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.

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.

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.

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.

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.

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.

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.

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.

A proteome analysis of freezing tolerance in red clover (Trifolium pratense L.).

BACKGROUND: Improvement of freezing tolerance of red clover (Trifolium pratense L.) would increase its persistence under cold climate. In this study, we assessed the freezing tolerance and compared the proteome composition of non-acclimated and cold-acclimated plants of two initial cultivars of red clover: Endure (E-TF0) and Christie (C-TF0) and of populations issued from these cultivars after three (TF3) and four (TF4) cycles of phenotypic recurrent selection for superior freezing tolerance. Through this approach, we wanted to identify proteins that are associated with the improvement of freezing tolerance in red clover. RESULTS: Freezing tolerance expressed as the lethal temperature for 50 % of the plants (LT50) increased markedly from approximately -2 to -16 °C following cold acclimation. Recurrent selection allowed a significant 2 to 3 °C increase of the LT50 after four cycles of recurrent selection. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to study variations in protein abundance. Principal component analysis based on 2D-DIGE revealed that the largest variability in the protein data set was attributable to the cold acclimation treatment and that the two genetic backgrounds had differential protein composition in the acclimated state only. Vegetative storage proteins (VSP), which are essential nitrogen reserves for plant regrowth, and dehydrins were among the most striking changes in proteome composition of cold acclimated crowns of red clovers. A subset of proteins varied in abundance in response to selection including a dehydrin that increased in abundance in TF3 and TF4 populations as compared to TF0 in the Endure background. CONCLUSION: Recurrent selection performed indoor is an effective approach to improve the freezing tolerance of red clover. Significant improvement of freezing tolerance by recurrent selection was associated with differential accumulation of a small number of cold-regulated proteins that may play an important role in the determination of the level of freezing tolerance.

Establishment and effectiveness of inoculated arbuscular mycorrhizal fungi in agricultural soils.

Arbuscular mycorrhizal fungi (AMF) are promoted as biofertilizers for sustainable agriculture. So far, most researchers have investigated the effects of AMF on plant growth under highly controlled conditions with sterilized soil, soil substrates or soils with low available P or low inoculum potential. However, it is still poorly documented whether inoculated AMF can successfully establish in field soils with native AMF communities and enhance plant growth. We inoculated grassland microcosms planted with a grass-clover mixture (Lolium multiflorum and Trifolium pratense) with the arbuscular mycorrhizal fungus Rhizoglomus irregulare. The microcosms were filled with eight different unsterilized field soils that varied greatly in soil type and chemical characteristics and indigenous AMF communities. We tested whether inoculation with AMF enhanced plant biomass and R. irregulare abundance using a species specific qPCR. Inoculation increased the abundance of R. irregulare in all soils, irrespective of soil P availability, the initial abundance of R. irregulare or the abundance of native AM fungal communities. AMF inoculation had no effect on the grass but significantly enhanced clover yield in five out of eight field soils. The results demonstrate that AMF inoculation can be successful, even when soil P availability is high and native AMF communities are abundant.

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.

Transgenerational effects of land use on offspring performance and growth in Trifolium repens.

Central European grasslands vary widely in productivity and in mowing and grazing regimes. The resulting differences in competition and heterogeneity among grasslands might have direct effects on plants, but might also affect the growth and morphology of their offspring through maternal effects or adaptive evolution. To test for such transgenerational effects, we grew plants of the clonal herb Trifolium repens from seeds collected in 58 grassland sites differing in productivity and mowing and grazing intensities in different treatments: without competition, with homogeneous competition, and with heterogeneous competition. In the competition-free treatment, T. repens from more productive, less frequently mown, and less intensively grazed sites produced more vegetative offspring, but this was not the case in the other treatments. When grown among or in close proximity to competitors, T. repens plants did not show preferential growth towards open spaces (i.e., no horizontal foraging), but did show strong vertical foraging by petiole elongation. In the homogeneous competition treatment, petiole length increased with the productivity of the parental site, but this was not the case in the heterogeneous competition treatment. Moreover, petiole length increased with mowing frequency and grazing intensity of the parental site in all but the homogeneous competition treatment. In summary, although the expression of differences between plants from sites with different productivities and land-use intensities depended on the experimental treatment, our findings imply that there are transgenerational effects of land use on the morphology and performance of T. repens.

Effect of nanoparticles on red clover and its symbiotic microorganisms.

BACKGROUND: Nanoparticles are produced and used worldwide and are released to the environment, e.g., into soil systems. Titanium dioxide (TiO2) nanoparticles (NPs), carbon nanotubes (CNTs) and cerium dioxide (CeO2) NPs are among the ten most produced NPs and it is therefore important to test, whether these NPs affect plants and symbiotic microorganisms that help plants to acquire nutrients. In this part of a joint companion study, we spiked an agricultural soil with TiO2 NPs, multi walled CNTs (MWCNTs), and CeO2 NPs and we examined effects of these NP on red clover, biological nitrogen fixation by rhizobia and on root colonization of arbuscular mycorrhizal fungi (AMF). We also tested whether effects depended on the concentrations of the applied NPs. RESULTS: Plant biomass and AMF root colonization were not negatively affected by NP exposure. The number of flowers was statistically lower in pots treated with 3 mg kg(-1) MWCNT, and nitrogen fixation slightly increased at 3000 mg kg(-1) MWCNT. CONCLUSIONS: This study revealed that red clover was more sensitive to MWCNTs than TiO2 and CeO2 NPs. Further studies are necessary for finding general patterns and investigating mechanisms behind the effects of NPs on plants and plant symbionts.

Physiological and biochemical responses of the forage legume Trifolium alexandrinum to different saline conditions and nitrogen levels.

Salinity stress reduces plant productivity, but low levels of salinity often increase plant growth rates in some species. We herein describe the effects of salinity on plant growth while focusing on nitrogen use. We treated Trifolium alexandrinum with two nitrogen concentrations and salinity levels and determined growth rates, mineral concentrations, nitrogen use efficiency, photosynthesis rates, and nitrate reductase (NR, E.C. 1.6.6.1) and glutamine synthetase (GS, EC 6.3.1.2) activities. The T. alexandrinum growth rate increased following treatment with 100 mM NaCl in low nitrogen (LN) and high nitrogen (HN) conditions. Salt treatment also increased root volume, intrinsic water use efficiency, and nitrogen use efficiency in LN and HN conditions. These changes likely contributed to higher biomass production. Salinity also increased accumulations of sodium, chloride, and phosphate, but decreased potassium and calcium levels and total nitrogen concentrations in all plant organs independently of the available nitrogen level. However, the effect of salt treatment on magnesium and nitrate concentrations in photosynthetic organs depended on nitrogen levels. Salt treatment reduced photosynthesis rates in LN and HN conditions because of inhibited stomatal conductance. The effects of salinity on leaf NR and GS activities depended on nitrogen levels, with activities increasing in LN conditions. In saline conditions, LN availability resulted in optimal growth because of low chloride accumulation and increases in total nitrogen concentrations, nitrogen use efficiency, and NR and GS activities in photosynthetic organs. Therefore, T. alexandrinum is a legume forage crop that can be cultivated in low-saline soils where nitrogen availability is limited.

Interactions between biochar and mycorrhizal fungi in a water-stressed agricultural soil.

Biochar may alleviate plant water stress in association with arbuscular mycorrhizal (AM) fungi but research has not been conclusive. Therefore, a glasshouse experiment was conducted to understand how interactions between AM fungi and plants respond to biochar application under water-stressed conditions. A twin chamber pot system was used to determine whether a woody biochar increased root colonisation by a natural AM fungal population in a pasture soil ('field' chamber) and whether this was associated with increased growth of extraradical AM fungal hyphae detected by plants growing in an adjacent ('bait') chamber containing irradiated soil. The two chambers were separated by a mesh that excluded roots. Subterranean clover was grown with and without water stress and harvested after 35, 49 and 63 days from each chamber. When biochar was applied to the field chamber under water-stressed conditions, shoot mass increased in parallel with mycorrhizal colonisation, extraradical hyphal length and shoot phosphorus concentration. AM fungal colonisation of roots in the bait chamber indicated an increase in extraradical mycorrhizal hyphae in the field chamber. Biochar had little effect on AM fungi or plant growth under well-watered conditions. The biochar-induced increase in mycorrhizal colonisation was associated with increased growth of extraradical AM fungal hyphae in the pasture soil under water-stressed conditions.

Bumble bee nest abundance, foraging distance, and host-plant reproduction: implications for management and conservation.

Recent reports of global declines in pollinator species imply an urgent need to assess the abundance of native pollinators and density-dependent benefits for linked plants. In this study, we investigated (1) pollinator nest distributions and estimated colony abundances, (2) the relationship between abundances of foraging workers and the number of nests they represent, (3) pollinator foraging ranges, and (4) the relationship between pollinator abundance and plant reproduction. We examined these questions in an alpine ecosystem in the Colorado Rocky Mountains, focusing on four alpine bumble bee species (Bombus balteatus, B. flavifrons, B. bifarius, and B. sylvicola), and two host plants that differ in their degrees of pollinator specialization (Trifolium dasyphyllum and T. parryi). Using microsatellites, we found that estimated colony abundances among Bombus species ranged from ~18 to 78 colonies/0.01 km2. The long-tongued species B. balteatus was most common, especially high above treeline, but the subalpine species B. bifarius was unexpectedly abundant for this elevation range. Nests detected among sampled foragers of each species were correlated with the number of foragers caught. Foraging ranges were smaller than expected for all Bombus species, ranging from 25 to 110 m. Fruit set for the specialized plant, Trifolium parryi, was positively related to the abundance of its Bombus pollinator. In contrast, fruit set for the generalized plant, T. dasyphyllum, was related to abundance of all Bombus species. Because forager abundance was related to nest abundance of each Bombus species and was an equally effective predictor of plant fecundity, forager inventories are probably suitable for assessing the health of outcrossing plant populations. However, nest abundance, rather than forager abundance, better reflects demographic and genetic health in populations of eusocial pollinators such as bumble bees. Development of models incorporating the parameters we have measured here (nest abundance, forager abundance, and foraging distance) could increase the usefulness of foraging worker inventories in nionitoring, managing, and conserving pollinator populations.

Nitric oxide contributes to minerals absorption, proton pumps and hormone equilibrium under cadmium excess in Trifolium repens L. plants.

Nitric oxide (NO) is a stress-signaling molecule in plants that mediates a wide range of physiological processes and responses to metal toxicity. In this work, various NO modulators (NO donor: SNP; NO scavenger: cPTIO; NO synthase inhibitor: l-NAME; and SNP analogs: sodium nitrite/nitrate and sodium ferrocyanide) were investigated to determine the role of NO in Trifolium repens L. plants exposed to Cd. Cd (100µM) markedly reduced biomass, NO production and chlorophyll (Chl a, Chl b and total Chl) concentration but stimulated reactive oxygen species (ROS) and Cd accumulation in plants. SNP (50µM) substantially attenuated growth inhibition, reduced hydrogen peroxide (H2O2) and malonyldialdehyde (MDA) levels, stimulated ROS-scavenging enzymes/agents, and mitigated the H(+)-ATPase inhibition in proton pumps. Interestingly, SNP considerably up-regulated the levels of jasmonic acid (JA) and proline in plant tissues but down-regulated the levels of ethylene (ET) in both shoots and roots and the level of salicylic acid (SA) in roots only, which might be related to the elevated NO synthesis. Additionally, SNP (25-200µM) regulated mineral absorption and, particularly at 50µM, significantly enhanced the uptake of shoot magnesium (Mg) and copper (Cu) and of root calcium (Ca), Mg and iron (Fe). Nevertheless, the effects of SNP on plant growth were reversed by cPTIO and l-NAME, suggesting that the protective effect of SNP might be associated with NO synthesis in vivo. Moreover, SNP analogs did not display roles similar to that of SNP. These results indicated that NO depleted Cd toxicity by eliminating oxidative damage, enhancing minerals absorption, regulating proton pumps, and maintaining hormone equilibrium.

Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover.

In stolon of white clover (Trifolium repens L.), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS-PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS-PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress.

Aridity shapes cyanogenesis cline evolution in white clover (Trifolium repens L.).

Adaptive differentiation between populations is often proposed to be the product of multiple interacting selective pressures, although empirical support for this is scarce. In white clover, populations show adaptive differentiation in frequencies of cyanogenesis, the ability to produce hydrogen cyanide after tissue damage. This polymorphism arises through independently segregating polymorphisms for the presence/absence of two required cyanogenic components, cyanogenic glucosides and their hydrolysing enzyme. White clover populations worldwide have evolved a series of recurrent, climate-associated clines, with higher frequencies of cyanogenic plants in warmer locations. These clines have traditionally been hypothesized to reflect a fitness trade-off between chemical defence in herbivore-rich areas (warmer climates) and energetic costs of producing cyanogenic components in areas of low herbivore pressure (cooler climates). Recent observational studies suggest that cyanogenic components may also be beneficial in water-stressed environments. We investigated fitness trade-offs associated with temperature-induced water stress in the cyanogenesis system using manipulative experiments in growth chambers and population surveys across a longitudinal precipitation gradient in the central United States. We find that plants producing cyanogenic glucosides have higher relative fitness in treatments simulating a moderate, persistent drought stress. In water-neutral treatments, there are energetic costs to producing cyanogenic components, but only in treatments with nutrient stress. These fitness trade-offs are consistent with cyanogenesis frequencies in natural populations, where we find clinal variation in the proportion of plants producing cyanogenic glucosides along the precipitation gradient. These results suggest that multiple selective pressures interact to maintain this adaptive polymorphism and that modelling adaptation will require knowledge of environment-specific fitness effects.

Plant population differentiation and climate change: responses of grassland species along an elevational gradient.

Mountain ecosystems are particularly susceptible to climate change. Characterizing intraspecific variation of alpine plants along elevational gradients is crucial for estimating their vulnerability to predicted changes. Environmental conditions vary with elevation, which might influence plastic responses and affect selection pressures that lead to local adaptation. Thus, local adaptation and phenotypic plasticity among low and high elevation plant populations in response to climate, soil and other factors associated with elevational gradients might underlie different responses of these populations to climate warming. Using a transplant experiment along an elevational gradient, we investigated reproductive phenology, growth and reproduction of the nutrient-poor grassland species Ranunculus bulbosus, Trifolium montanum and Briza media. Seeds were collected from low and high elevation source populations across the Swiss Alps and grown in nine common gardens at three different elevations with two different soil depths. Despite genetic differentiation in some traits, the results revealed no indication of local adaptation to the elevation of population origin. Reproductive phenology was advanced at lower elevation in low and high elevation populations of all three species. Growth and reproduction of T. montanum and B. media were hardly affected by garden elevation and soil depth. In R. bulbosus, however, growth decreased and reproductive investment increased at higher elevation. Furthermore, soil depth influenced growth and reproduction of low elevation R. bulbosus populations. We found no evidence for local adaptation to elevation of origin and hardly any differences in the responses of low and high elevation populations. However, the consistent advanced reproductive phenology observed in all three species shows that they have the potential to plastically respond to environmental variation. We conclude that populations might not be forced to migrate to higher elevations as a consequence of climate warming, as plasticity will buffer the detrimental effects of climate change in the three investigated nutrient-poor grassland species.