Noninvasive Measurement of Vulnerability to Drought-Induced Embolism by X-Ray Microtomography

Hydraulic failure induced by xylem embolism is one of the primary mechanisms of plant dieback during drought. However, many of the methods used to evaluate the vulnerability of different species to drought-induced embolism are indirect and invasive, increasing the possibility that measurement artifacts may occur. Here, we utilize x-ray computed microtomography (microCT) to directly visualize embolism formation in the xylem of living, intact plants with contrasting wood anatomy (Quercus robur, Populus tremula × Populus alba, and Pinus pinaster). These observations were compared with widely used centrifuge techniques that require destructive sampling. MicroCT imaging provided detailed spatial information regarding the dimensions and functional status of xylem conduits during dehydration. Vulnerability curves based on microCT observations of intact plants closely matched curves based on the centrifuge technique for species with short vessels (P. tremula × P. alba) or tracheids (P. pinaster). For ring porous Q. robur, the centrifuge technique significantly overestimated vulnerability to embolism, indicating that caution should be used when applying this technique to species with long vessels. These findings confirm that microCT can be used to assess the vulnerability to embolism on intact plants by direct visualization.Theory describing the physiological mechanism that allows plants to extract water from the soil and transport it many tens of meters in height has often been the subject of intense debate (Tyree, 2003). Plants have evolved a water-transport system that relies on water sustaining a tensile force; as a result, xylem sap is at negative absolute pressures (Dixon and Joly, 1895; Melcher et al., 1998; Wei et al., 1999). However, this transport mechanism comes with its own set of problems. Most notably, water under tension is prone to cavitation, which results in the formation of gas bubbles (emboli) that block xylem conduits. Embolism reduces the capacity of the xylem tissue to deliver water to the canopy, where it is required to maintain adequate levels of cellular hydration (Tyree and Sperry, 1989). The probability of embolism occurring in the xylem increases during drought, with increasing tension in the xylem sap. During prolonged and severe droughts, xylem embolism can reach lethal levels, causing branch dieback and, ultimately, plant death (Davis et al., 2002; Brodribb and Cochard, 2009; Hoffmann et al., 2011; Choat, 2013; Urli et al., 2013). Water stress-induced embolism is now recognized as one of the principal causes of plant mortality in response to extreme drought events (Anderegg, 2015). In the face of increasingly severe droughts expected with rising global temperatures, hydraulic failure due to embolism has the potential to cause widespread dieback of trees across all major forest biomes (Choat et al., 2012).The majority of techniques used to estimate cavitation resistance are indirect and/or invasive, increasing the possibility of artifacts occurring during measurement (Cochard et al., 2013). Artifacts relating to invasive techniques are particularly relevant in this case, since xylem sap under tension is in a metastable state and may easily vaporize as a result of disturbance. Noninvasive imaging techniques offer the potential to make direct observations of xylem function in intact plants at high resolution and in real time. Noninvasive techniques include magnetic resonance imaging (MRI; Holbrook et al., 2001; Kaufmann et al., 2009; Choat et al., 2010) and, more recently, x-ray computed microtomography (microCT; Brodersen et al., 2010; Charra-Vaskou et al., 2012; McElrone et al., 2012). MicroCT provides superior spatial resolution to MRI, with resolutions below 2 μm attainable for a plant stem of 4 to 5 mm in diameter. This allows for detailed analysis of embolism formation and repair in the xylem, including spatial patterns of embolism spread between conduits (Brodersen et al., 2013; Dalla-Salda et al., 2014).However, noninvasive imaging techniques have seldom been used to validate indirect or invasive techniques used to estimate cavitation resistance. At this stage, only a handful of studies have utilized imaging technology to measure cavitation resistance in trees (Torres-Ruiz et al., 2014; Cochard et al., 2015), and these studies employed a destructive mode of the technique in which small branches were cut off the plant before scanning took place. Thus far, noninvasive imaging on intact plants has only been used to measure cavitation resistance in two species, grapevine (Vitis vinifera; Choat et al., 2010; Brodersen et al., 2013) and Sequoia sempervirens (Choat et al., 2015). Further measurement of cavitation resistance using noninvasive imaging on intact plants across a range of species, therefore, is a high priority.These comparisons are particularly important because of the current debate surrounding the invasive techniques (Cochard et al., 2013). Specifically, evidence from a variety of experiments suggests that centrifuge and air injection techniques underestimate cavitation resistance in species with long xylem vessels (Choat et al., 2010; Cochard et al., 2010; Ennajeh et al., 2011; Martin-StPaul et al., 2014; Torres-Ruiz et al., 2014; Wang et al., 2014). This artifact occurs when samples placed into centrifuge rotors or air injection collars have a large proportion of vessels that are cut open at both ends of the segment. A number of studies have disputed this open-vessel hypothesis and suggested that some versions of the centrifuge and air injection techniques provide reliable estimates of cavitation resistance (Jacobsen and Pratt, 2012; Sperry et al., 2012; Tobin et al., 2013). Because there will always be uncertainties associated with indirect measurements, noninvasive imaging using intact plants provides the best option for resolving these methodological issues.In this study, synchrotron-based microCT was utilized to investigate the formation of drought-induced embolism in the xylem of intact, potted plants. Three species were selected to provide a range of contrasting xylem structures: Quercus robur (ring porous), Populus tremula × Populus alba (diffuse porous), and Pinus pinaster (tracheid bearing). Visualizations of xylem embolism in the stems of these species during a sequence of natural dehydration were used to construct embolism vulnerability curves. We hypothesized that (1) vulnerability curves based on microCT observations would match vulnerability curves based on the centrifuge technique for species with short vessels (P. tremula × P. alba) or tracheid-based xylem (P. pinaster) and (2) the centrifuge technique would overestimate vulnerability to embolism in the long-vesseled species (Q. robur) due to the open-vessel artifact.     

Herbaceous Angiosperms Are Not More Vulnerable to Drought-Induced Embolism Than Angiosperm Trees

The water transport pipeline in herbs is assumed to be more vulnerable to drought than in trees due to the formation of frequent embolisms (gas bubbles), which could be removed by the occurrence of root pressure, especially in grasses. Here, we studied hydraulic failure in herbaceous angiosperms by measuring the pressure inducing 50% loss of hydraulic conductance (P₅₀) in stems of 26 species, mainly European grasses (Poaceae). Our measurements show a large range in P₅₀ from −0.5 to −7.5 MPa, which overlaps with 94% of the woody angiosperm species in a worldwide, published data set and which strongly correlates with an aridity index. Moreover, the P₅₀ values obtained were substantially more negative than the midday water potentials for five grass species monitored throughout the entire growing season, suggesting that embolism formation and repair are not routine and mainly occur under water deficits. These results show that both herbs and trees share the ability to withstand very negative water potentials without considerable embolism formation in their xylem conduits during drought stress. In addition, structure-function trade-offs in grass stems reveal that more resistant species are more lignified, which was confirmed for herbaceous and closely related woody species of the daisy group (Asteraceae). Our findings could imply that herbs with more lignified stems will become more abundant in future grasslands under more frequent and severe droughts, potentially resulting in lower forage digestibility.Terrestrial biomes provide numerous ecosystem services to humans, such as biodiversity refuges, forage supply, carbon sequestration, and associated atmospheric feedback (Bonan, 2008). Drought frequency and severity are predicted to increase across various ecosystems (Dai, 2013), and its impact on the fate of terrestrial biomes has aroused great concern for stakeholders over the past decade. For instance, worldwide forest declines have been associated with drought events (Allen et al., 2010), and the sustainability of grasslands, one of the most important agro-ecosystems representing 26% of the world land area, is threatened due to increasing aridity in the light of climate change (Tubiello et al., 2007; Brookshire and Weaver, 2015). Since the maintenance of grasslands is of prime importance for livestock, and several of the most valuable crops are grasses, herbaceous species deserve more attention from a hydraulic point of view to understand how they will cope with shifts in precipitation and temperature patterns.During water deficit, hydraulic failure in trees has been put forward as one of the primary causes of forest decline (Anderegg et al., 2015, 2016). Drought exacerbates the negative pressure inside the water conducting cells, making the liquid xylem sap more metastable, and thus more vulnerable, to air entry (i.e. gas embolism; Lens et al., 2013a). Extensive levels of embolisms may lead to desiccation, leaf mortality, branch sacrifice, and ultimately plant death (Barigah et al., 2013; Urli et al., 2013). Plant resistance to embolism is therefore assumed to represent a key parameter in determining the drought tolerance of trees and is estimated using so-called vulnerability curves (VCs), from which the P₅₀, i.e. the sap pressure inducing 50% loss of hydraulic conductivity, can be estimated (Cochard et al., 2013). P₅₀ values are therefore good proxies for drought stress tolerance in woody plants and have been published for hundreds of angiosperm and gymnosperm tree species (Delzon et al., 2010; Choat et al., 2012), illustrating a wide range from −0.5 to −19 MPa (Larter et al., 2015).Studies focusing on P₅₀ values of herbs are limited to stems of ∼14 angiosperm species (see Supplemental Table S1 and references cited therein). Half of the herbaceous angiosperms studied so far (Supplemental Table S1) have a stem P₅₀ between 0 and −2 MPa, indicating that many herbs are highly vulnerable to embolism. Moreover, positive root pressure has been reported in various herbs, including many grasses (Poaceae) with hydathodes in their leaves (Evert, 2006), and root pressure is hypothesized to refill embolized conduits overnight when transpiration is low (Miller, 1985; Neufeld et al., 1992; Cochard et al., 1994; Macduff and Bakken, 2003; Saha et al., 2009; Cao et al., 2012). This could suggest that embolism formation and repair follow a daily cycle in herbs. In other words, the midday water potential that herbs experience in the field may often be more negative than P₅₀, which would result in an extremely vulnerable hydraulic pipeline characterized by a negative hydraulic safety margin (expressed as the minimum midday water potential minus P₅₀). In contrast to herbs, most trees operate at a slightly positive hydraulic safety margin (Choat et al., 2012), and woody plants are often too tall to allow refilling by positive root and/or stem pressure in the upper stems (Ewers et al., 1997; Fisher et al., 1997). Therefore, it could be postulated that herbaceous species possess a hydraulic system that is more vulnerable to embolism than that of woody species. In this study, we want to underpin possible differences in embolism resistance between stems of herbaceous and woody angiosperms.The scarcity of P₅₀ measures in herbaceous angiosperms, including grasses and herbaceous eudicots, is mainly due to their fragile stems and low hydraulic conductivity, making VCs technically more challenging. Using minor adaptations to existing centrifuge techniques (Supplemental Text S1), we obtained a P₅₀ stem data set of 26 herbaceous angiosperm species (mainly grasses) from various collection sites in France and Switzerland. In addition, we compared our data set with published data from woody (gymnosperm and angiosperm) species, confronted some of our herbaceous eudicot measurements with original P₅₀ data from derived, woody relatives, and performed anatomical observations in grasses to investigate a possible link between stem anatomical characters and differences in P₅₀ among the species studied. Three main research questions are central in our article: (1) Are stems of herbaceous angiosperms more vulnerable to embolism than those of woody angiosperms? (2) Do grasses operate with highly vulnerable, negative hydraulic safety margins? (3) Do grasses show structure-function trade-offs in their stems with respect to embolism resistance?     

Allometry in Anisian (Middle Triassic) segminiplanate conodonts and its implications for conodont taxonomy

Conodonts are a clade of chordates and are valuable indicator fossils for biostratigraphy. The segminiplanate (neogondolelliform) conodonts represent a major morphological group ranging from upper Carboniferous to Upper Triassic marine sediments. However, the morphological similarity of segminiplanate P₁ elements generates problems for taxonomy, especially in the Permian and Triassic clades. This paper represents the first study of morphological variation in Triassic segminiplanate conodonts using a geometric morphometric approach. The laminar microstructures observed in conodont cross‐sections indicate that, within our analysed specimens, smaller conodonts with fewer laminae are generally from an earlier ontogenetic stage while larger conodonts with more laminae are from a later stage of ontogeny. Using linear regressions between relative warp scores from both upper and lateral views and conodont length, we demonstrate strongly allometric growth patterns for the species Paragondolella bifurcata Budurov & Stefanov. Our results indicate that the species‐group taxon Pg. praeszaboi bystrickyi (Kovacs et al.) is an early growth stage of Pg. bifurcata and thus synonymous. We suggest that the allometry of conodonts should be considered seriously, especially when there are numerous transitional morphologies between large‐ and small‐sized conodonts. Reconstructing the ontogenetic series and using larger‐sized conodonts within the numerous transitional morphologies in the population of a rock sample for the definition of new species are suggested for future studies.     

Old and unmowed saltmarsh patches provide attractive habitats for breeding passerines

Although the conservation stakes of saltmarshes are widely documented, these areas are still subjected to strong anthropic pressures, including land reclamation, leading to their conversion into arable lands, and agricultural exploitation (mainly cattle grazing and mowing), which modifies their floral and faunal composition. Through the example of one of the largest French saltmarshes, we first assessed how the age of the saltmarsh patches and the mowing intensity determined the spatial distribution of the different saltmarsh habitats. We then tested how the five commonest breeding passerines were distributed in accordance with the mowing activity and the distribution of these habitats. We found that the oldest and the unmowed patches promote the development of habitats dominated by Elymus pungens and Atriplex portulacoides, and also host the highest abundance of four of the five bird species studied. In the current context of an intense artificialization of the littoral area, this study highlights the importance of maintaining the oldest and the least human-impacted patches of natural habitats to conserve their associated biodiversity.     

Next-generation bis-locked nucleic acids with stacking linker and 2′-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes

Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson–Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson–Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2′-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.     

Modeling spatial expansion of invasive alien species: relative contributions of environmental and anthropogenic factors to the spreading of the harlequin ladybird in France

Species distribution models (SDM) have often been used to predict the potential ranges of introduced species and prioritize management strategies. However, this approach assumes equilibrium between occurrences and environmental gradients, an assumption which is violated during the invasion process, where many suitable sites are empty because the species has not yet reached them. Here we considered the invasive ladybird Harmonia axyridis as a case study to show the benefits of using a dynamic colonization–extinction model that does not assume equilibrium. We used a multi‐year occupancy model incorporating environmental, anthropogenic and neighborhood effects, to identify factors that explained spreading variation of this species in France from 2004, when only a few occupied sites were detected, to 2011. We found that anthropogenic factors (urbanization, agriculture, vineyards, and presence/absence of highways) explained more variation in the diffusion process than environmental factors (winter and summer temperatures, wind‐speed, and rainfall). The surface of urbanization was the major anthropogenic factor increasing the probability of colonization. The average summer temperature was the main environmental factor affecting colonization, with a negative effect when high or low. The neighborhood effect revealed that colonization was mostly influenced by contributions coming from a radius of 24 km around the focal cell. The contribution of neighborhood decreases over time, suggesting that H. axyridis is reaching its equilibrium in France. This is confirmed by the small discrepancy observed between the performance of our approach and a SDM approach when predicting a single year occupancy pattern at the end of the study period. Our approach has the advantage of explicitly modelling the state of the biological system during the spatial expansion and identifying colonization constraints. This allows managers to explore the effect of different actions on the system at key moments of the invasion process, hence providing a powerful approach to prioritize management strategies.     

Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: C_L1, C_L2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (C_L1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages C_L1, C_L2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages C_L1, C_L2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts.     

Phylogeography of the heavily poached African common pangolin (Pholidota,Manis tricuspis) reveals six cryptic lineages as traceable signatures of Pleistocene diversification

Knowledge on faunal diversification in African rainforests remains scarce. We used phylogeography to assess (i) the role of Pleistocene climatic oscillations in the diversification of the African common pangolin (Manis tricuspis) and (ii) the utility of our multilocus approach for taxonomic delineation and trade tracing of this heavily poached species. We sequenced 101 individuals for two mitochondrial DNA (mtDNA), two nuclear DNA and one Y‐borne gene fragments (totalizing 2602 bp). We used a time‐calibrated, Bayesian inference phylogenetic framework and conducted character‐based, genetic and phylogenetic delineation of species hypotheses within African common pangolins. We identified six geographic lineages partitioned into western Africa, Ghana, the Dahomey Gap, western central Africa, Gabon and central Africa, all diverging during the Middle to Late Pleistocene. MtDNA (cytochrome b + control region) was the sole locus to provide diagnostic characters for each of the six lineages. Tree‐based Bayesian delimitation methods using single‐ and multilocus approaches gave high support for ‘species’ level recognition of the six African common pangolin lineages. Although the diversification of African common pangolins occurred during Pleistocene cyclical glaciations, causative correlation with traditional rainforest refugia and riverine barriers in Africa was not straightforward. We conclude on the existence of six cryptic lineages within African common pangolins, which might be of major relevance for future conservation strategies. The high discriminative power of the mtDNA markers used in this study should allow an efficient molecular tracing of the regional origin of African common pangolin seizures.     

Engineering stilbene metabolic pathways in microbial cells

Numerous in vitro and in vivo studies on biological activities of phytostilbenes have brought to the fore the remarkable properties of these compounds and their derivatives, making them a top storyline in natural product research fields. However, getting stilbenes in sufficient amounts for routine biological activity studies and make them available for pharmaceutical and/or nutraceutical industry applications, is hampered by the difficulty to source them through synthetic chemistry-based pathways or extraction from the native plants. Hence, microbial cell cultures have rapidly became potent workhorse factories for stilbene production. In this review, we present the combined efforts made during the past 15?years to engineer stilbene metabolic pathways in microbial cells, mainly the Saccharomyces cerevisiae baker yeast, the Escherichia coli and the Corynebacterium glutamicum bacteria. Rationalized approaches to the heterologous expression of the partial or the entire stilbene biosynthetic routes are presented to allow the identification and/or bypassing of the major bottlenecks in the endogenous microbial cell metabolism as well as potential regulations of the genes involved in these metabolic pathways. The contributions of bioinformatics to synthetic biology are developed to highlight their tremendous help in predicting which target genes are likely to be up-regulated or deleted for controlling the dynamics of precursor flows in the tailored microbial cells. Further insight is given to the metabolic engineering of microbial cells with “decorating” enzymes, such as methyl and glycosyltransferases or hydroxylases, which can act sequentially on the stilbene core structure. Altogether, the cellular optimization of stilbene biosynthetic pathways integrating more and more complex constructs up to twelve genetic modifications has led to stilbene titers ranging from hundreds of milligrams to the gram-scale yields from various carbon sources. Through this review, the microbial production of stilbenes is analyzed, stressing both the engineering dynamic regulation of biosynthetic pathways and the endogenous control of stilbene precursors.