetics, angiogenesis, and wound-related growth factor expression were measured. RESULTS: The application of sNAG membranes to wounds 24 hours before application of the VAC device was associated with a significant activation of wounds (expression of PDGF, TGFβ, EGF), superior granulation tissue formation rich in Collagen I as well as superior wound epithelialization (8.6% ± 0.3% vs. 1.8% ± 1.1% of initial wound size) and wound contraction. CONCLUSIONS: The application of sNAG fiber-containing membranes before the application of the polyurethane foam interface of VAC devices leads to superior healing in db/db mice and represents a promising wound healing adjunct that can also reduce the risk of bleeding complications.

Organic material decomposition and nutrient dynamics in a mulch system enriched with leguminous trees in the Amazon

The new techniques proposed for agriculture in the Amazon region include rotational fallow systems enriched with leguminous trees and the replacement of biomass burning by mulching. Decomposition and nutrient release from mulch were studied using fine-mesh litterbags with five different leguminous species and the natural fallow vegetation as control. Samples from each treatment were analyzed for total C, N, P, K, Ca, Mg, lignin, cellulose content and soluble polyphenol at different sampling times over the course of one year. The decomposition rate constant varied with species and time. Weight loss from the decomposed litter bag material after 96 days was 30.1 % for Acacia angustissima, 32.7 % for Sclerolobium paniculatum, 33.9 % for Iinga edulis and the Fallow vegetation, 45.2 % for Acacia mangium and 63.6 % for Clitoria racemosa. Immobilization of N and P was observed in all studied treatments. Nitrogen mineralization was negatively correlated with phenol, C-to-N ratio, lignin + phenol/N ratio, and phenol/phosphorus ratios and with N content in the litterbag material. After 362 days of field incubation, an average (of all treatments), 3.3 % K, 32.2 % Ca and 22.4 % Mg remained in the mulch. Results confirm that low quality and high amount of organic C as mulch application are limiting for the quantity of energy available for microorganisms and increase the nutrient immobilization for biomass decomposition, which results in competition for nutrients with the crop plants.

Green manure in coffee systems in the region of Zona da Mata, Minas Gerais: characteristics and kinetics of carbon and nitrogen mineralization

The use of green manure may contribute to reduce soil erosion and increase the soil organic matter content and N availability in coffee plantations in the Zona da Mata, State of Minas Gerais, in Southeastern Brazil. The potential of four legumes (A. pintoi, C. mucunoides, S. aterrimum and S. guianensis) to produce above-ground biomass, accumulate nutrients and mineralize N was studied in two coffee plantations of subsistence farmers under different climate conditions. The biomass production of C. mucunoides was influenced by the shade of the coffee plantation. C. mucunoides tended to mineralize more N than the other legumes due to the low polyphenol content and polyphenol/N ratio. In the first year, the crop establishment of A. pintoi in the area took longer than of the other legumes, resulting in lower biomass production and N2 fixation. In the long term, cellulose was the main factor controlling N mineralization. The biochemical characteristics, nutrient accumulation and biomass production of the legumes were greatly influenced by the altitude and position of the area relative to the sun.