Effects of nitrogen source and ectomycorrhizal association on growth and δ15N of two subtropical Eucalyptus species from contrasting ecosystems.

Ectomycorrhizal (EM) associations facilitate plant nitrogen (N) acquisition, but the contribution of EM associations to tree N nutrition is difficult to ascertain in ecosystems. We studied the abilities of subtropical EM fungi and nutritionally contrasting Eucalyptus species, Eucalyptus grandis W.Hill ex Maiden and Eucalyptus racemosa Cav, to use N sources in axenic and soil cultures, and determined the effect of EM fungi on plant N use and plant 15N natural abundance (δ15N). As measured by seedling growth, both species showed little dependence on EM when growing in the N-rich minerotrophic soil from E. grandis rainforest habitat or in axenic culture with inorganic N sources. Both species were heavily dependent on EM associations when growing in the N-poor, organotrophic soil from the E. racemosa wallum habitat or in axenic culture with organic N sources. In axenic culture, EM associations enabled both species to use organic N when supplied with amide-, peptide- or protein-N. Grown axenically with glutamine- or protein-N, δ15N of almost all seedlings was lower than source N. The δ15N of all studied organisms was higher than the N source when grown on glutathione. This unexpected 15N enrichment was perhaps due to preferential uptake of an N moiety more 15N-enriched than the bulk molecular average. Grown with ammonium-N, the δ15N of non-EM seedlings was mostly higher than that of source N. In contrast, the δ15N of EM seedlings was mostly lower than that of source N, except at the lowest ammonium concentration. Discrimination against 15N was strongest when external ammonium concentration was high. We suggest that ammonium assimilation via EM fungi may be the cause of the often observed distinct foliar δ15N of EM and non-EM species, rather than use of different N sources by species with different root specialisations. In support of this notion, δ15N of soil and leaves in the rainforest were similar for E. grandis and co-occurring non-mycorrhizal Proteaceae. In contrast, in wallum forest, E. racemosa leaves and roots were strongly 15N-depleted relative to wallum soil and Proteaceae leaves. We conclude that foliar δ15N may be used in conjunction with other ecosystem information as a rapid indicator of plant dependency on EM associations for N acquisition.

Global aspects of C/N interactions determining plant-environment interactions.

The atomic C:N ratio in photolithotrophs is a function of their content of nucleic acids, proteins, lipids, polysaccharides, and other organic materials, and varies from about 5 in some protein-rich microalgae to much higher values in macroalgae and in higher plants with relatively more structural and energy storage materials. These differences in C:N ratios among organisms means that there is more N assimilation by photosynthetic organisms in the oceans than on land despite the near equality of global photosynthetic C assimilation rates in the two environments. Aquatic organisms obtain inorganic C and inorganic N from the surrounding water. Terrestrial photolithotrophs obtain inorganic C, dinitrogen (by diazotrophy) and some combined N from the atmosphere, with the remaining combined N coming from the soil. The nitrogen cost of growth (biomass production rate per unit plant N) varies with the C:N ratio and specific growth rate of the organism. The C:N ratio of plants can be increased with no, or minimal, decrease in growth rate by switching from N-containing to N-free solutes involved in, for example, UV-B screening or by reducing the content of particular proteins. The water cost of growth (water lost per unit biomass gain) in terrestrial plants is a function of N supply and of C supply; water cost is lower with higher N and C availability. Water supply is also important in determining denitrification rates on land and on N (and C) fluxes from terrestrial to aquatic systems.

Isolation of ammonium-N as 1-sulfonato-iso-indole for measurement of delta15N.

The conversion of ammonium (NH(4) (+)) to 1-sulfonato-iso-indole has been examined as a method for natural abundance measurement of delta(15)N of NH(4) (+). The reaction is complete within 2 h and is based on the derivatisation of NH(4) (+) by o-phthaldialdehyde and sodium sulfite at a high pH, 11.2. The product is readily concentrated from dilute solutions by reverse-phase solid-phase extraction (SPE). The method is compound-specific despite partial derivatisation of potentially interfering amino acids, as their derivatives are not extracted by SPE. delta(15)N values of NH(4) (+) in KCL soil extracts can be measured within 48 h by automated continuous-flow IRMS with a precision of 0.23 per thousand (1 SD). Parallel measurements of NH(4) (+) standards of known delta(15)N are made to allow correction for the isotopic dilution by non-sample NH(4) (+). The practicality of this method is demonstrated by measuring the changes in NH(4) (+) concentration and delta(15)N following the addition of urea as a nitrogen source to inorganic N-depleted soil.

Seaweeds in cold seas: evolution and carbon acquisition.

Much evidence suggests that life originated in hydrothermal habitats, and for much of the time since the origin of cyanobacteria (at least 2.5 Ga ago) and of eukaryotic algae (at least 2.1 Ga ago) the average sea surface and land surface temperatures were higher than they are today. However, there have been at least four significant glacial episodes prior to the Pleistocene glaciations. Two of these (approx. 2.1 and 0.7 Ga ago) may have involved a 'Snowball Earth' with a very great impact on the algae (sensu lato) of the time (cyanobacteria, Chlorophyta and Rhodophyta) and especially those that were adapted to warm habitats. By contrast, it is possible that heterokont, dinophyte and haptophyte phototrophs only evolved after the Carboniferous-Permian ice age (approx. 250 Ma ago) and so did not encounter low (

Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state.

The extent to which the N2-fixing bacterial endophyte Azoarcus sp. strain BH72 in the rhizosphere of Kallar grass can provide fixed nitrogen to the plant was assessed by evaluating inoculated plants grown in the greenhouse and uninoculated plants taken from the natural environment. The inoculum consisted of either wild-type bacteria or nifK- mutant strain BHNKD4. In N2-deficient conditions, plants inoculated with strain BH72 (N2-fixing test plants) grew better and accumulated more nitrogen with a lower delta15N signature after 8 months than did plants inoculated with the mutant strain (non-N2-fixing control plants). Polyadenylated or polymerase chain reaction-amplified BH72 nifH transcripts were retrieved from test but not from control plants. BH72 nifH transcripts were abundant. The inocula could not be reisolated. These results indicate that Azoarcus sp. BH72 can contribute combined N2 to the plant in an unculturable state. Abundant BH72 nifH transcripts were detected also in uninoculated plants taken from the natural environment, from which Azoarcus sp. BH72 also could not be isolated. Quantification of nitrogenase gene transcription indicated a high potential of strain BH72 for biological N2 fixation in association with roots. Phylogenetic analysis of nitrogenase sequences predicted that uncultured grass endophytes including Azoarcus spp. are ecologically dominant and play an important role in N2-fixation in natural grass ecosystems.

Mechanistic interpretation of carbon isotope discrimination by marine macroalgae and seagrasses.

The literature, and previously unpublished data from the authors' laboratories, shows that the δ13C of organic matter in marine macroalgae and seagrasses collected from the natural environment ranges from -3 to -35‰. While some marine macroalgae have δ13C values ranging over more than 10‰ within the thallus of an individual (some brown macroalgae), in other cases the range within a species collected over a very wide geographical range is only 5‰ (e.g. the red alga Plocamium cartilagineum which has values between -30 and -35‰). The organisms with very negative δ13C (lower than -30‰) are mainly subtidal red algae, with some intertidal red algae and a few green algae; those with very positive δ13C values (higher than -10‰) are mainly green macroalgae and seagrasses, with some red and brown macroalgae. The δ13C value correlates primarily with taxonomy and secondarily with ecology. None of the organisms with δ13C values lower than -30‰ have pyrenoids. Previous work showed a good correlation between δ13C values lower than -30‰ and the lack of CO2 concentrating mechanisms for several species of marine red algae. The extent to which the low δ13C values are confined to organisms with diffusive CO2 entry is discussed. Diffusive CO2 entry could also occur in organisms with higher δ13C values if diffusive conductance was relatively low. The photosynthesis of organisms with δ13C values more positive than -10‰ (i.e. more positive than the δ13C of CO2 in seawater) must involve HCO3- use.

Transport and assimilation of inorganic carbon by Lichina pygmaea under emersed and submersed conditions.

Photosynthetic O2 evolution by the upper littoral lichen, Lichina pygmaea (Lightf.) C.Ag., under light-saturated conditions at 5 °C is saturated by the 2 mol m-3 inorganic C found in seawater at pH 8.0. Photosynthesis is not reduced when pH is increased to pH 9.4, and is slightly reduced at pH 10.0, when submersed in seawater with 2 mol m-3 inorganic C. The rate of photosynthesis at pH 10 greatly exceeds the rate of uncatalysed conversion of HCO3 - . It is concluded that HCO3 - is used in photosynthesis. Since extracellular carbonic anhydrase is present, it is possible that CO2 enters the photobiont (Calothrix) cells even during HCO3 use. pH drift experiments support the notion of HCO3 - use. Emersed photosynthesis at 5 °C is more than half-saturated by 35 Pa (normal atmospheric) CO2 ; the light- and CO2 -saturated emersed photosynthetic rate is not significantly different from the light and inorganic C-saturated photosynthetic rate when submersed. Inorganic C diffusion from the thallus surface to the photobiont needs, at least under some conditions, carbonic anhydrase activity which permits HCO3 - fluxes to supplement CO2 movement. The CO2 compensation partial pressure at 5 °C is 0.83 Pa, i.e. at the low range of values found for terrestrial cyanobacterial lichens. Dark 14 C-inorganic C assimilation when submersed is a small fraction of the dark respiratory rate, consistent with the observed absence of diel CAM-like variation in intracellular titratable acidity. The high value (-11.5%) of δ13 C, the low CO2 compensation partial pressure, and the relatively high affinity for inorganic C., are consistent with the operation of an inorganic C concentrating mechanism such as occurs in free-living cyanobacteria and probably occurs in terrestrial cyanobacterial lichens and in most intertidal algae.

The role of CO2 uptake by roots and CAM in acquisition of inorganic C by plants of the isoetid life-form: a review, with new data on Eriocaulon decangulare L.

The isoetid life-form was originally defined on morphological grounds; subsequent physiological investigations showed that all of the isoetids examined took up a large fraction of the inorganic C fixed in their leaves from the root medium under natural conditions, and that some of them carried out much of their assimilation of inorganic C via a CAM-like mechanism. Root-dominated uptake of inorganic C appeared to be unique to, and ubiquitous in, the isoetids. I However, a large capacity for CAM-like metabolism in submerged vascular plants is not universal in isoetids, nor is it restricted to this life-form, being also found in Crassulaa aquatica. The work described here shows that submerged specimens of the North American Eriocaulon decangulare have a high fraction of their dry weight in the root system, a trait characteristic of isoetids but uncommon in other submerged vascular plants. E. decangulare has vesicular-arbuscular mycorrhizas, as do other flowering plant isoetids hut not, generally, submerged Isoetes spp. Under conditions of natural supply of inorganic C, E. decangulare, like other isoetids, takes up most of its inorganic C through its roots. Uptake of inorganic C by both roots and shoots involves CO2 rather than HCO3 : photosynthesis at high external pH values does not exceed the rate of uncatalysed HCO3 - to CO2 conversion in the medium and there is no detectable extracellular carbonic anhydrase activity. Measurements of titratable acidity and of malate content of leaves sampled at dawn and at dusk showed that E. decangulare, growing and tested under either emersed or submersed conditions, did not exhibit CAM-like behaviour. CAM was also absent from three non-isoetid aquatic macrophytes (Amphibolic antarctica, Eeklonia radiata and Vallisneria spiralis) which were examined. E. decangulare thus resembles all other isoetids tested in acquiring much of its inorganic C via the root system. E. decangulare also resembles most of the isoetids which are not members of the Isoetaceae (e.g.) E. septangulare, Lobelia dortmanna and Subularia aquatica) but differs from submerged Isoetaceae and Littorella uniflora in lacking CAM. The ecological significance of uptake of CO2 via the roots and, where it occurs, of CAM in isoetids may be related to either inorganic C or, via improved N use efficiency, inorganic C as a limiting resource. The isoetid life-forms has evolved independently in at last five different families of vascular plants; it probably derived fairly immediately from terrestrial or amphibious ancestors with a similar rosette form. Emergent Isoetaceae with acquisition of CO2 via roots and CAM probably evolved from submerged isoetids. CONTENTS Summary 123 I. Introduction 126 II. Material and Methods 127 III. Results and Discussion 129 IV. Conclusions 142 Acknowledgements 142 References 143.