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.

The White Clover TrMYB33-TrSAMS1 Module Contributes to Drought Tolerance by Modulation of Spermidine Biosynthesis via an ABA-Dependent Pathway.

Spermidine is well known to accumulate in plants exposed to drought, but the regulatory network associated with its biosynthesis and accumulation and the underlying molecular mechanisms remain unclear. Here, we demonstrated that the Trifolium repens TrMYB33 relayed the ABA signal to modulate drought-induced spermidine production by directly regulating the expression of TrSAMS1, which encodes an S-adenosylmethionine synthase. This gene was identified by transcriptome and expression analysis in T. repens. TrSAMS1 overexpression and its pTRV-VIGS-mediated silencing demonstrated that TrSAMS1 is a positive regulator of spermidine synthesis and drought tolerance. TrMYB33 was identified as an interacting candidate through yeast one-hybrid library screening with the TrSAMS1 promoter region as the bait. TrMYB33 was confirmed to bind directly to the predicted TAACCACTAACCA (the TAACCA MYB binding site is repeated twice in tandem) within the TrSAMS1 promoter and to act as a transcriptional activator. Additionally, TrMYB33 contributed to drought tolerance by regulating TrSAMS1 expression and modulating spermidine synthesis. Additionally, we found that spermidine accumulation under drought stress depended on ABA and that TrMYB33 coordinated ABA-mediated upregulation of TrSAMS1 and spermidine accumulation. This study elucidated the role of a T. repens MYB33 homolog in modulating spermidine biosynthesis. The further exploitation and functional characterization of the TrMYB33-TrSAMS1 regulatory module can enhance our understanding of the molecular mechanisms responsible for spermidine accumulation during drought stress.

Exogenous silicon induces aluminum tolerance in white clover (Trifolium repens) by reducing aluminum uptake and enhancing organic acid secretion.

Excessive aluminum (Al) in acidic soils is a primary factor that hinders plant growth. The objective of the present study was to investigate the effect and physiological mechanism of exogenous silicon (Si) in alleviating aluminum toxicity. Under hydroponic conditions, 4 mM Al significantly impeded the growth of white clover; however, pretreatments with 1 mM Si mitigated this inhibition, as evidenced by notable changes in growth indicators and physiological parameters. Exogenous silicon notably increased both shoot and root length of white clover and significantly decreased electrolyte leakage (EL) and malondialdehyde (MDA) content compared to aluminum treatments. This positive effect was particularly evident in the roots. Further analysis involving hematoxylin staining, scanning electron microscopy (SEM), and examination of organic acids (OAs) demonstrated that silicon relieved the accumulation of bioactive aluminum and ameliorated damage to root tissues in aluminum-stressed plants. Additionally, energy-dispersive X-ray (EDX) analysis revealed that additional silicon was primarily distributed in the root epidermal and cortical layers, effectively reducing the transport of aluminum and maintaining the balance of exchangeable cations absorption. These findings suggest that gradual silicon deposition in root tissues effectively prevents the absorption of biologically active aluminum, thereby reducing the risk of mineral nutrient deficiencies induced by aluminum stress, promoting organic acids exudation, and compartmentalizing aluminum in the outer layer of root tissues. This mechanism helps white clover alleviate the damage caused by aluminum toxicity.

Invasion of Trifolium repens L. aggravated by biodegradable plastics: adjustable strategy for foraging N and P.

The invasion of alien plant and the pollution caused by soil microplastics have emerged as significant ecological threats. Recent studies have demonstrated aggravating effect of non-biodegradable microplastics on plant invasion. However, the impact of biodegradable microplastics (BMPs) on plant invasion remains unclear. Therefore, it is imperative to explore the impact of BMPs on plant invasion. In this study, a 30-day potting experiment with Trifolium repens L. (an invasive plant) and Oxalis corniculata L. (a native plant) was conducted to evaluate the influence of BMPs on T. repens's invasion. The findings revealed that BMPs results in a reduction in available N and P contents, thereby facilitating the colonization of arbuscular mycorrhizal fungi on T. repens 's roots. Consequently, T. repens adjusted its N and P foraging strategy by increasing P absorption ratio, and enhancing the accumulation of N and P in leaves. This ultimately led to the decrease of relative neighbor effect index of T. repens, indicating an aggravated invasion by T. repens. This study significantly enhances and expands the understanding of mechanisms by which microplastics aggravate plant invasion.

Allelopathic effect of the methanol extract of the weed species-red sorrel (Rumex acetosella L.) on the growth, phytohormone content and antioxidant activity of the cover crop - white clover (Trifolium repens L.).

Allelopathy is a biological process in which one organism releases biochemicals that affect the growth and development of other organisms. The current investigation sought to determine the allelopathic effect of Rumex acetosella on white clover (Trifolium repens) growth and development by using its shoot extract (lower IC50 value) as a foliar treatment. Here, different concentrations (25, 50, 100, and 200 g/L) of shoot extract from Rumex acetosella were used as treatments. With increasing concentrations of shoot extract, the plant growth parameters, chlorophyll and total protein content of Trifolium repens decreased. On the other hand, ROS, such as O2.- and H2O2, and antioxidant enzymes, including SOD, CAT, and POD, increased with increasing shoot extract concentration. A phytohormonal study indicated that increased treatment concentrations increased ABA and SA levels while JA levels were reduced. For the identification of allelochemicals, liquid‒liquid extraction, thin-layer chromatography, and open-column chromatography were conducted using R. acetosella shoot extracts, followed by a seed bioassay on the separated layer. A lower IC50 value was obtained through GC/MS analysis. gammaSitosterol was identified as the most abundant component. The shoot extract of Rumex acetosella has strong allelochemical properties that may significantly impede the growth and development of Trifolium repens. This approach could help to understand the competitive abilities of this weed species and in further research provide an alternate weed management strategy.

Plants as effective bioindicators for heavy metal pollution monitoring.

This study investigated the bioindicator potential of Amaranthus retroflexus L., Plantago lanceolata L., Rumex acetosa L., and Trifolium pratense L. including the use of Lolium multiflorum L. as a reference species, for heavy metal pollution monitoring, in particular Zinc (Zn), Cadmium (Cd), Nickel (Ni), and Lead (Pb). Controlled heavy metal contamination was applied through irrigation with metal nitrate solutions two levels of contamination (low and high). The study also focused on analyzing heavy metals concentration in plant tissues and related physiological responses. Distinct physiological responses to heavy metal stress were observed among the investigated species, highlighting unique variations in their reactions. Hydrogen peroxide, malondialdehyde content, and enzymatic activities emerged as reliable indicators of plant stress induced by heavy metal solutions. P. lanceolata displayed elevated Zn concentrations in both roots and leaves (3271 ± 337 and 4956 ± 82 mg kg-1). For Pb, L. multiflorum and P. lanceolata showed highest root concentrations (2964 ± 937 and 1605 ± 289 mg kg-1), while R. acetosa had higher leaf concentration (1957 ± 147 mg kg-1). For Ni, L. multiflorum had the highest root concentration (1148 ± 93 mg kg-1), and P. lanceolata exhibited the highest leaf concentration (2492 ± 28 mg kg-1). P. lanceolata consistently demonstrated the highest Cd concentrations in both roots (126 ± 21 mg kg-1) and leaves (163 ± 12 mg kg-1). These results provide valuable insights for selecting effective bioindicator species to establish control strategies for heavy metal pollution.

Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress.

BACKGROUND: White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. RESULTS: In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. CONCLUSION: These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

Water Stress Influences Phytoestrogen Levels in Red Clover (Trifolium pratense) but Not Kura Clover (T. ambiguum).

Some forage legumes synthesize phytoestrogens. We conducted a glasshouse study to investigate how water stress (drought and waterlogging) influences phytoestrogen accumulation in red clover and kura clover. Compared to the red clover control, the 20 day drought resulted in an over 100% increase in the phytoestrogens formononetin and biochanin A, which together accounted for 91-96% of the total phytoestrogens measured. Waterlogging resulted in elevated concentrations of daidzein, genistein, and prunetin but not formononetin or biochanin A. Concentrations of phytoestrogens in kura clover were low or undetectable, regardless of water stress treatment. Leaf water potential was the most explanatory single-predictor of the variation in concentrations of formononetin, biochanin A, and total phytoestrogens in red clover. These results suggest that drought-stressed red clover may have higher potential to lead to estrogenic effects in ruminant livestock and that kura clover is a promising alternative low- or no-phytoestrogen perennial forage legume.

Root uptake of cereal benzoxazinoids grants resistance to root-knot nematode invasion in white clover.

Plants synthesize a plethora of chemical defence compounds, which vary between evolutionary lineages. We hypothesize that plants evolved the ability to utilize defence compounds synthesized and released by neighbouring heterospecific plants. In two experiments, we incubated clover (Trifolium repens L.) seedlings with individual benzoxazinoid (BX) compounds (2,4-dihydroxy-1,4-benzoxazin-3-one, 2-hydroxy-1,4-benzoxazin-3-one, benzoxazolinone, and 6-methoxy- benzoxazolin-2-one), a group of bioactive compounds produced by cereals, to allow clover BX uptake. Subsequently, we transplanted the seedlings into soil and quantified BX root and shoot content and invasion of root-knot nematodes in clover roots up to 8 weeks after transplantation. We show that clover root uptake of BXs substantially enhanced clover's resistance against the root-knot nematode Meloidogyne incognita. This effect lasted up to 6 weeks after the clover roots were exposed to the BXs. BXs were absorbed by clover roots, and then translocated to the shoots. As a result of clover metabolization, we detected the parent BXs and a range of their transformation products in the roots and shoots. Based on these novel findings, we envisage that co-cultivation of crop species with complementary and transferable chemical defence systems can add to plant protection.

Reducing dietary protein and supplementation with starch or rumen-protected methionine and its effect on performance and nitrogen efficiency in dairy cows fed a red clover and grass silage-based diet.

The increasing cost of milk production, in association with tighter manure N application regulations and challenges associated with ammonia emissions in many countries, has increased interest in feeding lower crude protein (CP) diets based on legume silages. Most studies have focused on alfalfa silage, and little information is available on low-CP diets based on red clover silage. Our objectives were to examine the effects of dietary CP content and supplementing a low-CP diet with dietary starch or rumen-protected Met (RPMet) on the performance, metabolism, and nitrogen use efficiency (NUE; milk N output/N intake) in dairy cows fed a red clover and grass silage-based diet. A total of 56 Holstein-Friesian dairy cows were blocked and randomly allocated to 1 of 4 diets over a 14-wk feeding period. Diets were based on red clover and grass silages at a ratio of 50:50 on a dry matter (DM) basis and were fed as a total mixed ration, with a 53:47 ratio of forage to concentrate (DM basis). The diets were formulated to supply a similar metabolizable protein (MP) content, and had a CP concentration of either 175 g/kg DM (control [CON]) or 150 g/kg DM (low-protein [LP]), or LP supplemented with either additional barley as a source of starch (LPSt; +64 g/kg DM) or RPMet (LPM; +0.3 g/100 g MP). At the end of the 14-wk feeding period, 20 cows (5 per treatment) continued to be fed the same diets for a further 6 d, and total urine output and fecal samples were collected. We observed that dietary treatment did not affect DM intake, with a mean of 21.5 kg/d; however, we also observed an interaction between diet and week with intake being highest in cows fed LPSt in wk 4 and CON in wk 9 and 14. Mean milk yield, 4% fat-corrected milk, and energy-corrected milk were not altered by treatment. Similarly, we found no effect of dietary treatment on milk fat, protein, or lactose content. In contrast, milk and plasma urea concentrations were highest in cows fed CON. The concentration of blood plasma ß-hydroxybutyrate was highest in cows receiving LPM and lowest in LPSt. Apparent NUE was 28.6% in cows fed CON and was higher in cows fed any of the low-protein diets (LP, LPSt, or LPM), with a mean value of 34.2%. The sum of milk fatty acids with a chain length below C16:0 was also highest in cows fed CON. We observed that dietary treatment did not affect the apparent whole-tract nutrient digestibility of organic matter, N, neutral detergent fiber, and acid detergent fiber, with mean values of 0.785, 0.659, 0.660, and 0.651 kg/kg respectively, but urinary N excretion was approximately 60 g/d lower in cows fed the low-CP diets compared with CON. We conclude that reducing the CP content of red clover and grass silage-based diets from 175 to 150 g/kg DM while maintaining MP supply did not affect performance, but reduced the urinary N excretion and improved NUE, and that supplementing additional starch or RPMet had little further effect.

Feeding alfalfa-grass or red clover-grass mixture baleage: Effect on milk yield and composition, ruminal fermentation and microbiota taxa relative abundance, and nutrient utilization in dairy cows.

Our goal was to investigate the effect of diets containing baleages harvested from alfalfa-grass or red clover-grass mixture on production performance, ruminal fermentation and microbiota taxa relative abundance, milk fatty acid profile, and nutrient utilization in dairy cows. Twenty Jersey cows (18 multiparous and 2 primiparous) averaging (mean ± SD) 148 ± 45.2 days in milk and 483 ± 65.4 kg of body weight in the beginning of the study were used in a randomized complete block design with repeated measures over time. The experiment lasted 9 wk, with a 2 wk covariate period followed by 7 wk of data and sample collection (wk 4 and 7 used in the statistical analyses). Cows were fed diets containing (dry matter basis) 35% of a concentrate mash and the following forage sources: (1) 65% second- and third-cut (32.5% each) alfalfa-grass mixture baleages (ALF) or (2) 65% second- and third-cut (32.5% each) red clover-grass mixture baleages (RC). Diets did not affect dry matter intake, milk yield, and concentrations of milk fat and true protein. In contrast, milk fat yield tended to decrease and energy-corrected milk yield decreased with feeding RC versus ALF. The apparent total-tract digestibilities of dry matter, organic matter, and ash-free neutral detergent fiber, milk proportions of trans-10 18:1, cis-9,cis-12,cis-15 18:3, and total n-3 fatty acids, ruminal molar proportion of acetate, and plasma concentrations of Leu, Phe, and Val all increased in RC versus ALF. Diet × week interactions were found for several parameters, most notably ruminal molar proportions of propionate and butyrate, ruminal NH3-N, milk urea N, plasma urea N, and plasma His concentrations, urinary N excretion, enteric CH4 production, and all energy efficiency variables. Specifically, ruminal NH3-N and plasma urea N concentrations, urinary excretion of N, and CH4 production decreased in cows fed RC in wk 4 but not in wk 7. Milk urea N concentration decreased and that of plasma His increased with feeding RC during wk 4 and 7, although the magnitude of treatments difference varied between the sampling periods. Efficiency of energy utilization calculated as milk energy/metabolizable energy decreased and that of tissue energy/ME increased in RC versus ALF cows in wk 4, suggesting that ME was portioned toward tissue and not milk in the RC diet. Interactions were also observed for the relative abundance of the rumen bacterial phyla Verrucomicrobiota and Fibrobacterota, with cows offered RC showing greater values than those receiving ALF in wk 4 but no differences in wk 7. Several diet × week interactions were detected in the present study implying short-term treatment responses and warranting further investigations.

Comparative GC-MS based nutrients profiling of less explored legume seeds of Melilotus, Medicago, Trifolium, and Ononis analysed using chemometric tools.

Exploring novel sources of plant protein for nutrition of both humans and animals is motivated mainly by its growing demand worldwide, besides identifying healthy alternatives for animal protein. The present study evaluates metabolome diversity within 15 legume seed species. The examined samples comprised three Melilotus, four Medicago, four Trifolium, and four Ononis seed species. A holistic approach for metabolites profiling using gas chromatography-mass spectrometry (GC-MS) led to the annotation and quantification of 87 metabolites comprising alcohols, free amino acids, aromatics, fatty acids/esters, nitrogenous compounds, organic acids, sugar alcohols, sugars, terpenes, and steroids. Fatty acids represented the major metabolite class represented by palmitic, stearic, oleic, linoleic, and linolenic acids. Sucrose and pinitol were the major sugars and sugar alcohols among seeds. Ononis seeds (OR, OS and OA) were the most abundant in fatty acids, sugars, sugar alcohols, and free amino acids, whereas Melilotus species (MO and MS) were least enriched in these key nutrients posing Ononis as potential food source for humans and animals. The examined seeds were generally low in sulfur-containing free amino acids and lacking many of the essential free amino acids. Multivariate data analysis aided in the identification of Ononis metabolite markers belonging to various classes i.e., (alcohol) glycerol, (sugar) allofuranose, and (sugar alcohol) pinitol, although the differentiation between Medicago, Melilotus, and Trifolium genera was not attained suggestive for other analytical platforms for its classification.

Identification and Evolutionary Analysis of the Auxin Response Factor (ARF) Family Based on Transcriptome Data from Caucasian Clover and Analysis of Expression Responses to Hormones.

Caucasian clover (Trifolium ambiguum M. Bieb.) is an excellent perennial plant in the legume family Fabaceae, with a well-developed rhizome and strong clonal growth. Auxin is one of the most important phytohormones in plants and plays an important role in plant growth and development. Auxin response factor (ARF) can regulate the expression of auxin-responsive genes, thus participating in multiple pathways of auxin transduction signaling in a synergistic manner. No genomic database has been established for Caucasian clover. In this study, 71 TaARF genes were identified through a transcriptomic database of Caucasian clover rhizome development. Phylogenetic analysis grouped the TaARFs into six (1-6) clades. Thirty TaARFs contained a complete ARF structure, including three relatively conserved regions. Physical and chemical property analysis revealed that TaARFs are unstable and hydrophilic proteins. We also analyzed the expression pattern of TaARFs in different tissues (taproot, horizontal rhizome, swelling of taproot, rhizome bud and rhizome bud tip). Quantitative real-time RT-PCR revealed that all TaARFs were responsive to phytohormones (indole-3-acetic acid, gibberellic acid, abscisic acid and methyl jasmonate) in roots, stems and leaves. These results helped elucidate the role of ARFs in responses to different hormone treatments in Caucasian clover.

Induced mechanism of phosphatase hormesis by Cd ions and rhizosphere metabolites of Trifolium repens L.

Rhizosphere phosphatases can exhibit hormetic effects in response to cadmium (Cd) ion stimulation. However, understanding the mechanisms underlying hormesis effects on soil ecosystems is challenging as studies on hormesis are usually specific to an organism, cell, or organ. To comprehensively investigate the mechanism of phosphatase hormesis, this study utilized in situ zymography and metabolomics to analyze the rhizosphere of Trifolium repens L. (white clover). Zymograms showed that rhizosphere phosphatase displayed a hormetic effect in 10 mg kg-1 Cd contaminated soil, with a hotspot area 1.8 times larger than non-Cd contaminated soil and a slight increase in enzyme activity. Nevertheless, the phosphatase activity was substantially suppressed upon elevating the Cd concentration in the soil to 50 mg kg-1. Differential metabolite identification and KEEG pathway enrichment analysis revealed that both rhizosphere organic acids and amino acid compounds positively affected phosphatase activity, and both were able to stabilize complexation with Cd ions via carboxyl groups. Besides, molecular docking models suggested that Cd ions act as cofactors to induce the formation of hydrogen bonds between amino acids/organic acids and phosphatase residues to form a triplet complex with a more stable structure, thereby improving phosphatase activity. The results indicated that amino acids and organic acids are heavily enriched in the rhizosphere of white clover and form a particular structure with soil Cd ions and phosphatase, which is essential for inducing the phosphatase hormesis as a detoxification mechanism in the rhizosphere micro-ecosystem.

DESIGNER fraction concept unmasks minor bioactive constituents in red clover (Trifolium pratense L.).

In botanical extracts, highly abundant constituents can mask or dilute the effects of other, and often, more relevant biologically active compounds. To facilitate the rational chemical and biological assessment of these natural products with wide usage in human health, we introduced the DESIGNER approach of Depleting and Enriching Selective Ingredients to Generate Normalized Extract Resources. The present study applied this concept to clinical Red Clover Extract (RCE) and combined phytochemical and biological methodology to help rationalize the utility of RCE supplements for symptom management in postmenopausal women. Previous work has demonstrated that RCE reduces estrogen detoxification pathways in breast cancer cells (MCF-7) and, thus, may serve to negatively affect estrogen metabolism-induced chemical carcinogenesis. Clinical RCE contains ca. 30% of biochanin A and formononetin, which potentially mask activities of less abundant compounds. These two isoflavonoids are aryl hydrocarbon receptor (AhR) agonists that activate P450 1A1, responsible for estrogen detoxification, and P450 1B1, producing genotoxic estrogen metabolites in female breast cells. Clinical RCE also contains the potent phytoestrogen, genistein, that downregulates P450 1A1, thereby reducing estrogen detoxification. To identify less abundant bioactive constituents, countercurrent separation (CCS) of a clinical RCE yielded selective lipophilic to hydrophilic metabolites in six enriched DESIGNER fractions (DFs 01-06). Unlike solid-phase chromatography, CCS prevented any potential loss of minor constituents or residual complexity (RC) and enabled the polarity-based enrichment of certain constituents. Systematic analysis of estrogen detoxification pathways (ERα-degradation, AhR activation, CYP1A1/CYP1B1 induction and activity) of the DFs uncovered masked bioactivity of minor/less abundant constituents including irilone. These data will allow the optimization of RCE with respect to estrogen detoxification properties. The DFs revealed distinct biological activities between less abundant bioactives. The present results can inspire future carefully designed extracts with phytochemical profiles that are optimized to increase in estrogen detoxification pathways and, thereby, promote resilience in women with high-risk for breast cancer. The DESIGNER approach helps to establish links between complex chemical makeup, botanical safety and possible efficacy parameters, yields candidate DFs for (pre)clinical studies, and reveals the contribution of minor phytoconstituents to the overall safety and bioactivity of botanicals, such as resilience promoting activities relevant to women's health.

Effects of forage and grain legume-based silages supplemented with faba bean meal or rapeseed expeller on lactational performance, nitrogen utilization, and plasma amino acids in dairy cows.

The objective of this experiment was to investigate the effect of forage type [red clover (51%)-grass silage, i.e., RCG; vs. faba bean (66%)-grass silage, i.e., FBG] and concentrate type (faba bean, FB; vs. rapeseed expeller, RE) on lactational performance, milk composition and nitrogen (N) utilization in lactating dairy cows. Eight lactating multiparous Nordic Red cows were used in a replicated 4 × 4 Latin Square experiment, with 21-d periods, in a 2 × 2 factorial arrangement of treatments. The experimental treatments were as follows: (1) RCG with RE, (2) RCG with FB, (3) FBG with RE, and (4) FBG with FB. Inclusion rates of RE and FB were isonitrogenous. Crude protein contents of the experimental diets were 16.3, 15.9, 18.1, and 17.9% of dry matter, respectively. All diets included oats and barley and were fed ad libitum as total mixed rations with forage-to-concentrate ratio of 55:45. Dry matter intake and milk yield were recorded daily, and spot samples of urine, feces, and blood were collected at the end of each experimental period. Dry matter intake did not differ across diets, averaging 26.7 kg/d. Milk yield averaged 35.6 kg/d and was 1.1 kg/d greater for RCG versus FBG, and milk urea N concentration was lower for RCG compared with FBG. Milk yield was 2.2 kg/d and milk protein yield 66 g/d lower for FB versus RE. Nitrogen intake, urinary N, and urinary urea N excretions were lower, and milk N excretion tended to be lower for RCG compared with FBG. The proportion of the dietary N excreted as fecal N was larger in cows fed RCG than for those fed FBG, and the opposite was true for urinary N. We detected an interaction for milk N as percentage of N intake: it increased with RE compared with FB for RCG-based diet, but only a marginal increase was observed for FBG-based diet. Plasma concentration of His and Lys were lower for RCG than for FBG, whereas His tended to be greater and Lys lower for FB compared with RE. Further, plasma Met concentration was around 26% lower for FB than for RE. Of milk fatty acids, saturated fatty acids were decreased by RCG and increased by FB compared with FBG and RE, respectively, whereas monounsaturated fatty acids were increased by RCG versus FBG, and were lower for FB than for RE. In particular, 18:1n-9 concentration was lower for FB compared with RE. Polyunsaturated fatty acids, such as 18:2n-6 and 18:3n-3, were greater for RCG than for FBG, and 18:2n-6 was greater and 18:3n-3 was lower for FB versus RE. In addition, cis-9,trans-11 conjugated linoleic acid was lower for FB compared with RE. Faba bean whole-crop silage and faba bean meal have potential to be used as a part of dairy cow rations, but further research is needed to improve their N efficiency. Red clover-grass silage from a mixed sward, without inorganic N fertilizer input, combined with RE, resulted in the greatest N efficiency in the conditions of this experiment.

Antioxidant properties of Trifolium resupinatum and its therapeutic potential for Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is the most common cause of dementia and is characterized by a progressive deterioration in cognitive function, which typically begins with impairment in memory. Persian clover (Trifolium resupinatum) is an annual plant found in central Asia. Due to its contents (high flavonoid and isoflavones), extensive researches have been done on its therapeutic properties, such as multiple sclerosis (MS) treatment. In this study, we investigate the neuroprotective effects of this plant on Streptozotocin (STZ)-induced AD in rats. MATERIAL AND METHODS: This research aimed to evaluate the neuroprotective effect of Trifolium resupinatum on the spatial learning and memory, superoxide dismutase (SOD), expressions of ß amyloid 1-42 (Ab 1-42 ), and b amyloid 1-40 (Ab 1-40 ) in the hippocampus of STZ-induced Alzheimer rats. RESULTS: Our data showed that Trifolium resupinatum extract administration for two weeks before and one week after AD induction significantly improves maze escape latency ( p = 0.027, 0.001 and 0.02 in 100, 200, and 300 mg of the extract, respectively) and maze retention time ( p = 0.003, 0.04 and 0.001 in 100, 200, and 300 mg of the extract, respectively). Also, the administration of this extract significantly increases the SOD levels from 1.72+0.20 to 2.31+0.45 ( p = 0.009), 2.48+0.32 ( p = 0.001) and 2.33+0.32 ( p = 0.007) and decreases the expressions of Ab 1-42 ) ( p = 0.001 in all concentrations of the extract) and Ab 1-40 ) ( p = 0.001 in all concentrations of the extract) in the rat's hippocampus. CONCLUSIONS: This study suggests that the alcoholic extract of Trifolium resupinatum has anti-Alzheimer and neuroprotective effects on rats.

Methane emission, nutrient digestibility, and rumen microbiota in Holstein heifers fed 14 different grass or clover silages as the sole feed.

This experiment investigated the variation in enteric methane production and associated gas exchange parameters, nutrient digestibility, rumen fermentation, and rumen microbiome when a range of silages based on different forage types (grass or clover), and different species within the 2 types, were fed as the sole feed to heifers. Three grass species (perennial ryegrass, festulolium, and tall fescue) and 2 clover species (red clover and white clover) were included. Perennial ryegrass was harvested at 2 maturity stages in the primary growth, white clover was harvested once in the primary growth, and 4 cuts of festulolium and tall fescue and 3 cuts of red clover were harvested during the growing season, giving 14 different silage batches in total. Sixteen Holstein heifers 16 to 21 mo old and 2 to 5 mo in pregnancy were fed the silages ad libitum as the sole feed in an incomplete crossover design. Each silage was fed to 4 heifers, except for the 2 perennial ryegrass silages, which were fed to 8 heifers; in total 64 observations. The CH4 production was measured for 3 d in respiration chambers. Heifers fed clover silages had higher dry matter intake (DMI) compared with heifers fed grass silages, and heifers fed tall fescue silages had the numerically the lowest DMI. Compared with grass silages, feeding clover silages led to higher crude protein digestibility but lower neutral detergent fiber (NDF) digestibility. Rumen pH was higher in heifers fed clover silages compared with those fed grass silages. Based on composition analysis, the rumen microbiota of the heifers clustered clearly according to forage type and species. More specifically, 7 of the 34 dominating rumen bacterial genus-level groups showed higher relative abundances for the clover silages, whereas 7 genus-level groups showed higher abundances for the grass silages. Methane yield was higher for heifers fed grass silages than for those fed clover silages when methane production was related to dry matter and digestible organic matter intake, whereas the opposite was seen when related to NDF digestion. The gross energy lost as methane (CH4 conversion factor, %) reduced from 7.5% to 6.7%, equivalent to an 11% reduction. The present study gives the outlines for choosing the optimal forage type and forage species with respect to nutrient digestibility and enteric methane emission in ruminants.

A Trifolium repens flavodoxin-like quinone reductase 1 (TrFQR1) improves plant adaptability to high temperature associated with oxidative homeostasis and lipids remodeling.

Maintenance of stable mitochondrial respiratory chains could enhance adaptability to high temperature, but the potential mechanism was not elucidated clearly in plants. In this study, we identified and isolated a TrFQR1 gene encoding the flavodoxin-like quinone reductase 1 (TrFQR1) located in mitochondria of leguminous white clover (Trifolium repens). Phylogenetic analysis indicated that amino acid sequences of FQR1 in various plant species showed a high degree of similarities. Ectopic expression of TrFQR1 protected yeast (Saccharomyces cerevisiae) from heat damage and toxic levels of benzoquinone, phenanthraquinone and hydroquinone. Transgenic Arabidopsis thaliana and white clover overexpressing TrFQR1 exhibited significantly lower oxidative damage and better photosynthetic capacity and growth than wild-type in response to high-temperature stress, whereas AtFQR1-RNAi A. thaliana showed more severe oxidative damage and growth retardation under heat stress. TrFQR1-transgenic white clover also maintained better respiratory electron transport chain than wild-type plants, as manifested by significantly higher mitochondrial complex II and III activities, alternative oxidase activity, NAD(P)H content, and coenzyme Q10 content in response to heat stress. In addition, overexpression of TrFQR1 enhanced the accumulation of lipids including phosphatidylglycerol, monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and cardiolipin as important compositions of bilayers involved in dynamic membrane assembly in mitochondria or chloroplasts positively associated with heat tolerance. TrFQR1-transgenic white clover also exhibited higher lipids saturation level and phosphatidylcholine:phosphatidylethanolamine ratio, which could be beneficial to membrane stability and integrity during a prolonged period of heat stress. The current study proves that TrFQR1 is essential for heat tolerance associated with mitochondrial respiratory chain, cellular reactive oxygen species homeostasis, and lipids remodeling in plants. TrFQR1 could be selected as a key candidate marker gene to screen heat-tolerant genotypes or develop heat-tolerant crops via molecular-based breeding.

Removal of cadmium and polychlorinated biphenyls by clover and the associated microbial community in a long-term co-contaminated soil.

Legumes such as clover are cost-effective and environmentally friendly components of strategies for remediating soils contaminated with heavy metals or organic pollutants. However, the mechanisms by which clover remediates co-contaminated soils are unclear. The present study explored the effects of phytoremediation by clover on pollutant removal and the microbial community in soil co-contaminated with cadmium (Cd) and polychlorinated biphenyls (PCBs). After 18 months of phytoremediation, Cd removal increased from 20.25 % in the control to 40.65 % in soil planted with clover, while PCB removal increased from 29.81 % to 60.02 %. High-throughput sequencing analysis showed that the relative abundances of the bacterial phylum Proteobacteria and the diazotrophic genus Rhizobium increased significantly after phytoremediation. Random forest analysis showed that bacterial and diazotrophic diversity significantly influenced Cd and PCB removal. Furthermore, co-occurrence network and correlation analyses revealed that Rhizobiales and Micromonosporales were the main bacteria associated with Cd removal, while Rhizobiales, Burkholderiales, and Xanthomonadales were identified as the main degraders of PCBs. PICRUSt functional prediction demonstrated that the gene bphC, which is related to PCB degradation, was significantly increased in the rhizosphere soil in the presence of clover. These results provide a better understanding for further studies of remediation efficiency by clover, rhizosphere microbial response and remediation mechanisms of co-contaminated soils under in situ conditions in the field.