Improving nitrogen fixation in legumes by plant breeding; the relevance of host selection experiments in red clover (Trifolium pratense L.) and subterranean clover (T. subterraneum L.)

Summary This paper reviews (i) basic studies on the genetics of symbiosis in red clover (a self-sterile species) and subterranean clover (cleistogamous) and (ii) work on selection and plant breeding to increase nitrogen fixation in these hosts.Symbiotic effectiveness in red clover is influenced by many major and minor genes. The highly effective phenotype is inherited in a complex manner associated with early nodulation and the formation of large amounts of persistent bacteroid-containing tissue. Lines bred to fix more nitrogen with one strain ofRhizobium trifolii do so with most but not all other strains examined. They also show slightly increased vigour when grown on nitrate. The highly effective response is correlated with abundant nodulation and an early flowering habit, the evidence from breeding studies indicating that this correlation is not absolute. Normally effective and highly effective nodules have the same specific nitrogenase activities. The expression of the highly effective response is relatively little affected by environmental factors (temperature, light intensity, day length, supplementary carbon-di-oxide). Inbreeding substantially degrades the symbiotic response.Heterosis is shown in crosses between cultivars of subterranean clover but otherwise selection to increase effectiveness in this host was unsuccessful.The relevance of these results (and their physiological aspects) for the improvement of grain legumes is discussed.     

Effects of seed phosphorus concentration on the emergence and growth of subterranean clover (Trifolium subterraneum)

Subterranean clover seed (Trifolium subterraneum cv. Dalkeith) with phosphorus concentrations of 0.75% (high P seed) and 0.48% (low P seed) and of uniform size (2.0–2.4 mm diameter) was used to measure the effect of seed P concentrations on seedling emergence and growth.Seedling emergence numbers were 35% greater for the high P seed, and this effect was independent of external P supply. High P seed also emerged more quickly than low P seed.Leaf emergence was faster and shoot dry weight was greater for seedlings grown from high compared with low P seed, but only when external P supply was deficient for plant growth. Phosphorus concentrations in the shoots of two-week old seedlings were 32–51% higher for high P seed, although by four weeks plants grown from high and low P seed had similar concentrations of P in their shoots. We suggest that establishing pastures using high P seed would improve both early and late season pasture production.     

The potential for breeding white clover (Trifolium repens L.) with improved nodulation and nitrogen fixation when grown with combined nitrogen

Summary Sodium nitrate applications ranging from 0.36 to 22.84 mM N were shown to depress rates of nodule formation and reduce total nitrogen fixation (acetylene reduction) in white clover plants grown in aseptic test tube culture.Low nitrate levels gave an initial depression in symbiotic activity but the reduction was of short duration and these treatments were subsequently associated with enhanced rates of nodule formation and nitrogen fixation. As a result, phenotypic variation appeared to be strongly differentially affected by the amount of nitrate present. A subsequent experiment suggested that much of the variation was a consequence of early enhancement of plant growth rates by low levels of nitrate followed by rapid depletion thus giving a transitory inhibitory effect. This was confirmed in a third experiment in which the range of nitrate concentration was held constant. Differential effects on variability in nodule formation and nitrogen fixation were then greatly reduced but there was still a residual level of plant-to-plant variation. The results have clear implications for selecting genetic variants capable of fixing di-nitrogen in the presence of combined N. The provision of a single limiting dose of combined nitrogen to a population containing individuals with inherently different growth rates can bring about variations in the phenotypic expression of symbiotic characters. These variations are unlikely to be based on genetic factors which have a direct and stable effect on nodule development and nitrogenase activity. The implications of the results for plant breeding are discussed.     

Comparison of the growth rates of dinitrogen fixing subterranean clover swards with those assimilating nitrate ions

Summary Subterranean clover plants were grown as swards (about 2000 plants/m²) under controlled conditions with N provided either by N₂-fixation (NO ₃ ^– withheld) or by assimilation of NO ₃ ^– (NO ₃ ^– supplied). Crop growth rates were measured by dry matter sampling over periods of up to 70 days at PPFD values of 400–1000 mole quanta/m²/s. When NO ₃ ^– was supplied from sowing the swards grew more rapidly than when the swards were not supplied with NO ₃ ^– and plants had to establish an N₂-fixing apparatus. When inter-plant competition was reduced within the sward, a difference in growth rate in favour of NO ₃ ^– -fed plants continued for at least 50 days. When however, a closed canopy was allowed to form, the NO ₃ ^– -fed swards had more dry weight than the N₂-fed swards at the time of canopy closure but thereafter the two swards grew at similar rates at light flux densities of above about 800 mole quanta/m²/s. At light flux densities of about 400 mole quanta/m²/s N₂-fed swards had a growth rate 70–80% of that of NO ₃ ^– -fed plants. NO ₃ ^– -fed plants had a higher organic N content than did N₂-fed plants under all conditions.     

Response to soil aluminium of two white clover (Trifolium repens L.) genotypes

An aluminium (Al) tolerant genotype of white clover was compared with an Al susceptible genotype in artificial soil profiles in which exchangeable Al increased with depth. The tolerant genotype had a greater proportion of its root mass deeper in the soil than the susceptible genotype. Nitrogenase activity showed a similar pattern. Shoot Al concentration did not vary between the genotypes but root Al in the susceptible line was twice that in the tolerant genotype. Plant potassium content in the susceptible line was relatively less, probably in response to higher aluminium content.     

Pea responses to saline stress is affected by the source of nitrogen nutrition (ammonium or nitrate)

The effect of the source of nitrogen nutrition (ammonium or nitrate), onthe response of pea plants to a moderate saline stress (30 mMNaCl)was studied. Growth declined under saline stress but nitrate-fed plants wereless sensitive to salinity than ammonium-fed plants. This different sensitivitywas due mainly to a better maintenance of root growth in nitrate-fed plants.Organic nitrogen content decreased significantly in roots of ammonium-fedplants. Water relations changed slightly under saline stress leading to adecrease in stomatal conductance, which was correlated to a decline in carbonassimilation rates regardless of nitrogen source. Salinity affects the uptakeofseveral nutrients in a different way, depending on the nitrogen source. Thus,chloride was accumulated mainly in nitrate-fed plants, displacing nitrate,whereas sodium was accumulated mainly in ammonium-fed plants, especially inroots, displacing other cations such as ammonium and potassium. It is concludedthat the nitrogen source (ammonium or nitrate) is a major factor affecting pearesponses to saline stress, plants being more sensitive when ammonium is thesource used. The different sensitivity is discussed in terms of a competitionfor energy between nitrogen assimilation and sodium exclusion processes.     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

The influence of long-term tillage systems on symbiotic N2 fixation of pea (Pisum sativum L.) and red clover (Trifolium pratense L.)

Pea as a grain legume and red clover as a forage legume in the seeding year were cultivated in two long-term differentiated tillage systems on a loess soil in Germany. A continuous conventional tillage system (plow; CT) and a continuous minimum tillage system (rotary harrow; MT) were established in 1970. With pea and red clover dry matter accumulation and N parameters (N accumulation, Ndfa, N-harvest-index, N balance) were investigated in 1998 and 1999. Differences in the N₂ fixation of pea due to the tillage system could clearly be shown whereas grain yields and total N accumulation were equal in both tillage systems and years. In both years a significantly (P < 0.05) higher Ndfa in the MT system was found at least in the final harvest (maturity of pea): 1998/1999, 0.42/0.54 in CT, 0.62/0.75 in MT. The differences in N₂ fixation of pea may be explained by the delayed soil N supply in MT at the beginning of the vegetative period. Simplified N balances of pea were -18 and –25 kg N ha^–1 in CT and –5 and +1 kg N ha^–1 in MT for 1998 and 1999, respectively. Red clover showed no significant differences in the DM and N accumulation between both tillage systems but a year dependent effect caused by different stubble and root yields between the years was apparent. With red clover slightly, but also significantly (P < 0.05) increased Ndfa values were found in the MT system compared to the CT system with 0.55/0.62 in CT (1998/1999) and 0.64/0.71 in MT. However, the difference in Ndfa between the tillage systems (9 percentage points) was much smaller with red clover than with pea (20 and 21 percentage points in 1998 and 1999, respectively). Soil N uptake of red clover using the longer growing season reflected the more adjusted N supply in both long-term differentiated tillage systems, whereas pea in using only a short-term vegetative period reacted stronger to the lower N mineralization in the MT system in springtime.     

Flowering and gibberellins in a mutant red clover (Trifolium pratense L.)

Relationships between gibberellins and floral initiation were investigated in a conditional non-flowering mutant of red clover, Trifolium pratense. Untreated mutant plants will not flower under long-days, but will do so when certain GAs are applied. Gibberellins, A₃, A₁, A₇, and A₅ all resulted in both stem elongation and flowering whilst GA₄ produced the elongation only. Applications of GA₂₀, GA₈ and GA₁₃ under long-days had no detectable effect. Thus, by combining the use of the mutant with the application of different GAs, the correlation between the processes of stem elongation and floral initiation, which is normally strongly expressed in this species, was broken. Endogenous gibberellins shown to be present in normal plants were also found in the mutant genotype. Gibberellins alone were not sufficient to initiate floral development in the mutant, there being an essential element of interaction with long-days. These results are discussed in relation to the nature of the lesion in the mutant and the signal provided by the applied gibberellin.     

Inheritance of phosphorus response in white clover (Trifolium repens L.)

Genotypes of white clover that exhibited divergent responses to P were identified in a glasshouse pot trial. Six high P-responding genotypes were selected from previously identified high P-responding cultivars and 5 low P-responding genotypes were selected from previously identified low P-responding cultivars. These were crossed in a full diallel design without selfing and reciprocals were kept separate. The P-response of progeny lines was compared with parents. High P-response was dominant over low P-response with progeny from crosses between high and low P-response genotypes being similar to the high P-response parent. Reciprocal effects were not significant. The general combining abilities of high P-response genotypes were generally greater than that of the low P-response genotypes, although there were significant specific combining abilities. Narrow sense heritabilities for P response were moderate, 0.46 based on the linear coefficient and 0.33 based on the quadratic coefficient of the fitted response curves.The mode of inheritance, feasibility of manipulating differences in P response by breeding and future directions of this work are discussed.Deceased.Deceased.     

Changes in intracellular amino acids and organic acids induced by nitrogen starvation and nitrate or ammonium resupply in the cyanobacterium Phormidium laminosum

In Phormidium laminosum cells, nitrogen starvation caused a decrease in the intracellular levels of all amino acids, except glutamate, and an increase in the total level of the analyzed organic acids. The addition of nitrate or ammonium to N-starved cells resulted in substantial increases in the pool size of most amino acids. Upon addition of ammonium the total level of organic acids diminished, whereas it increased upon addition of nitrate, after a transient decay during the first minutes. Nitrogen resupply stimulated amino acid synthesis, the effect being faster and higher when ammonium was assimilated. The data indicate that nitrate and ammonium assimilation induced an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to amino acid biosynthesis, with a concurrent decrease in the carbohydrate reserves. The results suggest that the availability of carbon skeletons limited the rate of ammonium assimilation, whereas the availability of reducing equivalents limited the rate of nitrate assimilation.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) This work has been supported by grants from the Spanish Ministry of Education and Science (DGICYT and PB92-0464) and the University of the Basque Country (042.310-EC203/94) M.I.T. and J.A.G. were the recipients of fellowships from the Basque Government.     

Evidence for a polyamine-mediated control of soluble nitrogen mobilization during post-clipping re-growth of white clover (Trifolium repens L.)

A putative contribution of polyamines to the control of peptidase activity expression during re-growth was studied in source organs (roots and stolons) of defoliated white clover (Trifolium repens L.). Endopeptidase activity increased in roots during the first 6 days following complete defoliation, while exopeptidase expression seemed to be restricted to the early hours of re-growth. These changes correlated with an immediate 80% decline in the content of total free polyamines, mainly represented by the diamine cadaverine. The inhibitory capacities of cadaverine and spermine were tested on enzyme activity in vitro in order to elucidate whether the endogenous polyamine level was associated with the cut-induced endopeptidase expression. Cadaverine seemed to inhibit endopeptidase activity of stolons but not root endopeptidase activity. These data support the view that polyamines may play a role in the regulation of peptidase expression in source organs of white clover during post-clipping re-growth. The existence of different endopeptidase isoforms in roots and stolons is discussed in relation to the molecular mechanisms by which polyamines may regulate their activities.Abbreviations AP aminopeptidase - Cad cadaverine - CP carboxypeptidase - EP endopeptidase - PA(s) polyamine(s) - Spm spermine     

The growth and asymmetry of neighbouring plants of white clover (Trifolium repens L.)

Summary Transplants of white clover (Trifolium repens L.) were grown isolated from each other and in pairs placed at different distances apart. The paired plants developed asymmetrically and at the interface between paired clones both the density of nodes and of stolons appeared to reach ceiling values that were of the same order as those achieved in isolated clones. It is argued that the growth of plants of T. repens is controlled by the local conditions experienced by the plant parts and not by integrated growth of the whole. Transplants of three different genotypes of T. repens, which differed in growth form, were grown as neighbouring pairs and the calculated asymmetry of the plants was used to compare their mutual aggressivenes. The more compact (phalangeal) genotypes induced greater asymmetry in their neighbours than the more diffuse forms.     

Regulation of the appearance of glutamine synthetase in mustard (Sinapis alba L.) cotyledons by light,nitrate and ammonium

During transformation of mustard seedlings cotyledons from storage organs to photosynthetically competent leaves, a process which occurs during the first 4 d after sowing, total glutamine-synthetase (GS, EC 6.3.1.2) activity increases from zero to the high level usually observed in green leaves. In the present study we have used ion-exchange chromatography to separate possible isoforms of GS during the development of the cotyledons. The approach failed since we could only detect a single form of GS, presumably plastidic GS, under all circumstances tested. The technique of selective photooxidative destruction of plastids in situ was applied to solve the problem of GS localization. It was inferred from the data that the GS as detected by ion-exchange chromatography is plastidic GS.The regulatory role, if any, of light, nitrate and ammonium in the process of the appearance of GS in the developing cotyledons was investigated. The results show that nitrate and ammonium play only minor roles. Light, operating via phytochrome, is the major regulatory factor.Abbreviations c continuous - D darkness - FPLC fast protein liquid chromatography - GS glutamine synthetase (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) - FR far-red light (3.5 W·m^-2) - NF Norflurazon - R red light (6.8 W·m^-2, _R=0.8)) - RG9-light long-wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Genotype-dependent whole plant regeneration from protoplasts of red clover (Trifolium pratense L.)

Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.Research supported by USDA/CRGO Grant No. 81 CRCR-1-0613     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Effect of herbicide residues in a sandy loam on the growth,nodulation and nitrogenase activity (C2H2/C2H4) of Trifolium subterraneum

The herbicides 2,4-D, amitrole, atrazine, diclofop-methyl, diquat, paraquat and trifiluralin were applied at rates of 0, 2, 5 and 10 μg ai. g⁻¹ to a sandy loam soil and allowed to degrade for 120 days. After this period, subterranean clover seedlings were transplanted into treated soil and the effect of herbicide residues on plant growth, number of nodules formed and nitrogenase activity was investigated. At all rates of atrazine and chlorsulfuron, and at all rates of amitrole in excess of 2 mg ai g⁻¹ of soil, sufficient herbicide remained to be lethal to the seedlings. When amitrole was applied at the rate of 2 mg ai g⁻¹ of soil, plant growth, nodulation and nitrogenase activity of plants were reduced. Residues of diquat reduced all plant parameters studied while, residues of 2,4-D reduced plant growth and nodule formation, but plant nitrogenase activity was unaffected. Residues of trifluralin had no effect on plant growth parameters but the number of nodules formed per plant was reduced. Residues of paraquat and diclofop-methyl had no effect on any of the plant parameters studied.     

Effects of a cold treatment of the root system on white clover (Trifolium repens L.) morphogenesis and nitrogen reserve accumulation

The ability of white clover (Trifolium repens L.) to undergo cold acclimation is an important determinant of its persistence in mixed swards since growth rate at low temperatures sustains higher clover contents at the start of spring. During a re-growth period following defoliation, a gradual exposure of the root system (cv. Grasslands Huia) led to some physiological and morphological changes of cold-adaptive significance, similar to those developed by clover ecotypes originating in northern areas of Europe. Thus, cold exposure of the root system resulted in small-leaved prostrate forms of white clover after one month of re-growth. Similarly, cold exposure increased the ability of plants to store nitrogen since the application of low temperatures to the root system enhanced soluble protein accumulation in roots and in stolons. More specifically, cold exposure of the roots induced gene expression of a vegetative storage protein (17.3 kDa VSP) in both organs. These results demonstrate that the root system of clover plants should be a site of perception of the low-temperature stimulus, and gave rise to the question of the transduction of the cold signal from the roots to the aerial parts. On the basis of this study and taking into account molecular aspects concerning the clover VSP, it is suggested that this protein could participate in cold acclimation in addition to its role in nitrogen storage.