Seed dispersal by ungulates as an ecological filter: a trait‐based meta‐analysis

Plant communities are often dispersal‐limited and zoochory can be an efficient mechanism for plants to colonize new patches of potentially suitable habitat. We predicted that seed dispersal by ungulates acts as an ecological filter – which differentially affects individuals according to their characteristics and shapes species assemblages – and that the filter varies according to the dispersal mechanism (endozoochory, fur‐epizoochory and hoof‐epizoochory). We conducted two‐step individual participant data meta‐analyses of 52 studies on plant dispersal by ungulates in fragmented landscapes, comparing eight plant traits and two habitat indicators between dispersed and non‐dispersed plants. We found that ungulates dispersed at least 44% of the available plant species. Moreover, some plant traits and habitat indicators increased the likelihood for plant of being dispersed. Persistent or nitrophilous plant species from open habitats or bearing dry or elongated diaspores were more likely to be dispersed by ungulates, whatever the dispersal mechanism. In addition, endozoochory was more likely for diaspores bearing elongated appendages whereas epizoochory was more likely for diaspores released relatively high in vegetation. Hoof‐epizoochory was more likely for light diaspores without hooked appendages. Fur‐epizoochory was more likely for diaspores with appendages, particularly elongated or hooked ones. We thus observed a gradient of filtering effect among the three dispersal mechanisms. Endozoochory had an effect of rather weak intensity (impacting six plant characteristics with variations between ungulate‐dispersed and non‐dispersed plant species mostly below 25%), whereas hoof‐epizoochory had a stronger effect (eight characteristics included five ones with above 75% variation), and fur‐epizoochory an even stronger one (nine characteristics included six ones with above 75% variation). Our results demonstrate that seed dispersal by ungulates is an ecological filter whose intensity varies according to the dispersal mechanism considered. Ungulates can thus play a key role in plant community dynamics and have implications for plant spatial distribution patterns at multiple scales. Synthesis Plant communities are often dispersal‐limited and zoochory can be an efficient mechanism for plants to colonize new patches of potentially suitable habitat. Our analysis is the first synthesis of ungulate seed dispersal that compares characteristics from both non‐dispersed and dispersed diaspores, distinguishing the three zoochory mechanisms ungulates are involved in: endozoochory, hoof‐epizoochory and fur‐epizoochory. We confirmed that seed dispersal by ungulates is an ecological filter whose intensity increases from endozoochory, then hoof‐epizoochory to finally fur‐epizoochory. By filtering seed traits through dispersal, ungulates can thus play a key role in plant community dynamics and have implications for plant spatial distribution patterns at multiple scales.     

Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]_cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]_cyt of the pollen tubes in vivo, we show that [pH]_cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]_cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]_cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]_cyt and its effect on several components in the context of SI-induced events and PCD.Programmed cell death (PCD) in plants is relatively well documented and characterized (Jones and Dangl, 1996; van Doorn, 2011; van Doorn et al., 2011). There is considerable biochemical evidence for the involvement of caspase-like activities in plant PCD (van Doorn and Woltering, 2005). For example, the vacuolar processing enzyme has YVADase (caspase-1-like) activity (Hatsugai et al., 2004; Rojo et al., 2004; Hara-Nishimura et al., 2005), DEVDase (caspase-3-like) and YVADases are associated with PCD in several plant systems (del Pozo and Lam, 1998; Korthout et al., 2000; Danon et al., 2004), and VEIDase (caspase-6-like) is the main caspase-like activity involved in embryonic pattern formation (Bozhkov et al., 2004). However, because plants have no caspase gene homologs (Sanmartín et al., 2005), the nature of their caspase-like enzymes is the subject of considerable debate. Vacuolar cell death is one of two major classes of PCD in plants (van Doorn et al., 2011). It is thought that collapse of the vacuole is a key irreversible step in several plant PCD systems, including during tissue and organ formation, such as the classic differentiation of tracheary elements (Hara-Nishimura and Hatsugai, 2011). Exactly how this is achieved and what processes are involved remain unknown. Until very recently, it was generally thought that the rupturing vacuole releases proteases, hydrolases, and nucleases, allowing cellular disassembly by an autophagy-like process. Some PCD systems cannot be assigned to either class; these include PCD triggered by the hypersensitive response to biotrophic pathogens, PCD in cereal endosperm, and self-incompatibility (SI)-induced PCD (van Doorn et al., 2011).SI is a genetically controlled pollen-pistil cell-cell recognition system. Self-pollen is recognized by the stigma as being genetically identical, resulting in inhibition of pollen tube growth. Most SI systems use tightly linked polymorphic genes: the pollen (male) and pistil (female) S-determinants. In field poppy (Papaver rhoeas), the S-determinants are a 14-kD signaling ligand field poppy stigma S (PrsS) and a unique transmembrane protein field poppy pollen S (PrpS; Foote et al., 1994; Wheeler et al., 2010). These interact in an S-specific manner, and increases in cytosolic free calcium ([Ca2+]_cyt) are triggered in incompatible pollen tubes (Franklin-Tong et al., 1993), resulting in phosphorylation of soluble inorganic pyrophosphatases (sPPases; Rudd et al., 1996; de Graaf et al., 2006), activation of a Mitogen-Activated Protein Kinase (MAPK; Rudd et al., 2003), and increases in reactive oxygen species (ROS) and nitric oxide (Wilkins et al., 2011, 2014). Most of these components are integrated into a signaling network leading to PCD (Bosch et al., 2008; Wilkins et al., 2014). The actin cytoskeleton is a key target in the field poppy SI response, undergoing depolymerization (Snowman et al., 2002) followed by polymerization into highly stable F-actin foci decorated with the actin binding proteins (ABPs) Actin-Depolymerizing Factor (ADF) and Cyclase-Associated Protein (CAP; Poulter et al., 2010, 2011), with both processes being involved in mediating PCD (Thomas et al., 2006). A major player in SI-mediated PCD is a caspase-3-like/DEVDase-like activity (Thomas and Franklin-Tong, 2004; Bosch and Franklin-Tong, 2007). The SI-induced caspase-3-like/DEVDase exhibits maximum substrate cleavage in vitro at pH 5, with peak activity 5 h after SI induction in vivo (Bosch and Franklin-Tong, 2007). The low pH optimum for this caspase-3-like/DEVDase activity is unusual, because most of the cytosolic plant caspase-like activities identified to date have optimal activity close to normal physiological pH (approximate pH, 6.5–7.0; Korthout et al., 2000; Bozhkov et al., 2004; Coffeen and Wolpert, 2004). Because the SI-induced cytosolic-located DEVDase requires a low pH for activity, this suggested that, during SI, the pollen tube cytosol undergoes dramatic acidification. In vivo pH measurements of the cytosol at 1 to 4 h after SI induction confirmed this, when cytosolic pH ([pH]_cyt) had dropped from pH 6.9 to pH 5.5 (Bosch and Franklin-Tong, 2007). This fits the in vitro pH optimum of the caspase-3-like/DEVDase almost exactly, implicating pollen cytosolic acidification as playing a vital role in creating optimal conditions for the activation of the caspase-3-like/DEVDase-like activity and progression of PCD.Under normal cellular conditions, [pH]_cyt is between approximately 6.9 and 7.5 (Kurkdjian and Guern, 1989; Felle, 2001). Pollen tubes, like other tip-growing cells, have [pH]_cyt gradients (Gibbon and Kropf, 1994; Feijó et al., 1999). The [pH]_cyt of the pollen tube shank is an approximate pH of 6.9 to 7.11 (Fricker et al., 1997; Messerli and Robinson, 1998). There has been much debate about the [pH]_cyt gradient, comprising an apical domain with an approximate pH of 6.8 and a subapical alkaline band with an approximate pH of 7.2 to 7.8 in Lilium longiflorum and Lilium formosanum pollen tubes (Fricker et al., 1997; Messerli and Robinson, 1998; Feijó et al., 2001; Lovy-Wheeler et al., 2006). Oscillations of [pH]_cyt between approximate pH values of 6.9 and 7.3 have been linked to tip growth in L. formosanum pollen tubes (Lovy-Wheeler et al., 2006). The vacuole and the apoplast have a highly acidic pH between pH 5 and pH 6 (Katsuhara et al., 1989; Feijó et al., 1999). The majority of studies of pH changes in plant cells reports modest, transient changes in [pH]_cyt of approximately 0.4 and 0.7 pH units during development, gravitropic responses, decreases in light intensity, and addition of elicitors, hormones, and other treatments. For example, during root hair development in Arabidopsis (Arabidopsis thaliana), root [pH]_cyt was elevated from an approximate pH of 7.3 to 7.7 (Bibikova et al., 1998). Root gravitropic responses stimulate small transient [pH]_cyt alterations (Scott and Allen, 1999; Fasano et al., 2001; Johannes et al., 2001). More recently, it has been shown that the [pH]_cyt drops during PCD controlling root cap development; however, exactly how many units the [pH]_cyt decreased was not measured (Fendrych et al., 2014). Other studies investigating [pH]_cyt in response to physiologically relevant signals also report small transient alterations. Light-adapted cells respond to a decrease in light intensity with a rapid transient cytosolic acidification by approximately 0.3 pH units (Felle et al., 1986). Addition of nodulation factors resulted in an increase of 0.2 pH units in root hairs (Felle et al., 1998), and abscisic acid increased the [pH]_cyt of guard cells by 0.3 pH units (Blatt and Armstrong, 1993). Changes in [pH]_cyt are thought to activate stress responses (Felle, 2001). Elicitor treatments resulted in a [pH]_cyt drop of between 0.4 and 0.7 pH units in suspension cells (Mathieu et al., 1996; Kuchitsu et al., 1997), a drop of 0.2 pH units in Nitellopsis obtusa cells treated with salt (Katsuhara et al., 1989), and a drop of 0.3 to 0.7 pH units in Eschscholzia californica (Roos et al., 1998).Here, we investigate SI-induced acidification of the cytosol, providing measurements of physiologically relevant temporal alterations in [pH]_cyt, and identify key targets of this, providing mechanistic insights into these events. The SI-induced acidification plays a pivotal role in the activation of a caspase-3-like/DEVDase activity, the formation of punctate F-actin foci, and ABP localization during SI PCD. We investigate the vacuole as a potential contributor to SI-induced [pH]_cyt acidification.     

Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton

Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca^b1086 loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal–ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.     

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1^Cre) mouse. Embryonic Wt1^Cre;Alk3^fl/fl specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1^Cre;Alk3^fl/fl mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1^Cre;Alk3^fl/fl specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.     

A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves

Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C₃ leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO₂ concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis.Photosynthetic metabolism continues to be studied intensively because of its importance for crop performance and the global carbon cycle in relation to climate change. The metabolic pathways and enzymes involved in carbon fixation and related metabolic processes, such as the synthesis of Suc and starch, have been well-characterized. However, it is apparent that full appreciation of leaf metabolism requires these metabolic processes to be placed in the context of the wider metabolic network (Szecowka et al., 2013). This is particularly important for predicting how strategies for engineering improved photosynthesis (Maurino and Weber, 2013) may affect network properties, such as redox and energy balancing (Kramer and Evans, 2011).Flux balance analysis (FBA) has emerged as the method of choice for predicting fluxes in large metabolic network models (Sweetlove and Ratcliffe, 2011), and several flux balance models have explicitly considered photosynthetic metabolism in a variety of plants species and microorganisms, including cyanobacteria (Synechocystis sp. PCC 6803; Knoop et al., 2010, 2013; Montagud et al., 2010; Nogales et al., 2012; Saha et al., 2012), Chlamydomonas reinhardtii (Boyle and Morgan, 2009; de Oliveira Dal''Molin et al., 2011), Arabidopsis (Arabidopsis thaliana; de Oliveira Dal’Molin et al., 2010a), rapeseed (Brassica napus) embryos (Hay and Schwender, 2011), rice (Oryza sativa; Poolman et al., 2013), maize (Zea mays; Saha et al., 2011), and several C₄ plants (de Oliveira Dal’Molin et al., 2010b). These models successfully predicted the metabolic routes involved in the fixation of CO₂ into different biomass components in the light. However, one major feature of metabolism of photosynthetic organisms, namely the interaction between light and dark metabolism, is neglected in most of these studies. Effectively, most models assume that the organism grows in constant light, which is rarely true in natural conditions.Apart from the obvious switch from photoautotrophic to heterotrophic metabolism between day and night, interactions between the two phases can occur through the temporal separation of storage compound synthesis and subsequent mobilization. For example, it has been shown that the carbon skeletons used for nitrogen assimilation during the day are largely provided by carboxylic acids that were synthesized and stored during the previous night (Gauthier et al., 2010). Such temporal shifts of carbon and nitrogen metabolism have substantial implications for fluxes in the central metabolic network of leaves in the light (Tcherkez et al., 2009). Interactions between temporally separated metabolic events are also a critical feature of Crassulacean acid metabolism (CAM) photosynthesis, in which CO₂ is initially fixed at night by phosphoenolpyruvate carboxylase (PEPC), leading to night storage of carboxylic acids (mainly malic acid) that are decarboxylated during the day to provide CO₂ for the conventional photosynthetic carbon assimilation cycle. Although this is principally an adaptation to arid environments, there are unresolved questions as to whether CAM photosynthesis is energetically more efficient than C₃ photosynthesis (Winter and Smith, 1996). Such questions are becoming more important in the light of the proposed use of CAM plants as a source of biofuel (Yan et al., 2011).One recent study used FBA to consider both light and dark metabolism in Synechocystis sp. PCC 6803 over a complete diel cycle divided into 192 time steps (Knoop et al., 2013). Time courses of metabolic flux predictions over a diel cycle were simulated by altering the constraints on metabolic outputs (biomass composition) depending on the time point and based on empirical rules. This simulation led to a highly constrained model and did not allow the range of potential interactions between the day and night phases to be fully explored. We have developed an alternative modeling framework for integrated day-night FBA, in which the metabolic fluxes in the light and dark phases were simulated simultaneously in a single optimization problem. A predefined list of storage compounds that can accumulate freely over the diurnal cycle was made available to the model. The model was then free to choose among these storage compounds, the choice being dictated by the need to satisfy the objective function within the applied constraints. This diurnal modeling framework was used to explore the interactions between light and dark metabolism and to predict the metabolic fluxes in the light in both C₃ and CAM photosynthesis. We show that accounting for day-night interactions leads to an altered pattern of fluxes during the day that provides a better match with experimental observations. We were also able to simulate network flux distributions in CAM metabolism. The model successfully predicts the classic CAM cycle in the different CAM subtypes and allows a comparison of the energetic efficiency and metabolic costs between CAM and C₃ photosynthetic metabolism.     

Ethylene is differentially regulated during sugar beet germination and affects early root growth in a dose-dependent manner

Main conclusion

By integrating molecular, biochemical, and physiological data, ethylene biosynthesis in sugar beet was shown to be differentially regulated, affecting root elongation in a concentration-dependent manner. There is a close relation between ethylene production and seedling growth of sugar beet (Beta vulgaris L.), yet the exact function of ethylene during this early developmental stage is still unclear. While ethylene is mostly considered to be a root growth inhibitor, we found that external 1-aminocyclopropane-1-carboxylic acid (ACC) regulates root growth in sugar beet in a concentration-dependent manner: low concentrations stimulate root growth while high concentrations inhibit root growth. These results reveal that ethylene action during root elongation is strongly concentration dependent. Furthermore our detailed study of ethylene biosynthesis kinetics revealed a very strict gene regulation pattern of ACC synthase (ACS) and ACC oxidase (ACO), in which ACS is the rate liming step during sugar beet seedling development.     

Single versus multiple enemies and the impact on biological control of spider mites in cassava fields in West-Africa

To determine whether to use single or multiple predator species for biological pest control requires manipulative field experiments. We performed such tests in Benin (West Africa) in cassava fields infested by the cassava green mite Mononychellus tanajoa, and the cotton red mite Oligonychus gossypii. These fields also harboured the cassava apex-inhabiting predator Typhlodromalus aripo and either the leaf-inhabiting predator Amblydromalus manihoti or Euseius fustis. We manipulated predator species composition on individual plants to determine their effect on prey and predator densities. In fields with T. aripo plus A. manihoti, M. tanajoa densities were reduced by T. aripo alone or together with A. manihoti, but neither of these predators, alone or together, reduced O. gossypii densities. In fields with T. aripo plus E. fustis, T. aripo alone or together with E. fustis exerted significant control over O. gossypii, but weak control over M. tanajoa. Densities of any of the predator species were not affected by co-occurring predator species, suggesting a minor role for intraguild predation in the field, contrary to earlier experiments on small plants in the laboratory. We conclude that (1) T. aripo is the most effective predator species in suppressing M. tanajoa, (2) two predator species, T. aripo and E. fustis, are needed to suppress O. gossypii, and (3) predator species together on the same plant do not negatively affect each other nor the extent to which they control their prey. We argue that intraguild predation is reduced due to partial niche separation among predator species.     

Genotype,development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus

Background and Aims

Species and hybrids of the genus Miscanthus contain attributes that make them front-runners among current selections of dedicated bioenergy crops. A key trait for plant biomass conversion to biofuels and biomaterials is cell-wall quality; however, knowledge of cell-wall composition and biology in Miscanthus species is limited. This study presents data on cell-wall compositional changes as a function of development and tissue type across selected genotypes, and considers implications for the development of miscanthus as a sustainable and renewable bioenergy feedstock.

Methods

Cell-wall biomass was analysed for 25 genotypes, considering different developmental stages and stem vs. leaf compositional variability, by Fourier transform mid-infrared spectroscopy and lignin determination. In addition, a Clostridium phytofermentans bioassay was used to assess cell-wall digestibility and conversion to ethanol.

Key Results

Important cell-wall compositional differences between miscanthus stem and leaf samples were found to be predominantly associated with structural carbohydrates. Lignin content increased as plants matured and was higher in stem tissues. Although stem lignin concentration correlated inversely with ethanol production, no such correlation was observed for leaves. Leaf tissue contributed significantly to total above-ground biomass at all stages, although the extent of this contribution was genotype-dependent.

Conclusions

It is hypothesized that divergent carbohydrate compositions and modifications in stem and leaf tissues are major determinants for observed differences in cell-wall quality. The findings indicate that improvement of lignocellulosic feedstocks should encompass tissue-dependent variation as it affects amenability to biological conversion. For gene–trait associations relating to cell-wall quality, the data support the separate examination of leaf and stem composition, as tissue-specific traits may be masked by considering only total above-ground biomass samples, and sample variability could be mostly due to varying tissue contributions to total biomass.