Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

De novo genome assembly of white clover (Trifolium repens L.) reveals the role of copy number variation in rapid environmental adaptation.

BACKGROUND: White clover (Trifolium repens) is a globally important perennial forage legume. This species also serves as an eco-evolutionary model system for studying within-species chemical defense variation; it features a well-studied polymorphism for cyanogenesis (HCN release following tissue damage), with higher frequencies of cyanogenic plants favored in warmer locations worldwide. Using a newly generated haplotype-resolved genome and two other long-read assemblies, we tested the hypothesis that copy number variants (CNVs) at cyanogenesis genes play a role in the ability of white clover to rapidly adapt to local environments. We also examined questions on subgenome evolution in this recently evolved allotetraploid species and on chromosomal rearrangements in the broader IRLC legume clade. RESULTS: Integration of PacBio HiFi, Omni-C, Illumina, and linkage map data yielded a completely de novo genome assembly for white clover (created without a priori sequence assignment to subgenomes). We find that white clover has undergone extensive transposon diversification since its origin but otherwise shows highly conserved genome organization and composition with its diploid progenitors. Unlike some other clover species, its chromosomal structure is conserved with other IRLC legumes. We further find extensive evidence of CNVs at the major cyanogenesis loci; these contribute to quantitative variation in the cyanogenic phenotype and to local adaptation across wild North American populations. CONCLUSIONS: This work provides a case study documenting the role of CNVs in local adaptation in a plant species, and it highlights the value of pan-genome data for identifying contributions of structural variants to adaptation in nature.

Genetics and plasticity of white leaf mark variegation in white clover (Trifolium repens L.).

BACKGROUND AND AIMS: Leaf variegation is common in plants and confers diverse adaptive functions. However, its genetic underpinnings remain largely unresolved; this is particularly true for variegation that arises through modified leaf tissue structure that affects light reflection. White clover is naturally polymorphic for structure-based white leaf mark variegation. It therefore provides a useful system to examine the genetic basis of this phenotype, and to assess potential costs to photosynthetic efficiency resulting from modified leaf structures. This study sought to map the loci controlling the white leaf mark in white clover and evaluate the relationship between white leaf mark, leaf thickness, and photosynthetic efficiency. METHODS: We generated a high-density genetic linkage map from an F3 mapping population, employing reference genome-based SNP markers. White leaf mark was quantified through detailed phenotypic evaluations alongside leaf thickness to test how tissue thickness may affect the variegation phenotype. Quantitative trait locus (QTL) mapping was performed to characterize their genetic bases. Photosynthetic efficiency measurements were used to test for physiological trade-offs between variegation and photosynthetic output. KEY RESULTS: The V locus, a major gene responsible for the white leaf mark polymorphism, was mapped to the distal end of chromosome 5, and several modifier loci were also mapped that contribute additively to variegation intensity. The presence and intensity of white leaf mark was associated with greater leaf thickness; however, increased variegation did not detectably affect photosynthetic efficiency. CONCLUSIONS: We have successfully mapped the major locus governing the white leaf mark in white clover, along with several modifier loci, revealing a complex basis for this structure-based variegation. The apparent absence of compromised photosynthesis in variegated leaves challenges the notion that variegation creates fitness trade-offs between photosynthetic efficiency and other adaptive functions. This finding suggests that other factors may maintain the white leaf mark polymorphism in white clover.

Functional characterization of TrGSTF15, a glutathione S-transferase gene family member, on the transport and accumulation of anthocyanins and proanthocyanidins in Trifolium repens.

Anthocyanins and proanthocyanidins (PAs) are important secondary metabolites in plants, high contents of which are an important goal for quality breeding of white clover (Trifolium repens). However, the involvement of glutathione S-transferase (GST) in the transport of anthocyanins and PAs remains unexplored in white clover. This study identified 153 different TrGSTs in white clover. At the transcriptional level, compared to other TrGSTFs, TrGSTF10 and TrGSTF15 are highly expressed in the 'Purple' white clover, and they may work with the anthocyanin biosynthesis structural genes CHS and CHI to contribute to pigment buildup in white clover. Subcellular localization confirmed that TrGSTF10 and TrGSTF15 are located in the cytoplasm. Additionally, molecular docking experiments showed that TrGSTF10 and TrGSTF15 have similar binding affinity with two flavonoid monomers. Overexpression of TrGSTF15 complemented the deficiency of anthocyanin coloring and PA accumulation in the Arabidopsis tt19 mutant. The initial findings of this research indicate that TrGSTF15 encodes an important transporter of anthocyanin and PA in white clover, thus providing a new perspective for the further exploration of related transport and regulatory mechanisms.

Alfalfa Mosaic Virus and White Clover Mosaic Virus Combined Infection Leads to Chloroplast Destruction and Alterations in Photosynthetic Characteristics of Nicotiana benthamiana.

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO2 concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana.

Evaluating foliar insecticides and economic thresholds for Tychius picirostris (Coleoptera: Curculionidae) management in Oregon white clover seed production.

The clover seed weevil, Tychius picirostris Fabricius (Coleoptera: Curculionidae), is a major pest in Oregon white clover seed crops. Reliance on synthetic pyrethroid insecticides and limited availability of diverse modes of action (MoAs) has increased insecticide resistance selection in regional T. picirostris populations, emphasizing the need to evaluate novel chemistries and rotational strategies for effective insecticide resistance management (IRM). The efficacy of 8 foliar insecticide formulations for managing T. picirostris adult and larval life stages was determined in small and large-plot field trials across 2 crop years. In both years, bifenthrin (Brigade 2EC), the grower's standard, showed negligible adult and larval suppression. Insecticide formulations with isocycloseram and cyantraniliprole active ingredients reduced adult and larval populations when applied at BBCH 59-60 (prebloom) and BBCH 65-66 (full bloom) growth stages, respectively. While differences in T. picirostris abundance were observed among insecticide treatments, seed yield differences were not detected in large-plot trials. Larval abundance was correlated with reduced seed yield, and an economic threshold of ≥3 larvae per 30 inflorescences was determined as a conservative larval threshold to justify foliar applications of diamide insecticides. Additional commercial white clover seed fields were surveyed to compare larval scouting techniques, including a standard Berlese funnel and a grower's do-it-yourself funnel. Both larval extraction techniques were correlated and provided similar estimates of larval abundance. These findings demonstrate new MoAs, optimal insecticide application timing, and larval monitoring methods that can be incorporated into an effective T. picirostris IRM program in white clover seed crops.

Optimizing forage harvest and the nutritive value of Italian ryegrass-based mixed forage cropping under northwestern Himalayan conditions.

The scarcity of high-quality forage has a significant influence on the productivity and profitability of livestock. Addressing this concern, an investigation was undertaken to assess the effects of distinct Italian ryegrass genotypes, namely, Punjab ryegrass-1, Kashmir collection, and Makhan grass, in conjunction with varying seeding ratios of Italian ryegrass to Egyptian clover. The seeding ratios considered were 100:0 (Italian ryegrass to Egyptian clover), 75:25, 50:50, and 25:75. All possible combinations of Italian ryegrass and Egyptian clover with seeding ratios were set up in a randomized complete block design and replicated thrice. Co-cultivating Italian ryegrass and Egyptian clover at a 75:25 seeding ratio yields the best yield benefit, as determined by the land equivalent ratio. It is noteworthy that in this configuration, real yield loss is higher for Egyptian clover and for Italian ryegrass when the seeding ratio is 25:75. The higher competitiveness of Italian ryegrass in comparison to Egyptian clover is highlighted by the competitive ratio. Notably, the nutritive parameter, crude protein yield, was significantly higher in the Makhan grass-based 50:50 and 75:25 seeding ratio. Results of the study ascertained the compatibility of grass-legume co-cultivation with significantly higher quantity and quality forage harvested under mixed cropping systems whereas Makhan grass as the superior and dominant genotype in comparison to Kashmir collection. The outcomes of this study revealed that the 100:0 seeding ratio, coupled with the Makhan grass genotype, exhibited superior performance in terms of cumulative forage harvest, dry matter accumulation, net returns, and benefit-cost ratio.

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.

Investigating the involvement of the NLRP3/ASC/caspase-1 and NF-κb/MAPK pathways in the pathogenesis of gouty arthritis: Insights from irradiated and non-irradiated Trifolium alexandrium L. extracts and some metabolites.

ETHNOPHARMACOLOGICAL RELEVANCE: Trifolium alexandrinum L. (TA), has traditionally been used in folk medicine for its anti-inflammatory properties against hyperuricemia and gout. However, the specific mechanisms of action of TA have not been thoroughly studied. AIM OF THE WORK: This study aimed to evaluate the protective effects of irradiated (TR25) and non-irradiated (TR0) Trifolium alexandrinum L. aqueous extract (TAAE), along with two isolated compounds, caffeine (CAF) and saponin (SAP), in a rat model of acute gouty arthritis (GA). MATERIALS AND METHODS: The GA model was established by injecting a monosodium urate (MSU) suspension into the knee joint. Synovial tissue pathology was assessed, and levels of TNF-α, IL-6, IL-1ß, NF-κB, mTOR, AKT1, PI3K, NLRP3, and ASC were measured by ELISA. mRNA expression of ERK1, JNK, and p-38 MAPK was detected using qRT-PCR, and Caspase-1 protein expression was assessed by immunohistochemical analysis. Knee swelling, uric acid levels, liver and kidney function, and oxidative stress markers were also evaluated. RESULTS: TAAE analysis identified 170 compounds, with 73 successfully identified using LC-HR-MS/MS, including caffeine citrate and theasapogenol B glycoside as the main constituents. The studied materials demonstrated significant protective effects against GA. TR25 administration significantly mitigated knee joint circumference compared to other treatments. It demonstrated potential in alleviating hyperuricemia, renal and hepatic impairments induced by MSU crystals. TR25 also alleviated oxidative stress and reduced levels of IL1ß, IL-6, TNF-α, and NF-κB. Weak Caspase-1 immune-positive staining was observed in the TR25 group. TR25 decreased NLRP3 and ASC expression, reducing inflammatory cytokine levels in GA. It effectively inhibited the PI3K, AKT, and mTOR signaling pathways, promoting autophagy. Additionally, TR25 suppressed ERK1, JNK, and p-38 MAPK gene expression in synovial tissue. These effects were attributed to various components in TAAE, such as flavonoids, phenolic acids, tannins, alkaloids, and triterpenes. CONCLUSION: Importantly, irradiation (25 KGy) enhanced the antioxidant effects and phtchemical contents of TAAE. Additionally, TR0, TR25, CAF, and SAP exhibited promising protective effects against GA, suggesting their therapeutic potential for managing this condition. These effects were likely mediated through modulation of the NLRP3/ASC/Caspase-1 and ERK/JNK/p-38 MAPK signaling pathways, as well as regulation of the PI3K/AKT/mTOR pathway. Further research is warranted to fully elucidate the underlying mechanisms and optimize their clinical applications.

Composition of Proteins Associated with Red Clover (Trifolium pratense) and the Microbiota Identified in Honey.

The nutritional composition of honey is determined by environmental conditions, and botanical and geographical origin. In addition to carbohydrates, honey also contain pollen grains, proteins, free amino acids, and minerals. Although the content of proteins in honey is low, they are an important component that confirms the authenticity and quality of honey; therefore, they became a popular study object. The aim of the study was to evaluate protein content and composition of monofloral red clover and rapeseed honey collected from five different districts of Lithuania. Forty-eight proteins were identified in five different origin honey samples by liquid chromatography. The number of red clover proteins identified in individual honey samples in monofloral red clover honey C3 was 39 in polyfloral honey S22-36, while in monofloral rapeseed honey S5, S15, and S23 there was 33, 32, and 40 respectively. Aphids' proteins and lactic acid bacteria were identified in all honey samples tested. The linear relationship and the strongest correlation coefficient (r = 0.97) were determined between the content of Apilactobacillus kunkeei and Apilactobacillus apinorum, as well as between the number of faba bean (Vicia faba) pollen and lactic acid bacteria (r = 0.943). The data show a strong correlation coefficient between the amount of lactic acid and aphid protein number (r = 0.693). More studies are needed to evaluate the relationship between the pollination efficiency of red clover by bees and the multiplicity of red clover proteins in honey protein, as well as microbiota diversity and the influence of nature or plant diversity on the occurrence of microbiota in honey.

Modified Clover Technique Using Automated Suture Placement and Securing Technology in a Passive Beating Heart Model.

Tricuspid regurgitation (TR) is a prevalent finding in echocardiography and in case of severe disease is associated with impaired patient outcome. Clover repair offers a surgical solution that can be applied for the treatment of primary and secondary TR. An ex vivo passive beating porcine heart model was created to test a modified clover technique using automated suturing devices and to compare this approach to standard ring annuloplasty. Secondary TR was induced in 10 porcine hearts and the backflow of fluid was assessed. The primary endpoint of this study was regurgitant volume measured in mL at the site right atrial cannula. The baseline regurgitation was 43.3 ± 10.8 mL. The mean regurgitant volume was significantly reduced after all repair procedures to 22.2 ± 5.9 mL with isolated ring annuloplasty, 12 ± 3.9 mL with the modified clover, and 7.6 ± 3.4 mL with the combined procedure (p < 0.0001). The modified clover technique shows how to effectively reduce TR in an ex vivo model. This method may be suitable to facilitate tricuspid repair, especially for totally endoscopic valve surgery.

Beyond cyanogenesis: Temperature gradients drive environmental adaptation in North American white clover (Trifolium repens L.).

Species that repeatedly evolve phenotypic clines across environmental gradients have been highlighted as ideal systems for characterizing the genomic basis of local environmental adaptation. However, few studies have assessed the importance of observed phenotypic clines for local adaptation: conspicuous traits that vary clinally may not necessarily be the most critical in determining local fitness. The present study was designed to fill this gap, using a plant species characterized by repeatedly evolved adaptive phenotypic clines. White clover is naturally polymorphic for its chemical defence cyanogenesis (HCN release with tissue damage); climate-associated cyanogenesis clines have evolved throughout its native and introduced range worldwide. We performed landscape genomic analyses on 415 wild genotypes from 43 locations spanning much of the North American species range to assess the relative importance of cyanogenesis loci vs. other genomic factors in local climatic adaptation. We find clear evidence of local adaptation, with temperature-related climatic variables best describing genome-wide differentiation between sampling locations. The same climatic variables are also strongly correlated with cyanogenesis frequencies and gene copy number variations (CNVs) at cyanogenesis loci. However, landscape genomic analyses indicate no significant contribution of cyanogenesis loci to local adaptation. Instead, several genomic regions containing promising candidate genes for plant response to seasonal cues are identified - some of which are shared with previously identified QTLs for locally adaptive fitness traits in North American white clover. Our findings suggest that local adaptation in white clover is likely determined primarily by genes controlling the timing of growth and flowering in response to local seasonal cues. More generally, this work suggests that caution is warranted when considering the importance of conspicuous phenotypic clines as primary determinants of local adaptation.

Preparation and Evaluation of the Biological Properties of Ethanolic Extract of Red Clover: An In Vitro Study.

INTRODUCTION: Red clover, a perennial herbaceous plant, has been demonstrated to possess blood-purifying, expectorant, and calming properties. This research endeavors to create and evaluate the antimicrobial, antioxidant characteristics, and cytotoxic effects of the ethanolic extract derived from red clover. METHODS: A water-based solution of red clover was formulated and subjected to centrifugation. Various concentrations of the extract were applied to the wells of agar plates inoculated with E. coli, Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis, and Candida albicans and then left to incubate. The inhibition zones for each concentration were subsequently measured. The antioxidant properties were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, while the cytotoxicity of the extract was assessed through the brine shrimp lethality assay. RESULTS: Initially, the extract was tested with a volume of 10 µL, which was subsequently incremented to 20 µL, 30 µL, 40 µL, and 50 µL. According to the DPPH assay, as the concentration of the extract solution increased incrementally by 10 µL, its antioxidant activity also exhibited a corresponding rise. The cytotoxicity assay indicated that the mouthwash formulated with red clover had minimal cytotoxic effects within the range of 5-20 µL. Antibacterial analysis revealed a similar zone of inhibition between the test and control groups. CONCLUSION: The ethanolic extract obtained from red clover was identified as a powerful antioxidant, antibacterial, and biocompatible substance. Hence, it can be a potential candidate for application as a mouthwash.

In silico Analysis of Selected Four-Leaf Water Clover (Marsilea species) Constituents as Human Acetyl Cholinesterase (hAchE), Carbonic Anhydrase II (hCA-II), and Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitory Agents.

A four-leaf water clover (Marsilea species) has been reported to exhibit various biological activities. In the present study, we aimed to evaluate 23 selected constituents of a four-leaf water clover (Marsilea species) as potent inhibitory agents of human acetyl cholinesterase (hAchE), carbonic anhydrase II (hCA-II), and protein tyrosine phosphatase 1B (hPTP-1B) using an in silico method. The 23 selected constituents of the four-leaf water clover (Marsilea species) were studied on the docking behavior of hAchE, hCA-II, and hPTP-1B by using the Webina docking method. In addition to docking, toxicity analysis was also performed using the pkCSM web server. Toxicity analysis has shown that 10 ligands (44%) of the four-leaf water clover (Marsilea species) were predicted to have hERG II (human ether-a-go-go-related gene) inhibition activity. The docking analysis showed that marsilin has exhibited the maximum binding energy (-11.3 kcal/mol) with the hAchE, whereas it fails to dock with both the target enzymes (hCA-II and hPTP-1B). Thus, the present find provides a new understanding about the 23 selected ligands of the four-leaf water clover (Marsilea species) as potent inhibitory agents of human acetyl cholinesterase (hAchE), carbonic anhydrase II (hCA-II), and protein tyrosine phosphatase 1B (hPTP-1B).

Including marker x environment interactions improves genomic prediction in red clover (Trifolium pratense L.).

Genomic prediction has mostly been used in single environment contexts, largely ignoring genotype x environment interaction, which greatly affects the performance of plants. However, in the last decade, prediction models including marker x environment (MxE) interaction have been developed. We evaluated the potential of genomic prediction in red clover (Trifolium pratense L.) using field trial data from five European locations, obtained in the Horizon 2020 EUCLEG project. Three models were compared: (1) single environment (SingleEnv), (2) across environment (AcrossEnv), (3) marker x environment interaction (MxE). Annual dry matter yield (DMY) gave the highest predictive ability (PA). Joint analyses of DMY from years 1 and 2 from each location varied from 0.87 in Britain and Switzerland in year 1, to 0.40 in Serbia in year 2. Overall, crude protein (CP) was predicted poorly. PAs for date of flowering (DOF), however ranged from 0.87 to 0.67 for Britain and Switzerland, respectively. Across the three traits, the MxE model performed best and the AcrossEnv worst, demonstrating that including marker x environment effects can improve genomic prediction in red clover. Leaving out accessions from specific regions or from specific breeders' material in the cross validation tended to reduce PA, but the magnitude of reduction depended on trait, region and breeders' material, indicating that population structure contributed to the high PAs observed for DMY and DOF. Testing the genomic estimated breeding values on new phenotypic data from Sweden showed that DMY training data from Britain gave high PAs in both years (0.43-0.76), while DMY training data from Switzerland gave high PAs only for year 1 (0.70-0.87). The genomic predictions we report here underline the potential benefits of incorporating MxE interaction in multi-environment trials and could have perspectives for identifying markers with effects that are stable across environments, and markers with environment-specific effects.

Harnessing cold adaptation for postglacial colonisation: Galactinol synthase expression and raffinose accumulation in a polyploid and its progenitors.

Allotetraploid white clover (Trifolium repens) formed during the last glaciation through hybridisation of two European diploid progenitors from restricted niches: one coastal, the other alpine. Here, we examine which hybridisation-derived molecular events may have underpinned white clover's postglacial niche expansion. We compared the transcriptomic frost responses of white clovers (an inbred line and an alpine-adapted ecotype), extant descendants of its progenitor species and a resynthesised white clover neopolyploid to identify genes that were exclusively frost-induced in the alpine progenitor and its derived subgenomes. From these analyses we identified galactinol synthase, the rate-limiting enzyme in biosynthesis of the cryoprotectant raffinose, and found that the extant descendants of the alpine progenitor as well as the neopolyploid white clover rapidly accumulated significantly more galactinol and raffinose than the coastal progenitor under cold stress. The frost-induced galactinol synthase expression and rapid raffinose accumulation derived from the alpine progenitor likely provided an advantage during early postglacial colonisation for white clover compared to its coastal progenitor.

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.

Effect of carbohydrate type in silages and concentrates on feed intake, enteric methane and milk yield from dairy cows.

Dietary carbohydrate manipulation can be used to reduce enteric CH4 emission, but there is a lack of studies on the interaction of different types of carbohydrates that can affect feed intake and ruminal fermentation. Understanding this interaction is necessary to make the most out of CH4 mitigation feeding strategies using different dietary carbohydrates. The aim of this study was to test the effect on enteric CH4 emission, feed intake and milk production response when cows were fed either grass-clover (GCS) or corn silage (CS) as the sole forage source (55% of dry matter, DM), in combination with either barley (BAR) or dried beet pulp (DBP) as a concentrate (21.5% of DM). Twenty-four (half first and half second parity) cows were used in a crossover design with 2 periods of 21 d each, receiving 2 of 4 diets obtained from a 2 × 2 factorial arrangement of the experimental diet. Feed intake, CH4 emission metrics and milk production were recorded at the end of the experimental periods. The diets had NDF concentrations between 258 and 340 g/kg of DM, and starch concentrations between 340 and 7.45 g/kg of DM (CS-BAR and GCS-DBP, respectively). The effects of silage and concentrate on dry matter intake (DMI) were additive, with the highest feed intake in cows fed COR-BAR, followed by cows fed COR-DBP, GCS-BAR, and GCS-DBP (21.2, 19.9, 19.1, and 18.3 kg/d). Energy corrected milk (ECM) yield was not affected by silage source in first parity cows, but it was higher for cows fed CS than cows fed GCS in second parity. The effects of silage and concentrate on CH4 production (g/d), yield (g/kg of DMI) and intensity (g/kg of ECM) were not additive as cows fed GCS had similar responses regardless of the concentrate used, but cows fed CS had lower CH4 production, yield and intensity, when fed BAR instead of DBP. The lower CH4 production, yield and intensity in cows fed CS-BAR compared with other diets could be partially explained by the nonlinear relationship between ruminal VFA and carbohydrates (NDF and starch) concentration reported in literature, however, we observed a linear relationship between acetate:propionate ratio and CH4 yield, suggesting possible other effects. The effects of silage and concentrate on the ruminal VFA were additive in first parity cows, but not in second parity cows. The interaction between dietary CHO type and parity might indicate an effect of feed intake or the energy balance of the cow. Feeding cows silage and concentrate both rich in starch can result in the lowest enteric CH4 emission.

Estrogenic Isoflavones in Clover Plants, Flower Nectar, Unripe Honeys and Mature Honeys: A Natural Biochemical Transformation of Isoflavones by Honeybees.

This study is the first to report on the presence of oestrogenic compounds in different clover flower nectar samples, in bee-deposited nectars collected from hive combs (unripe honey) and in mature honeys harvested from the same hives. The clover species investigated were two red clover (Trifolium pratense) cultivars, bred specifically for high isoflavone content, alongside a sainfoin (Onobrychis viciifolia) and a purple clover (T. purpureum) cultivar. A total of eight isoflavones, four of them non-glycosidic (biochanin A, formononetin, genistein and daidzein) the others glycosidic (sissotrin, ononin, genistin and daidzin), were targeted for identification and quantification in this study using high-performance thin-layer chromatography (HPTLC). Leaves and flower bracts of the clover samples were also investigated. Different isoflavone profiles were found across the four clover species and also in the different samples collected from each species indicating that, most likely due to the activity of honeybee (Apis mellifera) salivary enzymes, biochemical conversions take place when these bioactive compounds transition from flower nectar into ripe honey. Among the four investigated clover species, the two red clover cultivars, including their honeys, were found to contain higher levels of estrogenic compounds compared to other two cultivars.

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.