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Ozone differentially affects physiological and biochemical responses of two clover species; Trifolium repens and Trifolium pratense.

The effect of acute ozone exposure (150 ppb for 3 h) on two clover species, white clover (Trifolium repens L.) and red clover (Trifolium pratense L.) was investigated through the analysis of 10 different physiological and biochemical parameters. Twenty-four hours after fumigation, visible symptoms of injury on leaves were observed only in red clover, but from the biochemical point of view, both species revealed significant ozone-induced modifications. A decrease in the photosynthetic efficiency as well as an increase in the de-epoxidation index and a decrease in the redox state of ascorbate were detected only in T. pratense leaves; no significant change in pigment content was found in either of the two species. On the other hand, both white and red clover showed, although to different extents, significant decreases in the activities of the antioxidant enzymes peroxidase and ascorbate peroxidase. Multivariate statistical analysis revealed not only that ozone affects both species, but also that they differentially respond to the pollutant, confirming the higher sensitivity of Trifolium pratense to ozone exposure.

Analysis of Variance↗

Cyanotypic frequencies in adjacent and mixed populations of Trifolium occidentale Coombe and Trifolium repens L. are regulated by different mechanisms.

The cyanogenic polymorphism in Trifolium repens is caused by the variation in two genes, the interaction of which produces four distinct cyanotypes. Along the Atlantic coasts of Bretagne, T. repens is sometimes found in populations mixed with the related species Trifolium occidentale, although the latter species usually occurs only in a narrow fringe along the coast, whereas T. repens is a more inland species. No plants of T. occidentale have ever been reported to have linamarase activity. Indeed, of 763 T. occidentale plants studied, none contained linamarase activity. However, the variation in the proportion of cyanotypes in T. repens was enormous, even between sites less than 2km apart. Our results confirm the presumption that T. repens and T. occidentale are indeed separate species. Both the fact that T. occidentale plants never contain linamarase activity, and the difference in proportion of plants with cyanoglucosides in mixed stands show that gene flow between the species must be rare. These dissimilar distributions strongly indicate that cyanotypic frequencies in adjacent and mixed populations of the very closely related species T. occidentale and T. repens are regulated by different mechanisms

Journal Article↗

Ileal amino acid digestibilities in pigs of barley-based diets with inclusion of lucerne (Medicago sativa), white clover (Trifolium repens), red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne).

Two experiments were performed with post-valve T-cannulated growing pigs, using five animals in each experiment in a change-over design to evaluate the effect of inclusion of four different dried forage meals on ileal crude protein (CP) and amino acid (AA) digestibilities. The control diets (C1 and C2) were barley-based and the experimental diets were formulated by replacing the barley with 100 or 200 g/kg of either lucerne (Medicago sativa) or white clover (Trifolium repens) meal in Expt 1 and red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne) meal in Expt 2. A decrease (P < 0.05) in the apparent ileal digestibility of CP and most of the essential and nonessential AA was found with the inclusion of luceme, white clover and perennial ryegrass meal in the barley-based diets. When red clover meal was included, only the apparent ileal digestibilities of CP, leucine, phenylalanine, tyrosine and glutamic acid were found to decrease (P < 0.05). The estimated apparent ileal digestibilities of most essential AA in the forage meals were lower than in the barley-based diets. The ileal flow of glucosamine and ornithine was found to increase (P < 0.05) with increasing proportion of fibre in the diet, suggesting an increase in endogenous N secretions and small-intestinal microbial activity. With the minor changes found for ileal essential AA digestibilities with forage meal inclusion in the diet the present data confirm the potential of forage meals as a source of protein in pig diets.

Amino Acids↗

Ancestors of white clover ( Trifolium repens L.), as revealed by isozyme polymorphisms.

Isozymes were used to study the putative ancestors of white clover ( Trifolium repens L.). Ten enzymes were examined, and 18 loci were resolved via starch-gel electrophoresis for accessions representing T. repens, Trifolium isthmocarpum, Trifolium nigrescens, Trifolium occidentale and Trifolium uniflorum, in addition to two more distantly related species, Trifolium alpinum and Trifolium purseglovei. Nei's genetic identities indicate that T. uniflorum and T. nigrescens are the closest relatives of T. repens. The isozyme data thus support a hypothesis that the two genomes of the tetraploid T. repens could have been derived from hybridization between T. nigrescens and T. uniflorum. This conclusion is further supported by shared alleles between T. repens, T. nigrescens and T. uniflorum. However, the origin of T. repens is somewhat obscured by the presence of shared alleles between T. repens and both T. occidentale and T. isthmocarpum, suggesting that introgression of genes from the latter two species into T. repens may also have taken place. High values of genetic identity are shared between T. occidentale with T. nigrescens and T. uniflorum, also indicative of introgression. Alternatively the presence of shared alleles among the five species may reflect their recent common ancestry.

Evolution, Molecular↗

Molecular phylogenetics of the clover genus (Trifolium--Leguminosae).

Trifolium, the clover genus, is one of the largest genera of the legume family. We conducted parsimony and Bayesian phylogenetic analyses based on nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL intron sequences obtained from 218 of the ca. 255 species of Trifolium, representatives from 11 genera of the vicioid clade, and an outgroup Lotus. We confirm the monophyly of Trifolium, and propose a new infrageneric classification of the genus based on the phylogenetic results. Incongruence between the nrDNA and cpDNA results suggests five to six cases of apparent hybrid speciation, and identifies the putative progenitors of the allopolyploids T. dubium, a widespread weed, and T. repens, the most commonly cultivated clover species. Character state reconstructions confirm 2n=16 as the ancestral chromosome number in Trifolium, and infer a minimum of 19 instances of aneuploidy and 22 of polyploidy in the genus. The ancestral life history is hypothesized to be annual in subgenus Chronosemium and equivocal in subgenus Trifolium. Transitions between the annual and perennial habit are common. Our results are consistent with a Mediterranean origin of the genus, probably in the Early Miocene. A single origin of all North and South American species is hypothesized, while the species of sub-Saharan Africa may originate from three separate dispersal events.

Bayes Theorem↗

First RFLP linkage map of red clover ( Trifolium pratense L.) based on cDNA probes and its transferability to other red clover germplasm.

We constructed a genetic linkage map of red clover ( Trifolium pratense L., 2n=2 x=14) using RFLP markers from cDNA probes of a backcrossed mapping population, and investigated the transferability of the markers to other red clover germplasm. The map contains 157 RFLP markers and one morphological marker on seven linkage groups. The total map distance was 535.7 cM and the average distance between two markers was 3.4 cM. All of the cDNA probes of the map were hybridized to the fragments of genomic DNA from 12 plants derived from three varieties, and 87% of the cDNA probes detected polymorphic bands that corresponded to those of mapping parents. This result indicated that RFLP markers on the present map were transferable to the genome analysis of other red clover germplasm. This is the first report to construct a linkage map of Trifolium species; it should provide fundamental and useful genetic information relevant to the breeding of red clover and genus Trifolium.

Chromosome Mapping↗

Floral Trifolium proanthocyanidins: polyphenol formation and compositional diversity.

Recent studies on the biosynthesis of proanthocyandins have identified key genes and enzymes in the formation of 2,3-cis-flavan-3-ols (epiafzelechin, epicatechin, and epigallocatechin). However, the enzymes that catalyze the polymerization of monomer units remain unknown. Studies of proanthocyanidin synthesis have involved the reference plant, Arabidopsis thaliana, forage legumes, tobacco, and grape. For this paper the floral proanthocyanidins of 10 Trifolium species were studied to identify candidates with contrasting proanthocyanidin chemistry, for the identification of factors involved in polymerization. Proanthocyandins were present in the floral portions (3.1-12.2 g/100 g of dry matter) of Trifolium spp. Thiolyic cleavage of proanthocyanidin fractions liberated flavan-3-ol extension units dominated by cis stereochemistry. The terminal units' stereochemistry of T. michelianum was exclusively trans (catechin), that of T. hirtum mixed trans (catechin and gallocatechin), and that of T. vesiculosum equal proportions of trans and cis (catechin and epicatechin). Compositional dispersion for oligomers was determined by MALDI-TOF MS, which showed a range of ions up to approximately 2200 Da. The three Trifolium spp. highlighted, all annuals, may warrant investigation for insights into proanthocyanidins biosynthesis.

Flavonoids↗

[Effect of copper pollution on seedling growth and activate oxygen metabolism of Trifolium pratense].

The effect of copper (Cu) pollution on the seedling growth and activate oxygen metabolism of Trifolium pratense was studied by water cultivation experiments. The results showed that under low concentration of Cu (< 10 mg.L-1), the growth of Trifolium pratense seedlings could be improved, their fresh/dry weight and the contents of soluble protein and chlorophylls in leaves increased slightly, the MDA content of leaf cells decreased, the activities of SOD, CAT and POD in the activate oxygen metabolism system slightly increased, and the balance of protective enzyme system was hold. However, with the increase of Cu concentration (10-100 mg.L-1), there existed an obvious negative effect on the growth of Trifolium pratense seedlings. The seedlings under high concentrations of Cu were shorter and smaller, their fibrous roots were shorter and fewer, and their fresh/dry weight and the contents of soluble protein and chlorophylls in leaves decreased drastically. With the increase of Cu concentration, the membrane penetration, electric conductivity and MDA content of leaf cells increased. Furthermore, the activate oxygen metabolism system was destroyed, the balance of protective enzyme system was broken, the activities of SOD and CAT decreased by 26.67% and 71.31%, respectively, while the POD activity increased by 10.6 times.

Catalase↗

Trifolium pratense for breast disease: a case series.

A series of cases in which Trifolium pratense was used in breast disease with hyperoestrogenic symptoms is reported. Several patients with cystic mastosis and breast cancer appeared to do well. The 'picture' of Trifolium is discussed.

Adult↗

Triterpene saponins and flavonoids in the seeds of Trifolium species.

Seeds of 57 species of the genus Trifolium have been studied for the occurrence and concentration of soyasapogenol B glycosides and flavonoids. It was shown that all tested species contained soyasaponin I and in some species astragaloside VIII and/or 22-O-glucoside and 22-O-diglucoside of soyasaponin I were also present. Total concentration of saponins ranged from trace amounts up to 10 mg/g(DM). It was suggested that soyasapogenol B glycosides could be recognized as chemotaxonomic character of Fabaceae family. All but three tested species contained flavonoids. The majority of species contained quercetin as a sole flavonoid or in the mixture with a number of unidentified flavonoid components. Concentration of quercetine in some species was at the level of about 3 mg/g(DM). This high quercetin concentration and soyasaponin occurrence makes the seeds of some Trifolium species a potential source of health beneficial phytochemicals, to be used in human nutrition.

Chromatography, Liquid↗

Chemical defenses of Trifolium glanduliferum against redlegged earth mite Halotydeus destructor.

Trifolium glanduliferum Boiss. was resistant to redlegged earth mite, Halotydeus destructor Tucker (Acari: Penthaleidae), feeding compared with Trifolium subterraneum L. Extracts from T. glanduliferum leaves, obtained with dichloromethane and methanol, acted as feeding deterrents to the redlegged earth mite. Bioassay-directed fractionation of the extracts led to the isolation of several metabolites, coumarin, alpha-and beta-ionone, linalool, medicarpin, that were shown to be deterrent in laboratory experiments. The compounds contributing to the defense of T. glanduliferum were different from those influencing mite feeding on T. subterraneum leaves.

Animals↗

A lineage-specific centromeric satellite sequence in the genus Trifolium.

We report the molecular structure, genomic organization, chromosomal distribution and evolutionary dynamics of TrR350, a satellite DNA isolated from the forage legume white clover (Trifolium repens L.; 2n = 4 x = 32). The basic repeating unit is an A+T rich 350 bp Hin dIII fragment with a complex dimeric structure consisting of an internal direct repeat of 156 bp packed between unrelated flanking sequences. Each 156 bp repeat has a conserved 24 bp motif repeating at two places. Most of the 24 bp short repeating units enclose a pentanucleotide CAAAA motif, presumed to be involved in breakage-reunion mechanism of tandemly repeating arrays. The dimers share high sequence homology among themselves while monomers within dimers show significant sequence divergence. Genomic Southern hybridization and/or fluorescence in situ hybridization (FISH) on 17 Trifolium species/subspecies revealed that it is a lineage-specific repeat confined to several species within the section Lotoidea originating in the Mediterranean region. The uniform length of the basic repeating unit and the centromeric localization in most of the species harbouring it reflects its extensive conservation in the lineage. However, the Hin dIII restriction profile in seven species also indicated independent evolution of this repeat.

Centromere↗

Assessment of enzyme induction and aerenchyma formation as mechanisms for flooding tolerance in Trifolium subterraneum 'Park'.

The objective of this study was to evaluate the role of enzyme induction and aerenchyma formation in prolonged tolerance to soil flooding in a variety of underground clover (Trifolium subterraneum 'Park') previously selected for resistance. Seedlings were grown in hydroponic tanks, initially with aeration for 3 weeks and subsequently in the absence of aeration for up to 3 weeks. After 1 h in the absence of aeration, the oxygen concentration in the hydroponic medium had decreased to 1.5 %. During the 3 weeks of extreme oxygen deficiency, primary roots died and were replaced by considerable numbers of adventitious roots. Activities of many glycolytic and fermentative enzymes increased in adventitious roots. Excised adventitious roots were capable of immediate induction of ethanol in the absence of lactate production, in association with energy charge higher than that in excised roots of aerobically maintained controls. Energy charge was even higher when measured in adventitious roots in planta. Interestingly, haemoglobin protein could be correlated with energy charge. Aerenchyma was readily visualized in adventitious roots by optical microscopy of longitudinal and transverse sections. We conclude that avoidance of root anoxia via aerenchyma is the major mechanism for prolonged root tolerance in Trifolium subterraneum 'Park'.

Adaptation, Physiological↗

Phyllobacterium trifolii sp. nov., nodulating Trifolium and Lupinus in Spanish soils.

Bacterial strain PETP02(T) was isolated from nodules of Trifolium pratense growing in a Spanish soil. Phylogenetic analysis of the 16S rRNA gene sequence showed that this strain represents a member of the genus Phyllobacterium. However, divergence found with the 16S rRNA gene sequence of the single recognized species of this genus, Phyllobacterium myrsinacearum, indicated that strain PETP02(T) belongs to a different species. The results of DNA-DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain represents a novel species of the genus Phyllobacterium, for which the name Phyllobacterium trifolii sp. nov. is proposed. The type strain is PETP02(T) (=LMG 22712(T)=CECT 7015(T)). This strain was strictly aerobic and used several carbohydrates as carbon source. It was not able to reduce nitrate. Aesculin hydrolysis was negative. It did not produce urease, arginine dihydrolase, gelatinase or beta-galactosidase. The DNA G+C content was 56.4 mol%. The nodD gene of this strain showed a sequence closely related to those of strains able to nodulate Lupinus. Infectivity tests showed that this strain is able to produce nodules in both Trifolium repens and Lupinus albus.

Alphaproteobacteria↗

Clovers (Trifolium spp.).

Legumes constitute one of the most important global groups of agricultural species, providing a major source of protein and oil for humans and animals as well as fixing nitrogen and improving the fertility of soils. Gene technology can assist plant improvement efforts in clovers (Trifolium spp.), aiming to improve forage quality, yield, and adaptation to biotic and abiotic stresses. An efficient and reproducible protocol for Agrobacterium-mediated transformation of a range of Trifolium species, using cotyledonary explants and different selectable marker genes, is described. The protocol is robust and allows for genotype-independent transformation of clovers. Stable meiotic transmission of transgenes has been demonstrated for selected transgenic clovers carrying single T-DNA inserts recovered from Agrobacterium-mediated transformation. This methodology can also be successfully used for 'isogenic transformation' in clovers: the generation of otherwise identical plants with and without the transgene from the two cotyledons of a single seed.

Adaptation, Physiological↗

The effect of Trifolium, Raphanus, and Cistus pollen grains on some blood parameters and mesentery mast cells.

Three kinds of pollen taxa belonging to 3 families (Fabaceae--Trifolium spp., Brassicaceae--Raphanus spp. and Cistaceae--Cistus spp.) and commonly collected by honeybees were fed to mature male rats separately, in the form of 60 mg/animal/day for a 30-day period. The objective of this study was to investigate any positive effects or possible side effects of the use of pollen on the immune system. This was achieved through blood analysis and cell count on blood, hemoglobin, erythrocyte and immune system cells. The cell concentration of mast cells, degranulization and cell localization were investigated in prepared mesentery tissue samples. Histological investigations of the stomach and duedenum sections of pollen-fed rats were carried out to learn the reason for eosinophil gastroenteritis in the alimentary canal. The eosinophil and lymphocyte levels of rats fed with pollen of Trifolium spp., Raphanus spp., and Cistus spp. were observed to have increased blood cell counts, while neutrophil and monocyte levels decreased; different values were found in basophil leucocytes between the pollen groups. Differing reductions in mesentery mast cell concentration, degranulization and cell localization were found. Within the three separate pollens, the rats having been fed with Cistus spp. pollen were observed to have higher blood lymphocyte, eosinophil, hemoglobin and hematocrit values than those fed with the others, as well as low mesentery mast cell concentration. Hemoglobin values were determined to increase at a proportion of between 10.0-11.3%. No difference was found in other blood parameters. The fat proportion of the male rats fed with the three taxa was between 4.03-8.75%, while that for protein proportion was between 16.11-24.25%. Male rats receiving these taxa did not experience allergic reactions and it is possible to argue that the low protein and fat content of these pollens have a strengthening effect on the immune systems by the increase in lymphocyte content and the amount of hemoglobin leads to an increase of oxygen transport capacity in the tissues.

Animals↗