Investigation of red clover (Trifolium pratense) isoflavonoid residual complexity by off-line CCS-qHNMR.

The importance of Trifolium pratense L. as a dietary supplement and its use in traditional medicine prompted the preparation of a thorough metabolite profile. This included the identification and quantitation of principal constituents as well as low abundant metabolites that constitute the residual complexity (RC) of T. pratense bioactives. The purity and RC of isoflavonoid fractions from standardized red clover extract (RCE) was determined using an off-line combination of countercurrent separation (CCS) and two orthogonal analytical methodologies: quantitative 1H NMR spectroscopy with external calibration (EC-qHNMR) and LC-MS. A single-step hydrostatic CCS methodology (Centrifugal Partition Chromatography [CPC]) was developed that fractionated the isoflavonoids with a hexanes-ethyl acetate-methanol-water (HEMWat) 5.5/4.5/5/5, v/v solvent system (SS) into 75 fractions containing 3 flavonolignans, 2 isoflavonoid glycosides, as well as 17 isoflavonoids and related compounds. All metabolites were identified and quantified by qHNMR spectroscopy. The data led to the creation of a complete isoflavonoid profile to complement the biological evaluation. For example, fraction 69 afforded 90.5% w/w biochanin A (17), with 0.33% w/w of prunetin (16), and 0.76% w/w of maackiain (15) as residual components. Fraction 27 with 89.4% w/w formononetin (13) as the major component had, in addition, a residual complexity consisting of 3.37%, 0.73%, 0.68% w/w of pseudobaptigenin (11), kaempferol (10) and pratensein (8), respectively. Despite the relatively high resolving power of CPC, and not unexpectedly, the chromatographic fractions retained varying degrees of the original metabolomic diversity. Collectively, the extent of metabolomic diversity should be recognized and used to guide the development of isolation strategies, especially when generating samples for bioactivity evaluation. The simultaneous structural and quantitative characterization enabled by qNMR, supported by LC-MS measurements, enables the evaluation of a relatively large number of individual fractions and, thereby, advances both the chemical and biological evaluation of active principles in complex natural products.

Morphological diversity within a core collection of subterranean clover (Trifolium subterraneum L.): Lessons in pasture adaptation from the wild.

Subterranean clover (Trifolium subterraneum L.) is a diploid self-pollinated annual pasture legume native to the Mediterranean region and widely sown in southern Australia and other countries with Mediterranean-type climates. This study utilised a core collection of 97 lines, representing around 80% of the genetic diversity of the species, to examine morphological diversity within subterranean clover. A total of 23 quantitative agro-morphological and 13 semi-quantitative morphological marker traits were assayed on the core collection and 28 diverse Australian cultivars as spaced plants in a replicated common garden experiment. Relationships between these traits and 24 climatic and edaphic parameters at their sites of origin were also examined within the core collection. Significant diversity was present for all traits. The Australian cultivars had similar levels of diversity to the core collection for several traits. Among the agro-morphological traits, time to flowering, leaf size and petiole diameter in mid-winter, plant area in late winter, maximum stem length, content of the oestogenic isoflavone biochanin A and total isoflavone content, were correlated with seven or more environmental variables. These can be considered highly adaptive, being the result of strong environmental selection pressure over time. For the first time in a clover species, morphological markers, including leaf mark, anthocyanin pigmentation and pubescence traits, have been associated with rainfall and soil parameters. This suggests they either have an adaptive role or the genes controlling them may be linked to other genes controlling adaptive traits. This study demonstrated the value of core collections to examine diversity within much larger global collections. It also identified adaptive traits from wild plants that can be utilised to develop more productive and persistent subterranean clover cultivars. The high heritability of these traits indicates that selection gains can be readily made.

Morphometric approaches to promote the use of exotic germplasm for improved food security and resilience to climate change: a kura clover example.

Adaptation of agriculture to climate change and its associated ecological pressures will require new crops, novel trait combinations, and previously unknown phenotypic attributes to deploy in climate resilient cropping systems. Genebanks, a primary source of exotic germplasm for novel crops and breeding materials, need comprehensive methods to detect novel and unknown phenotypes without a priori information about the species or trait under consideration. We demonstrate how persistent homology (PH) and elliptical Fourier descriptors (EFD), two morphometric techniques easily applied to image-based data, can serve this purpose by cataloging leaf morphology in the USDA NPGS kura clover collection and demarcating a leaf morphospace for the species. Additionally, we identify a set of representative accessions spanning the leaf morphospace and propose they serve as a kura clover core collection. The core collection will be a framework for monitoring the effects of climate change on kura clover in situ diversity and determining the role of ex situ accessions in modern agriculture.

Cutting reduces variation in biomass production of forage crops and allows low-performers to catch up: A case study of Trifolium pratense L. (red clover).

Re-growth of fodder plants after grazing and mowing drives the profitability of their cultivation and is therefore an important target trait for plant breeding and agricultural engineering. However, for some fodder plants little is known about their re-growth dynamics in response to grazing or mowing. We analysed the native response of plant architecture, leaf morphology and growth performance to experimental cutting in wild Trifolium pratense L. (red clover) plants. A total of 150 potted clover plants were established under controlled field conditions, and half of the plants were cut to 5 cm 3 months after sowing. Each plant was measured every week for 5 months. The cut and subsequently re-grown plants carried fewer main branches (-20%), as well as fewer (-13%) and smaller (-32%) leaves than the control plants. However, the cut plants produced an average of 17% more accumulated leaf area (cut + re-grown leaf area) than the control plants. This discrepancy was explained by variation in the growth strategy of the plants, where the cut plants invariably expressed a second growth phase, while almost half of the untreated plants did not. Our results suggest that cutting acted as an artificial trigger initiating a second growth phase in the cut plants and thereby contributed to yield increase. Exploiting this mechanism may set new goals for breeding and optimisation of the mowing regime.

Physiological and biochemical responses of the forage legume Trifolium alexandrinum to different saline conditions and nitrogen levels.

Salinity stress reduces plant productivity, but low levels of salinity often increase plant growth rates in some species. We herein describe the effects of salinity on plant growth while focusing on nitrogen use. We treated Trifolium alexandrinum with two nitrogen concentrations and salinity levels and determined growth rates, mineral concentrations, nitrogen use efficiency, photosynthesis rates, and nitrate reductase (NR, E.C. 1.6.6.1) and glutamine synthetase (GS, EC 6.3.1.2) activities. The T. alexandrinum growth rate increased following treatment with 100 mM NaCl in low nitrogen (LN) and high nitrogen (HN) conditions. Salt treatment also increased root volume, intrinsic water use efficiency, and nitrogen use efficiency in LN and HN conditions. These changes likely contributed to higher biomass production. Salinity also increased accumulations of sodium, chloride, and phosphate, but decreased potassium and calcium levels and total nitrogen concentrations in all plant organs independently of the available nitrogen level. However, the effect of salt treatment on magnesium and nitrate concentrations in photosynthetic organs depended on nitrogen levels. Salt treatment reduced photosynthesis rates in LN and HN conditions because of inhibited stomatal conductance. The effects of salinity on leaf NR and GS activities depended on nitrogen levels, with activities increasing in LN conditions. In saline conditions, LN availability resulted in optimal growth because of low chloride accumulation and increases in total nitrogen concentrations, nitrogen use efficiency, and NR and GS activities in photosynthetic organs. Therefore, T. alexandrinum is a legume forage crop that can be cultivated in low-saline soils where nitrogen availability is limited.

The presence of nodules on legume root systems can alter phenotypic plasticity in response to internal nitrogen independent of nitrogen fixation.

All higher plants show developmental plasticity in response to the availability of nitrogen (N) in the soil. In legumes, N starvation causes the formation of root nodules, where symbiotic rhizobacteria fix atmospheric N2 for the host in exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic responses to internal [N] of legumes are altered by their symbionts. Glasshouse experiments compared root phenotypes of three legumes, Medicago truncatula, Medicago sativa and Trifolium subterraneum, inoculated with their compatible symbiont partners and grown under four nitrate levels. In addition, six strains of rhizobia, differing in their ability to fix N2 in M. truncatula, were compared to test if plastic responses to internal [N] were dependent on the rhizobia or N2 -fixing capability of the nodules. We found that the presence of rhizobia affected phenotypic plasticity of the legumes to internal [N], particularly in root length and root mass ratio (RMR), in a plant species-dependent way. While root length responses of M. truncatula to internal [N] were dependent on the ability of rhizobial symbionts to fix N2 , RMR response to internal [N] was dependent only on initiation of nodules, irrespective of N2 -fixing ability of the rhizobia strains.

Differential bud activation by a net positive root signal explains branching phenotype in prostrate clonal herbs: a model.

Regulation of branching within perennial prostrate clonal herbs differs from the annual orthotropic species, Arabidopsis and pea, as the dominant signal transported from roots is a branching promoter, not an inhibitor. Trifolium repens, an exemplar of such prostrate species, was used to investigate the interaction between roots and branch development. This study tests whether or not current knowledge when synthesized into a predictive model is sufficient to simulate the branching pattern developing on the shoot distal to a basal root. The major concepts underpinning the model are: (i) bud outgrowth (activation) is stimulated in a dose-dependent manner by branching promoter signals from roots, (ii) the distribution of this net root stimulus (NRS) is uniform throughout the shoot system distal to the basal root but declines geometrically in intensity upon continued enlargement of this shoot system, and (iii) each bud has an outgrowth potential, equal to the activation level of the apical bud in which it forms, that moderates its response to NRS. The validity of these concepts was further tested by running simulations of the branching of a phylogenetically-distanced prostrate perennial monocotyledonous species, Tradescantia fluminensis. For both species the model reasonably accounted for the observed pattern of branching. The outgrowth potential of buds plays an important role in limiting the number of hierarchies of branching that can develop on a plant. In conclusion, for both species, the model accounted for the major factors involved in the correlative regulation of branching and is possibly also pertinent for all prostrate clonal species.

Branching the risks: architectural plasticity and bet-hedging in Mediterranean annuals.

It has been suggested that architectural plasticity in shoot size and number allows plants to manage environmental risks. Simpler structures require shorter development times and fewer resources, which secure minimal fitness even under risky and unfavourable conditions. Here we tested the hypothesis that the magnitude of such architectural plasticity depends on the species' developmental strategy. Specifically, species with late reproduction were expected to express the highest levels of architectural plasticity in response to environmental cues predicting high probability of abrupt deterioration in growth conditions. This hypothesis was tested by comparing Mediterranean and semi-arid populations of three species, which differed in growth strategy: Trifolium purpureum, a determinate and late flowerer, and Emex spinosa and Hippocrepis unisiliquosa that flower indeterminately throughout the season. All plants were exposed to varying levels of water availability and competition, but only T. purpureum displayed plastic architectural responsiveness to the experimental manipulations. In contrast, the early and extended step-by-step flowering of both E. spinosa and H. unisiliquosa reflected a relatively deterministic bet-hedging reproductive schedule, whereby minimum fitness is secured even under adverse conditions. These two opposing strategies gave contrasting results, with E. spinosa and H. unisiliquosa displaying reduced efficiency under favourable conditions under which T. purpureum had the highest reproductive efficiency. The evolutionary interplay between deterministic risk-averse and plastic risk-prone growth strategies might reflect contrasts in the probability and severity of environmental risks, and the costs of missed opportunities.

Architectural plasticity in a Mediterranean winter annual.

Size variability in plants may be underlain by overlooked components of architectural plasticity. In annual plants, organ sizes are expected to depend on the availability and reliability of resources and developmental time. Given sufficient resources and developmental time, plants are expected to develop a greater number of large branches, which would maximize fitness in the long run. However, under restrictive growth conditions and environmental reliability, developing large branches might be risky and smaller branches are expected to foster higher final fitness. Growth and architecture of Trifolium purpureum (Papilionaceae) plants from both Mediterranean (MED) and semi-arid (SAR) origins were studied, when plants were subjected to variable water availability, photoperiod cues and germination timing. Although no clear architectural plasticity could be found in response to water availability, plants subjected to photoperiod cuing typical to late spring developed fewer basal branches. Furthermore, plants that germinated late were significantly smaller, with fewer basal branches, compared with plants which grew for the same time, starting at the beginning of the growing season. The results demonstrate an intricate interplay between size and architectural plasticities, whereby size modifications are readily induced by environmental factors related to prevalent resource availability but architectural plasticity is only elicited following the perception of reliable anticipatory cues.

Morphological response to competition for light in the clonal Trifolium repens (Fabaceae).

PREMISE OF THE STUDY: Plant communities in temperate zones are dominated by clonal plants that can plastically modify their growth characteristics in response to competition. Given that plants compete with one another, and the implications this has for species coexistence, we conducted a study to assess how clonal species morphologically respond to competition for light depending on its intensity and heterogeneity, which are determined by the competitor species. METHODS: We assessed the morphological response to competition for light of the clonal species Trifolium repens L. by measuring its growth performance, and vertical and horizontal growth traits. We used five competitive environments, i.e., one without competitor and four differing by their competitor species creating different conditions of competition intensity and heterogeneity. KEY RESULTS: The morphological response of Trifolium repens to competition for light depended on the competitor identity. Competition intensity and heterogeneity, determined by competitor identity, had an interactive effect on most traits. The increase in petiole elongation and specific leaf area due to increased competition intensity was observed only at low to intermediate competition heterogeneity. Competition heterogeneity promoted the elongation of clone connections allowing space exploration. CONCLUSIONS: Our results demonstrated that the intensity and heterogeneity of competition, which depended on competitor identity, are of primary importance in determining the plastic response of Trifolium repens. This emphasizes that it is important to consider the fine-scale spatial distribution of individuals when studying their interactions within plant communities.

Comparison of morphological and molecular genetic quantification of relative abundance of arbuscular mycorrhizal fungi within roots.

Nested PCR amplicons of ribosomal RNA genes have been used to identify individuals within assemblages of arbuscular mycorrhizal (AM) fungi in roots and to estimate their relative abundance. Microscopy has also been used to identify their relative abundance in roots, but only at low resolution, usually the genus level. We evaluated the robustness of using nested PCR amplicons of ribosomal RNA genes to estimate the relative abundance of undefined AM fungi in uniformly aged roots in comparison to visual estimates. The relative abundance of AM fungi was assessed as per cent root length colonised by morphotypes and relative sequence type abundance in clone libraries. Plants were grown in coastal soil to obtain assemblages of unknown AM fungi at two times (spring and autumn). Relative abundance of dominant genera of AM fungi in roots (Archaeospora and Glomus) based on an analysis of ribosomal RNA genes did not consistently correspond with relative abundance of morphotypes. This microscopic vs. molecular genetic comparison supports previous conclusions that there can be limitations in using nested PCR amplicons for quantifying the relative abundance of AM fungi in roots, with a sampling bias likely to be of significance. Both molecular genetic and morphological methods are used to estimate relative abundance of AM fungi as a precursor to understanding mycorrhizal function in field soils, but they are rarely verified using alternative approaches although this may be necessary.

Response of Trifolium repens to UV-B radiation: morphological links to plant productivity and water availability.

This study used nine populations of Trifolium repens L. (white clover) to investigate possible relationships between plant morphological attributes and responses to ultraviolet-B (UV-B) radiation. Plants were exposed to 0 or 13.3 kJ·m(-2) ·day(-1) UV-B for 12 weeks. Drought was applied in parallel to these treatments during the last 4 weeks of the experiment to test whether limited moisture availability would alter morphological UV-B responses. UV-B affected plant morphology under well-watered conditions, reducing leaf size by 15%, leaf number by 5% and stolon elongation by 19%. The number of leaf primordia in the apical bud was decreased by 4% under UV-B, and by 12% under drought. In drought-exposed plants, leaf size was reduced by 50%, leaf number by 30% and stolon elongation by 60%. In addition, drought reduced specific leaf area (SLA) by 33% and increased leaf percentage dry mass (PDM) by 40%. UV-B-induced reduction in plant biomass in the T. repens populations was associated with higher plant productivity and this was further linked to larger leaf size as well as to lower PDM. In conclusion, the findings suggest that morphological attributes conferring fast potential growth under productive conditions carry a cost in the form of lower biomass accumulation under UV-B.

G-fibres in storage roots of Trifolium pratense (Fabaceae): tensile stress generators for contraction.

Root contraction has been described for many species within the plant kingdom for over a century, and many suggestions have been made for mechanisms behind these contractions. To move the foliage buds deeper into the soil, the proximal part of the storage root of Trifolium pratense contracts by up to 30%. Anatomical studies have shown undeformed fibres next to strongly deformed tissues. Raman imaging revealed that these fibres are chemically and structurally very similar to poplar (Populus) tension wood fibres, which are known to generate high tensile stresses and bend leaning stems or branches upright. Analogously, an almost pure cellulosic layer is laid down in the lumen of certain root fibres, on a thin lignified secondary cell wall layer. To reveal its stress generation capacities, the thick cellulosic layer, reminiscent of a gelatinous layer (G-layer) in tension wood, was selectively removed by enzymatic treatment. A substantial change in the dimensions of the isolated wood fibre bundles was observed. This high stress relaxation indicates the presence of high tensile stress for root contraction. These findings indicate a mechanism of root contraction in T. pratense (red clover) actuated via tension wood fibres, which follows the same principle known for poplar tension wood.

Systemic induced resistance: a risk-spreading strategy in clonal plant networks?

Clonal plant networks consist of interconnected individuals (ramets) of different sizes and ages. They represent heterogeneous ramet assemblages with marked differences in quality and attractiveness for herbivores. Here, feeding preferences of a generalist herbivore (Spodoptera exigua) for differently-aged ramets of Trifolium repens were studied, and changes in herbivore preference in response to systemic defense induction were investigated. Dual-choice tests were used to assess the preference of herbivores for young versus mature ramets of induced and uninduced plants, respectively. Additionally, leaf traits related to nutrition, biomechanics and chemical defense were measured to explain variation in tissue quality and herbivore preference. Young ramets were heavily damaged in control plants. After systemic defense induction, damage on young ramets was greatly reduced, while damage on mature ramets increased slightly. Defense induction increased leaf strength and thickness, decreased leaf soluble carbohydrates and substantially changed phenolic composition of undamaged ramets connected to attacked individuals. Systemic induced resistance led to a more dispersed feeding pattern among ramets of different ages. It is proposed that inducible defense acts as a risk-spreading strategy in clonal plants by equalizing herbivore preference within the clone, thereby avoiding extended selective feeding on valuable plant tissues.

Micromorphologic study of the seed of the genus Trifolium, section Lotoidea, in Iran.

The characteristics of the seeds of 7 annual and perennial species of Trifolium, Lotoidea section, in Iran were investigated by means of Scanning Electron Microscopy (SEM) and a stereomicroscope. Contrary to Zohary's belief who maintained that the micromorphology of the Trifolium's seed played no role in taxonomy, the results revealed that such characteristics can play a determining role in differentiating such species and distinguishing them from one another. The results of the micromorphologic study of the seed confirmed the morphological results of the species of the Lotoidea section to a great extent, but was involved in the separation and differentiation of the two species of T. repens and T. nigrescens. In general, the characteristics of the species' seeds proved quite effective in determining their phenetic relationship. Among the characteristics studied in the present research, mention can be made of seed shape, hilum shape, seed sculpture and seed length and width (L/W ratio). The results demonstrated that the characteristics of seed length and hilum shape showed the greatest diversity in the species. The longest seed length was that of the species T. radicosum and the shortest that of T. repens. The spindle shape of the hilum was an exclusive characteristic of T. radicosum which distinguished it more than any other characteristic from all the other species of this section. The difference in hilum shape in the two species of T. repens and T. nigrescens was an important distinctive characteristic.

The management of extracellular ice by petioles of frost-resistant herbaceous plants.

BACKGROUND AND AIMS: Some frost-tolerant herbaceous plants droop and wilt during frost events and recover turgor and posture on thawing. It has long been known that when plant tissues freeze, extracellular ice forms. Distributions of ice and water in frost-frozen and recovered petioles of Trifolium repens and Escholschzia californica were visualized. METHODS: Petioles of intact plants were cryo-fixed, planed to smooth transverse faces, and examined in a cryo-SEM. KEY RESULTS: With frost-freezing, parenchyma tissues shrank to approx. one-third of their natural volume with marked cytorrhysis of the cells, and massive blocks of extracellular icicles grew under the epidermis (poppy) or epidermis and subepidermis (clover), leaving these layers intact but widely separated from the parenchyma except at specially structured anchorages overlying vascular bundles. On thawing, the extracellular ice was reabsorbed by the expanding parenchyma, and surface tissues again contacted the internal tissues at weak junctions (termed faults). These movements of water into and from the fault zones occurred repeatedly at each frost/thaw event, and are interpreted to explain the turgor changes that led to wilting and recovery. Ice accumulations at tri-cellular junctions with intercellular spaces distended these spaces into large cylinders, especially large in clover. Xylem vessels of frozen petioles were nearly all free of gas; in thawed petioles up to 20 % of vessels were gas-filled. CONCLUSIONS: The occurrence of faults and anchorages may be expected to be widespread in frost-tolerant herbaceous plants, as a strategy accommodating extracellular ice deposits which prevent intracellular freezing and consequent membrane disruption, as well as preventing gross structural damage to the organs. The developmental processes that lead to this differentiation of separation of sheets of cells firmly cemented at determined regions at their edges, and their physiological consequences, will repay detailed investigation.

Proteolysis and cell death in clover leaves is induced by grazing.

Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 degrees C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 degrees C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms.

A developmentally based categorization of branching in Trifolium repens L.: influence of nodal roots.

This study describes the successive stages of development of branches from axillary buds in fully rooted plants of Trifolium repens grown in near optimal conditions, and the way in which this developmental pathway differs when nodal root formation is prevented as plants grow out from a rooted base. Cuttings of a single genotype were established in a glasshouse with nodal root systems on the two basal phytomers and grown on so that nodal rooting was either permitted (+R) or prevented (-R). In +R plants, axillary tissues could be assigned to one of four developmental categories: unemerged buds, emerged buds, unbranched lateral branches or secondarily branched lateral branches. In -R plants, branch development was retarded, with the retardation becoming increasingly pronounced as the number of -R phytomers on the primary stolon increased. Retarded elongation of the internodes of lateral shoots on -R plants resulted in the formation of a distinct fifth developmental category: short shoots (defined as branches with two or more leaves but with mean internode length equal to, or less than, 10% of that of the immediately proximal internode on the parent stolon) which had reduced phytomer appearance rates but retained the potential to develop into lateral branches. Transfer of +R plants to -R conditions, and vice versa, after 66 d demonstrated that subsequent branch development was wholly under the control of the youngest nodal root present, regardless of the age and number of root systems proximal to it.