Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules of Acacia spp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationshipsbetween acetylene reduction activity, hydrogen evolution andnitrogen fixation in nodules of Acacia spp.: Experimental backgroundto assaying fixation by acetylene reduction under field conditions.—J.exp. Bot. 38: 1–12 Glasshouse grown, symbiotically-dependent seedlings of Acaciaalata R.Br., .A. extensa Lindl., and A. pulchella R.Br. wereexamined for acetylene reduction in closed assay systems usingundisturbed potted plants, excavated whole plants, nodulatedroots or detached nodules. Nitrogenase activity declined sharplyover the first hour after exposure of detached nodules to acetylene(10% v/v in air), less steeply or not at all over a 3 h periodin assays involving attached nodules. Using detached nodules,rates of acetylene reduction, nitrogen (¹⁵N₂) fixation, andhydrogen evolution in air (¹⁵N₂) and acetylene-containing atmosphereswere measured in comparable 30 min assays. Total electron flowthrough nitrogenase in air was determined from rates of nitrogen(¹⁵N₂) fixation ( ? 3) plus hydrogen evolution, that in thepresence of acetylene from rates of acetylene reduction andhydrogen evolution in air: acetylene. Values for the ratio ofelectron flow in air: acetylene to that in air ranged from 0?43to 0?83 in A. pulcheila, from 0?44 to 0?66 in A. alala and from0?37 to 0?70 in A. extensa, indicating substantial inhibitionof electron flow through nitrogenase of detached nodules byacetylene. Relative efficiencies of nitrogenase functioningbased on hydrogen evolution and acetylene reduction were from0?15 to 0?79, those based on nitrogen (¹⁵N₂) fixation and hydrogenevolution from 0?53 to 0?87. Molar ratios of acetylene reducedto nitrogen (¹⁵N₂) fixed were 2?82 ? 0?24, 201 ? 0?15, and 1?91? 0?11 (?s.e.; n = 7) for A. pulcheila,A. extensa and A. alata respectively A standard 5–10 min acetylene reduction assay, conductedon freshly detached unwashed nodules in daytime (12.00–14.00h), was calibrated for field use by comparing total N accumulationof seedlings with estimated cumulative acetylene reduction overa 7-week period of glasshouse culture. Molar ratios for acetylenereduced: nitrogen fixed using this arbitrary method were 3?58for A. alata, 4?82 for A. extensa and 1?60 for A. pulchella.The significance of the data is discussed. Key words: Acacia spp, nitrogenase functioning     

Relationships Between Fire Response, Morphology, Root Anatomy and Starch Distribution in South-west Australian Epacridaceae

Selected epacrids (92 species in 15 genera) were examined withrespect to fire response type, morphology, root anatomy andstarch storage. Seeders, 75% of the species investigated, possesseda single main stem and a small root system with lateral rootswhich in most cases did not spread beyond the shoot canopy.Resprouter species were generally multi-stemmed with large lignotuberousroot stocks. Certain seeder and resprouter species were intermediatein form and showed small root systems and basally branched mainstems. Amounts of starch in roots of seeders (1.9±0.5mgstarch gd.wt per root) were much less than in resprouters (14.1±3.3)whereas amounts in shoots were similar (1.9±0.5 and 1.6±0.6mgstarch gd.wt per shoot, respectively). Starch storage in rootswas mostly confined to rays of xylem parenchyma and inter-rayxylem parenchyma and the greater storage capacity of resprouterswas generally due to broader rays. Growth zones in root xylemranged from clear, verifiable annual rings, as in many seederspecies, to indistinct growth zones, typical of many resprouterspecies. Shoot:root dry weight ratios were higher in seedersthan resprouters. The study suggests that speciation withinthe Epacridaceae into seeder and resprouter forms involved divergentdifferentiation in terms of morphology, shoot:root dry weightratio root storage of starch. Seeder; starch storage; growth rings; growth zones; south-west Australia; resprouter; Epacridaceae     

Characteristics of inorganic nitrogen assimilation of plants in fire-prone Mediterranean-type vegetation

A range of approaches was used to investigate how species within a fire-prone Banksia woodland in South West Australia exploited inorganic soil nitrogen sources and how this changes through the development of the fire chronosequence. Nitrate and ammonium were present in soil solution throughout the chronosequence but nitrate predominated in recently burnt sites. Mean shoot nitrate reductase activities were high for all species in recently burnt sites and showed little increase when nitrate was supplied via the transpiration stream. Nitrate reductase of shoots of most species was low at sites not burnt for several years, but following transpirational induction with nitrate, developed activities similar to those at recently burnt sites. The principal amino compounds transported in the xylem were species specific, including asparagine, glutamine and citrulline-dominated species, and changed little in relative composition across the chronosequence. Species most active in leaf nitrate reduction transported the largest amounts of nitrate in their xylem sap and proportional amounts of nitrate in xylem tended to be greatest in recently burnt sites. Most of the species examined appeared to be shoot rather than root nitrate assimilators, but marked differences were recorded in potential of leafy shoots of different species to reduce nitrate. As a general rule, shallow-rooted herbaceous, non-mycorrhizal or VAM-positive species had the highest capacity to reduce nitrate, whereas woody species with ericoid mycorrhizae or combined vesicular arbuscular/ectomycorrhizal associations exhibited little capacity to reduce nitrate in roots or shoots. It seems likely that this latter group utilize ammonium or even organic forms of nitrogen rather than nitrate. Some putative nitrogen-fixing species were active in reducing and transporting nitrate, others were virtually inactive in these respects.     

Efficiency and regulation of root respiration in a legume: Effects of the N source

A comparison was made of energy metabolism of nodulated N₂ fixing plants and non-nodulated NO₃-fed plants of Lupinus albus L. Growth, N-increment, root respiration (O₂ uptake and CO² production) and the contribution of a SHAM-sensitive oxidative pathway (the alternative pathway) in root respiration were measured. Both growth rate and the rate of N-increment were the same in both series of plants. The rate of root respiration, both O₂ uptake and CO₂ production, and the activity of the SHAM-sensitive pathway were higher in NO₃-fed plants than in N₂ fixing plants. The rate of ATP production in oxidative phosphorylation was computed also to be higher in NO₃-fed plants. It is concluded that both carbohydrate costings and ATP costings for synthesis + maintenance of root material were lower in N₂ fixing than in NO₃-fed plants. The respiratory quotient of root respiration was 1.6 in N₂-fixing plants and 1.4 in NO₃-fed plants. These values were slightly higher than the values calculated on the basis of CO₂ output due to N-assimilation and the experimental values of O₂ uptake, but showed the same trend: highest in N₂ fixing plants. Root respiration of NO₃-fed plants showed a diurnal pattern (both O₂ uptake, CO₂ production and the activity of the SHAM-sensitive pathway), whilst no diurnal variation in root respiration was found in N₂ fixing plants. However, C₂H₂ reduction did show a diurnal rhythm, which is suggested to be related to the diurnal variation in transpiration. Addition of NO₃ to N₂ fixing plants increased the rate of root respiration and the activity of the alternative pathway. This treatment did not decrease C₂H₂ reduction and H₂ evolution within 4 days. Withdrawal of NO₃-supply from NO₃-fed plants decreased the rate of root respiration but had no effect on the relative activity of the alternative pathway. It is suggested that the higher rate of root respiration and the higher activity of the SHAM-sensitive pathway in NO₃-fed plants is due to a larger supply of carbohydrates to the roots, partly due to a better photosynthetic performance of the shoots and partly due to a higher capacity of the roots to attract carbohydrates.     

Diurnal Patterns of Transport and Accumulation of Minerals in Fruiting Plants of Lupinus angustifolius L.

Fluctuations in mineral elements id xylem (tracheal) sap, fruitphloem sap, leaflets and dmloping fruits were studied in a fieldpopulation of Lupinus angustifolius L. by three-hourly samplingover a 39 h period. Elements usually reached maximum contentsor concentrations at or near noon, minimum levels during thenight. Amplitudes of diurnal fluctuations in minerals lay withinthe range ±4–33 per cent of the mean content ofleaflets, and ±17–157 per cent of the mean concentrationsin xylem and phloem sap. Most minerals elements fluctuatcd inphase with daily changes in sugar level of phloem sap and drymatter and carbohydrate fluctuations of leaflets, suggestinga coupling of translocation of photosynthate and minerals fromthe leaflets. Rates of import of minerals by shoots wereestimatedfrom shoot transpiration and mineral concentrations in trachealsap. Average day time rates of import of most elements were12–25 times those at night. Translocation of minerals,nitrogen and carbon to fruits also exhibited diurnal periodicity,average rates of import king three to seven times higher inthe day than at night. A model of transport based on the carbonand water economy of the fruit suggested that P, K, Fe, Zn,Mn and Cu were imported predominantly by phloem. Estimates ofvascular import accounted for 87–104 per cent of the fruit'sactual increment of these elements. Na and Ca were gauged tobe imported mainly by xylem, Mg almost equally by xylem andphloem. However, large discrepancies existed for these threeelements between estimated vascular import and actual intakeby the fruit. Lupinus angustifolius L., mineral transport, accumulation, fruits, xylem sap, phloem sap, transpiration     

Nitrate Assimilation in Higher Plants with Special Reference to the Cocklebur (Xanthium pennsylvanicum Wallr.)

A soluble NADH-dependent nitrate reductase is described forthe shoot system of Xanthium. Young leaves and immature stemtissues contain high levels of the enzyme. They are relativelyrich in free amino acids and amides but store little free nitrate.The specific activity of the enzyme is lower in fully expandedleaves, although these leaves exhibit higher rates of fixationof carbon in photosynthesis than do younger leaves. Neithernitrate nor free amino acids accumulate in the mesophyll ofthe leaf. Older parts of the stem axis accumulate large amountsof soluble nitrogen, almost entirely as free nitrate. Reservesof nitrate in the shoot and root are rapidly depleted if nitrateis removed from the external medium. Nitrate reductase is apparently absent from roots of Xanthium.This finding is supported by analyses of bleeding sap from nitrate-fedplants which show that 95 per cent of the nitrogen exportedfrom roots is present as free nitrate. However, roots are capableof synthesizing and exporting large amounts of amino nitrogenif supplied with reduced nitrogen such as urea or ammonium. A scheme is presented summarizing the main features of the metabolismof nitrate in Xanthium and this is compared with the situationin nitrate-fed plants of the field pea (Pisum arvense L.), aspecies previously shown to be capable of reducing nitrate inits root system.     

The Carbon Balance of a Legume and the Functional Economy of its Root Nodules

Budgets for carbon and nitrogen in shoot, root, and nodulesof garden pea (Pisum sativum L.) are drawn up for a 9-d intervalin the life cycle, from data on nitrogen fixation, carbon accumulationin dry matter, respiratory output of plant organs, and organicsolute exchange between shoot and nodulated root. Of the carbon gained photosynthetically by the shoot from theatmosphere 26 per cent is incorporated directly into its drymatter, 32 per cent translocated to the nodules, and 42 percent to the supporting root. Of the nodules’ share, 5per cent is consumed in growth, 12 per cent in respiration,and 15 per cent returned to the shoot via the xylem, as aminocompounds generated in nitrogen fixation. Growth and respirationof the root utilize, respectively, 7 and 35 per cent. The respiratory efficiency of a nodulated root in terms of nitrogenfixation (5.9mg C per mg N₂-N fixed) is found to be very similarto that of an uninoculated root assimilating nitrate (6.2 mgC per mg NO₃-N reduced). The nodules require in growth, respiration,and export 4.1 mg C ( 10.3 mg carbohydrate) for each mg N whichthey fix. The nodules consume 3 ml O₂ for every 1 ml N₂ utilized in fixation. In exporting a milligram of fixed nitrogen the nodules requireat least 0.35 ml of water. Almost half of this requirement mightbe met by mass flow into the nodules via the phloem.     

The Effect of Nitrogen Source on Transport and Metabolism of Nitrogen in Fruiting Plants of Cowpea (Vigna unguiculata (L.) Walp.)

Nitrogen metabolism and transport were studied during reproductivedevelopment of cowpea (Vigna unguiculata (L.) Walp. cv. Vita3) under three contrasting nitrogen regimes: (1) nitrate suppliedcontinuously (plants non-nodulated), (2) symbiotic N₂ fixation(no combined nitrogen), (3) nitrogenstarvation post-anthesisof previously N₂-fixing plants. The last treatment involveddaily flushing of the root systems with 100% oxygen which suppressedpost-anthesis N₂-fixation by 76–79%, thereby making fruitgrowth almost entirely reliant upon mobilization of previouslyaccumulated nitrogen. The bulk of the xylem nitrogen (root bleedingsap or peduncle tracheal sap) of nitrate-fed plants was nitrateand amide, that of symbiotic and O₂-treated plants largely ureide.The composition of fruit cryopuncture phloem sap, however, wasclosely similar in all treatments, with most nitrogen as amidesand amino acids. The evidence suggested intense metabolic transferof root derived nitrate-N or ureide-N to amino acids by vegetativeplant parts prior to translocation to fruits. All tissues offruits showed patterns of development of enzymic activitiesconsistent with release of nitrogen from both ureides and amidesand re-assimilation of ammonia to form amino acids. Althoughthe levels of enzyme activities varied between treatments thedifferences could not be readily associated with individualpatterns of nitrogen transport in the treatments. Nitrogen sufficiencyin the NO₃-fed plants was marked by elevated vegetative biomassand low harvest indices for dry matter and nitrogen, while nitrogendeficiency of the O₂-treated plants was associated with seedabortion, small seed size and low seed nitrogen concentration,and efficient mobilization of nitrogen from vegetative partsto fruits. Key words: Nitrogen, Translocation, Cowpea