Phosphate and Membrane Electropotentials in Trifolium repens L.

In Trifolium repens L. there were immediate transient depolarizationsof the membrane electropotential (E_vo) when KH₂PO₄ was addedto phosphate-free media, but these were of the same magnitudeas the controls (K²SO⁴ and KCI). Furthermore, the extents ofdepolarization were the same as the expected effect of the addedK⁺ calculated using the Goldman equation. There was no significantdepolarization on adding H₃PO₄ to buffered media. Consequently,there was no evidence for a depolarization caused by phosphate.This result provides evidence that the H⁺–H₂PO₄ symportin roots of T. repens operates with a stoichiometry of 1: 1. In a group of control plants ( + P plants) and a group whichwere stressed by reducing the supply of phosphate (– Pplants), the – P plants had lower values for E_vo than+P plants (– 118 mV and – 130 mV, respectively).The absence of phosphate from the measurement media also reducedE_vo (mean effect = 9 mV). A significant difference in E_vo between– P and + P plants persisted when phosphate was addedto – P plants. The electropotential difference acrossthe tonoplast (E_vo) in – P plants became more positivewith time. Key words: White clover, membrane transport, roots, tonoplast, symport     

Detection of selection in populations of white clover (Trifolium repens L.)

Seed populations of white clover polymorphic for the presence/absence of both ovariogenic glucosides and the hydrolysing enzyme linamarase, were introduced into three natural populations. Over the first six months of life a significant increase in the frequency of linamarase containing individuals occurred. Estimated selection coefficients against plants lacking linamarase were in the region of 0.3. This result may have been due to selection at the enzyme locus alone, or to selection favouring cyanogenic individuals which possess both cyanogenic glucosides and enzyme.     

Variation in growth attributes of contrasting populations of Trifolium repens

A range of growth attributes was measured in seedlings of 10 Trifolium repens populations, differing in leaf size and origin, grown in three temperature and two glasshouse environments. Growth rates of large leaf types of Mediterranean origin were higher than those of smaller leaf types at 10°C. However, the greater temperature response of the smaller leaf types resulted in higher growth rates for S.100 and S.184 than for a large leaf type from Israel at 20°C. The increase of growth rate with temperature was associated with changes in leaf area ratio and net assimilation rate between 10° and 15°C but only with changes in net assimilation rate between 15° and 20°C. Within each temperature environment, population differences in growth rate were related to differences in net assimilation rate rather than leaf expansion. At low temperature a greater proportion of dry matter was distributed to leaf tissue in large leaf types particularly those of Mediterranean origin but they showed a proportionately smaller increase in allocation to leaves with increasing temperature compared with small leaf types. In the glasshouse environments growth rates in spring were more than double those in the autumn. This difference was associated with net assimilation rates which were about five times greater in the spring environment. However, leaf area ratios in the spring were only half those in the autumn. These differences in leaf area ratio between the glasshouse environments were closely related to differences in specific leaf area but not to differences in distribution of dry matter to leaf tissue which was greater in the spring environment.     

Variation in growth and ion accumulation between two selected populations of Trifolium repens L. differing in salt tolerance

Two divergent populations of T. repens cv. Haifa developed from two generations of recurrent selection for shoot chloride concentration, were grown in the greenhouse at 0 and 40 mol m^–3 NaCl. Over two harvest cycles at 40 mol m^–3 NaCl, the population selected for a low concentration of chloride in the shoot maintained a significantly lower chloride and sodium concentration compared with those plants selected for a high shoot chloride concentration. The distribution of chloride in the shoots was further examined in a subsample of plants from both populations. In all plants, concentrations of chloride were lower in the expanding and fully expanded leaves than in the older leaf tissue or petioles.While there were no significant differences in the photosynthetic rates between lines, shoot yields and relative leaf expansion rates were higher in the low chloride population. Plant death was greater in plants selected for high shoot chloride. These results suggest that selections based on measurements of low shoot chloride concentrations may be successful in developing a cultivar of T. repens with improved salt tolerance.     

Genetic analysis of shikimate dehydrogenase allozymes in Trifolium repens L.

 A genetic analysis was carried out on progeny families from pair crosses among plants polymorphic for shikimate dehydrogenase (SDH) isozymes in white clover (Trifolium repens L.). SDH was controlled by two independently assorting disomic loci. This result is consistent with the presence of a single gene (Sdh) represented in this putative allotetraploid by one locus in each of the genomes. One of the Sdh loci is linked (6.0±2.0 cM) to the linamarase (Li) locus. There was no evidence for differentiation of the duplicate Sdh loci (both carry common alleles). White clover behaves genetically as a diploidised allotetraploid but the possibility of a low frequency of multivalent formation and homoeologous pairing has not been ruled out. The SDH locus is likely to be useful for the marker-assisted selection of agronomic traits. Received: 5 June 1997 / Accepted: 19 November 1997     

Accumulation of Metabolites during Seed Development in Trifolium repens L.

Metabolite deposition during seed development was examined histochemicallyin Trifolium repens by light- and fluorescence microscopy. Allendosperm haustorium at the chalazal pole of the embryo sacand wall protrusions in cell walls of the suspensor and theembryo sac suggest that transfer of metabolites from maternalto offspring tissue takes place primarily at these sites. Thisis further supported by prominent cutinization of the interpolarregion of the embryo sac wall, accumulation of starch in integumentaltissue at the embryo sac poles, and breakdown of interpolarendothelial cells. Decomposition of osteosclereid starch isfollowed by accumulation in the cellular endosperm and subsequentlyin the embryo parallel to endosperm degradation. The starchaccumulates gradually inward from the subepidermal cells ofthe embryo to the stele. Protein bodies are formed in the vacuolesalong the tonoplast, later to be cut off in vesicles releasedinto the cytoplasm. At maturity the embryo is packed with proteinand starch, but without lipid reserves. Phytin is observed inthe protein bodies. The mature embryo is surrounded by a proteinand starch containing aleurone layer which originates from theendosperm.Copyright 1994, 1999 Academic Press White clover, protein, starch, cuticle, embryo sac wall     

Physiological integration in the clonal perennial herb Trifolium repens L.

Summary Translocation of ¹⁴C-labelled carbohydrates between the parent stolon and branches, and among branches, of Trifolium repens plants was investigated in two glasshouse experiments to determine patterns of physiological organisation in this clonal species. Differential defoliation treatments were applied to the parent stolon and/or branches to test the sensitivity of translocation to the short-term carbon needs of defoliated sinks. Strong reciprocal exchange of carbohydrate between the parent stolon and branches was observed, with 18 41% of the ¹⁴C exported from leaves on the parent stolon moving to branches, while branches simulta-neously exported 25% (for old source branches) to 54% (for young source branches) of the ¹⁴C they assimilated to the parent plant, including translocation to other branches. Branch-to-branch translocation occurred both acropetally and basipetally. Parent-to-branch, branch-to-parent and branch-to-branch carbon fluxes all increased in response to defoliation of the sink, at the expense of carbon supply to stolon tissue or roots of the source module. Reduced export to stolon tissue of the parent axis played a major role in facilitating C reallocation from leaves on the parent stolon to defoliated branches. The observed patterns of C allocation and translocation could be adequately explained by accepted source-sink theory, and are consistent with a high degree of intra-plant physiological integration in resource supply and utilisation. This information provides mechanistic explanations for aspects of the growth dynamics and ecological interactions of T. repens in the patchy environment of a grazed pasture.     

Cumulative activation of axillary buds by nodal roots in Trifolium repens L

In Trifolium repens the rate of outgrowth of an axillary bud was closely correlated with its duration of exposure to a nearby nodal root. The dose-dependent nature of this relationship, over 0-22 d, is consistent with the concept that axillary buds are cumulatively activated by a root signal (RS) such that the longer they receive the signal the higher is their level of activation and hence their rate of outgrowth. Furthermore, the activation level attained by a bud was subsequently retained following the excision of the nodal root providing the source of its activation: its rate of growth 3-6 weeks after root excision still reflected the initial level of activation of the bud. Thus, once activated, a bud required relatively little RS to maintain its rate of outgrowth, implying that activation involves the establishment of an autonomous control mechanism within the bud itself. This provides an explanation of how a strongly activated apical bud can continue growth at relatively low RS levels when it is distanced from its nearest root system, while at the same time the prevailing low RS environment leads to weak activation of the axillary buds emerging from it.     

On the origin of the cyanogenic polymorphism in Trifolium repens L.

Trifolium repens L. and Trifolium nigrescens Viv. are two of the approximately six cyanogenic species known in the genus Trifolium. The two species are closely related: T. nigrescens is considered to be one of the diploid ancestors of the amphidiploid T. repens . We studied morphology, meiosis and the cyanogenic system in T. repens (amphidiploid), T. nigrescens (diploid) and their reciprocal hybrids. A comparison of the enzyme linamarase in the species and hybrids shows that there is a general resemblance between their linamarases. Immunological studies indicate that the linamarases must have a somewhat different three-dimensional structure. These facts are consistent with the view that T. nigrescens (or an ancestral form of this species) has donated the Li gene of T. repens . The other putative parent, T. occidentale has probably not donated an active Li gene. The hypothesis of the origin of the Li gene does not explain its polymorphism in European populations of T. repens , as T. nigrescens is monomorphic for cyanogenesis and amphidiploids do not segregate for genes which are homozygous dominant in one of the parents. Segregation for Li could be caused by a gene mutation or a small exchange between homeologous chromosomes. The latter event is more probable. A nigrescens-repens exchange would give rise to a chromosomal region with reduced homology to both parental chromosomes. The genes in the region of exchange will be tightly linked due to diminished cross-over frequency. It has been known for years that Li has effects on the vegetative and reproductive characters in T. repens and we have recently shown that these effects must be the result of genes linked to Li . As the associated characters influence the fitness of the cyanotypes, not only the origin but also the maintenance of the cyanogenic polymorphism is closely related to the evolutionary history of T. repens .     

Self fertilization and seed set in Trifolium repens L. by in situ and in vitro pollination

Summary The frequency of seed formation has been determined from self-pollination in situ (by hand) and in vitro for Trifolium repens. Selfing in situ was measured over a period of 3 years in which plants were held either at 35 °C for 24 h post-pollination (1984 and 1985) or held at ambient temperatures (1986). Mean yield of self-seed per 100 florets was 2.8 in 1984, 5.2 in 1985 and 2.2 in 1986. This was based on over 15,000 selfings per year with seven varieties and a total of 166 genotypes. In general, seed set following self-pollination was low; 53% of all genotypes set less than one seed per 100 florets selfed. Selfing of 340 excised florets in vitro with six genotypes gave a mean of 30.6 seeds per 100 florets. Temperature treatments (post-pollination) had no significant effect on seed yield in vitro. Treatment of florets in vitro for 24 h post-pollination with 0.1% CO₂ increased the yield of self seed with three genotypes but had no effect on a fourth genotype.     

Direct measurement of nitrogen fixation by Trifolium repens L. and Alnus glutinosa L. using 15N2

A closed-system flow-through enclosure apparatus was used tomeasure symbiotic nitrogen fixation directly. A legume-basedsystem comprising 6-week-old Trifolium repens L. (white clovercv. Blanca) growing with Lolium perenne L. (perennial ryegrasscv. Trani) in an agricultural soil was incubated for 19 d ina ¹⁵N-enriched atmosphere (mean value 3.663 atom%). An actinorhizal-basedsystem comprising 1 -year-old Alnus glutinosa L. (alder) saplingsgrowing with Festuca rubra L. (red fescue) in open-cast coalspoil was incubated for 21 d in a ¹⁵N-enriched atmosphere (meanvalue 3.265 atom%). Indirect estimates of N2 fixation were carriedout concurrently using N difference and ¹⁵N isotope dilutiontechniques. The theory underlying the three techniques and modificationswhich were adopted for comparative purposes are discussed. Thedirect measurements of N2 fixation were then compared with theindirect estimates using P_inc, the proportion of the N incrementduring the measurement period that was derived from fixation.The simple N difference method gave similar values for Pinc(0.94 and 0.97) as those derived from more complicated isotopemethodologies, both indirect (0.91) and direct (0.90). Valuesfor alder were far more variable, ranging from 0.16 to 0.92;this was due largely to variability within the trees and a verysmall N increment during the measurement period. Key words: N₂ fixation, ¹⁵N₂, white clover, alder, enclosure apparatus     

Effects of nitrogen on leaves,dry matter allocation and regrowth dynamics in Trifolium repens L. and Lolium perenne L. in pure and mixed swards

The effects of applied nitrogen (N) on dynamics of regrowth, dry matter (DM) allocation and leaf characteristics of grass and clover were investigated. Binary mixtures and monocultures of the diploid perennial ryegrass cultivars Barlet (erect) and Heraut (prostrate) and the white clovers cvs. Alice (large-leaved) and Gwenda (small-leaved) were established in a field experiment. Grass monocultures received three levels of N application (0, 140 or 280 kg N ha^–1), and mixtures 150 kg N ha^–1 (+N) or no N (–N). N was applied split over the season. Application of N reduced the average clover content in the DM of the mixtures from 43 to 12%. Due to defoliation, clover lost relatively more leaf area and less DM than grass, leading to a lower clover fraction in the leaf area index (LAI) of the stubble at the start of the next regrowth. In the –N mixtures, the clover fraction of the biomass and of the LAI increased within successive regrowth periods. In the +N mixtures, large-leaved Alice maintained its content during summer, mainly due to its greater petiole length which increased in response to N. The opposite was observed for Gwenda. At each harvest, the content of small-leaved Gwenda in the LAI and DM was lower than in the stubble at the start of regrowth. The allocation of DM to the petioles of Alice led to a decrease in the leaf weight ratio (LWR) in the +N mixtures, while Gwenda had a higher LWR and specific leaf area (SLA) in the +N mixtures than in the –N mixtures. There was little or no effect of ryegrass cultivar on competition with white clover.     

Influence of Temperature and Floret Age on Nectar Secretion in Trifolium repens L.

The influence of temperature on nectar secretion in non-pollinatedflorets of Trifolium repens was investigated in growth chambersat 10, 14, 18 and 22°C. The effect of temperature on therate of nectar secretion was significant in all clones. Theoptimum temperature for secretion in three clones varied from10°C for a clone of Icelandic origin, to 18°C in a cloneselected from a Danish variety. Similarly, the average nectaryield varied significantly among clones of different geographicalorigin. One clone secreted two to four times more than othersat 10°C. The optimum day temperature for nectar secretionwas higher when the plants were exposed to low night temperature,presumably a result of decreased night respiration. Nectar accumulatedat the floret base until senescence. Evidence for reabsorptionof nectar was obtained in four clones. Sucrose, fructose andglucose were identified as the major sugars in the nectar. Highnight temperatures led to decreased sucrose percentage in favourof glucose and fructose. The frequency of new florets openingper day was not influenced by temperatures between 10 and 22°Cin one clone, whereas low temperatures significantly decreasedthe number of new florets in another. Few or no modified stomatawere observed in the epidermis of the nectary. The high variationwith respect to nectar secretion at low temperatures, alongwith the high heritability of this quality, suggests that breedingfor high nectar production at low temperature is plausible.The significance of nectar yield in pollination biology is discussed.Copyright1994, 1999 Academic Press Trifolium repens, white clover, nectar, temperature, floret age, flowering, nectary     

Photosynthetic 14CO2 fixation products in altitudinal ecotypes of Trifolium repens L. with different temperature requirements

Summary Two Trifolium repens clones from natural meadows at 600 m and 2030 m above sea level, and with differing dependence on temperature of their rate of apparent photosynthesis, were grown under controlled environments. Radioactive products in detached leaves were examined after 20 and 40 s periods of steady state photosynthesis in ¹⁴CO₂ at 3° C and 24° C. Glycine and serine were hardly labeled at 3° C. At 24° C, the leaves of the alpine clone showed significantly, (P<0.025) more activity in these amino acids than those from the low altitude clone. It is suggested that the alpine clone has a higher photorespiration. This is supported by the labeling pattern of glucose, fructose, sucrose, and glucose-6-phosphate.The high altitude clone requires lower temperatures for photosynthesis than the low altitude clone. It is suggested, that this is caused by its higher photorespiration, which reduces net photosynthesis at high temperatures. The lower photorespiration activity of the low altitude clone can be interpreted as an adaptation to its warmer habitat.     

Functional plasticity of Trifolium repens L. in response to sulphur and nitrogen availability

Recent control of atmospheric SO₂ pollution is leading to important soil sulphur impoverishment. Plasticity could be a mechanism allowing species to adapt to this rapid global change. Trifolium repens L. is a key grassland species whose performances in community are strongly linked to nitrogen availability. Plasticity of three white clover lines contrasting in their ability to use atmospheric N₂ or soil N was assessed by analysing a set of functional traits along a gradient of nitrogen and sulphur fertilisation applied on a poor soil. White clover traits showed high morphological and physiological plasticity. Nitrogen appeared to be the most limiting factor for the VLF (Very Low Fixation) line. S was the element that modulated the most traits for the nitrogen fixing lines NNU (Normal Nitrate Uptake) and LNU (Low Nitrate Uptake). As expected, N fertilisation inhibited white clover fixation, but we also observed that N₂ fixation was enhanced when S was added. S fertilisation increased nodule length as well as the proportion of nodules containing leghaemoglobin. S fertilisation, with a direct effect and an indirect effect through N₂ fixation, increases white clover performances particularly with regards to photosynthesis and potential vegetative reproduction. The important plasticity in response to S availability should allow it to adapt to a large range of abiotic conditions, but its sensitivity to S nutrition would be a disadvantage for competition in a situation of soil sulphur impoverishment. In contrast, S fertilisation could help maintain this species when nitrogen status is against it.     

Studies on the {beta}-glucosidase System of Trifolium repens L.

Using DEAE-cellulose two distinct ß-glucosidase enzymeshave been identified in white clover; one form is primarilyassociated with the seed, the other with young leaves. Evidenceis presented for the separate nature of ß-glucosidaseand ß-galactosidase activity in dry seed flour andin young leaf tissue. These results are considered in relationto the function of the Li locus in white clover, which is shownto control both ß-glucosidase and ß-galactosidaseactivity in expanding leaves.     

Differences between Natural Populations of Trifolium repens L. in Response to Mineral Nutrients

The response to phosphate of samples of natural local populationsof T. repens, collected from soils of different phosphate contents,was determined over a wide range of phosphate levels in sandculture. A marked correlation (r = +0.96) was found betweenresponse of populations to phosphate at low phosphate levelsand the amount of phosphate in the native soil of the population;populations from soils high in phosphate showed larger reductionsin growth at low phosphate levels than did those from soilslow in phosphate. It is concluded that response to phosphateis one aspect of the physiological adaptation of the naturalpopulations to particular soil conditions. Populations alsodiffered in response to very high levels of phosphate, and inthe range of phosphate levels tolerated; the possible adaptivesignificance of this is discussed in relation to the phosphatebuffering effects of the native soils. Populations tolerantof low phosphate levels had higher concentrations of phosphorusin the shoot material, and a higher uptake of phosphorus perunit weight of roots, than did intolerant populations. The differencesbetween populations in response to phosphate are therefore believedto be due to difference in uptake rather than to differencein metabolic requirement of phosphate.     

Reproductive capacity of bacteroids in nodules of Trifolium repens L. and Glycine max (L.) Merr.

Reproductive growth of intracellular bacteria from isolated protoplasts in nodules of clover and soybean was directly investigated using a microchamber with visual and video recording. Differentiated bacteriods from clover nodules uniformly failed to reproduce. Such growth as occurred came from undifferentiated rhizobia from within the protoplast or extracellularly in the nodule. Plating investigation gave results in agreement with this conclusion. Osmoprotective media failed to secure the reproduction of differentiated clover bacteroids. Reproductive growth of bacteroids from protoplasts and crushed nodules of soybean was regularly observed in the microchamber and determined as proportionate colony-forming ability (CFA) on laboratory media. The CFA markedly increased with age of nodule and with the addition of nodule or root extract. The promoting effect of such extracts was reduced after heating for 60 min at 100°C, and lost completely after 20 min at 121°C. High osmolarity in the suspending and culture media was detrimental to bacteroid recovery.Abbreviations BMM Bergersen's modified medium - B^+m BMM with additional mannitol - CDB Chlamydomonas dilution buffer - PDB protoplast dilution buffer - PDB PDB without mannitol or sorbitol - RMM Rhizobium minimal medium - R^+m RMM with mannitol instead of sucrose - YMA, YMB yeast mannitol agar and broth, respectively. For details, see Materials and methods     

Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.