Lipid remodelling and the conversion of lipids into sugars associated with tolerance to cadmium toxicity during white clover seed germination.

Cadmium (Cd) is a leading environmental issue worldwide. The current study was conducted to investigate Cd tolerance of 10 commercial white clover (Trifolium repens) cultivars during seed germination and to further explore differences in lipid remodelling, glycometabolism, and the conversion of lipids into sugars contributing to Cd tolerance in the early phase of seedling establishment as well as the accumulation of Cd in seedlings and mature plants. The results show that Cd stress significantly reduced seed germination of 10 cultivars. Compared to Cd-sensitive Sulky, Cd-tolerant Pixie accelerated amylolysis to produce more glucose, fructose, and sucrose by maintaining higher amylase and sucrase activities under Cd stress. Pixie maintained higher contents of various lipids, higher DGDG/MGDG ratio, and lower unsaturation levels of lipids, which could be beneficial to membrane stability and integrity as well as signal transduction in cells after being subjected to Cd stress. In addition, Pixie upregulated expression levels of key genes (TrACX1, TrACX4, TrSDP6, and TrPCK1) involved in the conversion of lipids into sugars for early seedling establishment under Cd stress. These findings indicate that lipid remodelling, enhanced glycometabolism, and accelerated conversion of lipids into sugars are important adaptive strategies for white clover seed germination and subsequent seedling establishment under Cd stress. In addition, Pixie not only accumulated more Cd in seedlings and mature plants than Sulky but also had significantly better growth and phytoremediation efficiency under Cd stress. Pixie could be used as a suitable and critical germplasm for the rehabilitation and re-establishment of Cd-contaminated areas.

Winter survival in red clover: experimental evidence for interactions among stresses.

BACKGROUND: There is a lack of knowledge on the combined effects of different stresses on plants, in particular different stresses that occur during winter in temperate climates. Perennial herbaceous plants in temperate regions are exposed to many different stresses during winter, but except for the fact that cold temperatures induce resistance to a number of them, very little is known about their interaction effects. Knowledge about stress interactions is needed in order to predict effects of climate change on both agricultural production and natural ecosystems, and to develop adaptation strategies, e.g., through plant breeding. Here, we conducted a series of experiments under controlled conditions to study the interactions between cold (low positive temperature), clover rot infection (caused by Sclerotinia trifoliorum) and freezing, in red clover (Trifolium pratense) accessions. We also compared our results with winter survival in field experiments and studied associations between stress and shoot growth. RESULTS: Exposure to low positive temperatures (cold acclimation) induced resistance to clover rot. There was a clear negative interaction effect between freezing stress and clover rot infection, resulting in up to 37% lower survival rate compared to what would have been expected from the additive effect of freezing and infection alone. Freezing tolerance could continue to improve during incubation under artificial snow cover at 3 °C in spite of darkness, and we observed compensatory shoot growth following freezing after prolonged incubation. At the accession level, resistance to clover rot was negatively correlated with growth in the field during the previous year at a Norwegian location. It was also negatively correlated with the shoot regrowth of control plants after incubation. Clover rot resistance tests under controlled conditions showed limited correlation with clover rot resistance observed in the field, suggesting that they may reveal variation in more specific resistance mechanisms. CONCLUSIONS: We here demonstrate, for the first time, a strong negative interaction between freezing and infection with a winter pathogen. We also characterize the effects of cold acclimation and incubation in darkness at different temperatures on winter stress tolerance, and present data that support the notion that annual cycles of growth and stress resistance are associated at the genetic level.

Global urban environmental change drives adaptation in white clover.

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale.

Effect of soil spatial configuration on Trifolium repens varies with resource amount.

Soil spatial heterogeneity involves nutrients being patchily distributed at a range of scales and is prevalent in natural habitats. However, little is known about the effect of soil spatial configurations at the small scale on plant foraging behavior and plant growth under different resource amounts. Here, we experimentally investigated how a stoloniferous species, Trifolium repens, responded to varied resource amounts and spatial configuration combinations. Plant foraging behavior (i.e., the orientation of the primary stolon, mean length of the primary stolon, foraging precision, and foraging scale) and plant growth (i.e., total biomass, root biomass, shoot biomass, and root/shoot) were compared among differently designed configurations of soil resources in different amounts. The relationships of foraging behavior and plant biomass were analyzed. The results showed that the effect of the spatial configuration of soil resources on Trifolium repens depended on the resource amount. Specifically, when the total resource amount was low, fragmented soil patches promoted root foraging and increased Trifolium repens plant biomass; however, when the total resource amount was high, the soil spatial configuration did not affect foraging behavior or plant growth. Our results also showed that plant growth was facilitated by root foraging scale to adapt to low resource amounts. We conclude that the spatial configuration of soil resources at small scales affects whole plant growth, which is mediated by a distinct foraging strategy. These findings contribute to a better understanding of how the growth strategy of clonal plants responds to heterogeneous environments caused by different resource amounts and its spatial configurations.

A physiological approach to study the competition ability of the grassland species Trifolium pratense and Agrostis capillaris.

The response of plant species to external factors depends partly on the interaction with the environment and with the other species that coexist in the same ecosystem. Several studies have investigated the main traits that determine the competitive capacity of plant species, and although the relevance of the traits is not clear, traits both from belowground and aboveground have been observed. In this paper, we grew Trifolium pratense and Agrostis capillaris in intra- and interspecific competition, analyzing the photosynthetic metabolism and nitrogen uptake, among other variables. The results indicated that T. pratense possesses better competition ability due to the higher competitive performance for soil resources compared to A. capillaris, explained by a higher root biomass and a higher nitrogen uptake rate in the former than in the latter. These traits permitted T. pratense to show higher photosynthetic rate than A. capillaris when both species were grown in mixture. Furthermore, the interspecific competition provoked A. capillaris to activate its antioxidant metabolism, through SOD activity, to detoxify the reactive oxygen species generated due to its lower capacity for using the photochemical energy absorbed. In this experiment, we conclude that the competitiveness seems to be more related with soil resources competition than with light competition, and that the photosynthetic rate decline in A. capillaris is more a secondary effect as a consequence of nitrogen limitation.

Spermine Regulates Water Balance Associated with Ca2+-Dependent Aquaporin (TrTIP2-1, TrTIP2-2 and TrPIP2-7) Expression in Plants under Water Stress.

Spermine (Spm) regulates water balance involved in water channel proteins, aquaporins (AQPs), in plants. An increase in endogenous Spm content via exogenous Spm application significantly improved cell membrane stability, photosynthesis, osmotic adjustment (OA) and water use efficiency (WUE) contributing to enhanced tolerance to water stress in white clover. Spm upregulated TrTIP2-1, TrTIP2-2 and TrPIP2-7 expressions and also increased the abundance of TIP2 and PIP2-7 proteins in white clover under water stress. Spm quickly activated intracellular Ca2+ signaling and Spm-induced TrTIP2-2 and TrPIP2-7 expressions could be blocked by Ca2+ channel blockers and the inhibitor of Ca2+-dependent protein kinase in leaves of white clover. TrSAMS in relation to Spm biosynthesis was first cloned from white clover and the TrSAMS was located in the nucleus. Transgenic Arabidopsis overexpressing the TrSAMS had significantly higher endogenous Spm content and improved cell membrane stability, photosynthesis, OA, WUE and transcript levels of AtSIP1-1, AtSIP1-2, AtTIP2-1, AtTIP2-2, AtPIP1-2, AtPIP2-1 and AtNIP2-1 than wild type in response to water stress. Current findings indicate that Spm regulates water balance via an enhancement in OA, WUE and water transport related to Ca2+-dependent AQP expression in plants under water stress.

Biochemical and molecular responses during overwintering of red clover populations recurrently selected for improved freezing tolerance.

Low freezing tolerance reduces the persistence of red clover under northern climate. The incidence of winter damages in perennial crops could increase in the future due to the adverse effects of the predicted warmer fall temperature on plant cold acclimation. To accelerate breeding progress, two cultivars of red clover Christie (C-TF0) and Endure (E-TF0) were exposed to a recurrent selection protocol for freezing tolerance performed indoor. New populations were obtained after five (C-TF5 and E-TF5), six (C-TF6 and E-TF6), and seven (C-TF7 and E-TF7) cycles of recurrent selection. These populations were overwintered under natural conditions and monitored for freezing tolerance and cold-induced molecular traits. Freezing tolerance was improved by up to 6 °C in recurrently selected populations when compared to initial cultivars confirming that further progress are achieved with advanced cycles of selection. Monthly analysis of biochemical changes shows that higher starch concentrations at the onset of the fall hardening period are contributing to the acquisition of superior freezing tolerance through its impact on sucrose accumulation. They also contribute to the vigor of spring regrowth by sustaining more pinitol and proline synthesis. Larger concentrations of these metabolites in populations with higher levels of freezing tolerance (TF7) hint at their involvement in winter survival of red clover. Among genes differentially expressed in response to both cold acclimation and recurrent selection, a concomitant cold induction of APPR9 and cold repression of 1-aminocyclopropane-carboxylate synthase suggests a link between the repression of a pathway regulated by ethylene and the improvement of freezing tolerance in red clover.

Lead toxicity induced phytotoxic impacts on rapeseed and clover can be lowered by biofilm forming lead tolerant bacteria.

Lead (Pb+2) is a heavy metal and one of the main environmental pollutant, toxic to plants, animals and humans. Present study was conducted to evaluate ten plant growth promoting bacteria strains (B1-10) for biofilm production and their effect on growth indices, physiology, yield, antioxidant profile and lead uptake in rapeseed (Brassica napus) and clover (Trifolium repens) in lead polluted soil under nutrient broth medium and pot condition. Three pre-characterized biofilm forming lead tolerant growth promoting strains (B3: Pseudomonas fluorescens), B6: Pseudomonas putida and (B8: Bacillus safensis) were used to inoculate rapeseed and clover growing in the soil polluted with different levels (400, 800 and 1200 mg kg-1) of Pb arranged in completely randomized design with factorial arrangement. Results from screening experiment exhibited that more biofilm was produced by B3, B6 and B8 under highest level of lead contamination (1200 mg kg-1). Further, lead contamination decreased rapeseed and clover growth, physiology and yield at all levels of lead stress. But biofilm forming lead tolerant growth promoting bacteria application in lead contaminated soil enhanced rapeseed and clover growth, physiology, yield, antioxidant profile, proline and decreased malanodialdehyde content (which was decreased by different strains application under lead stress) of rapeseed and clover over no inoculation. Inoculation with all strains also increased the lead uptake in roots, shoots and decreased lead uptake in seeds of rapeseed and clover than plants in lead stress without inoculation.

Metabolomics and physiological analyses reveal ß-sitosterol as an important plant growth regulator inducing tolerance to water stress in white clover.

MAIN CONCLUSION: ß-sitosterol influences amino acids, carbohydrates, organic acids, and other metabolite metabolism and homeostasis largely contributing to better tolerance to water stress in white clover. ß-sitosterol (BS) could act as an important plant growth regulator when plants are subjected to harsh environmental conditions. Objective of this study was to examine effects of BS on growth and water stress tolerance in white clover based on physiological responses and metabolomics. White clover was pretreated with or without BS and then subjected to water stress for 7 days in controlled growth chambers. Physiological analysis demonstrated that exogenous application of BS (120 µM) could significantly improve stress tolerance associated with better growth performance and photosynthesis, higher leaf relative water content, and less oxidative damage in white clover in response to water stress. Metabolic profiling identified 78 core metabolites involved in amino acids, organic acids, sugars, sugar alcohols, and other metabolites in leaves of white clover. For sugars and sugar alcohol metabolism, the BS treatment enhanced the accumulation of fructose, glucose, maltose, and myo-inositol contributing to better antioxidant capacity, growth maintenance, and osmotic adjustment in white clover under water stress. The application of BS was inclined to convert glutamic acid into proline, 5-oxoproline, and chlorophyll instead of going to pyruvate and alanine; the BS treatment did not significantly affect intermediates of tricarboxylic acid cycle (citrate, aconitate, and malate), but promoted the accumulation of other organic acids including lactic acid, glycolic acid, glyceric acid, shikimic acid, galacturonic acid, and quinic acid in white clover subjected to water stress. In addition, cysteine, an important antioxidant metabolite, was also significantly improved by BS in white clover under water stress. These altered amino acids and organic acids metabolism could play important roles in growth maintenance and modulation of osmotic and redox balance against water stress in white clover. Current findings provide a new insight into BS-induced metabolic homeostasis related to growth and water stress tolerance in plants.

Complementary gene interaction and xenia effect controls the seed coat colour in interspecific cross between Trifolium alexandrinum and T. apertum.

Trifolium alexandrinum (Egyptian clover) is a widely cultivated winter annual fodder. Present work deals with inheritance of the seed coat colour in segregating progenies of the interspecific cross between T. alexandrinum and T. apertum. Although, both the parent species possessed yellow seed coat, the F1 seeds were black coloured in the reciprocal cross (T. apertum × T. alexandrinum). Seeds borne on individual F2 plants and the advancing generations segregated in yellow and black seed coat colour, which confirmed xenia effect. F2 seeds collected from individual F1 plants exhibited nine black and seven yellow segregation ratio. The segregation of the seed coat colour recorded from F3 to F5 generations revealed that yellow seed coat was true breeding (i.e. non-segregating) in this interspecific cross (including the reciprocal crosses). However, the black seeded progenies were either true breeding or segregated in nine black: seven yellow ratio or three black: one yellow ratio suggesting a complementary gene interaction or duplicate recessive epistasis. It indicated that the seed coat colour is controlled by complementary gene interaction along with xenia effect in interspecific crosses between T. alexandrinum and T. apertum. Occurrence of the complementary genes across the species could suggest T. apertum to be the progenitor of T. alexandrinum. Inheritance of seed coat colour in reference to its importance in Egyptian clover breeding is also discussed.

Dose-dependence of growth and ecophysiological responses of plants to biochar.

Charcoal is a ubiquitous legacy of wildfire in terrestrial systems that often contributes to rapid revegetation following disturbance; the use of charcoal soil amendments, or "biochars", to promote plant growth has received recent research attention and increasing applied use. Despite its widespread use, well-resolved quantitative estimates of dose-response relationships for biochar effects on plant growth are nonexistent, and studies of biochar dosage effects on plant ecophysiology are minimal. We investigated the effects of biochar dosage on plant growth and ecophysiology in a glasshouse experiment involving two common early-successional plants, Abutilon theophrasti and Trifolium repens. Plants were grown in disturbed temperate soils with increasing dosages of wood biochars: 0, 2, 4, 6, 8, 10, 20, 30, 40, 50 t/ha. We measured leaf-level gas-exchange traits (Amax, gs, WUE), chlorophyll concentration, and leaf area growth throughout the experiment. At the end of the experiment, we measured biomass, foliar nutrition, and soil properties (pH, EC, C and N). Responses of biomass and physiological traits were highly dose-dependent, followed primarily unimodal forms, and differed in some traits between species. Increases in the uptake of K, P, and Mg, were responsible for accelerated growth. Biochars also generally increased the concentration of micronutrients, especially B. As a result, nutrient stoichiometry shifted substantially: in A. theophrasti, biochars increased C:N, P:N, and K:N ratios, suggesting nitrogen dilution or induced deficiency at higher dosages. This work supports the general hypothesis that ecophysiological responses to biochar are dose-dependent and driven mainly by changes in nutrient availability. Additional work is necessary to understand the broader ecological impacts of heterogeneity in soil pyrogenic C levels to succession and ecosystem function.

Nitric oxide, γ-aminobutyric acid, and mannose pretreatment influence metabolic profiles in white clover under water stress.

Nitric oxide (NO), γ-aminobutyric acid (GABA), and mannose (MAS) could be important regulators of plant growth and adaptation to water stress. The application of sodium nitroprusside (SNP, a NO donor), GABA, and MAS improved plant growth under water-sufficient conditions and effectively mitigated water stress damage to white clover. The metabonomic analysis showed that both SNP and GABA application resulted in a significant increase in myo-inositol content; the accumulation of mannose was commonly regulated by SNP and MAS; GABA and MAS induced the accumulation of aspartic acid, quinic acid, trehalose, and glycerol under water deficit. In addition, citric acid was uniquely up-regulated by SNP associated with tricarboxylic acid (TCA) cycle under water stress. GABA specially induced the accumulation of GABA, glycine, methionine, and aconitic acid related to GABA shunt, amino acids metabolism, and TCA cycle in response to water stress. MAS uniquely enhanced the accumulation of asparagine, galactose, and D-pinitol in association with amino acids and sugars metabolism under water stress. SNP-, GABA-, and MAS-induced changes of metabolic profiles and associated metabolic pathways could contribute to enhanced stress tolerance via involvement in the TCA cycle for energy supply, osmotic adjustment, antioxidant defense, and signal transduction for stress defense in white clover.

A neglected alliance in battles against parasitic plants: arbuscular mycorrhizal and rhizobial symbioses alleviate damage to a legume host by root hemiparasitic Pedicularis species.

Despite their ubiquitous distribution and significant ecological roles, soil microorganisms have long been neglected in investigations addressing parasitic plant-host interactions. Because nutrient deprivation is a primary cause of host damage by parasitic plants, we hypothesized that beneficial soil microorganisms conferring nutrient benefits to parasitized hosts may play important roles in alleviating damage. We conducted a pot cultivation experiment to test the inoculation effect of an arbuscular mycorrhizal fungus (Glomus mosseae), a rhizobium (Rhizobium leguminosarum) and their interactive effects, on alleviation of damage to a legume host (Trifolium repens) by two root hemiparasitic plants with different nutrient requirements (N-demanding Pedicularis rex and P-demanding P. tricolor). Strong interactive effects between inoculation regimes and hemiparasite identity were observed. The relative benefits of microbial inoculation were related to hemiparasite nutrient requirements. Dual inoculation with the rhizobium strongly enhanced promotional arbuscular mycorrhizal effects on hosts parasitized by P. rex, but reduced the arbuscular mycorrhizal promotion on hosts parasitized by P. tricolor. Our results demonstrate substantial contribution of arbuscular mycorrhizal and rhizobial symbioses to alleviating damage to the legume host by root hemiparasites, and suggest that soil microorganisms are critical factors regulating host-parasite interactions and should be taken into account in future studies.

Understanding the Complexity of Cold Tolerance in White Clover using Temperature Gradient Locations and a GWAS Approach.

White clover ( L.) is the most important grazing perennial forage legume in temperate climates. However, its limited capacity to survive and restore growth after low temperatures during winter constrains the productivity and wide adoption of the crop. Despite the importance of cold tolerance for white clover cultivar development, the genetic basis of this trait remains largely unknown. Hence, in this study, we performed the first genome-wide association study (GWAS) analyses in white clover to identify quantitative trait loci (QTL) for cold-tolerance-related traits. Seeds from 192 divergent genotypes from six populations in the Patagonia region of South America were collected and seed-derived plants were further clonally propagated. Clonal trials were established in three locations representing temperature gradient associated with elevation. Given the allotetraploid nature of the white clover genome, distinct genetic models (diploid and tetraploid) were tested. Only the tetraploid parameterization was able to detect the 53 loci associated with cold-tolerance traits. Out of the 53 single nucleotide polymorphism (SNP) trait associations, 17 controlled more than one trait or were stable across multiple sites. This work represents the first report of QTL for cold-tolerance-related traits, providing insights into its genetic basis and candidate genomic regions for further functional validation studies.

Indole-3-acetic acid modulates phytohormones and polyamines metabolism associated with the tolerance to water stress in white clover.

Endogenous hormones and polyamines (PAs) could interact to regulate growth and tolerance to water stress in white clover. The objective of this study was to investigate whether the alteration of endogenous indole-3-acetic acid (IAA) level affected other hormones level and PAs metabolism contributing to the regulation of tolerance to water stress in white clover. Plants were pretreated with IAA or L-2-aminooxy-3-phenylpropionic acid (L-AOPP, the inhibitor of IAA biosynthesis) for 3 days and then subjected to water-sufficient condition and water stress induced by 15% polyethylene glycol 6000 for 8 days in growth chambers. Exogenous application of IAA significantly increased endogenous IAA, gibberellin (GA), abscisic acid (ABA), and polyamine (PAs) levels, but had no effect on cytokinin content under water stress. The increase in endogenous IAA level enhanced PAs anabolism via the improvement of enzyme activities and transcript level of genes including arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase. Exogenous application of IAA also affected PAs catabolism, as manifested by an increase in diamine oxidase and a decrease in polyamine oxidase activities and genes expression. More importantly, the IAA deficiency in white clover decreased endogenous hormone levels (GA, ABA, and PAs) and PAs anabolism along with decline in antioxidant defense and osmotic adjustment (OA). On the contrary, exogenous IAA effectively alleviated stress-induced oxidative damage, growth inhibition, water deficit, and leaf senescence through the maintenance of higher chlorophyll content, OA, and antioxidant defense as well as lower transcript levels of senescence marker genes SAG101 and SAG102 in leaves under water stress. These results indicate that IAA-induced the crosstalk between endogenous hormones and PAs could be involved in the improvement of antioxidant defense and OA conferring tolerance to water stress in white clover.

Micro- and macroevolutionary adaptation through repeated loss of a complete metabolic pathway.

There is growing evidence for the convergent evolution of physically linked gene clusters encoding chemical defense pathways. Metabolic clusters are proposed to evolve because they ensure co-inheritance of all required genes where the defense is favored, and prevent inheritance of toxic partial pathways where it is not. This hypothesis rests on the assumption that clusters evolve in species where selection favors intraspecific polymorphism for the defense; however, they have not been examined in polymorphic species. We examined metabolic cluster evolution in relation to an adaptive polymorphism for cyanogenic glucoside (CNglc) production in clover. Using 163 accessions, we performed CNglc assays, BAC sequencing, Southern hybridizations and molecular evolutionary analyses. We find that the CNglc pathway forms a 138-kb cluster in white clover, and that the adaptive polymorphism occurs through presence/absence of the complete cluster. Component genes are orthologous to those in the distantly related legume Lotus japonicus. These findings provide empirical support for the co-inheritance hypothesis, and they indicate that adaptive CNglc variation in white clover evolves through recurrent deletions of the entire pathway. They further indicate that the shared ancestor of many important legume crops was likely cyanogenic and that this defense was lost repeatedly over the last 50 Myr.

The interaction between drought and elevated CO2 in water relations in two grassland species is species-specific.

Climate change can have major consequences for grassland communities since the different species of the community utilize different mechanisms for adaptation to drought and elevated CO2 levels. In addition, contradictory data exist when the combined effects of elevated CO2 and drought are analyzed because the soil water content is not usually similar between CO2 concentrations. Thus, the objectives of this work have been to examine the effect of water stress on plant water relations in two grassland species (Trifolium pratense and Agrostis capillaris), analyzing the possible differences between the two species when soil water content is equal in all treatments, and to elucidate if development under elevated CO2 increases drought tolerance and if so, which are the underlying mechanisms. At ambient CO2, when soil volumetric water content was 15%, both species decreased their water potential in order to continue taking up water. Trifolium pratense performed osmotic adjustment, while Agrostis capillaris decreased the rigidity of its cell wall; moreover, both species increased the root to shoot ratio and decreased leaf area. However, these mechanisms were not sufficient to maintain cell turgor. Elevated CO2 partially mitigated the negative impact of drought on turgor potential in Trifolium pratense through a higher osmotic adjustment and root to shoot ratio and in Agrostis capillaris through a higher leaf relative water content caused by higher hydraulic conductance, but the impact of drought was not mitigated in either species by higher soil water conservation.

Effects of different soil remediation methods on inhibition of lead absorption and growth and quality of Dianthus superbus L.

Heavy metal pollution in soil poses a serious threat to the growth of plants used in traditional Chinese medicine. Therefore, a pot experiment was conducted to study the effects of various soil remediation methods on the performance of Herba Dianthi (Dianthus superbus L.) grown on Pb-contaminated soil. The results show that inoculation of Herba Dianthi with arbuscular mycorrhizal fungi (AMF) led to a significant reduction in Pb uptake (P< 0.05), and increased root development and root-to-shoot ratio compared to untreated control plants, along with the highest content of active components. When planting with Trifolium repens, the reduction effect of Pb absorption was insignificant. Herba Dianthi showed improved growth and active ingredients, and the lowest Pb content, with AMF inoculation. The addition of EDTA decreased the growth of Herba Dianthi, but promoted the absorption of Pb. The inhibition of tumor cells was highest in E2. In conclusion, inoculation with AMF can ensure that plant lead content meets testing standards, helping to improve the quality of medicinal herbs.

Flight of the bumble bee: Buzzes predict pollination services.

Multiple interacting factors drive recent declines in wild and managed bees, threatening their pollination services. Widespread and intensive monitoring could lead to more effective management of wild and managed bees. However, tracking their dynamic populations is costly. We tested the effectiveness of an inexpensive, noninvasive and passive acoustic survey technique for monitoring bumble bee behavior and pollination services. First, we assessed the relationship between the first harmonic of the flight buzz (characteristic frequency) and pollinator functional traits that influence pollination success using flight cage experiments and a literature search. We analyzed passive acoustic survey data from three locations on Pennsylvania Mountain, Colorado to estimate bumble bee activity. We developed an algorithm based on Computational Auditory Scene Analysis that identified and quantified the number of buzzes recorded in each location. We then compared visual and acoustic estimates of bumble bee activity. Using pollinator exclusion experiments, we tested the power of buzz density to predict pollination services at the landscape scale for two bumble bee pollinated alpine forbs (Trifolium dasyphyllum and T. parryi). We found that the characteristic frequency was correlated with traits known to affect pollination efficacy, explaining 30-52% of variation in body size and tongue length. Buzz density was highly correlated with visual estimates of bumble bee density (r = 0.97), indicating that acoustic signals are predictive of bumble bee activity. Buzz density predicted seed set in two alpine forbs when bumble bees were permitted access to the flowers, but not when they were excluded from visiting. Our results indicate that acoustic signatures of flight can be deciphered to monitor bee activity and pollination services to bumble bee pollinated plants. We propose that applications of this technique could assist scientists and farmers in rapidly detecting and responding to bee population declines.

Stress-induced memory alters growth of clonal offspring of white clover (Trifolium repens).

PREMISE OF THE STUDY: The phenotype of an individual can be modified by the environment experienced by its predecessors, a phenomenon called transgenerational or maternal effects. These effects are studied mostly across sexual generations and are thought to be mediated also by epigenetic variation. However, we do not know how important transgenerational effects are across asexual generations of clonal plants. METHODS: We investigated the role of different drought intensities and durations experienced by parental plants of Trifolium repens on the growth of offspring ramets after transplantation of clonal cuttings to control conditions. We also treated half of the plants with 5-azacytidine, which is a demethylating agent, to test the potential role of DNA methylation on transgenerational effects. KEY RESULTS: Transgenerational effects were manifested as increased biomass of offspring ramets if parental plants experienced medium drought applied for a short period and decreased biomass of offspring ramets if parental plants experienced intense drought for a short period. These transgenerational effects were not observed for offspring of parents from the same treatments if these were treated with 5-azacytidine, whose application significantly decreased the amount of 5-methyl-2'-deoxycytidine in plants. CONCLUSIONS: Transgenerational effects might play an important role in the clonal plant Trifolium repens and are probably mediated by epigenetic variation. The growth and behavior of clonal plants might be affected not only by the ambient environment but also by environments that are no longer present at the time of clonal reproduction. This phenomenon can have yet unacknowledged ecological and evolutionary implications for clonal plants.