Nitrogen Turnover and Assimilation during Regrowth in Trifolium subterraneum L. and Bromus mollis L

Subterranean clover (Trifolium subterraneum L. cv Woogenellup) and soft chess grass (Bromus mollis L. cv Blando) were grown in monocultures with ¹⁵NH₄Cl added to the soil to study nitrogen movement during regrowth following shoot removal. Four clipping treatments were imposed. Essentially all available ¹⁵N was assimilated from the soil prior to the first shoot harvest. Measurements of total reduced nitrogen and ¹⁵N contained within that nitrogen fraction in roots, crowns, and shoots at each harvest showed large, significant (P ≤ 0.001) declines in excess ¹⁵N of crowns and roots in both species between the first and fourth harvests. There was no significant decline in total reduced nitrogen in the same organs over that period. Similar responses were evident in plants defoliated three times. The simplest interpretation of these data is that reduced nitrogen compounds turn over in plant roots and crowns during shoot regrowth. Calculations for grass and clover plants clipped four times during the growing season indicated that 100 to 143% of the nitrogen present in crowns and roots turned over between the first and fourth shoot harvest in both species, assuming nitrogen in those organs was replaced with nitrogen containing the lowest available concentration of ¹⁵N. If other potential sources of nitrogen were used for the calculations, it was necessary to postulate that larger amounts of total nitrogen flowed through the crown and root to produce the measured dilution of ¹⁵N compounds. These data provide the first quantitative estimates of the amount of internal nitrogen used by plants, in addition to soil nitrogen or N₂, to regenerate shoots after defoliation.     

The Assimilation of Nitrogen from Ammonium Salts and Nitrate by Fungi

1. The assimilation of inorganic nitrogen by Scopulariopsis brevicaulisand some physiologically similar species has been studied. Theirfailure to assimilate completely from ammonium sulphate hasbeen shown to be due to the fall in pH of the medium inducedby the initial uptake of ammonia.
2. Complete assimilation ofammonia takes place in the presenceof the neutral salts ofeach of thirteen organic acids investigated.The organic acidsact primarily through their buffering effectwhich preventsor slows down the fall in pH. They are not specificallyrequiredfor ammonia assimilation by these fungi and can beeffectivelyreplaced by certain inorganic buffers.
3. The influence of severalexternal factors on the rate of assimilationof ammonia, nitrate,and nitrite has been studied in S. brevicaulis.In correspondingconditions the mycelium assimilates ammoniamore rapidly thannitrate over a wide range of conditions.
4. Ammonia, even invery low concentration, completely suppressesnitrate assimilationwhen both sources of nitrogen are presenttogether. Nitrite,however, is assimilated simultaneously withammonia. It is thereforeconcluded that ammonia blocks the reductionof nitrate to nitriteby the fungus.
5. The suppression of nitrate assimilation inthe presence of ammoniais common to many mould fungi besidesS. brevicaulis, and isbelieved to have adaptive significancein natural habitats.
6. The nitrate-reducing and assimilatingsystem is formed, evenwhen S. brevicaulis is grown in completeabsence of nitrate(ammonia medium with organic acid). It comesinto action rapidlywhen the inhibiting effect of ammonia isremoved. Similarly,nitrate-grown mycelium is capable of assimilatingammonia atmaximal rate without any adaptive lag.

     

Effects of Nitrogen Nutrition on Leaf Expansion and Photosynthesis of Trifolium subterraneum L. 1. Comparison between Different Levels of Nitrogen Supply

Growth analysis showed that reductions in the relative growth-rateof subterranean clover plants (cv. Mt. Barker), even those dueto moderate nitrogen deficiencies, were reflected in reductionsof the leaf-area ratio and particularly of the net assimilationrate. A decline in nitrogen supply in the culture solutions was foundto depress net rates of carbon dioxide uptake per unit leafarea and leaf expansion per plant to about the same extent,even at moderate levels of nitrogen stress. Four days aftertransfer of plants grown with adequate nitrogen to solutionswithout nitrogen, leaf area and net carbon dioxide uptake haddeclined to 84 per cent and 89 per cent of the values for thecontrol plants. After a further 4 days these values had decreasedto 71 per cent and 52 per cent respectively. When net carbon dioxide uptake was expressed per unit weightof chlorophyll, the effect of changes in nitrogen supply onnet photosynthesis largely disappeared, indicating a close relationshipwith the chlorophyll content of the leaves. However, anotherand perhaps more direct effect of nitrogen on photosynthesiswas suggested by the fact that, during the early stages of recoveryfrom a severe nitrogen stress, photosynthesis began to increasebefore the chlorophyll content of the leaves.     

Nitrogenase Activity in Trifolium subterraneum L. in Relation to the Uptake of Nitrate Ions

An experiment was conducted to test the hypothesis that, when nitrogenase and nitrate reductase both contribute to the nitrogen nutrition of a nodulated legume, nitrogenase activity is inversely proportional to the rate of accumulation of organic nitrogen derived from the reduction of nitrate. Trifolium subterraneum L. plants, inoculated with Rhizobium trifolii and sown as small swards, were allowed to establish a closed canopy and steady rates of growth, dinitrogen fixation, and nitrogen accumulation. Swards were then supplied with nutrient solutions of 0, 0.5, 1.0, or 2.5 mm NO₃⁻ with a 29.69% enrichment of ¹⁵N and allowed to grow for a further 33 days. Harvests were made to measure dry weight, nitrogen accumulation, ¹⁵N accumulation, NO₃⁻ content and nitrogenase activity by acetylene reduction assay. Since the ¹⁵N of the plant organic matter could have been derived only from the NO₃⁻ of the nutrient solution, its rate of accumulation provided a measure of the rate of NO₃⁻ reduction. It was found that as this rate increased in response to external NO₃⁻ concentration the rate of nitrogenase activity decreased proportionately. It is concluded that the reduction of nitrate and the reduction of dinitrogen act in a complementary manner to supply a plant with organic nitrogen for growth.     

Metabolism of Glycollate by Lemna minor L. Grown on Nitrate or Ammonium as Nitrogen Source

Marques, I. A., Oberholzer, M. J. and Erismann, K. H. 1985.Metabolism of glycollate by Lemna minor L. grown on nitrateor ammonium as nitrogen source.—J. exp. Bot. 36: 1685–1697. Duckweed, Lemna minor L., grown on inorganic nutrient solutionscontaining either NH₄⁺ or NO₃^– as nitrogen source wasallowed to assimilate [1-¹⁴C]- or [2-¹⁴C]glycollate during a20 min period in darkness or in light. The incorporation ofradioactivity into water-soluble metabolites, the insolublefraction, and into the CO₂ released was measured. In additionthe extractable activity of phosphoenolpyruvate carboxylasewas determined. During the metabolism of [2-¹⁴C]glycollate in darkness, as wellas in the light, NH₄⁺ grown plants evolved more ¹⁴CO₂ than NO₃^–grown plants. Formate was labelled only from [2-¹⁴C]glycollateand in NH₄⁺ grown plants it was significantly less labelledin light than in darkness. In NO₃^– grown plants formateshowed similar radioactivity after dark and light labelling.The radioactivity in glycine was little influenced by the nitrogensource. Amounts of radioactivity in serine implied that thefurther metabolism of serine was reduced in darkness comparedwith its metabolism in the light under both nitrogen regimes.In illuminated NH₄⁺ plants, serine was labelled through a pathwaystarting from phosphoglycerate. After [1-¹⁴C]glycollate feedingNH₄⁺ grown plants contained markedly more radioactive aspartateand malate than NO₃^– plants indicating a stimulated phosphoenolpyruvatecarboxylation in plants grown on NH₄⁺. Key words: Photorespiration, glycollate, nitrogen, Lemna     

The Effect of Moisture Stress on Infection of Trifolium subterraneum L. by Rhizobium trifolii Dang

Moisture stress and method of inoculation greatly affected thenumber and distribution of infected root hairs and nodules ofyoung seedlings of Trifolium subterraneum. A reduction of soilmoisture from 5·5 to 3·5% (–0·36to –3·6 x 10⁵ Pa) significantly decreased the numberof infection threads and completely inhibited nodulation, althoughthe number of rhizobia in the rhizosphere was unaffected. Atlow soil moisture levels the root hairs were abnormally shortand swollen. Infection and nodulation were little affected between5·5 and 9·5% moisture (–0·36 to –0·089x 10⁵ Pa). Distribution of infected root hairs depended on the initialplacement of the inoculum; with the inoculum mixed evenly throughthe soil, infection threads occurred at discrete foci alongthe root. With seedlings inoculated at planting, infection threadswere restricted to the top 1–2 cm of root, even at thehighest soil moisture tested. Watering increased the number of infections in plants grownat 3·5% moisture; nodules were formed at a rate equivalentto non-stressed plants. Watering also enabled movement of theseedling-borne inocula; new infections were formed along theroot surface bearing mature root hairs.     

Regulation of Expression of Nitrate and Dinitrogen Assimilation by Anabaena Species

Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more ¹³NO₃⁻ into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species.     

Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress

The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO₃⁻. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO₃⁻. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO₃⁻, while with rewatering, leaf NRA recovery was quite important especially in the NO₃⁻-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO₃⁻ and in those without NO₃⁻ contrary to the behavior of the leaves. Beyond −15.10⁵ pascal, nodular NRA began to decrease in plants watered with NO₃⁻. This phenomenon was not observed in nodules of plants given water only.     

Sodium Stimulates Growth of Amaranthus tricolor L. Plants through Enhanced Nitrate Assimilation

Effects of Na application on the capacity of NO₃⁻ assimilation were studied in Na-deficient Amaranthus tricolor L. cv Tricolor plants. On day 30 after germination, Na-deficient A. tricolor plants received either 0.5 millimolar NaCl or KCl. The level of nitrate reductase activity doubled within 24 hours by the addition of Na and the enhanced level was maintained thereafter. When the plants were exposed to 2 millimolar ¹⁵NO₃⁻, total ¹⁵N taken up by the plants was greater in the Na-treated plants than in the K-treated plants within 24 hours of the Na treatment. Incorporation of ¹⁵N into the 80% ethanol-insoluble nitrogen fraction of the Na-treated plants in the light period was about 260% of those of the K-treated plants indicating greater capacity of NO₃⁻ assimilation in the Na-treated plants. From these results, it was demonstrated that Na application to the Na-deficient A. tricolor plants promoted NO₃⁻ reduction and its subsequent assimilation into protein, resulting in growth enhancement.     

Some Effects of Potassium and Magnesium on the Growth of Subterranean Clover (Trifolium subterraneum)

Subterranean clover plants (Trifolium subterraneum L., cv. Mt.Barker) were grown in culture solutions at optimum nutrientlevels and on the 23rd day after sowing transferred to solutionswithout magnesium or potassium. A third group remained at thesame nutrient levels as before (controls). Magnesium deficiency caused a particularly rapid decline inroot growth, followed later by a net loss of root dry matter.This, and the fact that leaf expansion declined relatively morethan the increase in laminae dry matter, resulted in an accumulationof assimilates per unit leaf area. Transfer of magnesium-deficientplants to complete solutions on day 35 caused a preferentialdistribution of dry matter to the roots, then to petioles andrelatively less to the laminae. These changes caused a pronouncedfall in the root: shoot ratio as the deficiency became moresevere and a rise in the ratio during recovery. Plants in solutions without potassium showed no marked shiftsin dry matter distribution between plant parts. The root: shootratio remained close to that for control plants, except duringthe recovery, when there was a decrease in the ratio. Net rates of CO₂ uptake by laminae from potassium-deficientplants showed little change during the first 10 days of thedeficiency although values were somewhat lower than those forcorresponding control laminae. After transfer to complete solutionsthere was a marked response in photosynthesis, rising to a finalvalue close to that for control laminae. Laminae of plants placedin solutions without magnesium showed a rapid decline in photosynthesisonly 4 days later; there was little response when plants weretransferred to complete solutions a week later. Trifolium subterraneum L., subterranean clover, growth, root: shoot ratio, potassium deficiency, magnesium deficiencies     

Effects of Nitrogen Deficiency on the Absorption of Nitrate and Ammonium by Barley Plants

When young barley plants which had been supplied with nitratewere deprived of this source of N, an enhanced capacity forabsorption of either nitrate or ammonium ions developed, reachinga maximum in about 3 d under the particular experimental conditionsused. The net uptake rate of either nutrient was then approximatelythree times that in plants which had received nitrate throughout.Likewise, withholding external N from plants previously growingwith ammonium caused a 2.4-fold increase in their subsequentcapacity to absorb that ion, compared with control plants grownwith an uninterrupted ammonium supply. Accelerated nitrate uptakein N-starved plants was not accompanied by additional phosphateor sulphate absorption, but the plants had the capacity to absorbmore potassium, whether or not ammonium was also present inthe solution. Indirect evidence from analyses of root tissuesuggests that these responses to mild N-stress may depend onsome property of an N fraction which does not include nitrateor ammonium. Hordeum vulgare, barley, nitrogen, ammonium, nitrate, N-deficiency, absorption     

Growth, fecundity and competitive ability of transgenic Trifolium subterraneum subsp. subterraneum cv. Leura expressing a sunflower seed albumin gene

Ecological risk assessment is an important step in the production and commercialisation of transgenic plants. To date, however, most risk assessment studies have been performed on crop plants, and few have considered the ecological consequences associated with genetic modification of pasture species. In this study we compared the growth, yield, population dynamics and competitive ability of transgenic Trifolium subterraneum subsp. subterraneum cv. Leura (subclover) expressing a nutritive sunflower seed albumin (ssa) gene with the equivalent non-transgenic commercial line in a glasshouse competition trial. Plants were grown in low-fertility soil typical of unimproved native southeastern Australian grasslands. We measured survivorship, seed production rate, seed germination rate, seed weight, dry weight yield and the intrinsic rate of population increase (lambda) of plants grown in mixtures and monocultures over a range of densities (250 to 2000 plants m(-2)), and also determined intragenotypic and intergenotypic competition coefficients for each line. There were no significant differences between transgenic and non-transgenic plants in any of the measured variables except survivorship; transgenic plants had a significantly lower survival rate than non-transgenic plants when grown at high densities (p<0.01). However, density-dependent effects were observed for all measured variables, and in all models plant density affected the response variables more than the presence of the transgene. Based on these results, we conclude that the ssa gene construct appears to confer no advantage to transgenic T. s. subterraneum cv. Leura growing in mixed or pure swards under the fertility and density regimes examined in the trial. Our data also suggest that transgenic subterranean clover expressing the ssa gene is unlikely to exhibit a competitive advantage over associated non-transgenic commercial cultivars when grown in dense swards in low-fertility pastures.     

The Effect of Nitrate and Ammonium Feeding on Carbon Dioxide Assimilation in Maize

The effect of nitrogen nutrition on the pattern of ¹⁴CO₂ fixationduring photosynthesis by young maize plants was studied. Nitratefeeding increased the incorporation of ¹⁴C into malate and certainamino acids (particularly aspartate) and decreased that intosucrose and starch. Ammonium nutrition caused similar, but notidentical, changes, the major difference being a much smallereffect on incorporation into malate. Tungstate applied to theplants prior to nitrate almost completely inhibited the formationof nitrate reductase and eliminated most, but not all, of theeffects of nitrate on the fixation pattern. The results obtainedare discussed in terms of: (i) more reduced nitrogen as substratefor amino acid synthesis; (ii) the changes in pH and ionic balancecaused by the process of nitrate reduction to ammonium; and(iii) specific enzyme activation or inhibition by nitrate orammonium ions.     

Interrelations Between Seeds and Associated Fruiting Structures During Their Development in Trifolium subterraneum L.

Developing burrs of T. subterraneum (cv. Daliak) were subjectedto dark and light treatments in experiments conducted in a controlledtemperature glasshouse with day/night air temperatures of 18/13°C. Post fertilization development of burr components wasfollowed and the effect of removal of seeds or sterile calyoeson the development of the remaining burr components studied. Peduncle elongation took place mainly between days 0 (anthesis)and 7, when the burr would normally have buried. Seed developmentwas completed by day 42. Pod walls attained near maximum weightat about day 22. The perianth increasd in weight between days12 and 22 due to enlargement of the calyx. Sterile calyces wereprduced continuously until day 42. The pattern of burr develompentwas similar in both dark and light, but the weight of the maincomponents were considerably reduced in the light treatment. Normal peduncle development depended on the presence of at leastone seed in the inflorescence. Pod walls did not develop, neitherdid the perianth enlarge unless these structures were associatedwith a developing seed. In the absence of seeds fewer sterilecalyces were produced in the dark treatment and fewer stillwere produced in the light, indicating a direct inhibitory effectof light on the production of sterile calyces. Within matureburrs the weight of pod walls and sterile calyces was closelyrelated to the weight of seeds. Removal of sterile calyces reducedseed production but interpretation of this effect is not clear. Trifolium subterraneum L., fruit development, seed production     

The Effect of Water Stress on the Vegetative Growth of White Clover (Trifolium repens L): Comparison of Long-term Water Deficit and a Short-term Developing Water Stress

White clover plants were subjected to either a short-term developingwater stress or long-term stable levels of water deficit on‘water stress columns’. The short-term stress reducedplant water status to –2?0 MPa over 15 d. The water stresscolumns imposed only mild levels of water stress (a reductionof 0?35 MPa in leaf water potential for the more severe treatment)but these were maintained for several weeks. The absolute growthof plants on the control columns was maintained throughout theexperimental period. Vegetative growth was measured. Stolon, petiole, and laminagrowth were all reduced to some extent when plants were grownsymbiotically. The two regimes gave comparable results. Whennitrate was supplied there was no effect of water stress. Aconsiderably reduced absolute growth rate did not result ina similar decrease in final organ size. Stolon growth was mostreduced by water stress. Leaf death during water stress wasas important as changes in growth in determining final dry matteryield. Consequently, the yield of petiole and lamina from plantsgrown without supplied nitrate on the water stress columns waslower than that of stolon at the end of the treatment period. The merits of the water stress column system for imposing long-termwater deficit are discussed. Key words: Trifolium repens, white clover, water stress, vegetative growth     

不同氮素形态及水分胁迫对水稻苗期水分吸收、光合作用及生长的影响

采用室内营养液培养, 聚乙二醇(PEG6000)模拟水分胁迫处理、HgCl2抑制水通道蛋白活性的方法, 在3种供氮形态下(NH4+-N/ NO 3--N为100/0、50/50和0/100), 研究了水稻苗期水分吸收、光合及生长的状况。结果表明, 在非水分胁迫下, 水稻单位干重吸水量以单一供NO3--N处理最高, 加HgCl2抑制水通道蛋白活性后, 单一供NO3--N、NH4+-N和NH4+-N/ NO3--N为50/50处理的水稻水分吸收分别下降了9.6%、20.7%和16.0%; 但在水分胁迫下, 单一供NO3--N的处理水分吸收量显著降低, 低于其它2个处理, 加HgCl2抑制水通道蛋白活性后, 水分吸收量分别降低了1.0%、18.8%和23.5%。在2种水分条件(水分胁迫与非水分胁迫)下, 净光合速率、气孔导度、蒸腾速率和细胞间隙CO2浓度等指标均以单一供NH4+-N处理最大,NH4+-N/ NO3--N为50/50处理次之, 单一供NO3--N处理最小。HgCl2处理结果表明, 不同形态氮素营养能够影响水稻幼苗根系水通道蛋白活性。在2种水分条件下, NH4+-N/ NO3--N为50/50处理的生物量(干重)均最大。本研究为水稻苗期合理施肥以壮苗提供了理论依据。     

The Use of a Closed System Flow-Through Enclosure Apparatus for Studying the Effects of Partial Pressure of Dinitrogen in the Atmosphere on Growth of Trifolium repens L. and Lolium perenne L.

A closed system flow-through enclosure apparatus was constructedand used to enclose mixtures ofLolium perenne L. (perennialryegrass cv. Trani) and nodulatedTrifolium repens L. (whiteclover cv. Blanca) growing in soil in pots. There were no significantdifferences between the shoot growth, in terms of dry matteraccumulation and nitrogen content, of mixtures in the systemcompared to that of mixtures grown in a standard growth cabinet.This demonstrated that stable ambient conditions could be achievedby the closed system and its control circuits and that therewere no apparent side-effects of the recirculatory gases.Reducingthe partial pressure of dinitrogen in the atmosphere affectedwhite clover, but not perennial ryegrass. A fairly rapid effectwas observed 4 h after reduction in dinitrogen partial pressureas some of the clover leaves folded downwards along the petiole.These same effects were observed at two different partial pressuresof dinitrogen (22% and 39%) and with two different replacementgases (argon and helium). In the longer term (11 to 16 d) drymatter accumulation and nitrogen content of the clover shootswere significantly reduced. These effects of reduced partialpressure of dinitrogen were observed in both nodulated (NOD⁺)and nitrate-dependent (NOD^–) clover. Possible reasonsfor these effects were discussed with particular reference toimpurities in the gases used, stomatal responses and plant waterrelations. It was concluded that the closed system flow-throughapparatus provides a useful tool for studying whole plant-soilsystems and, in particular, the cycling of nitrogen. However,the use of a replacement gas to reduce the cost of labelleddinitrogen was obviously not a viable proposition. Key words: Dinitrogen, flow-through system, Lolium perenne, partial pressure, Trifolium repens