Fitness trade-offs and the origins of endosymbiosis.

Endosymbiosis drives evolutionary innovation and underpins the function of diverse ecosystems. The mechanistic origins of symbioses, however, remain unclear, in part because early evolutionary events are obscured by subsequent evolution and genetic drift. This Essay highlights how experimental studies of facultative, host-switched, and synthetic symbioses are revealing the important role of fitness trade-offs between within-host and free-living niches during the early-stage evolution of new symbiotic associations. The mutational targets underpinning such trade-offs are commonly regulatory genes, such that single mutations have major phenotypic effects on multiple traits, thus enabling and reinforcing the transition to a symbiotic lifestyle.

Phytase catalysis of dephosphorylation studied using isothermal titration calorimetry and electrospray ionization time-of-flight mass spectroscopy.

Phytases are important commercial enzymes that catalyze the dephosphorylation of myo-inositol hexakisphosphate (phytate) to its lower inositol phosphate (IP) esters, IP6 to IP1. Digestion of phytate by Citrobacter braakii 6-phytase deviates significantly from monophasic Michaelis-Menten kinetics. Analysis of phytate digestion using isothermal titration calorimetry (ITC) using the single injection method produced a thermogram with two peaks consistent with two periods of high enzyme activity. Continuous-flow electrospray ionization time-of-flight mass spectroscopy (ESI-ToF-MS) provided real-time analysis of phytase catalysis. It was able to show that the first two cleavage steps were rapid and concurrent but the third cleavage step from IP4 to IP3 was slow. The third (IP4 to IP3), fourth (IP3 to IP2) and fifth (IP2 to IP1) cleavages were effectively sequential due to the preferred association of the more phosphorylated species with the phytase catalytic site. This created a bottleneck during the cleavage of IP4 to IP3 until the point at which IP4 was exhausted and was followed by the rapid cleavage of IP3 to IP2, which was observed as the second peak in the ITC thermogram. This work illustrates the importance of an orthogonal approach when studying non-specific or complex enzyme catalyzed reactions.

Functional complementarity of ancient plant-fungal mutualisms: contrasting nitrogen, phosphorus and carbon exchanges between Mucoromycotina and Glomeromycotina fungal symbionts of liverworts.

Liverworts, which are amongst the earliest divergent plant lineages and important ecosystem pioneers, often form nutritional mutualisms with arbuscular mycorrhiza-forming Glomeromycotina and fine-root endophytic Mucoromycotina fungi, both of which coevolved with early land plants. Some liverworts, in common with many later divergent plants, harbour both fungal groups, suggesting these fungi may complementarily improve plant access to different soil nutrients. We tested this hypothesis by growing liverworts in single and dual fungal partnerships under a modern atmosphere and under 1500 ppm [CO2 ], as experienced by early land plants. Access to soil nutrients via fungal partners was investigated with 15 N-labelled algal necromass and 33 P orthophosphate. Photosynthate allocation to fungi was traced using 14 CO2 . Only Mucoromycotina fungal partners provided liverworts with substantial access to algal 15 N, irrespective of atmospheric CO2 concentration. Both symbionts increased 33 P uptake, but Glomeromycotina were often more effective. Dual partnerships showed complementarity of nutrient pool use and greatest photosynthate allocation to symbiotic fungi. We show there are important functional differences between the plant-fungal symbioses tested, providing new insights into the functional biology of Glomeromycotina and Mucoromycotina fungal groups that form symbioses with plants. This may explain the persistence of the two fungal lineages in symbioses across the evolution of land plants.

Variation and asymmetry in host-symbiont dependence in a microbial symbiosis.

BACKGROUND: Symbiosis is a major source of evolutionary innovation and, by allowing species to exploit new ecological niches, underpins the functioning of ecosystems. The transition from free-living to obligate symbiosis requires the alignment of the partners' fitness interests and the evolution of mutual dependence. While symbiotic taxa are known to vary widely in the extent of host-symbiont dependence, rather less is known about variation within symbiotic associations. RESULTS: Using experiments with the microbial symbiosis between the protist Paramecium bursaria and the alga Chlorella, we show variation between pairings in host-symbiont dependence, encompassing facultative associations, mutual dependence and host dependence upon the symbiont. Facultative associations, that is where both the host and the symbiont were capable of free-living growth, displayed higher symbiotic growth rates and higher per host symbiont loads than those with greater degrees of dependence. CONCLUSIONS: These data show that the Paramecium-Chlorella interaction exists at the boundary between facultative and obligate symbiosis, and further suggest that the host is more likely to evolve dependence than the algal symbiont.

Reducing Stomatal Density in Barley Improves Drought Tolerance without Impacting on Yield.

The epidermal patterning factor (EPF) family of secreted signaling peptides regulate the frequency of stomatal development in model dicot and basal land plant species. Here, we identify and manipulate the expression of a barley (Hordeum vulgare) ortholog and demonstrate that when overexpressed HvEPF1 limits entry to, and progression through, the stomatal development pathway. Despite substantial reductions in leaf gas exchange, barley plants with significantly reduced stomatal density show no reductions in grain yield. In addition, HvEPF1OE barley lines exhibit significantly enhanced water use efficiency, drought tolerance, and soil water conservation properties. Our results demonstrate the potential of manipulating stomatal frequency for the protection and optimization of cereal crop yields under future drier environments.

A novel root-to-shoot stomatal response to very high CO2 levels in the soil: electrical, hydraulic and biochemical signalling.

Investigations were undertaken in the context of the potential environmental impact of carbon capture and storage (CCS) transportation in the form of a hypothetical leak of extreme levels of CO2 into the soil environment and subsequent effects on plant physiology. Laboratory studies using purpose built soil chambers, separating and isolating the soil and aerial environments, were used to introduce high levels of CO2 gas exclusively into the rhizosphere. CO2 concentrations greater than 32% in the isolated soil environment revealed a previously unknown whole plant stomatal response. Time course measurements of stomatal conductance (gs ), leaf temperature and leaf abscisic acid (ABA) show strong coupling between all three variables over a specific period (3 h following CO2 gassing) occurring as a result of CO2 -specific detection by roots. The coupling of gs and ABA subsequently breaks down resulting in a rapid and complete loss of turgor in the shoot. Root access to water is severely restricted as evidenced by the inability to counter turgor loss, however, the plant regains some turgor over time. Recovery of full turgor is not achieved over the longer term. Results suggest an immediate perception and whole plant response as changes in measured parameters (leaf temperature, gs and ABA) occur in the shoot, but the response is solely due to detection of very high CO2 concentration at the root/soil interface which results in loss of stomatal regulation and disruption to control over water uptake.

Understanding metabolism of arginine in biological systems via MALDI imaging.

Arginine is an important amino acid but has been barely studied in plants. The little research that has been done indicates that the pathways of synthesis are similar to those found in animals and procaryotes. However little is known about the cellular and tissue localization of the amino acid in plants. The research reported in this paper was designed to examine whether MALDI-MSI was sufficiently sensitive to examine the distribution of this amino acid in plant material, and whether the synthetic pathways were co-located. In wheat and orchid roots, the amount of arginine in tissues varies greatly and the pathways for its synthesis were not always detected with the amino acid.

Resolving the 'nitrogen paradox' of arbuscular mycorrhizas: fertilization with organic matter brings considerable benefits for plant nutrition and growth.

Arbuscular mycorrhizal fungi (AMF) can transfer nitrogen (N) to host plants, but the ecological relevance is debated, as total plant N and biomass do not generally increase. The extent to which the symbiosis is mutually beneficial is thought to rely on the stoichiometry of N, phosphorus (P) and carbon (C) availability. While inorganic N fertilization has been shown to elicit strong mutualism, characterized by improved plant and fungal growth and mineral nutrition, similar responses following organic N addition are lacking. Using a compartmented microcosm experiment, we determined the significance to a mycorrhizal plant of placing a (15) N-labelled, nitrogen-rich patch of organic matter in a compartment to which only AMF hyphae had access. Control microcosms denied AMF hyphal access to the patch compartment. When permitted access to the patch compartment, the fungus proliferated extensively in the patch and transferred substantial quantities of N to the plant. Moreover, our data demonstrate that allowing hyphal access to an organic matter patch enhanced total plant N and P contents, with a simultaneous and substantial increase in plant biomass. Furthermore, we demonstrate that organic matter fertilization of arbuscular mycorrhizal plants can foster a mutually beneficial symbiosis based on nitrogen transfer, a phenomenon previously thought irrelevant.

Host control and nutrient trading in a photosynthetic symbiosis.

Photosymbiosis is one of the most important evolutionary trajectories, resulting in the chloroplast and the subsequent development of all complex photosynthetic organisms. The ciliate Paramecium bursaria and the alga Chlorella have a well established and well studied light dependent endosymbiotic relationship. Despite its prominence, there remain many unanswered questions regarding the exact mechanisms of the photosymbiosis. Of particular interest is how a host maintains and manages its symbiont load in response to the allocation of nutrients between itself and its symbionts. Here we construct a detailed mathematical model, parameterised from the literature, that explicitly incorporates nutrient trading within a deterministic model of both partners. The model demonstrates how the symbiotic relationship can manifest as parasitism of the host by the symbionts, mutualism, wherein both partners benefit, or exploitation of the symbionts by the hosts. We show that the precise nature of the photosymbiosis is determined by both environmental conditions (how much light is available for photosynthesis) and the level of control a host has over its symbiont load. Our model provides a framework within which it is possible to pose detailed questions regarding the evolutionary behaviour of this important example of an established light dependent endosymbiosis; we focus on one question in particular, namely the evolution of host control, and show using an adaptive dynamics approach that a moderate level of host control may evolve provided the associated costs are not prohibitive.

Manipulating stomatal density enhances drought tolerance without deleterious effect on nutrient uptake.

Manipulation of stomatal density was investigated as a potential tool for enhancing drought tolerance or nutrient uptake. Drought tolerance and soil water retention were assessed using Arabidopsis epidermal patterning factor mutants manipulated to have increased or decreased stomatal density. Root nutrient uptake via mass flow was monitored under differing plant watering regimes using nitrogen-15 ((15) N) isotope and mass spectrometry. Plants with less than half of their normal complement of stomata, and correspondingly reduced levels of transpiration, conserve soil moisture and are highly drought tolerant but show little or no reduction in shoot nitrogen concentrations especially when water availability is restricted. By contrast, plants with over twice the normal density of stomata have a greater capacity for nitrogen uptake, except when water availability is restricted. We demonstrate the possibility of producing plants with reduced transpiration which have increased drought tolerance, with little or no loss of nutrient uptake. We demonstrate that increasing transpiration can enhance nutrient uptake when water is plentiful.

Integrating ecology and physiology of root-hemiparasitic interaction: interactive effects of abiotic resources shape the interplay between parasitism and autotrophy.

Root hemiparasites are green photosynthetic plants, which parasitically acquire resources from host xylem. Mineral nutrients and water, two principal below-ground abiotic resources, were assumed to affect the interaction between hemiparasites and their hosts. The shape of these effects and the underlying physiological mechanisms have, however, remained unclear. We conducted a glasshouse experiment with root-hemiparasitic Rhinanthus alectorolophus, in which we manipulated the availability of mineral nutrients and water. Biomass production and Chl fluorescence of the hemiparasites and hosts were recorded, together with proportion of host-derived carbon in hemiparasite biomass. The abiotic resources had profound interactive effects on the performance of both the hemiparasite and the hosts, as well as the balance of above-ground biomass between them. These effects were mainly based on an increase of growth and photosynthetic efficiency under high nutrient concentrations, on the hemiparasite's ability to induce strong water stress on the hosts if water is limiting, and on release of the host from parasitism by simultaneous abundance of both resources. Hemiparasitism is a highly variable interaction, in which environmental conditions affect both the parasitic and autotrophic (and thus competitive) components. A hemiparasite's own photosynthesis plays a crucial role in the assimilation of parasitized mineral resources and their transformation into growth and fitness.

From mycoheterotrophy to mutualism: mycorrhizal specificity and functioning in Ophioglossum vulgatum sporophytes.

Mycorrhizal functioning in the fern Ophioglossum is complex and poorly understood. It is unknown whether mature O. vulgatum sporophytes form mutualistic associations with fungi of the Glomeromycota and with what specificity. Are green sporophytes able to 'repay' fungal carbon (C) invested in them by mycorrhizal partners during the initially heterotrophic gametophyte and early sporophyte stages of the lifecycle? We identified fungal partners of O. vulgatum sporophytes using molecular techniques and supplied them with (33) P-orthophosphate and O. vulgatum sporophytes with (14) CO2 . We traced the movement of fungal-acquired nutrients and plant-fixed C between symbionts and analysed natural abundance (13) C and (15) N isotope signatures to assess nutritional interactions. We found fungal specificity of O. vulgatum sporophytes towards a mycorrhizal fungus closely related to Glomus macrocarpum. Our radioisotope tracers revealed reciprocal C-for-phosphorus exchange between fern sporophytes and fungal partners, despite competition from surrounding vegetation. Monocultures of O. vulgatum were enriched in (13) C and (15) N, providing inconclusive evidence of mycoheterotrophy when experiencing competition from the surrounding plant community. We show mutualistic and specific symbiosis between a eusporangiate fern and fungi of the Glomeromycota. Our findings suggest a 'take now, pay later' strategy of mycorrhizal functioning through the lifecycle O. vulgatum, from mycoheterotrophic gametophyte to mutualistic aboveground sporophyte.

First evidence of mutualism between ancient plant lineages (Haplomitriopsida liverworts) and Mucoromycotina fungi and its response to simulated Palaeozoic changes in atmospheric CO2.

The discovery that Mucoromycotina, an ancient and partially saprotrophic fungal lineage, associates with the basal liverwort lineage Haplomitriopsida casts doubt on the widely held view that Glomeromycota formed the sole ancestral plant-fungus symbiosis. Whether this association is mutualistic, and how its functioning was affected by the fall in atmospheric CO2 concentration that followed plant terrestrialization in the Palaeozoic, remains unknown. We measured carbon-for-nutrient exchanges between Haplomitriopsida liverworts and Mucoromycotina fungi under simulated mid-Palaeozoic (1500 ppm) and near-contemporary (440 ppm) CO2 concentrations using isotope tracers, and analysed cytological differences in plant-fungal interactions. Concomitantly, we cultured both partners axenically, resynthesized the associations in vitro, and characterized their cytology. We demonstrate that liverwort-Mucoromycotina symbiosis is mutualistic and mycorrhiza-like, but differs from liverwort-Glomeromycota symbiosis in maintaining functional efficiency of carbon-for-nutrient exchange between partners across CO2 concentrations. Inoculation of axenic plants with Mucoromycotina caused major cytological changes affecting the anatomy of plant tissues, similar to that observed in wild-collected plants colonized by Mucoromycotina fungi. By demonstrating reciprocal exchange of carbon for nutrients between partners, our results provide support for Mucoromycotina establishing the earliest mutualistic symbiosis with land plants. As symbiotic functional efficiency was not compromised by reduced CO2 , we suggest that other factors led to the modern predominance of the Glomeromycota symbiosis.

Stomatal density and aperture in non-vascular land plants are non-responsive to above-ambient atmospheric CO2 concentrations.

BACKGROUND AND AIMS: Following the consensus view for unitary origin and conserved function of stomata across over 400 million years of land plant evolution, stomatal abundance has been widely used to reconstruct palaeo-atmospheric environments. However, the responsiveness of stomata in mosses and hornworts, the most basal stomate lineages of extant land plants, has received relatively little attention. This study aimed to redress this imbalance and provide the first direct evidence of bryophyte stomatal responsiveness to atmospheric CO2. METHODS: A selection of hornwort (Anthoceros punctatus, Phaeoceros laevis) and moss (Polytrichum juniperinum, Mnium hornum, Funaria hygrometrica) sporophytes with contrasting stomatal morphologies were grown under different atmospheric CO2 concentrations ([CO2]) representing both modern (440 p.p.m. CO2) and ancient (1500 p.p.m. CO2) atmospheres. Upon sporophyte maturation, stomata from each bryophyte species were imaged, measured and quantified. KEY RESULTS: Densities and dimensions were unaffected by changes in [CO2], other than a slight increase in stomatal density in Funaria and abnormalities in Polytrichum stomata under elevated [CO2]. CONCLUSIONS: The changes to stomata in Funaria and Polytrichum are attributed to differential growth of the sporophytes rather than stomata-specific responses. The absence of responses to changes in [CO2] in bryophytes is in line with findings previously reported in other early lineages of vascular plants. These findings strengthen the hypothesis of an incremental acquisition of stomatal regulatory processes through land plant evolution and urge considerable caution in using stomatal densities as proxies for paleo-atmospheric CO2 concentrations.

Parasitic plant litter input: a novel indirect mechanism influencing plant community structure.

Parasitic plants have major impacts on plant community structure through their direct negative influence on host productivity and competitive ability. However, the possibility that these parasites may also have indirect impacts on community structure (via the mechanism of nutrient-rich litter input) while long hypothesized, has remained unsupported until now. Using the hemiparasite Rhinanthus minor, we established experimental grassland mesocosms to quantify the impacts of Rhinanthus litter and parasitism across two soil fertility levels. We measured the biomass and tissue nutrient concentration of three functional groups within these communities to determine their physiological response to resource abstraction and litter input by the parasite. We show that Rhinanthus alters the biomass and nutrient status of co-occurring plants with contrasting effects on different functional groups via the mechanism of nutrient-rich litter input. Critically, in the case of grass and total community biomass, this partially negates biomass reductions caused directly by parasitism. This demonstrates that the influence of parasitic plant litter on plant community structure can be of equal importance to the much-reported direct impacts of parasitism. We must consider both positive indirect (litter) and negative direct (parasitism) impacts of parasitic plants to understand their role in structuring plant communities.

How does elevated grassland productivity influence populations of root hemiparasites? Commentary on Borowicz and Armstrong (Oecologia 2012).

In their recent study, Borowicz and Armstrong (Oecologia 169:783-792, 2012) investigated effects of nutrient availability and competition for light on a perennial root hemiparasite Pedicularis canadensis. Their study showed a reduction of community productivity as a result of hemiparasite infection independently of a clear positive effect of increased nutrients. In contrast, there was a minimal effect of increased competition for light on growth of the parasite. Here, we summarize the available data on the influence of nutrient availability (closely related to productivity) on temperate grassland root hemiparasites thus expanding the discussion presented by Borowicz and Armstrong (Oecologia 169:783-792, 2012). Most studies show that root hemiparasites are highly sensitive to elevated competition for light in productive environments, which is manifested as an increase in mortality coupled to a decrease in population density. Such responses reflect increased mortality of hemiparasite seedlings that are physiologically inefficient in terms of photosynthesis and nutrient acquisition owing to a limited root network and consequently, are highly sensitive to competition for light. However, the susceptibility of hemiparasites to competition for light tends to decrease for individuals that survive the critical seedling stage. Moreover, survivors benefit from elevated nutrient availability, resulting in increased growth and fecundity. Elevated productivity can thus have opposing effects on the survival and growth of hemiparasites depending on life stage. We conclude that the findings by Borowicz and Armstrong (Oecologia 169:783-792, 2012) are not in conflict with this general view that root hemiparasite population ecology is strongly influenced by competition for light in highly productive environments.

Space controlled environment agriculture offers pathways to improve the sustainability of controlled environmental agriculture on Earth.

Terrestrial controlled environment agriculture (CEA) will have an increasingly important role in food production. However, present CEA systems are energy- and resource-hungry and rarely profitable, requiring a step change in design and optimization. Here we argue that the unique nature of space controlled environment agriculture (SpaCEA), which needs to be both highly resource efficient and circular in design, presents an opportunity to develop intrinsically circular CEA systems. Life-cycle analysis tools should be used to optimize the provision and use of natural or electrical light, power, nutrients and infrastructure in CEA and/or SpaCEA systems, and to guide research and development into subsystems that bring strong environmental advantages. We suggest that SpaCEA public outreach can also be used to improve the perception of terrestrial CEA on Earth by using space as a gateway for exhibiting CEA food growing technologies. A substantial focus on SpaCEA development should be viewed as an efficient contribution to addressing major current CEA challenges.

Untangling the Complexities of Processing and Analysis for Untargeted LC-MS Data Using Open-Source Tools.

Untargeted metabolomics is a powerful tool for measuring and understanding complex biological chemistries. However, employment, bioinformatics and downstream analysis of mass spectrometry (MS) data can be daunting for inexperienced users. Numerous open-source and free-to-use data processing and analysis tools exist for various untargeted MS approaches, including liquid chromatography (LC), but choosing the 'correct' pipeline isn't straight-forward. This tutorial, in conjunction with a user-friendly online guide presents a workflow for connecting these tools to process, analyse and annotate various untargeted MS datasets. The workflow is intended to guide exploratory analysis in order to inform decision-making regarding costly and time-consuming downstream targeted MS approaches. We provide practical advice concerning experimental design, organisation of data and downstream analysis, and offer details on sharing and storing valuable MS data for posterity. The workflow is editable and modular, allowing flexibility for updated/changing methodologies and increased clarity and detail as user participation becomes more common. Hence, the authors welcome contributions and improvements to the workflow via the online repository. We believe that this workflow will streamline and condense complex mass-spectrometry approaches into easier, more manageable, analyses thereby generating opportunities for researchers previously discouraged by inaccessible and overly complicated software.