Phytochemical Profile, Bioactive Properties, and Se Speciation of Se-Biofortified Red Radish (Raphanus sativus), Green Pea (Pisum sativum), and Alfalfa (Medicago sativa) Microgreens.

The impact of selenium (Se) enrichment on bioactive compounds and sugars and Se speciation was assessed on different microgreens (green pea, red radish, and alfalfa). Sodium selenite and sodium selenate at a total concentration of 20 µM (1:1) lead to a noticeable Se biofortification (40-90 mg Se kg-1 DW). In green pea and alfalfa, Se did not negatively impact phenolics and antioxidant capacity, while in red radish, a significant decrease was found. Regarding photosynthetic parameters, Se notably increased the level of chlorophylls and carotenoids in green pea, decreased chlorophyll levels in alfalfa, and had no effect on red radish. Se treatment significantly increased sugar levels in green pea and alfalfa but not in red radish. Red radish had the highest Se amino acid content (59%), followed by alfalfa (34%) and green pea (28%). These findings suggest that Se-biofortified microgreens have the potential as functional foods to improve Se intake in humans.

Inflammatory mediators, oxidative stress and genetic disturbance in rheumatoid arthritis rats supported by alfalfa seeds metabolomic constituents via blocking interleukin-1receptor.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by aggressive cartilage and bone erosion. This work aimed to evaluate the metabolomic profile of Medicago sativa L. (MS) (alfalfa) seeds and explore its therapeutic impact against RA in rats. Arthritis was induced by complete Freund's adjuvant (CFA) and its severity was assessed by the arthritis index. Treatment with MS seeds butanol fraction and interlukin-1 receptor antagonist (IL-1RA) were evaluated through measuring interlukin-1 receptor (IL-1R) type 1 gene expression, interlukin-1 beta (IL-1ß), oxidative stress markers, C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), caspase-3 (Cas-3), intracellular adhesion molecule-1 (ICAM-1), DNA fragmentation, and chromosomal damage. Total phenolics/ flavonoids content in the ethyl acetate, butanol fraction and crude extract of MS seeds were estimated. The major identified compounds were Quercetin, Trans-taxifolin, Gallic acid, 7,4'-Dihydroxyflavone, Cinnamic acid, Kudzusaponin SA4, Isorhamnetin 3-O-beta-D-2'',3'',4''-triacetylglucopyranoside, Apigenin, 5,7,4'-Trihydroxy-3'-methoxyflavone, Desmethylxanthohumol, Pantothenic acid, Soyasapogenol E, Malvidin, Helilandin B, Stigmasterol, and Wairol. Treatment with MS seeds butanol fraction and IL-1RA enhanced all the biochemical parameters and the histopathological features of the ankle joint. In conclusion, Trans-taxifolin was isolated for the first time from the genus Medicago. MS butanol fraction seeds extract and IL-1 RA were considered as anti-rheumatic agents.

Converging alfalfa (Medicago sativa L.) and petroleum hydrocarbon acclimated ACC-deaminase containing bacteria for phytoremediation of petroleum hydrocarbon contaminated soil.

Plant assisted bioremediation of petroleum hydrocarbon contaminated soil is considered an effective green technology whereby accelerated degradation occurs due to converged effect of microorganisms and plants. However, survival and growth of microbes and plants under stress conditions is challenging task for success of the technology. In this study, plant growth promoting bacteria containing 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity and tolerance to petroleum hydrocarbon contamination were used in association with alfalfa for bioremediation of petroleum hydrocarbon contaminated soil. Eight pre-isolated bacterial isolates from soil having previous history of petroleum contamination were used in convergence with alfalfa on sand soil which was artificially contaminated (10 g crude oil per kg-1 of coarse textured soil). Combined effect of bacteria and plants on the degradation of petroleum hydrocarbons under controlled conditions of light and temperature was observed for a period of 60 days. The results of the study revealed that four bacterial isolates Bacillus subtilis strain PM32Y, Bacillus cereus strain WZ3S1, Bacillus sp. strain SM73 and Bacillus sp. strain WZ3S3 in association with alfalfa significantly degraded petroleum hydrocarbons. The most significant biodegradation (47%) of petroleum hydrocarbons was recorded in the experimental unit receiving PM32Y inoculation in association with alfalfa. Biodegradation of petroleum hydrocarbons was 33% with alone inoculation (without alfalfa) of PM32Y. The study revealed that combined use of bacteria and alfalfa plant is more efficient than alone application of either bacteria or plants for degradation of petroleum hydrocarbons.

This study provides the evidence for phytoremediation and significant degradation of petroleum hydrocarbons by using plant growth promoting bacteria (PGPB), containing 1-aminocyclopropane-1-carboxylate deaminase (ACC-deaminase) in association with alfalfa (Medicago sativa L.). The most significant biodegradation of petroleum hydrocarbons was recorded with a new combination of Bacillus subtilis strain PM32Y in association with alfalfa.

A 24-epibrassinolide treatment and intercropping willow with alfalfa increase the efficiency of the phytoremediation of cadmium-contaminated soil.

Cadmium contamination in agricultural soils threatens food security and human health, and that has caused widespread concern worldwide. Willow and alfalfa are widely used for the phytoremediation of cadmium (Cd)-contaminated soil, and willow NJU513 is the promising plant for remediating Cd-contaminated soil. In order to discuss the effect of intercropping willow NJU513 with alfalfa on the phytoremediation of Cd-contaminated soil, a pot-culture experiment was conducted in the greenhouse. The result showed that the phytoremediation of Cd-contaminated soil was enhanced by this intercropping because of the 25.90 % increase in the available Cd content. In order to increase the phytoremediation efficiency of Cd in the intercropping treatment, a 24-epibrassinolide (Brs) treatment was designed in the current study. The results showed that the phytoremediation of Cd-contaminated soil by willow and alfalfa improved following a Brs treatment because of the 16.32-74.15 % and 16.91-44.48 % increases in the plant biomass and available Cd content, respectively. Additionally, the extracted Cd by plants in the intercropping treatments with and without Brs was 0.56 and 0.31 mg pot-1, respectively. Transcriptome analyses of willow leaves revealed that Brs up-regulated the expression of genes related to calcium channel activity, calcium and zinc transmembrane transport, photosynthesis, catalase/antioxidant activity, glutathione metabolic processes and detoxification, phagosomes, and vacuoles, and that these upregulated genes promoted plant remediation efficiency and resistance to Cd stress. Brs promoted the phosphate ion transporter activity in willow leaves, which may have enhanced the solubilization of insoluble phosphate minerals by bacterial species (e.g., Vicinamibacterales, Bacillus, and Gaiella) to release Cd, ultimately leading to increased phytoremediation efficiency. In addition, plants with and without Brs treatments induced the bacteria-mediated transformation of available Cd to stable Cd. The study findings may be useful for improving the phytoremediation of Cd-contaminated paddy soil.

Growth performance, nutrient digestibility, and ruminal fermentation of dairy calves fed starter diets with alfalfa hay versus corn silage as forage and soybean oil versus palm fatty acids as fat source.

The present study was intended to evaluate the effect of forage source (alfalfa hay; ALF vs. corn silage; CS) along with a supplemental fat source (soybean oil; SO vs. rumen-inert palm fatty acids; PF) on growth performance, nutrient digestibility, and ruminal fermentation in dairy calves. Forty-eight new-born Holstein female calves (3 d old) were assigned to one of 4 treatments: (1) alfalfa hay with soybean oil (ALF-SO); (2) alfalfa hay with palm fatty acids (ALF-PF); (3) corn silage with soybean oil (CS-SO); (4) corn silage with palm fatty acids (CS-PF). Starter diets had equal amounts of forage (100 g/kg dry matter; DM) and fat source (30 g/kg DM). Calves were fed a constant amount of milk (d 1 to 63) and had ad libitum access to water and starters (d 1 to 83). The lowest and greatest starter intakes during the preweaning period occurred in ALF-SO and CS-PF, respectively. This coincided with forage × fat source interaction for average daily gain (ADG) during preweaning. The forage source affected total DM intake and ADG over the entire period, body weight (BW) at weaning, and final BW with greater values in calves that received CS compared with ALF. The concentrations of total short-chain fatty acids and butyrate were increased, whereas concentration of acetate and acetate:propionate ratio were decreased in the rumen of calves fed CS compared with ALF. Feeding CS increased urinary excretion of allantoin and, as a trend, total purine derivatives (PD) and estimated microbial protein synthesis in comparison with ALF. The fat source affected starter intake, ADG, and BW postweaning with the highest values in PF. The digestibility of neutral detergent fiber, crude protein and, as a trend, organic matter were higher in calves fed PF compared with SO. Calves fed PF had lower ruminal ammonia-N concentration and urinary N excretion and greater urinary excretion of allantoin and total PD. Calves receiving SO had a lower ruminal protozoa population. In conclusion, supplementing starter diets with CS and PF is superior to ALF and SO. Interaction of the positive effects of CS and PF on performance underlines that concurrent supplementation of CS with PF is especially recommendable in young calves before weaning.

Effects of nitrogen and phosphorus addition on growth and leaf nitrogen metabolism of alfalfa in alkaline soil in Yinchuan Plain of Hetao Basin.

Alkaline soil is widely distributed in China. Its rational utilization is an effective measure to solve land shortage and improve the environment. Alfalfa is characterized by strong salt and alkali tolerance and high yield and protein content. Nitrogen (N) and phosphorus (P) are the main nutrients for plant growth, and N metabolism is one of the primary forms of plant metabolism, which plays a vital role in quality and yield formation. The exploration of the effect of N and P on N metabolism and alfalfa growth will provide a theoretical basis for scientific fertilization for alfalfa in the alkaline soil of the Yinchuan Plain of the Hetao Basin. Therefore, a 2-year experiment of N and P addition was conducted. Six treatments were set up with a randomized block design, including without N (WN), medium N (MN), high N (HN), without P (WP), medium P (MP), and high P (HP). It was found that the MN and MP treatments increased plant height, stem diameter, stem/leaf, dry/fresh, and dry matter of alfalfa. The HN and HP treatments inhibited alfalfa biomass formation. The MN and MP treatments increased key products and enzymes of leaf N metabolism of alfalfa and promoted activities of leaf nitrate reductase (NR), glutamine synthase (GS), glutamate synthase (GOGAT), glutamic-oxalacetic transaminase (GOT), and glutamic-pyruvate transaminase (GPT), and inhibited activities of leaf protease of alfalfa. The MN and MP treatments increased contents of leaf N, P, ammonium nitrogen (NH4 +-N), nitrate nitrogen (NO3 --N), total chlorophyll, and protein and reduced leaf chlorophyll a/b and amino acid, results after HN and HP treatments were opposite. The correlation among leaf P, N, NO3 --N, amino acid, and protein reached significant levels (P < 0.01). It is suggested that MN and MP treatments can improve the yield and quality of alfalfa by increasing key products and enzymes of N metabolism and can be adopted to promote alfalfa production in the alkaline soil of the Yinchuan Plain of the Hetao Basin.

Impact of Tannin Supplementation on Proteolysis during Post-Ruminal Digestion in Wethers Using a Dynamic In Vitro System: A Plant (Medicago sativa) Digestomic Approach.

The aim of this study was to characterize the effects of tannins on plant protein during sheep digestion using a digestomic approach combining in vivo (rumen) conditions and an in vitro digestive system (abomasum and small intestine). Ruminal fluid from wethers infused with a tannin solution or water (control) was introduced into the digester, and protein degradation was followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tannin infusion in the rumen led to a clear decrease in protein degradation-related fermentation end-products, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) protein was more abundant than in control wethers. In the simulated abomasum, peptidomic analysis showed more degradation products of RuBisCo in the presence of tannins. The effect of RuBisCo protection by tannins continued to impact Rubisco digestion into early-stage intestinal digestion but was no longer detectable in late-stage intestinal digestion. The peptidomics approach proved a potent tool for identifying and quantifying the type of protein hydrolyzed throughout the gastrointestinal tract.

Transcriptome analysis revealed accumulation-assimilation of selenium and physio-biochemical changes in alfalfa (Medicago sativa L.) leaves.

BACKGROUND: Selenium (Se) is an increasing concern for investigators predominantly because of its consumption in the human body mainly from crops. As the fourth largest plant crop globally, alfalfa is one of the most important forages. Alfalfa was fertilized with selenium(IV) (Se(IV)) under field conditions to study the accumulation and assimilation of Se(IV) and to assess the impact of Se fertilization. RESULTS: It was analyzed that the physio-biochemistry, Se species, combined with transcriptome after spraying Se(IV) at different times (0, 12, and 48 h). 9402 and 12 607 differentially expressed genes (DEGs) were identified at 12 h (versus 0 h) and 48 h (versus 12 h). DEG functional enrichments proposed two time-specific biological processes: Se(IV) accumulation was the primary process at 0-12 h, and its assimilation mainly occurred during 12-48 h. This was further proved by the separation of various Se speciation at different times. It showed that Se-supplementation also affected the soluble protein, soluble sugar, pigment contents and antioxidant capacity. Selenium-biofortification could improve the stress resistance of alfalfa by enhancing antioxidant system to scavenge reactive oxygen species (e.g. hydrogen peroxide) and boosting carbohydrate metabolism. CONCLUSION: By integrating physio-biochemistry, Se-related metabolites, and transcriptome under Se(IV) treatment, this study provides data to guide further work on Se-fortification in alfalfa. © 2022 Society of Chemical Industry.

Effect of incremental proportions of Desmanthus spp. in isonitrogenous forage diets on growth performance, rumen fermentation and plasma metabolites of pen-fed growing Brahman, Charbray and Droughtmaster crossbred beef steers.

Desmanthus (Desmanthus spp.), a tropically adapted pasture legume, is highly productive and has the potential to reduce methane emissions in beef cattle. However, liveweight gain response to desmanthus supplementation has been inconclusive in ruminants. This study aimed to evaluate weight gain, rumen fermentation and plasma metabolites of Australian tropical beef cattle in response to supplementation with incremental levels of desmanthus forage legume in isonitrogenous diets. Forty-eight Brahman, Charbray and Droughtmaster crossbred beef steers were pen-housed and fed a basal diet of Rhodes grass (Chloris gayana) hay supplemented with 0, 15, 30 or 45% freshly chopped desmanthus forage on dry matter basis, for 140 days. Varying levels of lucerne (Medicago sativa) hay were added in the 0, 15 and 30% diets to ensure that all diets were isonitrogenous with the 45% desmanthus diet. Data were analyzed using the Mixed Model procedures of SAS software. Results showed that the proportion of desmanthus in the diet had no significant effect on steer liveweight, rumen volatile fatty acids molar proportions and plasma metabolites (P ≥ 0.067). Total bilirubin ranged between 3.0 and 3.6 µmol/L for all the diet treatments (P = 0.67). All plasma metabolites measured were within the expected normal range reported for beef cattle. Rumen ammonia nitrogen content was above the 10 mg/dl threshold required to maintain effective rumen microbial activity and maximize voluntary feed intake in cattle fed low-quality tropical forages. The average daily weight gains averaged 0.5 to 0.6 kg/day (P = 0.13) and were within the range required to meet the target slaughter weight for prime beef markets within 2.5 years of age. These results indicate that desmanthus alone or mixed with other high-quality legume forages can be used to supplement grass-based diets to improve tropical beef cattle production in northern Australia with no adverse effect on cattle health.

Effects of feeding a live yeast on rumen fermentation and fiber degradability of tropical and subtropical forages.

BACKGROUND: The effect of live yeast Saccharomyces cerevisiae strain CNCM I-1077 (SC) on the ruminal degradability of different forages commonly found in dairy diets in South America was evaluated. We also assessed if SC supplementation interacts with forage group to affect ruminal fiber degradability. Four non-lactating rumen-cannulated Holstein cows were randomly assigned to two treatment sequences: Control-SC-Control or SC-Control-SC, in a switchback design, with three 30-day periods. Cows in the SC treatment were supplied with 1 × 1010 colony-forming units of yeast daily via rumen cannula. In situ degradability of dry matter (DM) and neutral detergent fiber (aNDF) was measured in 15 forages collected in South America. Forages were assigned to one of three groups: corn silages; tropical grasses (sugarcane silages and tropical grass silages); and temperate grasses and alfalfa (oat silages, ryegrass silages, alfalfa silage, and alfalfa hay). RESULTS: Cows supplemented with SC had higher (P = 0.05) counts of yeasts and lower (P = 0.03) concentration of lactate in rumen fluid. There was no interaction between forage group and yeast supplementation (P > 0.10) on in situ degradability. The SC increased DM (by 4.6%) and aNDF degradation (by 10.3%) at 24 h of incubation (P < 0.05). Metabolomics revealed that a chemical entity (C17 H29 N6 O3 , m/z 365.2284 [M + H]+ ) from the family of lipids and related molecules was suppressed in the rumen fluid of cows supplemented with SC. CONCLUSION: The SC supplementation improved DM and aNDF degradability regardless of the forage group. © 2021 Society of Chemical Industry.

Magnetite nanoparticles coated with citric acid are not phytotoxic and stimulate soybean and alfalfa growth.

In this work, the internalization and distribution of citric acid-coated magnetite nanoparticles (here, Fe3O4-NPs) in soybean and alfalfa tissues and their effects on plant growth were studied. Both legumes were germinated in pots containing an inert growing matrix (vermiculite) to which Hoagland solution without (control, C), with Fe3O4-NPs (50 and 100 mgironL-1, NP50 and NP100), or with the same amount of soluble iron supplied as Fe-EDTA (Fe50, Fe100) was added once before sowing. Then, plants were watered with the standard nutrient solution. The observation of superparamagnetic signals in root tissues at harvest (26 days after emergence) indicated Fe3O4-NPs uptake by both legumes. A weak superparamagnetic signal was also present in the stems and leaves of alfalfa plants. These findings suggest that Fe3O4-NPs are readily absorbed but not translocated (soybean) or scarcely translocated (alfalfa) from the roots to the shoots. The addition of both iron sources resulted in increased root weight; however, only the addition of Fe3O4-NPs resulted in significantly higher root surface; shoot weight also increased significantly. As a general trend, chlorophyll content enhanced in plants grown in vermiculite supplemented with extra iron at pre-sowing; the greatest increase was observed with NP50. The only antioxidant enzyme significantly affected by our treatments was catalase, whose activity increased in the roots and shoots of both species exposed to Fe3O4-NPs. However, no symptoms of oxidative stress, such as increased lipid peroxidation or reactive oxygen species accumulation, were evidenced in any of these legumes. Besides, no evidence of cell membrane damage or cell death was found. Our results suggest that citric acid-coated Fe3O4-NPs are not toxic to soybean and alfalfa; instead, they behave as plant growth stimulators.

Feeding various forages and live yeast culture on weaned dairy calf intake, growth, nutrient digestibility, and ruminal fermentation.

The objective of this study was to determine effects of various forages and live yeast culture on intake, growth, nutrient digestibility, and ruminal fermentation of weaned dairy calves. Holstein calves (n = 45) were randomly assigned to 2 × 3 factorial treatments: live yeast culture or no yeast and alfalfa haylage (AH), corn silage (CS), or grass hay (GH). Calves were weaned at 6 wk of age, housed individually, and studied from 7 to 16 wk of age. Rations, consisting of an 18% crude protein texturized grower (yeast or no yeast) and assigned forage, were offered as separate components until 9 wk of age. After 9 wk, diets were offered as a total mixed ration (TMR). Concentrate intake was capped at 2.25 kg of dry matter (DM)/d, and forage was offered ad libitum. The TMR contained equal forage neutral detergent fiber (8 ± 0.5%) on a DM basis for each basal diet. Calves were fed TMR to limit concentrate intake, and additional forage was offered ad libitum after 8 h if the entire TMR allotment was consumed. Total fecal collection (12 calves) was conducted for 4 d at 11 and 15 wk of age. Feeds and feces were evaluated for DM, neutral detergent fiber, acid detergent fiber, and starch to calculate digestibility. On the last day of fecal collection, rumen samples were collected to evaluate pH and volatile fatty acid (VFA) profile. Metabolizable energy and DM intake was least for calves consuming GH compared with other forages. Forage intake (% of DM intake) increased as calves aged (AH = 20 to 44.4%, CS = 24.5 to 37.6%, GH = 11.3 to 32.3% at 10 and 16 wk of age, respectively). Calves on CS had the greatest average daily gain and empty body weight gain, and calves on GH had the least. Calves on GH tended to have the lowest final body weight. There were no differences in structural growth. Digestibility of DM decreased with age. Fiber digestibility was least for calves on CS, and starch digestibility was least for calves on AH. Mean and minimum rumen pH increased with age. Forage offered changed VFA profile and there was an interaction between yeast and forage on VFA profile. These results indicated that recently weaned calves perform well on AH, CS, or GH and have minimal benefit from yeast supplementation. Feeding GH reduced weight gain, but all calves achieved a level of gain to meet growth goals for breeding and freshening. Furthermore, the ability to consume large portions of the ration as forage allows for more economical diets to be fed.

Comparison of the Polyphenolic Profile of Medicago sativa L. and Trifolium pratense L. Sprouts in Different Germination Stages Using the UHPLC-Q Exactive Hybrid Quadrupole Orbitrap High-Resolution Mass Spectrometry.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

Influences of arbuscular mycorrhizae, phosphorus fertiliser and biochar on alfalfa growth, nutrient status and cadmium uptake.

The objective of the study was to explore the influences of arbuscular mycorrhizae (AM), phosphorus (P) fertiliser, biochar application (BC) and their interactions on Medicago sativa growth, nutrient, Cd content and AM fungi-plant symbioses. Applications of both P fertiliser and BC significantly increased total biomass and P and potassium (K) uptake, regardless of AM. When no P fertiliser or BC was used, the shoot biomass and nitrogen (N), P, and K contents in the +AM treatments were 1.39, 1.54, 4.53 and 2.06 times higher than those in the -AM treatments, respectively. AM fungi only elevated the total P uptake by 44.03% when P fertiliser was applied at a rate of 30 mg P kg-1 in the absence of BC addition. With BC application or high-P fertiliser input (100 mg P kg-1), the soil available P was significantly higher than that in the other treatments, and AM fungi significantly reduced the shoot biomass. The minimum Cd concentration occurred in the shoots of alfalfas treated with BC and high-P fertiliser inputs; this concentration was lower than the maximum permitted concentration in China. Although the BC and high-P inputs could eliminate the positive mycorrhizal response, the results suggested that BC application in combination with high-P fertiliser input could not only increase forage yields but also lower Cd concentrations to meet the forage safety standards by the dilution effect.

Arbuscular Mycorrhizal Symbiosis Mitigates Iron (Fe)-Deficiency Retardation in Alfalfa (Medicago sativa L.) Through the Enhancement of Fe Accumulation and Sulfur-Assisted Antioxidant Defense.

Iron (Fe)-deficiency is one of the major constraints affecting growth, yield and nutritional quality in plants. This study was performed to elucidate how arbuscular mycorrhizal fungi (AMF) alleviate Fe-deficiency retardation in alfalfa (Medicago sativa L.). AMF supplementation improved plant biomass, chlorophyll score, Fv/Fm (quantum efficiency of photosystem II), and Pi_ABS (photosynthesis performance index), and reduced cell death, electrolyte leakage, and hydrogen peroxide accumulation in alfalfa. Moreover, AMF enhanced ferric chelate reductase activity as well as Fe, Zn, S and P in alfalfa under Fe-deficiency. Although Fe-transporters (MsIRT1 and MsNramp1) did not induce in root but MsFRO1 significantly induced by AMF under Fe deficiency in roots, suggesting that AMF-mediated Fe enhancement is related to the bioavailability of Fe at rhizosphere/root apoplast rather than the upregulation of Fe transporters under Fe deficiency in alfalfa. Several S-transporters (MsSULTR1;1, MsSULTR1;2, MsSULTR1;3, and MsSULTR3;1) markedly increased following AMF supplementation with or without Fe-deficiency alfalfa. Our study further suggests that Fe uptake system is independently influenced by AMF regardless of the S status in alfalfa. However, the increase of S in alfalfa is correlated with the elevation of GR and S-metabolites (glutathione and cysteine) associated with antioxidant defense under Fe deficiency.

Investigation of Chemical Composition, Antioxidant Activity, and the Effects of Alfalfa Flavonoids on Growth Performance.

The flavonoids were extracted from alfalfa using ethanol assisted with ultrasonic extraction and purified by D101 macroporous resin column chromatography. The chemical composition and content of ethanol elution fractions (EEFs) were assessed by ultrahigh-performance liquid chromatography and hybrid quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS) and aluminum nitrate-sodium nitrite-sodium hydroxide colorimetric method. The in vitro antioxidant activity of two EEFs was conducted by scavenging DPPH free radical, and the main antioxidants of 75% EEFs were screened using DPPH-UHPLC. Moreover, the in vivo antioxidant activity of 75% EEFs and the growth performance of broilers were studied. The results showed that the content of 30% and 75% EEFs was 26.20% and 62.57%. Fifteen compounds were identified from 75% EEFs, and five of them were reported in alfalfa for the first time. The scavenging activity of 75% and 30% EEFs (200 µg/mL) against DPPH was 95.51% and 78.85%. The peak area of 5,3',4'-trihydroxyflavone and hyperoside was decreased by 82.69% and 76.04%, which exhibited strong scavenging capacities. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) level of three treated groups against the normal control group (NC) fed with basal diet significantly increased by 3.89-24.49%, 0.53-7.39%, and 0.79-11.79%, respectively. While the malondialdehyde (MDA) decreased by 0.47-18.27%. Compared with the NC, the feed to gain ratio (F : G) of three treated groups was lowered by 2.98-16.53% and survival rate of broilers significantly increased. Consequently, 75% EEFs extracted from alfalfa exhibited powerful antioxidant activities and might be a potential feed additive to poultry and livestock.

Chemometric evaluation of alfalfa sprouting impact on its metabolic profile using HPTLC fingerprint-efficacy relationship analysis modelled with partial least squares regression.

Sprouting is a commonly applied food processing practice specially in Western countries. Tracking the impact of sprouting of Medicago sativa L. (alfalfa) seeds on their phytochemical composition and curative efficacy was implemented in the current study. Sprouting of alfalfa seeds under controlled conditions for eleven days was performed in a biochemical incubator and three samples were randomly taken each day. A total of thirty-six samples (three ungerminated seeds and thirty-three sprouts samples) were collected, extracted and their cytotoxic, antioxidant and antimicrobial activities against five pathogenic microbial strains were measured. Samples were subjected to High performance thin layer chromatography (HPTLC) as a pattern-oriented strategy for metabolite fingerprinting to discover the fluctuations occurring during the sprouting process accompanied by multivariate chemometric analysis. Unsupervised pattern recognition was carried out using Principal Component Analysis (PCA) after extracting the chromatographic fingerprints from HPTLC chromatograms using ImageJ® software. PCA- loading plots demonstrated that luteolin-7-O-glucoside, ferulic acid and P-coumaric acid were the metabolically significant markers. Thus, simultaneous quantification of these crucial three markers in different aged alfalfa seeds/ sprouts extracts was performed using a newly developed and validated HPTLC-image analysis method. The results of the biological activities together with the quantitative data were further subjected to a Partial Least Squares Regression (PLSR) model for implementing HPTLC fingerprint-efficacy relationship analysis. The results obtained from metabolic pool profiling revealed that sprouting can cause remarkable changes in the phytochemical, nutritional and efficacy characteristics of alfalfa seeds.

Optimisation and kinetic study of the ultrasonic-assisted extraction of total saponins from alfalfa (Medicago sativa) and its bioaccessibility using the response surface methodology.

The ultrasonic-assisted extraction of total saponins from alfalfa leaves was optimised by the simultaneous maximization of the yield and bioaccessibility as a factor with increasingly great relevance in the biological activity by Response Surface Methodology. The kinetics of total saponins and bioaccessibility were investigated for the optimum ultrasound-assisted method compared to conventional method by the pseudo-first order model. The optimum extraction conditions were of solvent/raw material ratio of 11.4 mL/g, extraction time of 2.84 h, extraction temperature of 76.8 °C, ultrasound power of 112.0 w and ethanol concentration of 78.2%. The yield of total saponins and bioaccessibility was 1.61 and 18.6%, respectively. The yield rate constant for the ultrasound extraction was almost two times more than that of the heat-reflux method. Ultrasonic-assisted extraction, comparing to conventional method, had greater efficiency for the extraction yield and bioaccessibility of total saponins.

Soil C, N, and P distribution as affected by plant communities in the Yellow River Delta, China.

Soil carbon (C), nitrogen (N) and phosphorus (P) are important soil properties linked to nutrient limitation and plant productivity in terrestrial ecosystems. Up to 90% of the Yellow River Delta (YRD), China has been affected by soil salination due to groundwater overdraft, improper irrigation, land use and land cover change. The objective of this study is to evaluate the impact of different plant communities on soil quality in a saline-alkaline system of the YRD. We investigated the vertical distribution and seasonal variation of soil C, N, and P, and C:N ratio by choosing four dominant plant communities, namely, alfalfa grassland (AG), Chinese tamarisk (CT), locust forest (LF) and cotton field (CF). The results showed that the concentrations of soil organic carbon (SOC) and total nitrogen (TN) in CT and LF were always higher than that in AG and CF, especially in the topsoil layer (p<0.05), then gradually decreased with soil depth increasing (p<0.05). The C:N ratio was generally lower, and the average C:N ratio was higher in LF (11.55±1.99) and CT (11.03±0.47) than in CF (10.05±1.25) and AG (9.11±1.11) (p<0.05). The available phosphorus (AP) was highest in CT in Spring, while it was highest in CF in Summer and Autumn. It is worth noting that the soil AP concentrations were always low, particularly in AG (< 6.29 mg kg-1) and LF (< 4.67 mg kg-1), probably linked to P poorly mobile in the saline-alkaline region. In this study, soil nutrients in natural plant communities are superior to farmland, and are significantly affected by the types of plant community; therefore, we suggest that protection of natural vegetation and development of optimal vegetation are critical to restoring land degradation in the YRD.

Ectopic expression of tea MYB genes alter spatial flavonoid accumulation in alfalfa (Medicago sativa).

Flavonoids are one of the largest secondary metabolite groups, which are widely present in plants. Flavonoids include anthocyanins, proanthocyanidins, flavonols and isoflavones. In particular, proanthocyanidins possess beneficial effects for ruminant animals in preventing lethal pasture bloat. As a major legume forage, alfalfa (Medicago sativa) contains little proanthocyanidins in foliage to combat bloat. In an attempt to improve proanthocyanidin content in alfalfa foliage, we over-expressed two MYB transcription factors (CsMYB5-1 and CsMYB5-2) from tea plant that is rich in proanthocyanidins. We showed that, via targeted metabolite and transcript analyses, the transgenic alfalfa plants accumulated higher levels of flavonoids in stems/leaves than the control, in particular anthocyanins and proanthocyanidins. Over-expression of CsMYB5-1 and CsMYB5-2 induced the expression levels of genes involved in flavonoid pathway, especially anthocyanin/proanthocyanidin-specific pathway genes DFR, ANS and ANR in stems/leaves. Both anthocyanin/proanthocyanidin content and the expression levels of several genes were conversely decreased in flowers of the transgenic lines than in control. Our results indicated that CsMYB5-1 and CsMYB5-2 differently regulate anthocyanins/proanthocyanidins in stems/leaves and flowers. Our study provides a guide for increasing anthocyanin/proanthocyanidin accumulation in foliage of legume forage corps by genetic engineering. These results also suggest that it is feasible to cultivate new varieties for forage production to potentially solve pasture bloat, by introducing transcription factors from typical plants with high proanthocyanidin level.