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Several lichens and the terrestrial alga Trentepohlia were found to have extremely depleted 15N signatures at two sites near the Rotorua geothermal area, New Zealand. Values, typically -20 per thousand, with several extreme cases of -24 per thousand, are more isotopically depleted than any previously quoted delta15N signature for vegetation growing in natural environments. For Trentepohlia, distance from a geothermal source did not affect isotopic signature. A 100-km transect showed that the phenomenon is widespread and the discrimination is not related to substrate N, or to elevation. Rainfall NHx and atmospheric gaseous NH3 (NH3(g)) were shown to be isotopically depleted in the range -1 per thousand to -8 per thousand and could not, of themselves, be responsible for the plant values obtained. A simulation of Trentepohlia thallus was created using an acidified fiberglass mat and was allowed to absorb NH3(g) from the atmosphere. Mats exposed at the geothermal sites and on farm-land showed a significant further depletion of 15N to -17 per thousand. We hypothesize that the extreme isotopic depletion is due to dual fractionation: firstly by the volatilization of NH3(g) from aqueous sources into the atmosphere; secondly by the diffusive assimilation of that NH3(g) into vegetation. We further hypothesize that lithophytes, epiphytes, and higher plants, growing on strongly N-limited substrates, will show this phenomenon more or less, depending on the proportion of diffusively assimilated NH3(g) utilized as a N source. Many of the isotopically depleted delta15N signatures in vegetation, previously reported in the literature, especially epiphytes, may be due to this form of uptake depending on the concentration of atmospheric NH3(g), and the degree of reliance on that form of N.

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Using stable N isotopes, the fate of effluent-derived N has been determined within a land based municipal effluent irrigation scheme. Over 900 metric tonnes(t) of effluent-derived N have been applied to 192 ha of production conifer forest near Rotorua (NZ) over the past 11 years. The effluent N has a natural isotopic signal, generated by the treatment process, allowing it to be traced into various components of the system. Using this isotopic signal, a realistic approximation of storage capacity of various components of the system has been generated, including a calculation of the contribution of effluent N exiting the catchment via stream flow. Forest storage accounts for 50% of the applied N with a considerable proportion of that immobilized in wood and soil. The wetland, although not intensively sampled, retains 115 t, (13%) of the applied N. Denitrification, including that occurring within the wetland, accounts for 23 t (3%). Nitrogen isotope data confirm that the rise in NO3 concentrations is directly attributable to effluent N. Currently 88% of NO3-N in the stream is effluent-derived. Using current N isotope values for the stream and extrapolating over the discharge period, export of effluent N via the stream is estimated as 263 t (29%) of the applied N. Overall the forest and wetland ecosystem has intercepted or denitrified 65% of applied N, with 29% lost to the stream, and 50 t (5%) unaccounted for. The forest ecosystem is currently over-supplied with N and a number of management implications flows from these findings. In the long term the continued application of effluent N to the current irrigation area is not sustainable.

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Gunnera manicata L. glands consist of up to nine separate papillae. Surgical removal of papillae showed that more than two papillae were needed for successful infection with Nostoc. Infection occurs only in the enclosed space between adjacent papillae. Dividing Gunnera cells in the enclosed space are the sites of infection.

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To study the global diversity of plant-symbiotic nitrogen-fixing Frankia strains, a rapid method was used to isolate DNA from these actinomycetes in root nodules. The procedure used involved dissecting the symbiont from nodule lobes; ascorbic acid was used to maintain plant phenolic compounds in the reduced state. Genes for the small-subunit rRNA (16S ribosomal DNA) were amplified by the PCR, and the amplicons were cycle sequenced. Less than 1 mg (fresh weight) of nodule tissue and fewer than 10 vesicle clusters could serve as the starting material for template preparation. Partial sequences were obtained from symbionts residing in nodules from Ceanothus griseus, Coriaria arborea, Coriaria plumosa, Discaria toumatou, and Purshia tridentata. The sequences obtained from Ceonothus griseus and P. tridentata nodules were identical to the sequence previously reported for the endophyte of Dryas drummondii. The sequences from Frankia strains in Coriaria arborea and Coriaria plumosa nodules were identical to one another and indicate a separate lineage for these strains. The Frankia strains in Discaria toumatou nodules yielded a unique sequence that places them in a lineage close to bacteria that infect members of the Elaeagnaceae.

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Although herbaceous species generally show little within plant variation in delta(13)C, trees show large spatial and temporal differences. We found that the aspect of exposure and branch length accounted for up to 6 per thousand delta(13)C difference within the foliage of individual trees of Pinus radiata D. Don. The foliage on branches 0.5 m in length was as much as 4 per thousand more depleted in (13)C than foliage on 10-m long branches, and an additional 2 per thousand more depleted on the shaded side than on the exposed side. We confirmed that on clear days, relative branch hydraulic conductivity, defined as the ratio of transpiration to the water potential gradient, was much higher in short branches than in long branches. Stomatal conductance remained high in foliage on short branches during the day, whereas it declined progressively in long-branch foliage under similar conditions. These differences were sufficient to explain the observed variation in delta(13)C in foliage on long and short branches.

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Frankia strains are N2-fixing actinomycetes whose isolation and cultivation were first reported in 1978. They induce N2-fixing root nodules on diverse nonleguminous (actinorhizal) plants that are important in ecological successions and in land reclamation and remediation. The genus Frankia encompasses a diverse group of soil actinomycetes that have in common the formation of multilocular sporangia, filamentous growth, and nitrogenase-containing vesicles enveloped in multilaminated lipid envelopes. The relatively constant morphology of vesicles in culture is modified by plant interactions in symbiosis to give a diverse array of vesicles shapes. Recent studies of the genetics and molecular genetics of these organisms have begun to provide new insights into higher-plant-bacterium interactions that lead to productive N2-fixing symbioses. Sufficient information about the relationship of Frankia strains to other bacteria, and to each other, is now available to warrant the creation of some species based on phenotypic and genetic criteria.

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Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S. Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O(2) partial pressure (pO(2)) rise, to the proximity of the starting and ending oxygen tensions to the pO(2) optimum, and to the time for which the plant was exposed to the lower pO(2). Oxygen-induced transients, induced both by step jumps in pO(2) and by O(2) pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance.

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When Frankia HFPCcI3 was grown in culture at oxygen O(2) levels ranging from 2 to 70 kilopascals O(2), under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO(2) and showed a marked optimum close to growth pO(2). Vesicles were thin walled at low pO(2) and very thick walled at high pO(2). Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO(2).

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Nitrogenase activity in mangrove forests at two locations in the North Island, New Zealand, was measured by acetylene reduction and N(2) uptake. Nitrogenase activity (C(2)H(2) reduction) in surface sediments 0 to 10 mm deep was highly correlated (r = 0.91, n = 17) with the dry weight of decomposing particulate organic matter in the sediment and was independent of light. The activity was not correlated with the dry weight of roots in the top 10 mm of sediment (r = -0.01, n = 13). Seasonal and sample variation in acetylene reduction rates ranged from 0.4 to 50.0 mumol of C(2)H(4) m h under air, and acetylene reduction was depressed in anaerobic atmospheres. Nitrogen fixation rates of decomposing leaves from the surface measured by N(2) uptake ranged from 5.1 to 7.8 nmol of N(2) g (dry weight) h, and the mean molar ratio of acetylene reduced to nitrogen fixed was 4.5:1. Anaerobic conditions depressed the nitrogenase activity in decomposing leaves, which was independent of light. Nitrogenase activity was also found to be associated with pneumatophores. This activity was light dependent and was probably attributable to one or more species of Calothrix present as an epiphyte. Rates of activity were generally between 100 and 500 nmol of C(2)H(4) pneumatophore h in summer, but values up to 1,500 nmol of C(2)H(4) pneumatophore h were obtained.

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Exogenous pyruvate added to cultures of the blue-green alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO2 (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with, or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O2, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O2. Malate, citrate, alpha-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of 14C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.

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