Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Quantifying the effect of genetic, environmental and individual demographic stochastic variability for population dynamics in Plantago lanceolata.

Simple demographic events, the survival and reproduction of individuals, drive population dynamics. These demographic events are influenced by genetic and environmental parameters, and are the focus of many evolutionary and ecological investigations that aim to predict and understand population change. However, such a focus often neglects the stochastic events that individuals experience throughout their lives. These stochastic events also influence survival and reproduction and thereby evolutionary and ecological dynamics. Here, we illustrate the influence of such non-selective demographic variability on population dynamics using population projection models of an experimental population of Plantago lanceolata. Our analysis shows that the variability in survival and reproduction among individuals is largely due to demographic stochastic variation with only modest effects of differences in environment, genes, and their interaction. Common expectations of population growth, based on expected lifetime reproduction and generation time, can be misleading when demographic stochastic variation is large. Large demographic stochastic variation exhibited within genotypes can lower population growth and slow evolutionary adaptive dynamics. Our results accompany recent investigations that call for more focus on stochastic variation in fitness components, such as survival, reproduction, and functional traits, rather than dismissal of this variation as uninformative noise.

Effects of Arbuscular Mycorrhiza on Primary Metabolites in Phloem Exudates of Plantago major and Poa annua and on a Generalist Aphid.

Arbuscular mycorrhiza (AM), i.e., the interaction of plants with arbuscular mycorrhizal fungi (AMF), often influences plant growth, physiology, and metabolism. Effects of AM on the metabolic composition of plant phloem sap may affect aphids. We investigated the impacts of AM on primary metabolites in phloem exudates of the plant species Plantago major and Poa annua and on the aphid Myzus persicae. Plants were grown without or with a generalist AMF species, leaf phloem exudates were collected, and primary metabolites were measured. Additionally, the performance of M. persicae on control and mycorrhizal plants of both species was assessed. While the plant species differed largely in the relative proportions of primary metabolites in their phloem exudates, metabolic effects of AM were less pronounced. Slightly higher proportions of sucrose and shifts in proportions of some amino acids in mycorrhizal plants indicated changes in phloem upload and resource allocation patterns within the plants. Aphids showed a higher performance on P. annua than on P. major. AM negatively affected the survival of aphids on P. major, whereas positive effects of AM were found on P. annua in a subsequent generation. Next to other factors, the metabolic composition of the phloem exudates may partly explain these findings.

Directly quantifying multiple interacting influences on plant competition.

When plants compete what influences that interaction? To answer this we measured belowground competition directly, as the simultaneous capture of soil ammonium and nitrate by co-existing herbaceous perennials, Dactylis glomerata and Plantago lanceolata, under the influence of: species identity; N uptake and biomass of focal and neighbour plants; location (benign lowland versus harsher upland site); N availability (low or high N fertilizer); N ion, ammonium or nitrate production (mineralisation) rate, and competition type (intra- or interspecific), as direct effects or pairwise interactions in linear models. We also measured biomass as an indirect proxy for competition. Only three factors influenced both competitive N uptake and biomass production: focal species identity, N ion and the interaction between N ion and neighbour N uptake. Location had little effect on N uptake but a strong influence on biomass production. N uptake increased linearly with biomass only in isolated plants. Our results support the view that measuring resource capture or biomass production tells you different things about how competitors interact with one another and their environment, and that biomass is a longer-term integrative proxy for the outcomes of multiple separate interactions-such as competition for N-occurring between plants.

The novel features of Plantago ovata seed mucilage accumulation, storage and release.

Seed mucilage polysaccharide production, storage and release in Plantago ovata is strikingly different to that of the model plant Arabidopsis. We have used microscopy techniques to track the development of mucilage secretory cells and demonstrate that mature P. ovata seeds do not have an outer intact cell layer within which the polysaccharides surround internal columellae. Instead, dehydrated mucilage is spread in a thin homogenous layer over the entire seed surface and upon wetting expands directly outwards, away from the seed. Observing mucilage expansion in real time combined with compositional analysis allowed mucilage layer definition and the roles they play in mucilage release and architecture upon hydration to be explored. The first emergent layer of hydrated mucilage is rich in pectin, extremely hydrophilic, and forms an expansion front that functions to 'jumpstart' hydration and swelling of the second layer. This next layer, comprising the bulk of the expanded seed mucilage, is predominantly composed of heteroxylan and appears to provide much of the structural integrity. Our results indicate that the synthesis, deposition, desiccation, and final storage position of mucilage polysaccharides must be carefully orchestrated, although many of these processes are not yet fully defined and vary widely between myxospermous plant species.

Resident and phytometer plants host comparable rhizosphere fungal communities in managed grassland ecosystems.

Plants are known to modulate their own rhizosphere mycobiome. However, field studies that use resident plants to relate the microbiome assemblage to environmental factors such as land-use suffer from the problem that confounding factors such as plant age and performance may override the targeted effects. In contrast, the use of even-aged phytometer plants pre-cultivated under uniform conditions helps to reduce such random variation. We investigated the rhizosphere mycobiomes of phytometer and resident plants of two common grassland species, Dactylis glomerata L. s. str. and Plantago lanceolata L. along a land-use intensity gradient using ITS rRNA Illumina amplicon sequencing. Remarkably, we did not detect effects of the plant types (resident vs. phytometer plant, even though some fungal taxa exhibited plant species specificity), indicating that phytometer plants hosted a comparable rhizosphere mycobiome as resident plants. Our data indicate that the plant species harbor distinct fungal communities, with fungal richness in the rhizosphere of P. lanceolata being substantially higher than that of D. glomerata. Land-use intensity had a clear impact on the mycobiome of both plant species, with specific fungal genera showing differential tolerance to high intensities. Overall, the phytometer approach has a high potential to reveal environmental impacts on rhizosphere communities.

Moderate plant water stress improves larval development, and impacts immunity and gut microbiota of a specialist herbivore.

While host plant drought is generally viewed as a negative phenomenon, its impact on insect herbivores can vary largely depending on the species involved and on the intensity of the drought. Extreme drought killing host plants can clearly reduce herbivore fitness, but the impact of moderate host plant water stress on insect herbivores can vary, and may even be beneficial. The populations of the Finnish Glanville fritillary butterfly (Melitaea cinxia) have faced reduced precipitation in recent years, with impacts even on population dynamics. Whether the negative effects of low precipitation are solely due to extreme desiccation killing the host plant or whether moderate drought reduces plant quality for the larvae remains unknown. We assessed the performance of larvae fed on moderately water-stressed Plantago lanceolata in terms of growth, survival, and immune response, and additionally were interested to assess whether the gut microbial composition of the larvae changed due to modification of the host plant. We found that larvae fed on water-stressed plants had increased growth, with no impact on survival, up-regulated the expression of one candidate immune gene (pelle), and had a more heterogeneous bacterial community and a shifted fungal community in the gut. Most of the measured traits showed considerable variation due to family structure. Our data suggest that in temperate regions moderate host plant water stress can positively shape resource acquisition of this specialized insect herbivore, potentially by increasing nutrient accessibility or concentration. Potentially, the better larval performance may be mediated by a shift of the microbiota on water-stressed plants, calling for further research especially on the understudied gut fungal community.

Progressive programmed cell death inwards across the anther wall in male sterile flowers of the gynodioecious plant Plantago lanceolata.

MAIN CONCLUSION: A cell death signal is perceived and responded to by epidermal cells first before being conveyed inwards across the anther wall in male sterile Plantago lanceolata flowers. In gynodioecious plants, floral phenotype is determined by an interplay between cytoplasmic male sterility (CMS)-promoting factors and fertility-restoring genes segregating in the nuclear background. Plantago lanceolata exhibits at least four different sterilizing cytoplasms. MS1, a "brown-anther" male sterile phenotype, segregates with a CMSI cytoplasm and a non-restoring nuclear background in P. lanceolata populations. The aim of this study was to investigate the cytology of early anther development in segregating hermaphrodite and male sterile flowers sharing the same CMSI cytoplasm, and to determine if the sterility phenotype correlates with any changes to the normal pattern of programmed cell death (PCD) that occurs during anther development. Cytology shows cellular abnormalities in all four anther wall layers (epidermis, endothecium, middle layer and tapetum), the persistence and enlargement of middle layer and tapetal cells, and the failure of microspore mother cells to complete meiosis in male sterile anthers. In these anthers, apoptotic-PCD occurs earlier than in fertile anthers and is detected in all four cell layers of the anther wall before the middle layer and tapetal cells become enlarged. PCD is separated spatially and temporally within the anther wall, occurring first in epidermal cells before extending radially to cells in the inner anther wall layers. This is the first evidence of a cell death signal being perceived and responded to by epidermal cells first before being conveyed inwards across the anther wall in male sterile plants.

Life-history trade-offs vary with resource availability across the geographic range of a widespread plant.

Trade-offs between reproduction, growth and survival arise from limited resource availability in plants. Environmental stress is expected to exacerbate these negative correlations, but no studies have evaluated variation in life-history trade-offs throughout species geographic ranges. Here we analyse the costs of growth and reproduction across the latitudinal range of the widespread herb Plantago coronopus in Europe. We monitored the performance of thousands of individuals in 11 populations of P. coronopus, and tested whether the effects of growth and reproduction on a set of vital rates (growth, probability of survival, probability of reproduction and fecundity) varied with local precipitation and soil fertility. To account for variation in internal resources among individuals, we analysed trade-offs correcting for differences in size. Growth was negatively affected by previous growth and reproduction. We also found costs of growth and reproduction on survival, reproduction probability and fecundity, but only in populations with low soil fertility. Costs also increased with precipitation, possibly due to flooding-related stress. In contrast, growth was positively correlated with subsequent survival, and there was a positive covariation in reproduction between consecutive years under certain environments, a potential strategy to exploit temporary benign conditions. Overall, we found both negative and positive correlations among vital rates across P. coronopus geographic range. Trade-offs predominated under stressful conditions, and positive correlations arose particularly between related traits like reproduction investment across years. By analysing multiple and diverse fitness components along stress gradients, we can better understand life-history evolution across species' ranges, and their responses to environmental change.

The female advantage in natural populations of gynodioecious Plantago coronopus: seed quantity vs. offspring quality.

In gynodioecious plant species, females can only persist when they have a reproductive advantage in comparison with hermaphrodites. However, several studies have shown that females do not necessarily produce more seeds than hermaphrodites, since seed production can be affected by population characteristics, such as female frequency or population size. The aim of this study was to quantify the female advantage across a large number of natural populations, examine its relationship with population sex ratio and size, and to assess the role of competition on the magnitude of the female advantage. We sampled 27 populations of Plantago coronopus (nuclear-cytoplasmic gynodioecy) along the Belgian and Dutch coast. In each population, we estimated population sex ratio and size, and assessed seed production per flower and seed production per plant. Subsequently, germination, growth, and competition experiments were performed in the greenhouse to determine the female advantage regarding offspring quality. Females produced fewer seeds per plant than hermaphrodites (FA = 0.90), and seed production was negatively related to female frequency. Since both sex morphs were equally affected by pollen availability, the female advantage was not related to population sex ratio. On the other hand, offspring of females showed higher germination and growth rates, resulting in higher competitive abilities when seeds of a female and a hermaphrodite were grown together. Overall, these results indicate that differences in competitive abilities between the offspring of females and hermaphrodites may have contributed to the maintenance of females in relatively high frequencies in populations of this short-lived gynodioecious plant species.

Physiological characteristics of Plantago major under SO2 exposure as affected by foliar iron spray.

Sulfur dioxide (SO2) is considered as a main air pollutant in industrialized areas that can damage vegetation. In the present study, we investigated how exposure to SO2 and foliar application of iron (Fe) would affect certain physiological characteristics of Plantago major. The plant seedlings exposed or unexposed to SO2 (3900 µg m-3) were non-supplemented or supplemented with Fe (3 g L-1) as foliar spray. Plants were exposed to SO2 for 6 weeks in 100 × 70 × 70 cm chambers. Fumigation of plants with SO2 was performed for 3 h daily for 3 days per week (alternate day). Lower leaf Fe concentration in the plants exposed to SO2 at no added Fe treatment was accompanied with incidence of chlorosis symptoms and reduced chlorophyll concentration. No visible chlorotic symptoms were observed on the SO2-exposed plants supplied with Fe that accumulated higher Fe in their leaves. Both at with and without added Fe treatments, catalase (CAT) and peroxidase (POD) activity was higher in the plants fumigated with SO2 in comparison with those non-fumigated with SO2. Foliar application of Fe was also effective in increasing activity of antioxidant enzymes CAT and POD. Exposure to SO2 led to reduced cellulose but enhanced lignin content of plant leaf cell wall. The results obtained showed that foliar application of Fe was effective in reducing the effects of exposure to SO2 on cell wall composition. In contrast to SO2, application of Fe increased cellulose while decreased lignin content of the leaf cell wall. This might be due to reduced oxidative stress induced by SO2 in plants supplied with Fe compared with those unsupplied with Fe.

Photosynthesis and biochemical responses to elevated O3 in Plantago major and Sonchus oleraceus growing in a lowland habitat of northern China.

A field experiment was carried out to compare the responses to ozone (O3) in two common herbaceous plant species, Plantago major L. and Sonchus oleraceus L., by building open-top growth chambers in situ to simulate O3 stress (+O3, 85±5ppb, 9hr/day for 30days) in a lowland habitat in Inner Mongolia, Northern China. Responses to O3 of gas exchange, chlorophyll a fluorescence, leaf pigment content, antioxidant capability, soluble protein content, membrane lipid peroxidation and dark respiration (Rd) were analyzed. Results showed that elevated O3 exposure significantly reduced the light-saturated net photosynthesis (PNsat), stomatal conductance (gs) and transpiration rate (E) in both species. Although non-significant interactive effect between species and O3 on PNsat was analyzed, the reduction in PNsat in S. oleraceus might be due primarily to the higher fraction of close PSII reaction centers and impaired activities of plant mesophyll cells as evidences by decreased maximum efficiency of PSII photochemistry after dark adapted state (Fv/Fm) and unchanged intercellular CO2 concentration (Ci). Besides, biochemical analysis showed that S. oleraceus had lower antioxidant ability compared to P. major. As a result, S. oleraceus was damaged to the larger extent in terms of lipid peroxidation and visible O3 injury, indicating that S. oleraceus was more sensitive to O3 than P. major. Our results indicated that wild herbaceous plant species growing in a lowland habitat in sandy grassland were sensitive to O3 stress and S. oleraceus can be considered as one of the bio-indicators for high O3 concentration in semi-arid grassland of northern China.

Differences in glycosyltransferase family 61 accompany variation in seed coat mucilage composition in Plantago spp.

Xylans are the most abundant non-cellulosic polysaccharide found in plant cell walls. A diverse range of xylan structures influence tissue function during growth and development. Despite the abundance of xylans in nature, details of the genes and biochemical pathways controlling their biosynthesis are lacking. In this study we have utilized natural variation within the Plantago genus to examine variation in heteroxylan composition and structure in seed coat mucilage. Compositional assays were combined with analysis of the glycosyltransferase family 61 (GT61) family during seed coat development, with the aim of identifying GT61 sequences participating in xylan backbone substitution. The results reveal natural variation in heteroxylan content and structure, particularly in P. ovata and P. cunninghamii, species which show a similar amount of heteroxylan but different backbone substitution profiles. Analysis of the GT61 family identified specific sequences co-expressed with IRREGULAR XYLEM 10 genes, which encode putative xylan synthases, revealing a close temporal association between xylan synthesis and substitution. Moreover, in P. ovata, several abundant GT61 sequences appear to lack orthologues in P. cunninghamii. Our results indicate that natural variation in Plantago species can be exploited to reveal novel details of seed coat development and polysaccharide biosynthetic pathways.

Effects of Arbuscular Mycorrhiza on Plant Chemistry and the Development and Behavior of a Generalist Herbivore.

Arbuscular mycorrhiza (AM) formed between plants and AM fungi (AMF) can alter host plant quality and thus influence plant-herbivore interactions. While AM is known to affect the development of generalist chewing-biting herbivores, AM-mediated impacts on insect behavior have been neglected until now. In this study, the effects of Rhizophagus irregularis, a generalist AMF, on phenotypic and leaf metabolic traits of Plantago major plants were investigated. Further, the influence of AM-mediated host plant modifications on the development and on seven behavioral traits of larvae of the generalist Mamestra brassicae were recorded. Tests were carried out in the third (L3) and fourth (L4) larval instar, respectively. While shoot water content, specific leaf area, and foliar concentrations of the secondary metabolite aucubin were higher in AM-treated compared to non-mycorrhized (NM) plants, lower concentrations of the primary metabolites citric acid and isocitric acid were found in leaves of AM plants. Larvae reared on AM plants gained a higher body mass and tended to develop faster than individuals reared on NM plants. However, plant treatment had no significant effect on any of the behavioral traits. Instead, differences between larvae of different ages were detected in several behavioral features, with L4 being less active and less bold than L3 larvae. The results demonstrate that AM-induced modifications of host plant quality influence larval development, whereas the behavioral phenotype seems to be more fixed at least under the tested conditions.

Photosynthetic activity of vascular bundles in Plantago media leaves.

Photosynthetic processes in the leaf lamina and midribs of Plantago media were investigated using plants grown in high light (HL) or low light (LL) conditions. The fluorescence parameters, which indicate photochemical/photosynthetic activity, were different in HL and LL grown plants, but no major differences between lamina and midribs were found. An OJIP test (chlorophyll a fluorescence transient induction) of LL grown plants, indicative of the chloroplast electron transport chain, also showed both tissues to be similar. In HL plants, a partial blockage of electron flow between QA (the primary plastoquinone electron acceptor of PSII) and QB (the secondary plastoquinone acceptor of PSII) was found, and this was less visible in midribs. The effective dissipation of quantum energy per reaction center (DI0/RC) was similar in both tissues of HL grown plants, while in the midribs of LL leaves, this process seemed to be less effective. Measurements of 13C discrimination showed that the midrib tissues of LL and HL leaves effectively used ß-carboxylation products to accumulate their biomass. Thus, the well protected activity of electron transport in midribs with their limited capacity to fix CO2 from the air may indicate the involvement of this tissue in ß-carboxylation, transport or signaling. Carbon accumulated in roots showed a lower 13C discrimination value (more negative) than the values observed in lamina. This could indicate that roots are supplied with assimilates mostly during the light phase of the day cycle with intensive C3 photosynthesis.

Plant-plant interactions mediate the plastic and genotypic response of Plantago asiatica to CO2: an experiment with plant populations from naturally high CO2 areas.

BACKGROUND AND AIMS: The rising atmospheric CO2 concentration ([CO2]) is a ubiquitous selective force that may strongly impact species distribution and vegetation functioning. Plant-plant interactions could mediate the trajectory of vegetation responses to elevated [CO2], because some plants may benefit more from [CO2] elevation than others. The relative contribution of plastic (within the plant's lifetime) and genotypic (over several generations) responses to elevated [CO2] on plant performance was investigated and how these patterns are modified by plant-plant interactions was analysed. METHODS: Plantago asiatica seeds originating from natural CO2 springs and from ambient [CO2] sites were grown in mono stands of each one of the two origins as well as mixtures of both origins. In total, 1944 plants were grown in [CO2]-controlled walk-in climate rooms, under a [CO2] of 270, 450 and 750 ppm. A model was used for upscaling from leaf to whole-plant photosynthesis and for quantifying the influence of plastic and genotypic responses. KEY RESULTS: It was shown that changes in canopy photosynthesis, specific leaf area (SLA) and stomatal conductance in response to changes in growth [CO2] were mainly determined by plastic and not by genotypic responses. We further found that plants originating from high [CO2] habitats performed better in terms of whole-plant photosynthesis, biomass and leaf area, than those from ambient [CO2] habitats at elevated [CO2] only when both genotypes competed. Similarly, plants from ambient [CO2] habitats performed better at low [CO2], also only when both genotypes competed. No difference in performance was found in mono stands. CONCLUSION: The results indicate that natural selection under increasing [CO2] will be mainly driven by competitive interactions. This supports the notion that plant-plant interactions have an important influence on future vegetation functioning and species distribution. Furthermore, plant performance was mainly driven by plastic and not by genotypic responses to changes in atmospheric [CO2].

Seasonal dynamic of morpho-physiological properties and the lipid composition of Plantago media (Plantaginaceae) in the Middle Volga region.

The changes in morpho-physiological properties and lipid composition have been studied in the leaves of the plant Plantago media collected from two different places in the Middle Volga region during the summer of 2010. The plants gathered from the first plot (P1 plants) grew on plain ground in the midst of typical meadow-steppe perennial plants. The plants of the second group (P2 plants) grew on a flat slope of the South-West exposition, in the grass community. The leaves of the plants Р1 had lower specific area densities but larger areas and masses; they accumulated more levels lipid peroxide products. The changes in lipid compositions depended on the growth phase and habitats. Correlations between morpho-physiological parameters and certain lipids have been established. The amounts of galactolipids (GL) have been shown to correlate with the leaf areas. When the leaf areas were reduced, a ratio between phosphatidylcholines (PC) and phosphatidylethanolamines (PE) decreased. The result of our study showed that gradual changes of morphometrical parameters were accompanied by the alterations in biomass structure and modifications in lipids and fatty acids (FA).

Selection on pollen and pistil traits during pollen competition is affected by both sexual conflict and mixed mating in a self-compatible herb.

PREMISE OF THE STUDY: Although much attention has focused on the diversity of plant mating systems, only a few studies have considered the joint effects of mating system and sexual conflict in plant evolution. In mixed-mating Collinsia heterophylla, a sexual conflict over timing of stigma receptivity is proposed: pollen with a capacity to induce early onset of stigma receptivity secures paternity for early-arriving pollen (at the expense of reduced maternal seed set), whereas late onset of stigma receptivity mitigates the negative effects of early-arriving pollen. Here we investigated whether selection on pollen and pistil traits involved in sexual conflict is affected by the presence of both outcross- and self-pollen (mixed mating) during pollen competition. METHODS: We conducted two-donor crosses at different floral developmental stages to explore male fitness (siring ability) and female fitness (seed set) in relation to male and female identity, pollen and pistil traits, and type of competitor pollen (outcross vs. self). KEY RESULTS: Late-fertilizing pollen rather than rapidly growing pollen tubes was most successful in terms of siring success, especially in competition with self-pollen after pollination at early floral stages. Late stigma receptivity increased seed set after early-stage pollinations, in agreement with selection against antagonistic pollen. CONCLUSIONS: Selection on pollen and pistil traits in C. heterophylla is affected by both sexual conflict and mixed mating, suggesting the importance of jointly considering these factors in plant evolution.