Chemical Basis for Determining the Allelopathic Potential of Invasive Plant Wall Barley (Hordeum murinum L. subsp. murinum).

The study investigated compounds present in the invasive grass Hordeum murinum L. subsp. murinum and tested the allelopathic potential of this plant against common meadow species Festuca rubra L. and Trifolium repens L. Gas chromatography-mass spectrometry (GC-MS) performed separately on the ears and stalks with leaves of wall barley revealed 32 compounds, including secondary metabolites, that may play an important role in allelopathy. Two compounds, N-butylbenzenesulfonamide (NBBS) and diphenylsulfone (DDS), were described for the first time for wall barley and the Poaceae family. The presence of 6,10,14-trimethylpentadecan-2-one (TMP) has also been documented. Aqueous extracts of H. murinum organs (ears and stalks with leaves) at concentrations of 2.5%, 5%, and 7.5% were used to evaluate its allelopathic potential. Compared to the control, all extracts inhibited germination and early growth stages of meadow species. The inhibitory effect was strongest at the highest concentration for both the underground and aboveground parts of the seedlings of the meadow species tested. Comparing the allelopathic effect, Trifolium repens proved to be more sensitive. In light of the results of the study, the removal of wall barley biomass appears to be important for the restoration of habitats where this species occurs due to its allelopathic potential.

Laboratory and Field Evaluation of the Phytotoxic Activity of Sapindus mukorossi Gaertn Pulp Extract and Identification of a Phytotoxic Substance.

Interest in finding plant-based herbicides to supplement synthesized herbicides is increasing. Although the extract of Sapindus mukorossi Gaertn has been reported to have herbicidal activity, little is known about phytotoxic substances and their efficacy of weed control in the field. To identify phytotoxic substances, the bioassay-guided fractionation by column chromatography and high-speed counter-current chromatography (HSCCC) was carried out. The phytotoxic activity assay, performed by the agar medium method, showed that the 70% ethanol fraction exhibited strong root growth inhibition against Trifolium pratense with an 50% inhibitory concentration (IC50) value of 35.13 mg/L. An active compound was isolated from the 70% ethanol fraction and identified as hederagenin 3-o-ß-D-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (Compound A). Compound A had an IC50 value of 16.64 mg/L. Finally, a new formulation was prepared based on the 70% ethanol fraction, which exhibited good efficacy against broadleaf weeds in a carrot field. The fresh weight control efficacy was 78.7% by 45 days after treatment at the dose of 1500 g a. i./ha. Hence, the extract of S. mukorossi pulp could be a promising supplement to the synthesized herbicides. Furthermore, compound A from S. mukorossi may be responsible for its phytotoxic activity.

Effect of drill cuttings addition on physicochemical and chemical properties of soil and red clover (Trifolium pretense L.) growth.

The most economical method of drill cuttings disposal may be their application in land reclamation which allows for the wastes recovery. However, the wastes application into the soil should ensure that the quality of the environment would not be deteriorated. These investigations were aimed at identifying the effect of drill cuttings, which were the mixture of different types of drilling wastes, on the physicochemical properties of acidic soil and growth of red clover (Trifolium pratense L.). The experimental design comprised 5 treatments, which differed in a dose of the drill cuttings: 0% (control), 2.5%, 5%, 10% and 15% of dry weight. A six-week pot experiment was conducted to determine the influence of the wastes on the plant growth. The results showed that the drill cuttings addition significantly changed the chemical and physicochemical properties of the soil, such as: electrical conductivity (EC), pH, base saturation, content of carbonate, alkaline cations (Ca2+, Na+, K+, Mg2+), organic matter, total organic carbon (TOC), and available phosphorus form. However, the most important factors that influenced the growth of red clover were pH, base saturation, content of Mg2+ and plant available phosphorus. The red clover biomass was increased from 1.5 to 2.5 times depending on the dose of wastes. We concluded that the examined wastes can be used for reclamation of the acid and unfertile degraded soils, but the amount of wastes should not exceed 5% of the soil, because the highest total clover biomass was observed just at this dose.

Responses of microbial communities and metabolic activities in the rhizosphere during phytoremediation of Cd-contaminated soil.

Phytoremediation is an effective way to repair heavy metal contaminated soil and rhizosphere microorganisms play an important role in plant regulation. Nevertheless, little information is known about the variation of microbial metabolic activities and community structure in rhizosphere during phytoremediation. In this study, the rhizosphere soil microbial metabolic activities and community structure of Trifolium repensL. during Cd-contaminated soil phytoremediation, were analyzed by Biolog EcoPlate™ and high-throughput sequencing. The uptake in the roots of Trifolium repensL. grown in 5.68 and 24.23 mg/kg Cd contaminated soil was 33.51 and 84.69 mg/kg respectively, causing the acid-soluble Cd fractions decreased 7.3% and 5.4%. Phytoremediation significantly influenced microbial community and Trifolium repensL. planting significantly increased the rhizosphere microbial population, diversity, the relative abundance of plant growth promoting bacteria (Kaistobacter and Flavisolibacter), and the utilization of difficultly metabolized compounds. The correlation analysis among substrate utilization and microbial communities revealed that the relative abundance increased microorganisms possessed stronger carbon utilization capacity, which was beneficial to regulate the stability of plant-microbial system. Collectively, the results of this study provide fundamental insights into the microbial metabolic activities and community structure during heavy metal contaminated soil phytoremediation, which may aid in the bioregulation of phytoremediation.

Toxicity of Ag+ on microstructure, biochemical activities and genic material of Trifolium pratense L. seedlings with special reference to phytoremediation.

The objective of this research was to evaluate Ag+ toxicity in Trifolium pratense L. seedlings subjected to increasing doses of Ag+ by determining photosynthetic pigment and malondialdehyde (MDA) contents, microstructure and hereditary substance alterations, changes in activities of antioxidase-superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) as well as the content of total Ag absorbed in vivo with evaluation of root growth. Doses of approximately 80 mg L-1 Ag+ severely affected photosynthetic efficiency in Trifolium pratense L. seedlings promoted by damages in photosynthetic apparatus evidenced by downward trend in photosynthetic pigment contents and obvious chlorosis. Alterations in enzymatic activity, lipid peroxidation, genic material damage and the presence of Ag+in vivo had impacted on photosynthetic machinery as well. A hormesis effect was observed at 60 mg L-1 Ag+ for the photosynthetic pigments and antioxidase for Trifolium pratense L. seedlings. Tissue changes (i.e., roots, stems and leaves) observed in fluorescence microscope with obvious chlorosis, roots blackening and formation of agglomerated black particles, were related to the lesion promoted by excessive ROS in vivo. Asynchronous change of antioxidase activity corresponded to the alteration in the MDA content, indicating the synchronization in the elimination of ROS. The changes occurred in RAPD profiles of treated samples following Ag+ toxicity containing loss of normal bands, appearance of new bands and variation in band intensity compared to the normal plants with a dose-dependent effect. On average, the roots of Trifolium pratense L. immobilized 92.20% of the total Ag absorbed as a metal exclusion response. Root growth was significantly sensitive to Ag+ stress with obvious hormesis, which corresponded to the changes in Ag uptake, demonstrating the functional alterations in plants. To sum up, we suggest that modulating the genotype of Trifolium pratense L. seedlings to bear higher proportion of pollutants is conducive to contamination site treatment.

Toxicity evaluation of olive oil mill wastewater and its polar fraction using multiple whole-organism bioassays.

Olive mill wastewater (OMW) as a by-product of olive oil extraction process has significant polluting properties mainly related to high organic load, increased COD/BOD ratio, high phenolic content and relatively acidic pH. Raw OMW from Slovenian Istria olive oil mill and its polar fraction were investigated in this study. Chemical characterization of OMW polar fraction identified tyrosol as the most abundant phenolic product, followed by catechol. Lethal and sub-lethal effects of OMW matrix and its polar fraction were tested using a battery of bioassays with model organisms: bacteria Vibrio fischeri, algae Chlorella vulgaris, water fleas Daphnia magna, zebrafish Danio rerio embryos, clover Trifolium repens and wheat Triticum aestivum. Raw OMW sample was the most toxic to V. fischeri (EC50 = 0.24% of OMW sample final concentration), followed by D. magna (EC50 = 1.43%), C. vulgaris (EC50 = 5.20%), D. rerio (EC50 = 7.05%), seeds T. repens (EC50 = 8.68%) and T. aestivum (EC50 = 11.58%). Similar toxicity trend was observed during exposure to OMW polar fraction, showing EC50 values 2.75-4.11 times lower comparing to raw OMW. Tested samples induced also sub-acute effects to clover and wheat (decreased roots, sprouts elongation); and to zebrafish embryos (increased mortality, higher abnormality rate, decreased hatching and pigmentation formation rate). A comprehensive approach using a battery of bioassays, like those used in this study should be applied during ecotoxicity monitoring of untreated and treated OMW.

Cigarette butts have adverse effects on initial growth of perennial ryegrass (gramineae: Lolium perenne L.) and white clover (leguminosae: Trifolium repens L.).

Cigarette filters (butts) are currently the most abundant form of anthropogenic litter on the planet, yet we know very little about their environmental impacts on terrestrial ecosystems, including plant germination and primary production. When discarded, filters contain a myriad of chemicals resulting from smoking tobacco and some still contain unsmoked remnants. A greenhouse experiment was used to assess the impacts of discarded filters of regular or menthol cigarette, either from unsmoked, smoked, or smoked cigarettes with remnant tobacco, on the growth and development of Lolium perenne (perennial ryegrass) and Trifolium repens (white clover). After 21 days, shoot length and germination success were significantly reduced by exposure to any type of cigarette filter for the grass and clover. Although total grass biomass was not measurably affected, the root biomass and root:shoot ratio were less in the clover when exposed to filters from smoked regular cigarettes and those with remnant tobacco. Cigarette filters caused an increase in chlorophyll-a in clover shoots and an increase in chlorophyll-b in grass shoots. Accordingly, whilst the chlorophyll a:b ratio was increased in the clover exposed to cigarette filters, it was decreased in grass. This study indicates the potential for littered cigarette filters to reduce growth and alter short-term primary productivity of terrestrial plants.

Legumes in catch crop mixtures: Effects on nitrogen retention and availability, and leaching losses.

Catch crop (CC) mixtures of non-legumes (nL) and legumes (L) have been promoted as a strategy to achieve two different goals: to decrease the risk of nitrate leaching and to enhance the nitrogen supply to the subsequent crop. To investigate if two-component mixtures of nL + L have advantages over pure nL stands experiments were carried out over a two year period (2013-2015) at two contrasting field sites in Denmark. Nitrogen (N) uptake by the CCs was measured by aboveground biomass sampling, and N leaching by ceramic suction cups. When grown in pure stands, white clover (Trifolium repens) on coarse sand and common vetch (Vicia sativa) on sandy loam were less effective at reducing N leaching than perennial ryegrass (Lolium perenne) and fodder radish (Raphanus sativus). When the proportion of the nL + L in mixtures was similar or favored the nL, leaching was not significantly different from the nL in the pure stand. However, during one of the years on the sandy loam L (vetch) almost outperformed nL (fodder radish), resulting in N leaching from nL + L similar to L. The yield of the following spring barley was only significantly different from the yield in the plots with previously bare soil in one of the years on the coarse sandy soil. It is concluded that in nL + L mixtures L can take over and thereby lower the effect of the CCs on N leaching while not necessarily enhancing the N supply for the subsequent crop.

Vanadium elicitation of Trifolium pratense L. cell culture and possible pathways of produced isoflavones transport across the plasma membrane.

KEY MESSAGE: Vanadium compounds increased the content and release of distinct isoflavones in a Trifolium pratense suspension culture. Regarding transport-mechanism inhibitors, the process was mostly facilitated by ABC proteins and vesicular transport. The transport of isoflavones and other secondary metabolites is an important part of metabolism within plants and cultures in vitro regarding their role in defence against various abiotic and biotic stressors. This research focuses on the way how to increase production and exudation of isoflavones by application of chemical elicitor and the basic identification of their transport mechanisms across cell membranes. The release of five isoflavones (genistin, genistein, biochanin A, daidzein, and formononetin) into a nutrient medium was determined in a Trifolium pratense var. DO-8 suspension culture after two vanadium compound treatments and cultivation for 24 and 48 h. The NH4VO3 solution caused a higher concentration of isoflavones in the medium after 24 h. This increased content of secondary metabolites was subsequently suppressed by distinct transport-mechanism inhibitors. The transport of isoflavones in T. pratense was mostly affected by ABC inhibitors from the multidrug-resistance-associated protein subfamily, but the genistein concentration in the medium was lower after treatment with multidrug-resistance protein subfamily inhibitors. Brefeldin A, which blocks vesicular transport, also decreased the concentration of some isoflavones in the nutrient medium.

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.

Combined toxic effects and DNA damage to two plant species exposed to binary metal mixtures (Cd/Pb).

Acute and long-term (3-, 10- and 56-day exposure) laboratory toxicity tests were carried out to assess the individual and combined toxic effects of cadmium (Cd) and lead (Pb) in Brassica oleracea and Trifolium repens. In addition to morphological parameters, this work also used comet assay to address endpoints in relation to genotoxicity. Bioaccumulation was measured to demonstrate the influence of the mixture on the concentrations of each metal in the plant. The statistical method reported by Ince et al. (1999) was used to evaluate the types of interaction between Cd and Pb in each treatment and concerning their combined effect. This study concludes that the combined effects of binary metal combinations of Cd/Pb on morphological parameters are most often additive, sometimes antagonistic and more rarely synergistic, thus extending the findings of previous publications on this subject. DNA damage analysis revealed concentration- and time-dependent interactions. Synergistic effects of mixed metals (more breaks than individually applied metals) are observed in T. repens after a short exposure. Antagonistic effects are statistically significant after 10 days-exposure, suggesting competition between metals. At 56 days, the rate of DNA damage observed in plants exposed to the Cd/Pb mixture was similar to that measured in plants exposed to lead only and was significantly lower than the rate of DNA damage induced by Cd. This supports the idea that there may be competition between metals and also strengthens the hypothesis that long-term reparation mechanisms may be implemented. Cd/Pb co-exposure does not significantly influence the bioaccumulation of each metal. It is nevertheless important to note that a statistically significant 'interaction' is not necessarily biologically relevant and should therefore be considered with caution when assessing heavy metals combined effects.

Pretreatment with NaCl Promotes the Seed Germination of White Clover by Affecting Endogenous Phytohormones, Metabolic Regulation, and Dehydrin-Encoded Genes Expression under Water Stress.

This study was designed to examine the effects of NaCl pretreatment on the seed germination of white clover (Trifolium repens cv. Ladino) under water stress induced by 19% polyethylene glycol (PEG) 6000. Lower concentrations of NaCl (0.5, 1, and 2.5 mM) pretreatment significantly alleviated stress-induced decreases in germination percentage, germination vigor, germination index, and radicle length of seedlings after seven days of germination under water stress. The soaking with 1 mM of NaCl exhibited most the pronounced effects on improving seed germination and alleviating stress damage. NaCl-induced seeds germination and growth could be associated with the increases in endogenous gibberellic acid (GA) and indole-3-acetic acid (IAA) levels through activating amylases leading to improved amylolysis under water stress. Seedlings pretreated with NaCl had a significantly lower osmotic potential than untreated seedlings during seed germination, which could be related to significantly higher soluble sugars and free proline content in NaCl-treated seedlings under water stress. For antioxidant metabolism, NaCl pretreatment mainly improved superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase activities, transcript levels of FeSOD, APX, and DHAR, and the content of ascorbic acid, reduced glutathione, and oxidized glutathione during seed germination under water stress. The results indicated that seeds soaking with NaCl could remarkably enhance antioxidant metabolism, thereby decreasing the accumulation of reactive oxygen species and membrane lipid peroxidation during germination under water stress. In addition, NaCl-upregulated dehydrin-encoded genes SK2 expression could be another important mechanism of drought tolerance during seeds germination of white clover in response to water stress.

Microsatellite polymorphism of Trifolium pratense population at the conditions of radioactive and chemical contamination of soil (Komi republic, Russia).

There is no clear understanding of microevolutionary changes in natural populations of plants and animals due to anthropogenic contamination of the environment with toxicants and mutagens. But such data are necessary to forecast long-term effects of human activity. In this research, we studied genetic polymorphism in T. pratense sampled from seven sites varying in radioactive and chemical soil contamination in the vicinity of Vodny settlement (Komi, Russia). Analysis of five SSR loci was shown to be similar in a whole (N), mean (Na) and effective (Ne) numbers of alleles, heterozygosity indexes (Ho and He), and the Shannon index (I). Difference in the private allele numbers was registered: the most contaminated site has 5 and others from 0 up 2 private alleles. No difference was found in the genetic structure of T. pratense population growing at the conditions of radioactive and chemical contamination. The Bayesian analysis provided evidence of a single cluster (K = 1) due to a similar genetic structure of samples, while AMOVA results demonstrated a high variability within individuals (75%) and a low variability (1%) among groups of T. pratense from sites that differ in the contamination level. Thus, the long-term radioactive and heavy metal contamination of soil did not result in significant microevolutionary changes in T. pratense population.

Essential Oil Composition, Antioxidant and Allelopathic Activities of Cleome droserifolia (Forssk.) Delile.

People rely on the synthetic chemical pesticides in most of the agricultural practices, which have a serious problem on human health and ecosystem. Essential oil (EO), derived from wild plants, offers a new opportunity to explore eco-friendly green biocides. This study aimed to characterize the chemical constituents of EO from Egyptian ecospecies of Cleome droserifolia (Forssk.) Delile and evaluate its antioxidant and allelopathic potential. The EO was extracted from aerial parts by hydrodistillation and analyzed by GC/MS. To assess the allelopathic potential, EO concentrations from 50 to 200 µL L-1 were tested on Trifolium repens and three weeds. Antioxidant activity was determined using DPPH. Hydrodistillation yielded 0.64 % of dark yellow oil, which comprises 35 compounds, in which sesquiterpene was a major class and represented by 61.97 % of the total essential oil. Moreover, the dominant sesquiterpenes are cis-nerolidol, α-cadinol, δ-cadinene, and γ-muurolene. The speed of germination index, shoot and root length of clover and weeds were reduced in a concentration-dependent manner. The IC50 values of C. droserifolia EO on germination of T. repens, Cuscuta trifolii, Melilotus indicus, and Chenopodium murale were 181.6, 183.5, 159.0, and 157.5 µL L-1 , respectively. From the obtained data, we concluded that C. droserifolia EO could provide a hope to produce environment-friendly bioherbicide as well as a natural resource of antioxidants.

Investigation on microbial community in remediation of lead-contaminated soil by Trifolium repensL.

Trifolium repensL. is a plant with strong adaptability and large biomass, which possess great potential for phytoremediation. However, little is known concerning its remediation effects and changes in rhizosphere microbial activity and community structure under heavy metal pressure. The aims of this study were to evaluate lead accumulation of Trifolium repensL., study microbial lead resistance, metabolism and community structure characteristics in rhizosphere soils. The accumulated Pb concentration of Trifolium repensL. was observed in 100 and 500 mg/kg Pb contained soil at 55.81 and 90.3 mg/kg, respectively, which cause the decrease of acid-soluble fractions in rhizosphere soil. In the progress of lead-contaminated soil phytoremediation by Trifolium repensL., Pb resistance and metabolic activities of microorganisms have been prompted gradually. In addition, the microbial community composition and abundance were investigated using Illumina sequencing and quantitative PCR. The result showed that after phytoremediation, beneficial microorganisms, such as Flavisolibacter, Kaistobacter, and Pseudomonas, increased, becoming the dominant genera. This study has provided insight into the distribution and activity of the microbial community.

The γ-Aminobutyric Acid (GABA) Alleviates Salt Stress Damage during Seeds Germination of White Clover Associated with Na⁺/K⁺ Transportation, Dehydrins Accumulation, and Stress-Related Genes Expression in White Clover.

The objective of this study was to determine the effect of soaking with γ-aminobutyric acid (GABA) on white clover (Trifolium repens cv. Haifa) seed germination under salt stress induced by 100 mM NaCl. Seeds soaking with GABA (1 µM) significantly alleviated salt-induced decreases in endogenous GABA content, germination percentage, germination vigor, germination index, shoot and root length, fresh and dry weight, and root activity of seedling during seven days of germination. Exogenous application of GABA accelerated starch catabolism via the activation of amylase and also significantly reduced water-soluble carbohydrate, free amino acid, and free proline content in seedlings under salt stress. In addition, improved antioxidant enzyme activities (SOD, GPOX, CAT, APX, DHAR, GR and MDHR) and gene transcript levels (Cu/ZnSOD, FeSOD, MnSOD, CAT, GPOX, APX, MDHR, GPX and GST) was induced by seeds soaking with GABA, followed by decreases in O2∙-, H2O2, and MDA accumulation during germination under salt stress. Seeds soaking with GABA could also significantly improve Na⁺/K⁺ content and transcript levels of genes encoding Na⁺/K⁺ transportation (HKT1, HKT8, HAL2, H⁺-ATPase and SOS1) in seedlings of white clover. Moreover, exogenous GABA significantly induced the accumulation of dehydrins and expression of genes encoding dehydrins (SK2, Y2K, Y2SK, and dehydrin b) in seedlings under salt stress. These results indicate that GABA mitigates the salt damage during seeds germination through enhancing starch catabolism and the utilization of sugar and amino acids for the maintenance of growth, improving the antioxidant defense for the alleviation of oxidative damage, increasing Na⁺/K⁺ transportation for the osmotic adjustment, and promoting dehydrins accumulation for antioxidant and osmotic adjustment under salt stress.

Use of Ethylenediurea (EDU) in identifying indicator cultivars of Indian clover against ambient ozone.

Three clover (Trifolium alexandrium L.) cultivars (Bundel, Wardan and JHB-146) were assessed for their responses to ambient ozone (O3) with respect to growth, physiological and biochemical parameters at two rural sites (R1 and R2) using ethylenediurea (EDU). EDU solution (300ppm) was applied as soil drench, 10 days after germination (DAG) at an interval of 10 days up to 80 DAG. The average O3 concentrations were 52.76 and 60.86 ppb at R1 and R2 sites, respectively during the experimental period. Ambient O3 induced visible symptoms in all the cultivars at both the sites, with more at R2 site having high ambient O3 levels. Visible injury was observed first in non-EDU treated plants of Wardan at R2 site. Wardan also showed maximum reduction in leaf injury under EDU treatment at both the sites with more at R2. Under EDU treatment, better adaptation to ambient O3 at initial age of observation and higher acquisition of resources at later ages of observation at both the sites led to better physiological and biochemical adaptations in Wardan. Bundel retained more biomass in shoot as is reflected with higher shoot/root ratio and thus focused more on repair and defense. Shoot/root ratio of JHB-146 did not respond to EDU treatment and thus showed insignificant variations except at initial age of observation at R1 site. This study clearly suggests that Wardan and Bundel are sensitive to ambient O3 and can be used as bioindicator species in areas having higher O3 levels using EDU as a research tool.

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.

Effect of Pseudomonas fluorescens and pyoverdine on the phytoextraction of cesium by red clover in soil pots and hydroponics.

With the aim of improving the phytoextraction rate of cesium (Cs), the effect of Pseudomonas fluorescens ATCC 17400 and its siderophore pyoverdine (PVD) on the uptake of Cs by red clover was studied in soil pots. This work also provides a mechanistic understanding of the Cs-bacteria (or PVD)-illite-plant interactions by using a simplified experimental design, i.e., hydroponics with either Cs in solution or Cs-spiked illite in suspension. For soil spiked with 11.2 mmol kg-1 (1480 mg kg-1) of Cs, 0.43% of total Cs was taken up by red clover in 12 days (119 µmol g-1 (16 mg g-1) of Cs dry matter in roots and 40 µmol g-1 (5 mg g-1) in shoots). In hydroponics with Cs in solution (0.1 mmol L-1 or 13 mg L-1), 75% of Cs was taken up vs. only 0.86% with Cs-spiked illite suspension. P. fluorescens and PVD did not increase Cs concentrations in aboveground parts and roots of red clover and even decreased them. The damaging effect of PVD on red clover growth was demonstrated with the biomass yielding 66% of the control in soil pots (and 100% mortality after 12 days of exposition) and only 56% in hydroponics (78% with illite in suspension). Nonetheless, PVD and, to a lesser extent, P. fluorescens increased the translocation factor up to a factor of 2.8. This study clearly showed a direct damaging effect of PVD and to a lower extent the retention of Cs by biofilm covering both the roots and illite, both resulting in the lower phytoextraction efficiency.

Signalling cross-talk between nitric oxide and active oxygen in Trifolium repens L. plants responses to cadmium stress.

The significant influence of •NO on the stress response is well established; however, the precise metabolic pathways of •NO and RNS under metal stresses remain unclear. Here, the key components of ROS and RNS metabolism under Cd stress were investigated with multi-level approaches using high-quality forage white clover (Trifolium repens L.) plants. For the studied plants, Cd disturbed the redox homeostasis, affected the absorption of minerals, and exacerbated the degree of lipid peroxidation, thus triggering oxidative stress. However, •NO was also involved in regulating mineral absorption, ROS-scavenger levels and mRNA expression in Cd-treated white clover plants. In addition, GSNOR activity was up-regulated by Cd with the simultaneous depletion of •NO generation and GSNO but was counteracted by the •NO donor sodium nitroprusside. Response to Cd-stressed SNOs was involved in generating ONOO- and NO2-Tyr in accordance with the regulation of •NO-mediated post-translational modifications in the ASC-GSH cycle, selected amino acids and NADPH-generating dehydrogenases, thereby provoking nitrosative stress. Taken together, our data provide comprehensive metabolite evidence that clearly confirms the relationships between ROS and RNS in Cd-stressed plants, supporting their regulatory roles in response to nitro-oxidative stress and providing an in-depth understanding of the interaction between two families subjected to metal stresses.