White Clover N2-Fixation in Response to Root Temperature and Nitrate: I GROWTH AND UPTAKE OF NITRATE FROM FLOWING NUTRIENT SOLUTIONS

Established, nodulated white clover plants (Trifolium repensL. cv. Blanca) were transferred to eight plant culture unitsof a system of flowing solution culture. Solution temperatureswere 5, 11, 17, and 25 ?C (two units per temperature), withshoot temperature of 25/15 ?C day/night and light regime commonto all plants. After 7 d, was supplied at 10 mmol m^–3 to one of each pair of culture units and thenet uptake of was monitored over 14 d. The remaining four culture units served as controls in which plantswere entirely dependent on N₂ fixation, as estimated by sequentialharvesting and the change in the amount of N in plants. Totalfresh and dry weights increased exponentially with time andwith increasing root temperature, between 5–25 ?C in nitrateplants and 5–17 ?C in control plants, respectively, byfactors of 2.9 and 1.8. Nodule dry weight of nitrate plantsshowed little increase after 6 d compared with control plants.Dry weight shoot: root ratios increased with time in all treatmentsexcept 5 ?C control plants. Total net uptake of over 13 d was 0.41, 4.27, 11.92, and 12.77 mmol plant-1, respectively,at 5, 11, 17, and 25 ?C. At all temperatures except 5 ?C, plantsaccumulated high concentrations of (10–40 mol m^–3) in leaflets and roots, within 2 or 3 days ofsupplying . Daily mean unit absorption rates of increased 12-fold with increasing temperature between 5 and 25 ?C, and showed little change with time at 5,17, and 25 ?C. The total N₂ fixed by nitrate plants over 14d increased 5-fold with temperature between 5 and 17 ?C, butwas always less than the amount fixed by control plants. Thepercentage contributed by N2 fixation to total N acquisitionby nitrate plants over 14 d decreased with increasing temperature,from 77% at 5 ?C to 11 % at 25 ?C. Mean daily rates of N₂ fixationper unit dry weight of nodule were lower in nitrate plants thanin control plants throughout treatment at 5 ?C and 25 ?C, butat 11 ?C and 17 ?C the rates for nitrate plants increased progressivelywith time and exceeded the rates for control plants after 8d. In both nitrate and control plants the effect of temperatureon N₂ fixation per nodule dry weight was proportionately lessthan that on unit absorption rate of . The results are discussed in terms of the overall regulation ofN accumulation by white clover and the adaptive significanceof differences in the sensitivities of uptake and N₂ fixation to root temperature. Key words: Trifolium repens, white clover, root temperature, N₂ fixation, nitrate uptake     

N2 Fixation and Nitrate Uptake by White Clover Swards in Response to Root Temperature in Flowing Solution Culture

Nodulated white clover plants (Trifolium repens L. cv. Huia)were grown as simulated swards for 71 d in flowing nutrientsolutions with roots at 11 C and shoots at 20/15 C, day/night,under natural illumination. Root temperatures were then changedto 3, 5, 7, 11, 13, 17 or 25 C and the total N₂, fixation over21 d was measured in the absence of a supply mineral N. Alltreatments were subsequently supplied with 10 mmol m^–2NO₂ ^– in the flowing solutions for 14 d, and the relativeuptake of N by N₂, fixation and NO₃ ^– uptake was compared.Net uptake of K⁺ was measured on a daily basis. Root temperature had little effect on root d. wt over the 35-dexperimental period, but shoot d. wt increased by a factor of3.5 between 3 and 25 C, with the sharpest increase occurringat 7–11 C. Shoot: root d. wt ratios increased from 25to 68 with increasing temperature at 7–25 C. N₂-fixationper plant (in the absence of NO₂ ^–) increased with roottemperature at 3–13C, but showed little change above13 C. The ratios of N₂ fixation: NO₂ ^– uptake over 14d (mol N: mol N) were 0.47–0.77 at 3–7 C, 092–154at 11–17 C, and 046 at 25 C, reflecting the dominanceof NO₃ ^– uptake over N₂ fixation at extremes of high andlow root temperature. The total uptake of N varied only slightlyat 11–25 –C (095–110 mmol N plant^–1),the decline in N₂ fixation as root temperature increased above11 C was compensated for by the increase in NO^– ₃ uptake.The % N in shoot dry matter declined with decreasing root temperature,from 32% at 13 C to 15% at 3 C. In contrast, concentrationsof N expressed on a shoot water content basis showed a modestdecrease with increasing temperature, from 345 mol m^–3at 3 C to 290 mol m^–3 at 25 C. Trifolium repens L, white clover, root temperature, N₂ fixation, potassium uptake, nitrate uptake, flowing solution culture     

Effect of Contrasting Patterns of Nitrate Application on the Nitrate Uptake, N2-Fixation, Nodulation and Growth of White Clover

White clover (Trifolium repens L.) plants were grown from seedin perlite, inoculated with effective rhizobia and exposed tothe same ‘concentration x days’ of ¹⁵N-labellednitrate in four contrasting patterns of doses. Acetylene reductionwas measured at intervals using an open, continuous-flow sytem.Mean dry weight per nodule and rates of acetylene reductionfell rapidly (2–3 d) during periods of exposure to highnitrate concentrations (> 7 mM N) and rose again, equallyrapidly, when nitrate was withdrawn or substantially reduced.The fall in mean dry weight per nodule (50–66 per cent)was almost certainly too large to be accounted for by loss ofsoluble or storage carbohydrate only. No new nodules were formedduring periods of high nitrate availability. When nitrate wassupplied continuously at a moderate concentration (5.7 mM N)nodule numbers stabilised although existing nodules increasedin dry weight by almost four-fold over the 30 d measurementperiod. Treatment had no effect on the percentage nitrogen in planttissues although there were large differences in the proportionsderived from nitrate and N₂-fixation. Plants exposed continuouslyor frequently to small doses of nitrate took up more nitrate,and hence relied less heavily on N₂-fixation, than those exposedto larger doses less often. Increased reliance on nitrate broughtwith it increased total dry weight and shoot: root ratios. Possiblemechanisms involved in bringing about these differences in nitrogennutrition and growth are discussed. White clover, Trifolium repens, nitrate, N₂-fixation, nodule, acetylene reduction, ¹⁵N     

Concurrent Rates of N2 Fixation, Nitrate and Ammonium Uptake by White Clover in Response to Growth at Different Root Temperatures

Nodulated white clover plants (Trifolium repens L. cv. Huia)were grown for 71 d in flowing nutrient solutions containingN as 10 mmol m_–3 NH₄NO₃, under artificial illumination,with shoots at 20/15°C day/night temperatures and root temperaturereduced decrementally from 20 to 5°C. Root temperatureswere then changed to 3, 7, 9, 11, 13, 17 or 25°C, and theacquisition of N by N₂ fixation, NH₄+ and NO₃^– uptakewas measured over 14 d. Shoot specific growth rates (d. wt)doubled with increasing temperature between 7 and 17°C,whilst root specific growth rates showed little response; shoot:root ratios increased with root temperature, and over time at11°C. Net uptake of total N per plant (N₂ fixation + NH₄++ NO₃^–) over 14 d increased three-fold between 3 and 17°C.The proportion contributed by N₂ fixation decreased with increasingtemperature from 51% at 5°C to 18% at 25°C. Uptake ofNH₄+ as a proportion of NH₄+ + NO₃^– uptake over 14 d variedlittle (55–62%) with root temperature between 3 and 25°C,although it increased with time at most temperatures. Mean ratesof total N uptake per unit shoot f. wt over 14 d changed littlebetween 9 and 25°C, but decreased progressively with temperaturebelow 9°C, due to the decline in the rates of NH₄+ and NO₃^–uptake, even though N₂ fixation increased. The results suggestthat N₂ fixation in the presence of sustained low concentrationsof NH₄+ and NO₄^– is less sensitive to low root temperaturethan are either NH₄+ or NO₃^– uptake systems. White clover, Trifolium repens L. cv. Huia, root temperature, nitrogen fixation, ammonium, nitrate     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

Growth of White Clover, Dependent on N2 Fixation, in Elevated CO2 and Temperature

Clonal plants of white clover (Trifolium repens L ), whollydependent on N₂ fixation, were grown for 6 weeks in controlledenvironments providing either (C680 regime) 23/18 °C day/nighttemperatures and a CO₂, concentration of 680 µmol mol^–1,or (C340 regime) 20/15 °C day/night temperatures and a CO₂,concentration of 340 µmol mol^–1 During the firsthalf of the experimental period the C680 plants grew fasterthan their C340 counterparts so that by week 3 they were twicethe weight this 2 1 superiority in weight persisted until theend of the experiment The faster initial growth of the C680plants was based on an approx 70 % increase in leaf numbersand an approx 30 % increase in their individual area Initially,specific leaf area (cm2 g^–1 leaf) was lower in C680 thanin C340 leaves but became similar in the latter half of theexperiment Shoot organ weights, including petioles and stolons,reflected the C680 plant's better growth in terms of photosyntheticsurface Throughout, C680 plants invested less of their weightin root than C340 plants and this disparity increased with timeAcetylene reduction assays showed that nitrogenase activityper unit nodule weight was the same in both C680 and C340 plantsBoth groups of plants invested about the same fraction of totalweight in nodules Nitrogen contents of plant tissues were similarirrespective of growth regime, but C680 expanded leaves containedslightly less nitrogen and their stolons slightly more nitrogenthan their C340 counterparts However, C680 leaves containedmore non-structural carbohydrate Young, unshaded C680 leavespossessed larger palisade cells, packed more tightly withinthe leaf, than equivalent C340 leaves The reason for the C680regime's loss of superiority in relative growth rate duringthe second half of the experiment was not clear, but more accumulationof non-structural carbohydrate, constriction of root growthand increased self-shading appear to be the most likely causes Trifolium repens, white clover, elevated CO₂, elevated temperature, growth, N₂ fixation, leaf structure     

Defoliation in White Clover: Regrowth, Photosynthesis and N2 Fixation

Single plants of white clover, grown in a controlled environmentand dependent for nitrogen on fixation in their root nodules,were defoliated once by removing approximately half their shoottissue. Their regrowth was compared with the growth of comparableundefoliated plants. Two similar experiments were carried out:in the first, plants were defoliated at 2.5 g, and in the secondat 1.2 g total plant d. wt. Defoliation reduced rate of N₂ fixation by > 70 per cent,rate of photosynthesis by 83–96 per cent, and rate ofplant respiration by 30–40 per cent. Nodule weights initiallydeclined following defoliation as a result of loss of carbohydratesand other unidentified components. No immediate shedding ofnodules was observed but nodules on the most severely defoliatedplants exhibited accelerated senescence. The original rates of N₂ fixation were re-attained after 5–6or 9 d regrowth, with increase in plant size at defoliation.In general, the rate of recovery of N₂ fixation was relatedto the re-establishment and increase of the plant's photosyntheticcapacity. Throughout the growth of both defoliated and undefoliatedplants nodule respiration (metabolism) accounted for at least23 ± 2 per cent of gross photosynthesis. The unit ‘cost’of fixing N₂ in root nodules, in terms of photosynthate, appearedto be unaffected by defoliation, except perhaps for plants veryrecently defoliated. Similarly, the percentage nitrogen contentsof shoot, root and nodules of defoliated plants became adaptedwithin a few days to those characteristic of undefoliated plants. Trifolium repens, white clover, N₂ fixation, defoliation, photosynthesis, respiration     

Short-Term Nitrate Effects on Hydroponically-Grown Soybean cv. Bragg and its Supernodulating Mutant: I. CARBON, NITROGEN AND MINERAL ELEMENT DISTRIBUTION, RESPIRATION AND THE EFFECT OF NITRATE ON NITROGENASE ACTIVITY

A comparison between two hydroponically-grown soybean genotypes(Glycine max [L.] Merr.) cv. Bragg and the supernodulating mutantnts 1007 was made in terms of dry matter accumulation, carbon,nitrogen, and mineral element distribution, ¹⁵N natural abundanceand the effect of short-term treatment with 4·0 mol m^–3KNO₃ on nitrogenase activity and respiration. Differences weremost pronounced in nodule dry weight and plant nitrogen content,both of which were recorded to be substantially elevated inthe mutant. Mineral element concentrations in different plantparts proved to be rather similar with the exception of Ca,found to be lower in leaves of the mutant, and Mn concentrationswhich were twice as high in roots of nts 1007. The values of¹⁵N natural abundance showed that both genotypes were equallydependent on nitrogen fixation when nitrate was absent. Theresults of the acetylene reduction assays indicated similarspecific nodule activity, while on a per plant basis nitrogenaseactivity of the mutant proved to be more than twice the amountof Bragg. This effect was also reflected in higher nodule respirationwhile root respiration remained below that of Bragg. Nitrate induced a substantial reduction in nitrogenase activitynot only in Bragg, but also in nts 1007. Nodule respiratoryactivity of Bragg was reduced by nitrate from 1·27 to0·34 mg C h^–1 plant^–1. In nts 1007 correspondingvalues were 2·70 to 1·52 mg C h^–1 plant^–1.Starch concentration in nodules was decreased in both genotypes,but nevertheless remained higher in nts 1007. Values for solublesugars in nodules even increased in the mutant in response tonitrate while the same treatment caused a reduction in Bragg.The data indicate that nitrogenase activities of Bragg and nts1007 are equally sensitive to short-term application of nitrate. Key words: Glycine max, C and N distribution, nitrate, root respiration, ¹⁵N natural abundance     

Low Root Temperature Retardation of the Mineral Nitrogen Induced Decline in N2Fixation by a Northern Ecotype of White Clover

Nodulated white clover plants (Trifolium repensL.) of a Norwegian ecotype from Pasvik (70°N) were grown in flowing solution culture. Root temperature was 17°C until 51d after sowing, when it was lowered decrementally over 5d to 7°C in four of the eight plant culture units. After a further 24h, mineral N was supplied automatically at 20μMNH₄NO₃in three culture units at each root temperature (7 and 17°C) over 17d. The remaining two units provided control plants solely dependent on N₂fixation at 7 and 17°C.The supply of NH₄NO₃greatly reduced the nodule biomass per plant at 17°C over 17d compared with control plants, but had little effect at 7°C. The nodule decline at 17°C accompanied an acute and progressive decrease in specific rate of N₂fixation, from 9mmolN d^-1g^-1nodule d.wt on day 0 to zero by day 10. Whilst initial rates of N₂fixation were lower at 7°C, the mineral N-induced decrease in fixation rates was also less severe than at 17°C and specific fixation rates recovered after reaching a minimum on day 11. N₂fixation accounted for 36% of the total uptake of N by +min.N plants during the treatment period at 7°C as opposed to only 13% at 17°C. The total N₂fixed at 7°C was 86% of that fixed at 17°C, although the specific growth rate (d.wt) at 7°C was only 55% of that at 17°C. Addition of NH₄NO₃at 7°C had little effect on the gross amount of N₂fixed subsequently. In contrast, total N₂fixation by +min.N plants at 17°C was only 24% of that fixed by the corresponding controls. The possible mechanisms by which mineral N affects N₂fixation are discussed.     

Floral Initiation of White Clover (Trifolium repens L.) in Response to Daylength and Temperature

The response to different combinations of daylength (14, 15and 16 h) and day/night temperature (20/10°C, 30/15°C)of four white clover (Trifolium repens) varieties in terms offloral initiation and development was studied. More plants flowered under long days (16 h) at 30°C thanin any other combination. Varietal differences in response todaylength were large but temperature was the factor controllinginitiation. Daylength was more important in regulating peduncle length,floret and ovule numbers than was temperature. The amount ofnectar produced was lowest at the higher temperature althoughnectar concentration was unaffected by temperature. Nectar concentrationwas lowest in the shortest daylength (14 h). White clover, floral initiation, temperature, daylength     

Response of Photosynthesis and Dark-CO2-Fixation to Light, CO2 and Temperature in Leaf Slices under Osmotic Stress

Photosynthesis and dark-CO₂-fixation were measured in vacuum-infiltratedleaf slices from the mesophyte Spinacia oleracea and the Mediterraneanxerophyte Arbutus unedo under hypertonic stress as a functionof light-intensity, CO₂-concentration and temperature, in theabsence of stomatal control. Under hypertonic stress, photosynthesis and dark-CO₂-fixationwere inhibited in leaf tissue from both plants. 50% inhibitionof photosynthesis in spinach occurred at about –3.0 MPa,and of dark-CO₂-fixation at about –3.5 MPa. 50% inhibitionof photosynthesis in Arbutus unedo was reached at about –4.0MPa (sorbitol as osmoticum). In both plants, osmotic dehydration decreased the slope andthe maximum of the CO₂- and light-response curves. The slopeof the CO₂-response curve of dark-CO₂-fixation was also decreasedunder hypertonic stress, but the inhibition of the maximal fixationrate was less obvious than for photosynthesis. Photosynthesis and dark-CO₂-fixation differed significantlyin their response to high temperature: under light- and CO₂-saturation,photosynthesis of spinach leaf slices had a temperature optimumat about 37 °C, and it was nearly completely inhibited at45 °C. The rate of dark-CO₂-fixation, however, increasedcontinuously up to 45 °C. Osmotic dehydration increasedthe resistance of photosynthesis to high temperatures. Key words: CO₂ response, Heat stress, Light response, Photosynthesis, Water stress     

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover: I. NITROGEN FIXATION AND THE RESPIRATION OF THE NODULATED ROOT

Soyabean, cowpea, and white clover, inoculated with effectiverhizobia, were grown singly with a standard mineral nutritionand light regime in controlled environments until seed maturation(in soyabean and cowpea) or late vegetative growth (white clover).Day/night temperature regimes were 23/18, 30/24, and 20/15 °Cin soyabean, cowpea, and white clover, respectively. The respiratorylosses of CO₂ from the nodulated root systems were studied inrelation to the concurrent rate of fixation of atmospheric nitrogen.Despite differences in development, the effectiveness of thesymbioses, and the temperature of growth, all three legumesexhibited similar respiratory losses from nodulated roots perunit of nitrogen fixed. During intense nitrogen fixation, theaverage respiratory losses for the three legumes varied between6·3 and 6·8 mg C mg^–1 N; within each species,the losses varied more widely at different stages of development.These respiratory burdens reflect the total cost to the plantof the nodule/nitrogen fixation syndrome including the subtendingroots. The results are discussed in relation to the respiratoryeffluxes from nodules and roots, and to biochemical investigationsof the costs of nitrogen fixation.     

Nitrogen Economy of the Main Shoot of Field-Grown Barley (Hordeum vulgare L.): II. IN VIVO NITRATE REDUCTASE ACTIVITY DURING GROWTH

The method for assay of in vivo nitrate reductase (NR) activitywas standardized for barley (Hordeum vulgare L.). NR activitywas determined in the various organs of the main shoot of field-grownJyoti barley at 40 kg N ha_–1. Total nitrate reductaseactivity (TNRA) of each organ for the period it was metabolicallyactive was calculated. The NR activity was highest in the laminae,followed by the sheaths, reproductive organs; and internodes.The NR activity was high in the first-formed laminae and itshowed a decline in the ones formed subsequently. The valuesvaried from 43.2 ± 4.33 to 7.2 ± 1.49 µmolNO^–₃ reduced g^–1 dry wt. h^–1. Maximum TNRAin the laminae, sheath, and internodes was at 49, 84, and 84–93d after sowing, respectively. The TNRA of the main shoot asa whole showed three peaks, one around 49 d, a second around63 d, and a third around 84 d after sowing. Correlation coefficient(r) between NR and NO3 concentration was highly significantin the laminae and sheath viz. 0.76^*** and 0.62^***, respectively.The results are discussed in relation to alteration in managementpractices to maximize nitrate assimilatory activity and theamount of reduced N harvested.     

Influence of Elevated CO2 and Temperature on the Photosynthesis and Respiration of White Clover Dependent on N2 Fixation

Single clonal plants of white clover (Trifolium repens L) grownfrom explants in a Perlite rooting medium, and dependent fornitrogen on N₂ fixation in root nodules, were grown for severalweeks in controlled environments which provided two regimesof CO₂, and temperature 23/18 °C day/night temperaturesat 680 µmol mol^–1 CO₂, (C680), and 20/15 °Cday/night temperatures at 340 µmol mol^–1 CO₂ (C340)After 3–4 weeks of growth, when the plants were acclimatedto the environmental regimes, leaf and whole-plant photosynthesisand respiration were measured using conventional infra-red gasanalysis techniques Elevated CO₂ and temperature increased ratesof photosynthesis of young, fully expanded leaves at the growthirradiance by 17–29%, despite decreased stomatal conductancesand transpiration rates Water use efficiency (mol CO₂ mol H₂O^–1)was also significantly increased Plants acclimated to elevatedCO₂, and temperature exhibited rates of leaf photosynthesisvery similar to those of C340 leaves ‘instantaneously’exposed to the C680 regime However, leaves developed in theC680 regime photosynthesised less rapidly than C340 leaves whenboth were exposed to a normal CO₂, and temperature environmentIn measurements where irradiance was varied, the enhancementof photosynthesis in elevated CO₂ at 23 °C increased graduallyfrom approx 10 % at 100 µmol m^–1 s^–1 to >27 % at 1170 µmol m^–2 s^–1 In parallel, wateruse efficiency increased by 20–40 % at 315 µmolm^–2 s^–1 In parallel, water use efficiency increasedby 20–40 % at 315 µmol m^–2 s^–1 In parallel,water use efficiency increased by 20–40 % at 315 µmolm^–2 s^–1 In parallel, water use efficiency increasedby 20–40 % at 315 µmol m^–2 s^–1 to approx100 % at the highest irradiance Elevated CO₂, and temperatureincreased whole-plant photosynthesis by > 40 %, when expressedin terms of shoot surface area or shoot weight No effects ofelevated CO₂ and temperature on rate of tissue respiration,either during growth or measurement, were established for singleleaves or for whole plants Dependence on N₂, fixation in rootnodules appeared to have no detrimental effect on photosyntheticperformance in elevated CO₂, and temperature Trifolium repens, white clover, photosynthesis, respiration, elevated CO₂, elevated temperature, water use efficiency, N₂ fixation     

Defoliation-Induced Stress in Nodules of White Clover: II. IMMUNOLOGICAL AND ENZYMIC MEASUREMENTS OF KEY PROTEINS

Changes in key nodule proteins following defoliation of whiteclover plants were assessed by measurements of enzyme activitiesand by use of antibodies to specific nodule proteins. Defoliation caused major declines in protein and leghaemoglobinlevels and in the activities of invertase, sucrose synthase,UDP glucose pyrophosphorylase, aspartate amino transferase,glutamine synthetase, phosphoenolpyruvate carboxylase, and malatedehydrogenase. In continuously defoliated plants the activities of these noduleenzymes continued to decline throughout the course of the 17d experiment. In plants defoliated once and then allowed toregrow new leaves, nodule enzyme activities declined for 7 to10 d before increasing again to control levels by the end ofthe experiment. In this single defoliation/recovery treatmentonly the activities of PEP carboxylase and glutamine synthetasedeclined to a greater extent than the general decline in proteincontent. Alcohol dehydrogenase, increased in specific activityfollowing a single defoliation and only declined in nodulesof continuously defoliated plants. Amino peptidase activity declined in concert with other enzymeactivities described above. Endopeptidase activity, in contrast,increased significantly after 4 d following either a singleor continuous defoliation. In the plants allowed to regrow newleaves endopeptidase activity declined to control levels again,whereas in plants continuously defoliated the activity rose5-fold. However, endopeptidase increased only after significantdeclines in protein and other enzyme activities had alreadyoccurred. Western immunoblotting confirmed that the declines in glutaminesynthetase activity and leghaemoglobin levels were due to thedisappearance of antigen. Declines in nitrogenase componentsI and II indicated that bacteroid proteins were affected bydefoliation over the same time-scale as host plant encoded proteins.As new leaves grew and nodule N₂ fixation was reestablished,these specific nodule proteins were again detectable by immunoblotting. Key words: Trifolium repens, white clover, defoliation, nodules, enzymes, antibodies     

N and P Cycling in Tanzanian Humid Savanna: Influence of Herbivores, Fire, and N2-Fixation

Availabilities of nitrogen (N) and phosphorus (P) have a strong influence on plant growth and the species composition of savannas, but it is not clear how these availabilities depend on factors such as fire, N₂-fixation, and activities of wild herbivores and cattle. We quantified soil N and P availabilities in various ways (extractable pools, mineralization, resin adsorption) along vegetation gradients within a recently abandoned cattle ranch and a former game reserve in Tanzania (both areas now part of the Saadani National Park). We also assessed annual N and P balances to evaluate how long-term availabilities of N and P are affected by large herbivores, symbiotic N₂-fixation, and fire. The results show that cattle ranching led to a spatial re-distribution of nutrients, with the local accumulation of P being stronger and more persistent than that of N. In the former game reserve, intensively grazed patches of short grass tended to have elevated soil N and P availabilities; however, because quantities of nutrients removed through grazing exceeded returns in dung and urine, the nutrient balances of these patches were negative. In dense Acacia stands, N₂-fixation increased N availability and caused a net annual N input. Fire was the major cause for nutrient losses from tallgrass savanna, and estimated N inputs from the atmosphere and symbiotic N₂-fixation were insufficient to compensate for these losses. Our results call into question the common assumption that N budgets in annually burned savanna are balanced; rather, these ecosystems are a mosaic of patches with both N enrichment and impoverishment, which vary according to the vegetation type.     

Responses of N2 Fixation-linked Respiration to Host-plant Energy Status in White Clover Acclimated to a Controlled Environment

Ryle, G. J. A., Powell, C. E. and Gordon, A. J. 1988. Responsesof N₂ fixation-linked respiration to host-plant energy statusin white clover acclimated to a controlled environment.—J.exp. Bot. 39: 879–887. Single plants of white clover, acclimated to a controlled environmentand dependent for nitrogen on N₂ fixation in their root nodules,were darkened, defoliated or exposed to enhanced CO₂ levelsto establish the quantitative relationships between the photosynthesisof the host plant and the N₂ fixation metabolism of root nodules. The nodule respiration associated with N₂ fixation (FLR) declinedrapidly to 10–15% of its normal rate following plant darkeningearly in the photoperiod. Darkening at progressively later intervalsduring the photoperiod demonstrated a positive, apparently linearrelationship between duration of illumination and total FLRduring the photoperiod and the following night period. Completeor partial defoliation reduced FLR according to the leaf arearemoved: again, there was a strong positive correlation betweencurrent rate of photosynthesis, whether of defoliated or undefoliatedplants and the FLR of root nodules. Doubling the current rateof photosynthesis, by enhancing CO₂ levels around the shoots,promoted FLR within 1–2 h when plants were stressed bylack of light. However, enhanced CO₂ levels increased FLR onlyslowly over a period of several hours in plants entrained tothe normal growing conditions. It is concluded that, in these plants acclimated to a uniformand favourable controlled environment, the supply and utilizationof photosynthetic assimilate in N₂ fixation was finely balancedand quantitatively linked during a single diurnal period andthat nodule functioning was not depressed by lack of energysubstrate. Key words: White clover, N₂ fixation, photosynthesis.     

Short-term Changes in CO2 Evolution Associated with Nitrogenase Activity in White Clover in Response to Defoliation and Photosynthesis

Direct, continuous measurements of the CO₂ evolution of rootnodules, calibrated by direct measurements of rate of ethyleneproduction, were utilized to determine the short-term responseof nitrogenase activity to defoliation and photosynthesis inwhite clover. Defoliation (removal of all expanded leaflets) generally resultedin a fall in nodulated root respiration within 10 min; mostrespiration associated with nitrogenase activity ceased within1–2 h. Darkening of the shoot also reduced nodulated root respirationwithin 10 min, but the subsequent fall in respiration, althoughof the same magnitude, was slower. The re-illumination of shootslargely reversed these effects. The inhibition of photosynthesisby DCMU largely simulated the effects of darkening the shoots. It is concluded that, in these white clover plants of 100 mgto 2.0 g total weight, current photosynthate provides the primarysource of energy for N₂ fixation. The mobilization of reserveenergy substrate appeared to play only a small role. The minimumtime interval of 10 min between onset of treatment and fallin nodule respiration probably reflects the time taken to exhaustthe assimilate in transit between leaf and nodule. Key words: White clover, N₂ fixation, Defoliation, Photosynthesis