Reaction wood – a key cause of variation in cell wall recalcitrance in willow

Background

The recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered.

Results

A pot trial was designed to test if the ‘RW response’ varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site.

Conclusions

Our findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a ‘RW response’) indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.

     

Expression of lauroyl-acyl carrier protein thioesterase in brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation

Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids.     

Persistence of native and exotic plants 10 years after prairie reconstruction

Prairie reconstructions are a critical component of preservation of the imperiled tallgrass prairie ecosystem in the Midwestern United States. Sustainability of this endeavor depends on establishment of persistent cover of planted native species and resistance to noxious weeds. The goal of this study was to understand the influence of early reconstruction practices on long‐term outcomes. Twelve replicates of three planting methods (dormant‐season broadcast, growing‐season broadcast, and growing‐season drill) and three seed mix richness levels (10, 20, or 34 species), fully crossed in a completely randomized design were planted in 2005 on nine former agricultural fields located in Iowa and Minnesota. Cover by species was estimated in 2005–2007, 2010, and 2015. In 2015, cover of planted species, native nonplanted species, and exotic species were similar to those recorded in 2010. Cover of the noxious weed Cirsium arvense had also declined by an average of 49% without herbicide from a peak in 2007 to low stable levels from 2010 to 2015. Richness of planted forbs, on the other hand, were still increasing in high‐richness broadcast treatments (e.g. 17–59% increase 2010–1015 in Minnesota). Two results in 2015 are reasons for concern: cover of planted species is only slightly over 50% in both Minnesota and Iowa, though with forbs still increasing, this may improve; and the cool‐season exotic grasses Poa pratensis and Bromus inermis are increasing at both Minnesota and Iowa sites. Control of these invasive grasses will be necessary, but care will be needed to avoid negative impacts of control methods on natives.     

A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.     

Separation of large quantities of chinese hamster chromosomes by velocity sedimentation at unit gravity followed by flow sorting (FACS II)

Separation of large quantities of isolated metaphase chromosomes of Chinese hamster cells was performed by velocity sedimentation at unit gravity in a specially designed sedimentation chamber. This simple and easy technique results in chromosome fractions of relatively high purity as determined by flow cytometry and microscopy. Up to 10¹⁰ chromosomes can be processed depending upon the size of the sedimentation device, and enrichments up to 10 times of individual chromosomes were achieved. In addition, further chromosome purification was performed by fluorescence activated flow sorting using fractions, pre-enriched at unit gravity. The flow sorted chromosomal fractions were pure according to flow cytometric analyses. The combination of l g sedimentation and flow-sorting opens the possibility for preparative chromosome sorting by reducing the flow sorting time considerably.     

Two new cytotypes reinforce that <Emphasis Type="Italic">Micronycteris hirsuta</Emphasis> Peters, 1869 does not represent a monotypic taxon

Background

The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n?=?26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data.

Results

We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n?=?26 and 25, NF?=?32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA.

Conclusions

The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

     

Triggers of phytoplankton bloom dynamics in permanently eutrophic waters of a South African estuary

The permanently eutrophic Sundays Estuary experiences recurrent harmful algal blooms (HABs) of Heterosigma akashiwo (Raphidophyceae). This study aimed to identify the environmental variables shaping phytoplankton community composition and succession patterns during a typical spring/summer harmful algal bloom (HAB) period. Monitoring of abiotic and phytoplankton variables was undertaken over the period of a month in 2016. Surface water salinity corresponding to mesohaline conditions (9 to 12) was a prerequisite for site selection. During the study, two HABs (>550 µg Chl a l^?1) of H. akashiwo occurred, each lasting for approximately a week in duration. Analyses highlighted nutrient depletion (i.e. nitrate and phosphate concentrations) as the key constraint on bloom duration. When the density of H. akashiwo decreased, the community composition became more diverse with species belonging to Bacillariophyceae and Dinophyceae becoming more abundant; albeit to a lesser degree (<180 µg Chl a l^?1). Dissolved oxygen shifted from super-saturated conditions (>14 mg l^?1) during peak HAB conditions, to instances of bottom water oxygen depletion (2–4 mg l^?1) during the decay phase. These findings highlight the potential severity of transforming a catchment from natural to one that is highly regulated by agricultural practices, while also emphasising the need for management intervention.     

Weight loss is not mandatory for exercise-induced effects on health indices in females with metabolic syndrome

The aim of this study was to investigate the impact of moderate aerobic training on functional, anthropometric, biochemical, and health-related quality of life (HRQOL) parameters on women with metabolic syndrome (MS). Fifteen untrained women with MS performed moderate aerobic training for 15 weeks, without modifications of dietary behaviours. Functional, anthropometric, biochemical, control diet record and HRQOL parameters were assessed before and after the training. Despite body weight maintenance, the patients presented decreases in waist circumference (P = 0.001), number of MS components (P = 0.014), total cholesterol (P = 0.049), HDL cholesterol (P = 0.004), LDL cholesterol (P = 0.027), myeloperoxidase activity (P = 0.002) and thiobarbituric acid-reactive substances levels (P = 0.006). There were no differences in total energy, carbohydrate, protein and lipid intake pre- and post-training. Furthermore, improvements in the HRQOL subscales of physical functioning (P = 0.03), role-physical (P = 0.039), bodily pain (P = 0.048), general health (P = 0.046) and social functioning scoring (P = 0.011) were reported. Despite the absence of weight loss, aerobic training induced beneficial effects on functional, anthropometric, biochemical and HRQOL parameters in women with MS.     

Cell kinetic characterization of cultured human keratinocytes from normal and psoriatic individuals

Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, disturbed differentiation, and inflammation. It is still a matter of debate whether the pathogenesis of psoriasis is based on immunological mechanisms, on defective growth control mechanisms, or possibly on a combination of both. Several in vivo cell biological differences between psoriatic lesional epidermis and normal epidermis have been reported. However, it is not clear whether these changes are causal or consequential. In case that keratinocytes from psoriatic patients have genetically determined deficiencies or polymorphisms with respect to autocrine growth regulation and the response to inflammatory cytokines, we hypothesize that these differences should be maintained in culture. Here we have started a systematic comparison of first passage keratinocytes cultured from normal skin and uninvolved psoriatic skin to address the question whether there are intrinsic differences in basic cell cycle parameters. In an established, defined culture system using keratinocyte growth medium (KGM) we have determined: (i) cell cycle parameters of exponentially growing keratinocytes, (ii) induction of quiescence by transforming growth factor β₁ (TGF-β₁), and (iii) restimulation from the G₀-phase of the cell cycle. Bivariate analysis of Iodo-deoxyuridine incorporation and relative DNA content was performed by flow cytometry. Within the limitations of this model no gross differences were found between normal and psoriatic keratinocytes with respect to S-phase duration (T_s), total cell cycle duration (T_c), responsiveness to TGF-β₁ and the kinetics for recruitment from G₀. In psoriatic keratinocytes we found a lower amount of cells in S-phase and a shorter duration of G₁, compared to normal keratinocytes. The methodology developed here provides us with a model for further studies on differences between normal and psoriatic keratinocytes in their response to immunological and inflammatory mediators. © 1996 Wiley-Liss, Inc.