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Cadmium (Cd) is a leading environmental issue worldwide. The current study was conducted to investigate Cd tolerance of 10 commercial white clover (Trifolium repens) cultivars during seed germination and to further explore differences in lipid remodelling, glycometabolism, and the conversion of lipids into sugars contributing to Cd tolerance in the early phase of seedling establishment as well as the accumulation of Cd in seedlings and mature plants. The results show that Cd stress significantly reduced seed germination of 10 cultivars. Compared to Cd‐sensitive Sulky, Cd‐tolerant Pixie accelerated amylolysis to produce more glucose, fructose, and sucrose by maintaining higher amylase and sucrase activities under Cd stress. Pixie maintained higher contents of various lipids, higher DGDG/MGDG ratio, and lower unsaturation levels of lipids, which could be beneficial to membrane stability and integrity as well as signal transduction in cells after being subjected to Cd stress. In addition, Pixie upregulated expression levels of key genes (TrACX1, TrACX4, TrSDP6, and TrPCK1) involved in the conversion of lipids into sugars for early seedling establishment under Cd stress. These findings indicate that lipid remodelling, enhanced glycometabolism, and accelerated conversion of lipids into sugars are important adaptive strategies for white clover seed germination and subsequent seedling establishment under Cd stress. In addition, Pixie not only accumulated more Cd in seedlings and mature plants than Sulky but also had significantly better growth and phytoremediation efficiency under Cd stress. Pixie could be used as a suitable and critical germplasm for the rehabilitation and re‐establishment of Cd‐contaminated areas. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi may alleviate water stress in plants. Although several protection mechanisms have already been described, little information is available on how these fungi influence the hormonal response to water stress at an organ‐specific level. In this study, we evaluated the physiological and hormonal responses to water stress in above and below‐ground tissues of the legume grass Trifolium repens colonized by the arbuscular mycorrhizal fungus Rhizoglomus irregulare. Plants were subjected to progressive water stress and recovery. Different leaf and root physiological parameters, as well as phytohormone levels, were quantified. Water‐stressed mycorrhizal plants showed an improved water status and no photoinhibition compared to uncolonized individuals, while some stress markers like α‐tocopherol and malondialdehyde content, an indicator of the extent of lipid peroxidation, transiently increased in roots, but not in leaves. Water stress protection exerted by mycorrhiza appeared to be related to a differential root‐to‐shoot redox signaling, probably mediated by jasmonates, and mycorrhization enhanced the production of the cytokinin trans‐zeatin in both roots and leaves. Overall, our results suggest that mycorrhization affects physiological, redox and hormonal responses to water stress at an organ‐specific level, which may eventually modulate the final protection of the host from water stress. [ABSTRACT FROM AUTHOR]

Labeling 13CO2 in steady-state condition was used to estimate quantitative mobilization of recently fixed carbon or stored sugar during water-deficit in white clover (Trifolium repens L.). Water-deficient gradually decreased leaf-water parameters and total amount of recently fixed carbon. Amount of 13C incorporated into glucose, sucrose and soluble sugars fraction rapidly decreased after 3 days of water-deficit treatment. In contrast, the previously stored soluble sugars significantly increased after 5 days of water-deficit with a coincidence of significant decrease in starch concentration. A highly significant (P ≤ 0.001) relationship between the decrease in leaf-water potential caused by water-deficit and the increase in ratio of soluble sugar/starch concentration was observed in water deficit-stressed plants. The data indicate that soluble carbohydrate accumulated by water-deficit treatment is mainly because of the hydrolysis of previously stored starch rather than to de novo synthesis. [ABSTRACT FROM AUTHOR]

To investigate the responses of pathogenesis-related (PR) proteins to the intensity of drought stress and their physiological significance in white clover ( Trifolium repens L.), the change of enzyme activity and its relationship with some physiological parameters were assessed for 28 days under well-watered (control) and water-deficit conditions. Water-deficit treatment gradually decreased leaf water potential (Ψw) to −2.33 MPa at day 28. Dry matter significantly decreased from 21 days of water-deficit treatment, while proline and ammonia concentration increased within 7 days. The increase in PR-protein activity was closely related with the decrease in Ψw. The β-1,3-glucanase (EC 3.2.1.39) activity in water-deficit leaves rapidly increased for the first 14 days (Ψw ≥ −1.67) and then slightly decreased, while the chitinase (EC 3.2.1.14) and cellulase (EC 3.2.1.4) activity continued to increase throughout the experimental period. The enhanced activation of β-1,3-glucanase, chitinase and cellulase for the period of days 0–14 was significantly ( P ≤ 0.01) related to the increase of proline and ammonia concentrations. The results indicate that the enhanced activity of β-1,3-glucanase, cellulase and chitinase for the early period might be an act of transient tolerance to drought stress, but the activation of these enzymes during terminal stress might be a drought-stress-induced injurious symptom. [ABSTRACT FROM AUTHOR]

The stolons of white clover (Trifolium repens L.) contain a predominant 17.3-kDa protein, previously characterized as a vegetative storage protein (VSP), which accumulates under autumn and winter conditions. Its full-length complementary DNA, TrVsp, was obtained and its 157 amino acid sequence deduced. This VSP has common characteristics to stress-responsive proteins (high neutral amino acid content and potential alpha helices in its secondary structure) and shows high homologies to abscisic acid-responsive and pathogenesis-related-10 proteins. The lack of any common amino acid sequence domains with known dehydrins or late embryogenesis abundant proteins suggests that clover VSP is not related to these proteins. Antibodies raised against the protein were produced and used in light and electron microscopic studies to show that it is localized to the cytosol of cortical parenchyma cells. This is in agreement with the VSP sequence, which does not contain any transit peptide signal. The accumulation of the transcript and the protein in roots is quickly induced by root chilling, suggesting a direct transcriptional regulation of TrVsp in response to low temperatures. Altogether, these results suggest that the 17.3-kDa protein may have an additional or alternative function to its role in nitrogen storage and may confer putative tolerance to chilling in white clover. [ABSTRACT FROM AUTHOR]

Reverse-transcriptase-dependent polymerase chain reaction (RT-PCR) was used to identify three distinct DNA sequences (designated TR-ACS1,TR-ACS2 and TR-ACS3), each with identity to 1-aminocyclopropane-1-carboxylate synthase (ACS). Southern analysis confirmed that these sequences represented three distinct genes, and phylogenetic analysis showed that each gene was more closely related to homologues from other plant species than to each other. The expression of these three ACS genes during leaf ontogeny in white clover was examined. Northern analysis revealed that TR-ACS1 was expressed in the apical structure of the stolon in mature-green leaf tissue and in leaf tissue at the onset of senescence; RT-PCR analysis showed that TR-ACS2 was expressed in the apical structure of the stolon and in newly initiated leaf tissue. Furthermore, the expression of TR-ACS1 was regulated by the exogenous application of auxin. The third gene, TR-ACS3, was expressed in senescent leaf tissue, but translation of the nucleotide sequence provided an enzyme with a deleted binding site for pyridoxal-phosphate and S-adenosyl methionine (AdoMet), suggesting that this was a pseudogene. Antibodies, raised to the protein product of TR-ACS1 expressed in Escherichia coli, recognized a protein of 55 kDa in leaf extracts of white clover using Western analysis, with the pattern of protein accumulation corresponding to the pattern of gene expression determined using Northern analysis. The significance of these results is discussed with respect to the developmental expression of ACS genes during leaf ontogeny. [ABSTRACT FROM AUTHOR]

The present study was to assess the effect of heavy metal stress on the DNA methylation of a metal-sensitive plant, Trifolium repens L. and of a metal-tolerant plant, Cannabis sativa L. The changes in the level of 5-methylcytosine (5mC) in the root DNA of plants grown on soils contaminated with different concentrations of Ni2+, Cd2+ and Cr6+ compared with that of untreated plants, were determined by immunolabelling with a monoclonal antibody, using the Slot-Blot technique. Results showed that DNA of hemp control plants was about three times more methylated than clover DNA, for the same amount of root DNA. Heavy metal treatments induced a global dose-dependent decrease of 5mC content, both in hemp and clover, ranging from 20 to 40%. Changes in methylation pattern of 5′-CCGG-3′ containing sequences were investigated by methylation-sensitive amplification polymorphism (MSAP) technique. Control plants of the same species showed a very similar pattern, suggesting that, in normal condition, methylation involves precise sites. Heavy metals induced DNA methylation changes mainly related to hypomethylation events. These variations were not randomly directed but involved specific DNA sequences, since the detected polymorphisms were the same in all the plants analysed for each treatment. [ABSTRACT FROM AUTHOR]

The effects of potassium (K) deficiency on growth, N2-fixation and photosynthesis in white clover ( Trifolium repens L.) were investigated using natural occurring gas fluxes on the nodules in real time of plants under three contrasting relative addition rates of K causing mild K deficiency, or following abrupt withdrawal of the K supply causing strong K deficiency of less than 0.65% in dry matter. A steady-state below-optimum K supply rate led to an increase in CO2-fixation per unit leaf surface area as well as per plant leaf surface. However, nitrogenase activity per unit root weight and per unit nodule weight was maintained, as was the efficiency with which electrons were allocated to the reduction of N2 in the nodules. Abrupt K removals stimulated nodule growth strongly without delay, but as K concentrations decreased in the plant tissue a significant decline in nitrogenase activity per unit root weight as well as per unit nodule mass occurred. Further, the rate of photosynthesis per unit leaf area was unaffected, while the CO2 acquisition for the plant as a whole increased due to an expansion of total leaf area whereas the leaf area per unit leaf weight was unaffected. The ratio between CO2-fixation and N2-fixation increased, although not statistically significant, under short-term K deprivation as well as under long-term low K supply indicating a downregulation of nodule activity following morphological and growth adjustments. This downregulation took place despite a partly substitution of the K by Na. It is concluded that N2-fixation does not limit the growth of K-deprived clover plants. K deprivation induces changes in the relative growth of roots, nodules, and shoots rather than changes in N and/or carbon uptake rates per unit mass or area of these organs. [ABSTRACT FROM AUTHOR]

Petiole response of white clover to variations in blue light (BL) was studied on the main axis and on primary and secondary branches. The objectives of the present work were to determine (1) the time course of petiole response to BL and (2) whether these responses were dependent on petiole location. Under BL, clover had shorter petioles, and the switch to conditions without BL increased the length of forthcoming petioles. The fitting of a logistic function was used to compare the effect of BL on final petiole length, maximum elongation rate and the duration of petiole elongation between axes and phytomers. Petiole response to BL was not dependent on its location within the plant (axis type or phytomer position along the axis). A reduction in BL induced a rapid increase in leaf elongation rate, despite a small decrease in the duration of petiole elongation. Moreover, petiole response was dependent on petiole stage of development: the increase in the maximum rate of petiole elongation was inversely proportional to the petiole stage of development at the time of the switch. We conclude that the effects of BL on petiole elongation were not dependent on its position within the plant, whereas internode elongation resulted from the integration of light environment at the plant level. The difference between the responses of orthotropic and plagiotropic organs of clover to BL is discussed in relation to their structural function and localisation in the canopy. [ABSTRACT FROM AUTHOR]

Two isoforms of ACC oxidase (ACO) (EC 1.4.3), expressed differentially during leaf ontogeny in white clover (Trifolium repens L.), have been identified and purified to homogeneity. One isoform, designated MGI, was purified from mature green leaf tissue while the second isoform, designated SEII, was purified from senescent leaf tissue. The isolation and purification of these isoforms were achieved using a combination of hydrophobic interaction chromatography, anion exchange chromatography, chromatofocusing and gel filtration column chromatography. The Mr of both MGI and SEII was determined to be 37.5 kDa by gel filtration, and 37 kDa (MGI), 35 kDa (SEII) by SDS-PAGE, indicating that both isoforms are active as monomers. During purification, both isoforms were recognised by a polyclonal antibody directed against a recombinant polypeptide derived from a white clover ACO gene expressed in mature green leaf tissue, TR-ACO2. In addition to molecular mass, differences between the two isoforms were observed in terms of pH optima, isoelectric point (pI), Km for ACC, optimal requirements for the co-substrate ascorbate, and NaHCO3 and Fe2+ as co-factors. The identification of distinct ACC oxidases from the same tissue at different developmental stages shows that the now widely observed transcriptional regulation of the ACO gene family in higher plants is also expressed in terms of differential regulation of enzyme isoforms. [ABSTRACT FROM AUTHOR]

Changes in biomass production and partitioning, together with morphology and growth of three white clover (Trifolium repens L.) cultivars after exposure to winter stress were recorded in a controlled environment study. The cultivars used were Grasslands Huia (H, New Zealand), AberHerald (AH, bred in UK from material of Swiss origin) and Sandra (S, Sweden), sub-populations of these (survival populations), and a wild ecotype from the Uppsala area. The sub-populations were collected after 2 (with the exception of S) and 4 years of winter stress, respectively, at Uppsala (59°49′ N, 17°39′ E). In the controlled environment study the photoperiod was 12 h and there were two temperature regimes, namely 24/18 and 12/9°C (day/night), respectively. The experimental plants were cloned from eight different plants of each population. There were few interactions between populations and temperature regimes. Biomass production was lowest in the Swedish populations and in the surviving populations of the other cultivars collected after 4 years of exposure to winter stress. However, biomass partitioning, with the exception of allocation to branches, remained unaffected. Morphology differed significantly between cultivars, but, with the exception of internode length, was unaffected by exposure to winter stress. Internode length was significantly reduced in surviving populations of both H and AH after exposure to four winters, when its value was similar to that of the Swedish populations. The reduced internode length was a result of a greater reduction in the stolon extension rate than in the leaf appearance rate of survival populations compared with the original populations. It is hypothesised that reduced internode length would have implications for the carbon economy of the plant. [ABSTRACT FROM AUTHOR]

The seasonal pattern of concentrations of nitrogen, starch and vegetative storage protein (VSP) in stolons of Trifolium repens L. grown in the field were studied. Two different genotypes, cv. Aran and cv. Rivendel, differing in their morphology (stolon thickness and branching rate) but with similar growth rates, were used. Maximum concentrations of starch were found in summer whereas hydrolysis of starch took place throughout winter, suggesting that C storage is more important for winter survival than for promotion of early spring growth. On the other hand, VSP and nitrogen accumulated in autumn and early winter and then decreased when growth was resumed during early spring. For both cultivars, an inverse relationship was found between VSP concentration in stolons and mean air temperature, suggesting that VSP accumulation may be triggered by low temperature. Further experiments with plants grown under different regimes of temperature and daylength, suggested that VSP synthesis is stimulated by low root temperatures, with a slight synergistic effect of short daylength. The effects of root temperature on growth, N2 fixation, NH4+ uptake and N allocation within Trifolium repens L., were studied under controlled conditions. The shoot growth rate was greatly reduced when root temperatures were lowered from 12 to 6°C, while the rate of stolon growth was less affected. Low root temperatures inhibited N2 fixation more than it did NH4+ uptake, but the relative allocation of N to stolons was increased. Lowering root temperature also increased the accumulation of VSP in stolons. These results are discussed in terms of the mechanism associated with low temperature stimulation of VSP accumulation and its coupling with changes in the source/sink relations for allocation of N, between growth and storage. [ABSTRACT FROM AUTHOR]

Successful winter survival of perennial plants, like white clover, is dependent on proper timing of both hardening and dehardening. The purpose of this study was to investigate the regulation of these processes in two cultivars (AberCrest and AberHerald) and two Norwegian ecotypes (Særheim collected at 58°46'N lat. and Bodø at 67°20'N lat.) of white clover (Trifolium repens L.). For hardening and dehardening, plants were exposed to controlled temperature conditions and frost hardiness of stoIons was tested by programmed freezing at the rate of 3°C per hour. In addition, stoIons were analysed for starch, soluble sugars and soluble amino acids. Cultivars AberCrest and AberHerald, selected for growth at low temperature and winter hardiness in the United Kingdom, were significantly less hardy than the Norwegian populations. After six weeks of hardening (2 weeks at 6°C and 4 weeks at 0.5°C), estimated LT50 values were -13.8, -13.0, -17.8 and -20.3°C for AberCrest, AberHerald, Særheim and Bodø, respectively. The rate of dehardening increased with increasing temperature. At low temperature (6°C), the northern ecotype from Bodø was more resistant to dehardening than AberHerald. However, at 18°C the absolute rate of dehardening (°C day-1) was twice as high in Bodø as in AberHerald plants. Stolon elongation during dehardening was initiated at lower temperatures in AberHerald than in plants of the Bodø ecotype. The content of total soluble sugars, sucrose and the amino acids proline and arginine were significantly higher in hardy plants of Bodø than in those of AberHerald. Sucrose levels decreased during dehardening and correlations between sucrose content and LT50 during this process were statistically highly significant for both Bodø and AberHerald. The least hardy populations of white clover were characterized by thick stolons, long internodes and large leaves. [ABSTRACT FROM AUTHOR]

The patterns and variability in the diurnal and ultradian fluctuations in net uptake of K+ by 85-day-old plants of white clover (Trifolium repens L. cv. Huia) were analysed during 21 days of vegetative growth under natural light conditions, at constant root temperatures of 13 and 25°C. Plants were established in flowing solution culture, and hourly rates of K+ uptake from solutions containing 2.5-6.5 µM K+ were automatically measured without physical disturbance. Total daily solar radiation ranged between 1.3 and 12.5 MJ m-2 day-1 over the 21 days of measurements. Substantial diurnal changes in rates of K+ uptake, both absolutely and proportionately, were recorded on all but the day of lowest radiation (day 19), during which there was a period of net efflux of K+ from the root system into the flowing nutrient solutions at both temperatures. Typically, uptake rates increased severalfold during the first half of the light period, followed by several hours of relative stability during the afternoon; then a rapid decline throughout the evening and first half of the night, and finally gradual decline during the second half of the night. The data from a sequence of typical days were normalised and indicated that the pattern could be resolved into 4 distinct and linear phases, successively: (1) rapid acceleration, (2) stable uptake, (3) rapid deceleration, and (4) slow deceleration. The full data set (days 1-21) of hourly K+ uptake rates was Fourier-transformed and subjected to time-series analysis to determine whether there were significant ultradian rhythms in K+ uptake. Periodograms indicated a strong 24 h periodicity for uptake at both root temperatures. In addition, there were strong ultradian periodicities of 7 h for uptake at 13°C and 4 h for uptake at 25°C. The results are interpreted in terms of the possible mechanisms responsible for diurnal fluctuations in nutrient uptake. [ABSTRACT FROM AUTHOR]

To assess genotypic variability in nutrient supply of shoot branches, the distribution of 32P and 45Ca exported from a source nodal root (24‐h uptake period) was measured within a genotype of a large‐leaved (Kopu) and a small‐leaved (Tahora) cultivar of Trifolium repens. Source‐sink relationships of plants were modified by root severance, defoliation, and shade treatments. In control plants of both genotypes distribution of 32P and 45Ca closely followed the pathways that could be predicted from the known phyllotactic constraints on the vascular system. As such there was little allocation of radioisotopes (3.1% and 2.5% of exported 32P and 45Ca, respectively) from the source root to branches on the apposite side of the parent axis (far‐side branches). However, genotypic differences in nutrient allocation were apparent, when treatments were imposed to alter intra‐plant source‐sink relationships. In the large‐leaved genotype, the imposed treatments had minor effects on the allocation to far‐side branches: whereas, in the small‐leaved genotype, root severance and defoliation treatments increased lateral transport to far‐side branches to 30% (32P) and 10% (45Ca) of exported radioisotopes. Genotypes with low (8–9) and high (12–13) numbers of vascular bundles were selected from within the large‐leaved cultivar. Distribution of 32P was then measured after plants had been pre‐treated by removal of all far‐side roots two days prior to labelling. Genotypes with low vascular bundle number allocated 20% and those with high vascular bundle number 3.2% of exported 32P to far‐side branches. It was concluded (1) that genotypic variation exists within T. repens for potential to alter intra‐plant allocation of mineral nutrients, in response to treatments that modify source‐sink relationships within plants; and (2) that this variation is correlated with differences among genotypes in the organisation of the vasculature of their stolons. [ABSTRACT FROM AUTHOR]

Nitrogenase (N2ase; EC 1.18.6.1) activity (H2 evolution) and root respiration (CO2 evolution) were measured under either N2:O2 or Ar:O2 gas mixtures in intact nodulated roots from white clover (Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 mM) inhibition of N2-fixation was promoted by competition for light between clover shoots, which reduced CO2 net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery paralleled that of nitrogenase activity. Rhizosphere pO2 was increased from 20 to 80 kPa under N2:O2. A simple mono-exponential model, fitted to the nodule permeability response to pO2, indicated NO-3 induced changes in minimum and maximum nodule O2-diffusion permeability. Peak H2 production rates at 80 kPa O2 and in Ar:O2 were close to the pre-decline rates at 20 kPa 07. At the end of the nitrate treatment, this O2-induced recovery in nitrogenase activity reached 71 and 82% for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed. [ABSTRACT FROM AUTHOR]

Patterns of translocation of recently-assimilated phosphorus (P) exported from ‘young’ source roots (located 3–4 nodes from the stolon apex) and ‘old’ source roots (located near the base of the stolon) on the primary stolon of clonal plants of the forage legume white clover (Trifolium repens L.) were determined using 32P. Plants of a small-leaved genotype and of a large-leaved genotype were grown in sand culture at two notionally limiting or near-limiting rates of P supply and one non-limiting rate of supply. The small-leaved genotype showed little response in growth rate to the full range of P treatments whereas growth of the large-leaved genotype at the non-limiting rate of P supply was 2.4 times greater than at the two low rates of P supply. Source roots of both genotypes exported only 26–30% of the P they acquired to the shoot within 24 h when P supply was limited whereas at the high-P rate 54% of recently-assimilated P was exported. Patterns of translocation of exported P to specific sinks differed little between the genotypes and the P treatments; branches were the main sink, accounting for nearly 80% of the estimated amounts of P (µg day-1) exported from young and old roots combined. Translocation patterns from individual roots were determined largely by the modular structure of plants and by the location of the root relative to the major sinks, and were therefore consistent with the same source-sink principles which govern carbohydrate translocation in clonally-growing species. There were strong suggestions that storage of P in stolons and roots played a much greater role in the growth of the small-leaved plants than of the large-leaved plants. [ABSTRACT FROM AUTHOR]

Effects of mineral nitrogen (2, 4, 6 and 8 mM NH4NO3) and nodulation with Rhizobium on frost hardiness in seedlings of white clover (Trifolium repens) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of praline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply. [ABSTRACT FROM AUTHOR]

Seeds of white clover (Trifolium repens L., cv. Podkowa) were germinated at water potential ψ = -0.3 MPa in darkness, at 25°C. A short exposure to blue light (B) inhibited germination in a manner similar to that described earlier for red (R) and far-red (FR) light (Niedźwiedź-Siegień and Lewak 1989). No reversibility of B, R and FR effects was observed. Saturation irradiance and energy was the lowest for R and the highest for B. The reciprocity of irradiance versus time of exposure was observed only for non-saturating irradiances of B, R and FR. [ABSTRACT FROM AUTHOR]

To study a possible adaptation of the symbiosis between white clover (Trifolium repens L..) and Rhizobium leguminosarum biovar trifolii with regard to light and temperature at northern latitudes, local seed populations of white clover and isolates of R. leguminosarum biovar trifolii from 3 different latitudes in Norway, 58° 48'N, 67° 20'N and 69° 22'N, were used. The commercial cultivar Undrom was used as a reference plant. The experiments were done at 18 and 9°C under controlled conditions in a phytotron during the natural growing season at 69° 39'N. Growth of the plants was evaluated by number and size of leaves, dry matter production and total Ncontent. At 18°C the white clover plants were harvested twice while at 9°C there was only one growth period. The results from first harvest at 18°C and total growth at 9°C, showed that white clover populations from northern Norway had a lower growth potential than the population from the south and cv. Undrom. This difference was not apparent in the second growth period at 18°C. Growth of the plants from seeds to first harvest was enhanced by mineral nitrogen compared to plants dependent on Rhizobium only. However, after a second growth period dry weight and total nitrogen content of the plants with nitrogen fixation were comparable to the plants receiving mineral nitrogen. Statistical analysis showed that the most important factor for the variation in dry matter production was the plant population. Within the populations at 9°C and at first harvest at 18°C, there were no significant differences in dry matter production with different Rhizobium inoculum. In the second growth period at 18°C, different inoculum gave significantly different amount of dry matter within a population. The results showed a significant interaction between plant population and Rhizobium inoculum, and the results indicated that plants from the north gave higher yield when nodulated by Rhizobium from the north than from the south. [ABSTRACT FROM AUTHOR]

The source:sink ratio of clonal white clover (Trifolium repens L.) plants was manipulated by shading or removing leaves, and the consequences for carbon export from, and for the weight, area and net photosynthesis of, developing leaves were determined. When treatments were imposed just before young leaves usually change from C sinks to C sources, no effect on the point at which the sink to source transition occurred was observed. Leaves exported a similar proportion of the C they fixed, irrespective of stresses imposed upon the rest of the plant. However, differences in the destination of exported C were observed. More C moved to the stolon apex, and less to the stolon tissue itself, from leaves at Carlson stage 0.8 (leaflets about 60% unfolded) when mature leaves were removed or shaded. When 2 out of 3 mature leaves were removed from a stolon, short-term responses such as a 10% increase in net photosynthesis of the residual mature leaf, and greater export of C from this leaf to the apex, partially compensated very young leaves for loss of C supply. The result was that, when these young leaves were fully unfolded, they had similar surface area to those which had developed on undefoliated plants, but weighed nearly 20% less. Thus the immediate response to defoliation was an increase in the specific leaf area [cm2 (g of dry weight)-1] of new leaves, and the assimilation rate (net photosynthesis × area) of these leaves remained unchanged. [ABSTRACT FROM AUTHOR]

Effects of photoperiod and temperature on frost resistance of seedling populations and clones of white clover (Trifolium repens L.) originating from various latitudes (58°48'-69°54'N) and altitudes (up to 1 100 m above sea level) were studied in a phytotron. Low-temperature-induced cold acclimation in all populations was significantly stimulated by short photoperiod and low temperature. The highest levels of host resistance were found in the northernmost seedling populations and in clones from northern coastal locations. [ABSTRACT FROM AUTHOR]

Effects of photoperiod and temperature on vegetative growth of seedling populations and clones of white clover (Trifolium repens L.) originating from various latitudes (58°48'-69°54'N) and altitudes (up to 1100 m above sea level) were studied in a phytotron. Dry matter production, stolon elongation, petiole elongation and leaf lamina size were enhanced by long photoperiod. The requirement for long photoperiod increased with decreasing . temperature. At 6°C the maximum growth was recorded under 24-h daylength. At 18°C already an increase in photoperiod from 12 to 1.5 h significantly enhanced growth, and maximum growth was obtained at 21-h photoperiod. The studied populations responded similarly to daylength, and the results did not indicate photoperiodic ecotypes in the material. The southernmost clones and populations generally had the highest dry matter production at all temperature treatments (6-18°C). Variation between dories within one location was, however, significant, and rapidly growing clones were found also in high-latitude locations. Dry matter production was poorly correlated with the morphological characters observed, but in some cases significant correlation with leaf lamina size was found. [ABSTRACT FROM AUTHOR]

Photosensitivity of germination of white clover (Trifolium repens L. cv. Podkowa) seeds was studied under water deficit (low water potential) conditions at 25°C. The seeds showed negative photoblastism, which was most pronounced at ‐0.03 MPa polyethylene glycol solution. Inhibition was observed at two different wavelength bands with maxima at 660 nm (R) and around 730 nm (FR). Red light acted identically to white light (maximum inhibition ca 50%). The effect of far‐red illumination was less inhibitory (20–30%). The photoresponse required long illuminations (3 h exposures); saturation level was at 0.1 W m−2, independently of the light quality. White clover seed germination showed no reversibility of the effects of R and FR light. Prolonged illumination with R and FR increased the inhibition, and intermittent illumination had a higher effect than a continuous one. It was concluded that the photoinhibition of germination of seeds of Trifolium repens involves a reaction dependent on the rate of phytochrome interconversion, a property that is characteristic for the high irradiance reaction. [ABSTRACT FROM AUTHOR]

Net photosynthesis, concurrent carbon export and starch, sucrose and inorganic phosphorus concentrations were measured in leaves of white clover (Trifolium repens L. cv. Grasslands Huia) grown at four levels of phosphorus supply in the presence or absence of mineral nitrogen. The nitrogen treatments had no effect on growth, photosyntheis or carbon export. At the three higher levels of phosphorus supply, the amount of carbon exported was about 77% of net fixation. Photosynthesis and export per leaf decreased with phosphorus supply, primarily through the effect of phosphorus supply on leaf area. The rate of photosynthesis was reduced only at the lowest level of phosphorus supply. Inorganic phosphorus rose with phosphorus supply but starch concentration was unaffected. Sucrose was reduced at the lowest level of phosphorus supply but not significantly affected at higher levels. The ratio between starch and sucrose concentration was also unaffected at the higher levels, but was increased at the lowest level of supply. There thus appeared to be direct effects of phosphorus supply on photosynthesis, partitioning of carbon to carbohydrates and, by implication, export, only at the lowest level of phosphorus supply. As leaf area and plant growth were affected over the whole range of phosphorus supply, factors other than photosynthesis per se must have determined the response of growth to phosphorus supply. [ABSTRACT FROM AUTHOR]

The differential response of white clover (Trifolium repens L. cv. Regal Ladino) and berseem clover (Trifolium alexandrinum L. cv. Mississippi ecotype) was investigated by treating greenhouse cultured plants with 4‐(2,4‐dichlorophenoxy)butyric acid (2,4‐DB). Berseem clover plants were significantly injured by a treatment concentration of 0.6 kg ha‐1 of 2,4‐DB, whereas white clover plants were not injured by treatment levels below 2.4 kg ha‐1. The metabolism of 2,4‐DB in cell suspension cultures of white clover and berseem clover was investigated using [ring‐14C]‐2,4‐DB and non‐labeled 2,4‐DB. White clover cell cultures metabolized ca 4‐fold more 2,4‐DB than berseem cultures over a 44‐h treatment period. The decrease in berseem cell population was 4‐fold greater than the decrease in white clover cell population in response to the 8 μM 2,4‐DB treatment. The herbicide and its [ring‐14C]‐labeled metabolites were isolated from treated cells and medium after 44 h by partition and thin‐layer chromatography. White clover cells metabolized 90% of the [14C]‐2,4‐DB and berseem clover cells metabolized 22% of the herbicide. The major portion of the radiolabel was in the glycoside fractions from extracts of both species. The differential response of Trifolium species to 2,4‐DB is implied to be due to the differential rate of 2,4‐DB metabolism to a glycoside by the clover plants. [ABSTRACT FROM AUTHOR]

'Grasslands Huia' white clover (Trifolium repens L.), 'Grasslands Maku' lotus (Lotus pedunclulatus Cav.) and suckling clover (T. dubium Sibth) were grown in a controlled environment at various levels of P supply. Dry weights and the concentration of inorganic-, lipid-, ester- and residual-P in trifoliate leaves were measure. Lotus grew better than white or suckling clover at low P. White clover and lotus responded steeply to increased P and had similar shoot dry weights at high P. Suckling clover had lower shoot weights than the other species at all P levels. The superior growth of lotus at low levels of P was probably due to better root growth and P uptake. Lotus had higher shoot P concentrations at low levels of P but lower concentration than the others at high levels. White clover and suckling clover total leaf P concentration rose with P supply. Of the P fractions, inorganic- and residual-P showed the largest rises in concentration. The increases in lipid- and ester-P were smaller. Increases- and residual-P. White clover is a vigorous species but requires high levels of P for best growth. Suckling clover has a relatively small response to improvements in P availability. The behaviour of the various P fractions is similar to that in white clover. Lotus grows well at low P but also shows rapid growth at high P supply. Whether efficiency is defined as the ability to extract P from the environment or to maintain low internal P concentrations, lotus makes efficient use of P over the whole range of P supply. [ABSTRACT FROM AUTHOR]

Contact of adjacent root hairs of seedlings of white clover (Trifolium repens L. cv. Ladino and Louisiana Nolin) led to cell‐cell adhesion of root hair tips. The involvement of the root lectin, trifoliin A, in this phenomen was examined in slide cultures of axenically grown seedlings. Trifoliin A was detected by indirect immunofluorescence on root hair tips, which had adhered to one another. Seedlings grown under conditions which specifically reduce the levels of this lectin on the root surface (e.g., in the presence of 15 mM NO3– or 5 mM 2‐deoxy‐d‐glucose) had significantly fewer adhesions of root hair tips. In addition, flushing the slide cultures with 20 mM 2‐deoxy‐d‐glucose resulted in an immediate 4‐fold reduction in frequency of tip adhesions. These results are consistent with the lectin cross‐bridging model, which predicts that cell‐cell adhesions would occur when trifoliin A on root hair tips contacts complementary glycosylated receptors on neighboring root hairs. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the behaviour of different legumes against salinity and water stress, thus trying to discover simultaneous adaptations to both stresses. The nitrogen fixation, transpiration, predawn leaf water potential, and stomatal response of Medicago sativa L. (cvs. Tierra de Campos and Aragon), Trifolium repens L. (cv. Aberystwyth S‐184) and T. brachycalycinum Katzn. et Morley (=T. subterraneum L. cv. Clare) were compared at three levels of stress (0.05, 0.3 and 0.5 MPa of either NaCl or polyethylene glycol 6000) in nutrient solution. The plants were stressed for three days and then returned to control nutrient solution. The changes in the parameters analyzed were dependent on the proportion of stress treatments and the nature of the species, always being greater in plants from PEG than from NaCl solutions. Transfer of lucerne and subclover plants from NaCl at 0.05 MPa to a non‐saline medium resulted in an increase of nitrogen fixation above the level of the non‐salinized control plants, especially significant in lucerne. Analysis of possible inorganic impurities in commercial PEG suggest that such type of impurities are not responsible for the toxic effects reported. Plant damage resulting from PEG treatment was apparently due to penetrations of PEG (as determined qualitatively by using the tetraiodinebismuthic acid technique) or low‐molecular organic impurities into the plant. – The results are discussed as part of the adaptation of the different species to salinity and water stress. The best performance was given by “Tierra de Campos”. [ABSTRACT FROM AUTHOR]

Proliferating shoot cultures of Trifolium repens L. cv. Grasslands Huia have been established on Murashige and Skoog's medium containing 3% sucrose and 0.2 mgl-1 benzylamino purine. The shoots could be readily rooted on media without cytokinin and transferred to potting mix with 100% survival. The cultured shoots have been stored for 10 months at 5°C in darkness without adversely affecting their potentiality for rapid multiplication. The significance of this approach for genotype storage is discussed. [ABSTRACT FROM AUTHOR]

Stomatal behaviour, transpiration and nitrogen fixation were investigated in Medicago sativa L. (cvs. Tierra de Campos and Aragon, Hidalgo-Maynar 1966), Trifolium repens L. (cv. Aberystwyth S-184) and Trifolium subterraneum L. (cv. Clare) subjected to drought by withholding water and then to three days' recovery after rewatering. Dawn leaf water potential was measured with pressure chamber, stomatal response with a diffusion porometer and nitrogen fixation by using acetylene reduction technique. At low water potentials, the leaf resistance was higher in Medicago than in Trifolium. As water stress developed all species decreased their transpiration, T. subterraneum being the one most affected by moderate deficits. During water stress 'Tierra de Campos' always maintained higher acetylene reduction levels than 'Aragon' and the Trifolium species, except for the lowest water potentials. During recovery from water stress only 'Tierra de Campos' reached predeficit transpiration rates. In 'Tierra de Campos' acetylene reduction recovery after rewatering was more rapid and intense than in 'Aragon'. It is concluded that, of the plants investigated, 'Tierra de Campos' was best adapted to water deficits. [ABSTRACT FROM AUTHOR]

Autoradiography was used to provide evidence for the transfer of Rhizobium produced moieties to the host nuclei of young clover nodule cells. Cells of Rhizobium trifolii Dangeard were labeled with 3H-adenine or 4,5-3H-l-leucine and 3,4-3H-L-proline, washed free of external label, and allowed to nodulate young seedlings of white clover (Trifolium repens L. cv. White Dutch). Sections (0.5-1.0 μm) of young nodules up to four days old were autoradiographed using the dipping technique. Grain counts indicated movement of tritium from the leucine-proline labeled rhizobia to the polyploid nuclei of two day old clover nodule cells. Acetylene reduction was not detected until approximately 24 hours after the transfer of tritium was observed. No transfer of tritium was observed with 3H-adenine labeled rhizobia. It is hypothesized that nodulating rhizobia may induce clover nodule cells to initiate leghemogiobin synthesis by the transfer of a bacterially produced inducer. [ABSTRACT FROM AUTHOR]