Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance

Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (g_s) can be ascribed to changes in guard cells functioning in amphistomateous leaves. g_s was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where g_s was at the highest. In contrast, genes encoding H⁺-ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca²⁺-vacuolar antiporters, K⁺ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.     

Antibacterial activity of sugar maple autumn-shed leaf extract: Identification of the active compound

Ethanolic crude extract prepared from autumn-shed leaves of sugar maple (Acer saccharum Marsh.) was recently shown to have antibacterial activity against Pseudomonas cichorii and Xanthomonas campestris pv. vitians, two bacteria causing diseases in lettuce production. In this study, antibacterial activity of sugar maple autumn-shed leaves (SMASL) extract was further investigated. SMASL ethanolic crude extract was fractionated using HPLC system and geraniin was identified as the antibacterial compound by UPLC/Q-Tof-MS system. Geraniin, an ellagitannin, was then purified from SMASL crude extract using a glass chromatographic C18-reversed phase silica gel column (purification Step 1) and a semi-preparative HPLC system equipped with 5 μm XTerra Prep MS C18 column (purification Step 2). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of purified geraniin (purity of 96%) against P. cichorii and X. campestris pv. vitians were determined. X. campestris pv. vitians (MIC of 0.024 mg ml⁻¹ and MBC of 3.125 mg ml⁻¹) was more sensitive to geraniin than P. cichorii (MIC of 0.781 mg ml⁻¹ and MBC of 6.25 mg ml⁻¹). In the present study, geraniin is reported for the first time as the main antibacterial compound present in SMASL.     

Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration

Aims

Define the chemical factors structuring plant communities of three copper-cobalt outcrops (Tenke-Fungurume, Katangan Copperbelt, D. R. Congo) presenting extreme gradients.

Methods

To discriminate plant communities, 172 vegetation records of all species percentage cover were classified based on NMDS and the Calinski criterion. Soil samples were analyzed for 13 chemical factors and means compared among communities with ANOVA. Partial canonical correspondence analysis (pCCA) was used to determine amount of variation explained individually by each factor and site effect.

Results

Seven communities were identified. Six of the studied communities were related to distinct sites. Site effect (6.0 % of global inertia) was identified as the most important factor related to plant communities’ variation followed by Cu (5.5 %), pH (3.6 %) and Co (3.5 %). Unique contribution of site effect (3.8 %) was higher than that of Cu (1.1 %) and Co (1.0 %).

Conclusions

In restoration, not only Cu and Co contents will be important to maintain vegetation diversity, attention should also be given to co-varying factors potentially limiting toxicity of metals: pH, organic matter, Ca and Mn. Physical parameters were also identified as important in the creation of adequate conditions for diverse communities. Further studies should focus on the effect of physical parameters and geology.     

Genetic Analysis of the Biosynthesis of 2-Methoxy-3-Isobutylpyrazine,a Major Grape-Derived Aroma Compound Impacting Wine Quality

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.The pleasure experienced while enjoying a glass of wine is the result of sophisticated sensory, neurophysiological, and psychological processes triggered by wine aroma. Wine flavor is the result of a complex mixture of volatile compounds in the headspace of the glass that induces feelings of pleasure at the brain level (Shepherd, 2006). During the last 40 years, over 800 volatile molecules have been formally identified in wines, in concentrations ranging from hundreds of milligrams per liter down to a few picograms per liter (Ebeler and Thorngate, 2009; Styger et al., 2011). Among all of them, a relatively limited number of compounds, called varietal (or primary) aromas, play a crucial role in wine flavor and typicality. These aromas, which are related to the grape variety, belong to a limited number of chemical families, including monoterpenes, C13 norisoprenoids, volatile sulfur compounds, and methoxypyrazines (MPs; Ebeler and Thorngate, 2009). Quite frequently, they exist mostly in the grape (Vitis vinifera) berry as nonvolatile, odorless, “bound” forms that can be released by chemical and enzymatic reactions occurring during the winemaking and wine aging processes, thus enhancing wine’s varietal expression (Styger et al., 2011). Two classical examples are the glycoside precursors of the monoterpenols (Strauss et al., 1986) and the cysteinylated or glutathionylated precursors of the volatile thiols (Tominaga et al., 1998; Peña-Gallego et al., 2012). Noticeable exceptions are the MPs, which are found in grape berries exclusively as free, volatile molecules.MPs are strongly odorant volatile heterocycles, with vegetable-like fragrances, that are widely occurring in the plant kingdom (Maga, 1982). In grape, they can be detected in fruits, leaves, shoots, and roots (Dunlevy et al., 2010). They are found in different grape varieties and are particularly abundant in the so-called Bordeaux cultivars (i.e. cv Cabernet Franc, Cabernet Sauvignon [CS], Sauvignon Blanc, Merlot, and Carménère [Car]; Bayonove et al., 1975; Lacey et al., 1991; Roujou de Boubée et al., 2002; Belancic and Agosin, 2007), whereas they are rarely detected in other cultivars, such as cv Pinot Noir (PN), Chardonnay, or Petit Verdot (PV). This finding indicates a strong genotype dependency of MP biosynthesis (Koch et al., 2010). MPs are accumulated in berries until bunch closure or véraison, and then their level declines after véraison (Hashizume and Samuta, 1999; Ryona et al., 2008). MP concentration in wine is highly correlated with the grape berry content at harvest (Roujou de Boubée et al., 2002). Three MPs are found in grape berries: 2-methoxy-3-isobutylpyrazine (IBMP), which is the most abundant, and two others, 2-methoxy-3-isopropylpyrazine (IPMP) and 2-methoxy-3-sec-butylpyrazine (SBMP; Ebeler and Thorngate, 2009). Both IBMP and IPMP display very low sensory detection thresholds in the wine matrix, ranging from 1 to 16 ng L^–1.MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols (Dubourdieu et al., 2006; Lund et al., 2009). Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these extremely powerful odorants will reduce consumer acceptance (Parr et al., 2007). In red wine, MPs are considered as off-flavor, and red wines can be depreciated by concentrations above 10 ng L^–1 (Allen et al., 1991; Roujou de Boubée et al., 2000; Belancic and Agosin, 2007). Given the importance of MPs, either as typical varietal aromas or as detrimental off-flavors, deciphering the genetic and molecular determinism of their accumulation is of high interest for viticulture.In spite of this, until recently little was known about the MP biosynthesis pathway or the MP biosynthetic genes, either in grapevine or other plant species. Theoretical biosynthesis pathways have been proposed since the mid-1970s. They all start by the addition of an α-dicarbonyl on a branched amino acid (Leu for IBMP, Val for IPMP) to form a 2-hydroxy-3-alkylpyrazine, which is subsequently transformed into the corresponding MP, by a methoxylation reaction (Murray and Whitfield 1975; Gallois et al., 1988). While the initial addition step remains to be demonstrated in plants, an S-adenosyl-l-Met (SAM)-dependent O-methyltransferase (OMT), capable of converting 2-hydroxy-3-isobutylpyrazine (IBHP) into IBMP, has been detected in CS shoots, partially purified and sequenced (Hashizume et al., 2001a, 2001b; Fig. 1). Recently, Dunlevy et al. (2010) characterized two OMTs, VvOMT1 and VvOMT2, capable of methylating IBHP in vitro, albeit with high apparent K_m values. To investigate the genetic bases of MP biosynthesis in grape berries, we performed a quantitative trait loci (QTL) analysis, which has led to the identification of two previously uncharacterized OMTs termed VvOMT3 and VvOMT4. Functional characterization of these two OMTs showed that VvOMT3 was highly specific and efficient for IBHP methylation. Based on its differential expression in high-MP and low-MP grapevine varieties, we propose that VvOMT3 and, to a lesser extent, VvOMT4 are key genes for MP biosynthesis in grapevine berries.Open in a separate windowFigure 1.Putative biosynthesis pathway for IBMP adapted from Hashizume et al. (2001a). SAHcy, S-Adenosyl-l-homo-Cys.     

Reassortment between Two Serologically Unrelated Bluetongue Virus Strains Is Flexible and Can Involve any Genome Segment

Coinfection of a cell by two different strains of a segmented virus can give rise to a “reassortant” with phenotypic characteristics that might differ from those of the parental strains. Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) segmented virus and the cause of bluetongue, a major infectious disease of livestock. BTV exists as at least 26 different serotypes (BTV-1 to BTV-26). Prompted by the isolation of a field reassortant between BTV-1 and BTV-8, we systematically characterized the process of BTV reassortment. Using a reverse genetics approach, our study clearly indicates that any BTV-1 or BTV-8 genome segment can be rescued in the heterologous “backbone.” To assess phenotypic variation as a result of reassortment, we examined viral growth kinetics and plaque sizes in in vitro experiments and virulence in an experimental mouse model of bluetongue disease. The monoreassortants generated had phenotypes that were very similar to those of the parental wild-type strains both in vitro and in vivo. Using a forward genetics approach in cells coinfected with BTV-1 and BTV-8, we have shown that reassortants between BTV-1 and BTV-8 are generated very readily. After only four passages in cell culture, we could not detect wild-type BTV-1 or BTV-8 in any of 140 isolated viral plaques. In addition, most of the isolated reassortants contained heterologous VP2 and VP5 structural proteins, while only 17% had homologous VP2 and VP5 proteins. Our study has shown that reassortment in BTV is very flexible, and there is no fundamental barrier to the reassortment of any genome segment. Given the propensity of BTV to reassort, it is increasingly important to have an alternative classification system for orbiviruses.     

Mineral and Bacterial Diversities of Desert Sand Grains from South-East Morocco

Mineralogy and microbiology of sand from Merzouga (Morocco) were simultaneously characterized, with the purpose of contributing to a better understanding of the geomicrobiology of deserts. In spite of very low measured bacterial biomass, bacterial diversity on each of the five defined mineralogical classes, was found high. An original grain by grain cultivation method enabled to obtain bacterial isolates with an unusually high recovery rate. The results of this study show that the genus Arthrobacter is well adapted to this environment with a preference for grains other than the dominant mineral quartz, and that the genera Chelatococcus and Saccharotrix are strongly attached to the grains.     

Shrub clearing adversely affects the abundance of Ixodes ricinus ticks

In order to get a better understanding of the importance of vertical forest structure as a component of Ixodes ricinus tick habitat, an experiment was set up in a coniferous forest on sandy soils in northern Belgium. Ticks were sampled in six control and six treatment plots on various sampling occasions in 2008–2010. In the course of the study period, a moderate thinning was carried out in all plots and shrub clearing was performed in the treatment plots. Thinning had no effect on tick abundance, while shrub clearing had an adverse affect on the abundance of all three life stages (larva, nymph, adult) up to 2 years post-clearing. Our findings are especially relevant in the light of the ongoing efforts to improve vertical forest structure in Belgium and many other parts of Europe, which might create suitable habitats for ticks and change the epidemiology of tick-borne diseases. Also, our results indicate that shrub clearing could be applied as a tick control measure in recreational areas where there is a high degree of human-tick contact.     

Global Analysis of DNA Methylation Variation in Adipose Tissue from Twins Reveals Links to Disease-Associated Variants in Distal Regulatory Elements

Epigenetic modifications such as DNA methylation play a key role in gene regulation and disease susceptibility. However, little is known about the genome-wide frequency, localization, and function of methylation variation and how it is regulated by genetic and environmental factors. We utilized the Multiple Tissue Human Expression Resource (MuTHER) and generated Illumina 450K adipose methylome data from 648 twins. We found that individual CpGs had low variance and that variability was suppressed in promoters. We noted that DNA methylation variation was highly heritable (h²_median = 0.34) and that shared environmental effects correlated with metabolic phenotype-associated CpGs. Analysis of methylation quantitative-trait loci (metQTL) revealed that 28% of CpGs were associated with nearby SNPs, and when overlapping them with adipose expression quantitative-trait loci (eQTL) from the same individuals, we found that 6% of the loci played a role in regulating both gene expression and DNA methylation. These associations were bidirectional, but there were pronounced negative associations for promoter CpGs. Integration of metQTL with adipose reference epigenomes and disease associations revealed significant enrichment of metQTL overlapping metabolic-trait or disease loci in enhancers (the strongest effects were for high-density lipoprotein cholesterol and body mass index [BMI]). We followed up with the BMI SNP rs713586, a cg01884057 metQTL that overlaps an enhancer upstream of ADCY3, and used bisulphite sequencing to refine this region. Our results showed widespread population invariability yet sequence dependence on adipose DNA methylation but that incorporating maps of regulatory elements aid in linking CpG variation to gene regulation and disease risk in a tissue-dependent manner.     

Potential for oilseed rape resistance in pollen beetle control

Breeding for plant resistance to insect pests is a classic strategy in integrated management, but it has never been developed for use against European pests of oilseed rape (Brassica napus) (OSR), especially one of the most damaging ones, the pollen beetle (Meligethes aeneus). In this paper we look at the three strategies that could be employed to improve OSR resistance (based on transgenes, relatives of B. napus or OSR natural variation) and review our current knowledge as to how these strategies could be put into practice. We identify the drawbacks which are specific to the pollen beetle that could impede breeding programs for resistance, and propose an approach to circumvent them. Finally, we detail the steps of the interaction between OSR and the pollen beetle that could be targeted in order to improve plant resistance (host plant location, adult survival, adult feeding, egg production and oviposition, larval development) and discuss their efficiency and durability potential.