Charles R. Hipkin

INTERACTIONS BETWEEN IRON, LIGHT, AMMONIUM, AND NITRATE: INSIGHTS FROM THE CONSTRUCTION OF A DYNAMIC MODEL OF ALGAL PHYSIOLOGY

A dynamic mechanistic mathematical model (FeLANIM) is described, capable of simulating the major documented interactions between iron, light, ammonium, and nitrate in phytoplankton. There are various points in the model where species‐ or group‐specific (e.g. eukaryote vs. prokaryote) details may be altered. Cellular Fe‐containing processes accounted for in the model are photosynthesis, oxidative phosphorylation, and nitrate assimilation; synthesis of photosystems and nitrate and nitrite reductase (NNiR) are functions of the surplus Fe quota (i.e. total Fe quota minus Fe in functional components). Model simulations were run using a range of different physiological parameters for Fe‐dependent processes and contrasting ammonium–nitrate interaction scenarios. Model output indicated the following interactions. Because the proportion of Fe in photosystems varies with irradiance, it is not possible to have a single Fe cost for nitrate assimilation. Fe stress can affect the relationship between ammonium and nitrate assimilations (the f‐ratio), with a more rapid depression of nitrate use at lower concentrations of ammonium. However, depending on the degree of the repression of nitrate assimilation at high N:C, such a result may not be universal, and further experimental studies are required to clarify this issue. Fe refeeding results in a rapid recovery of growth rate accompanied by a proportionately greater increase in the amount of chl a within 24– 48 h. Estimates of enhanced production in iron fertilization experiments based only on increases in pigment may thus be exaggerated. Stimulation of NNiR activity on Fe refeeding appears indirect, through enhancement of photosynthesis rather than relief of nitrate stress. During Fe refeeding at high light, N nutrient transport increases proportionately more in nitrate‐fed cells than in ammonium‐fed cells. Changing the N source supplied to simulated Fe‐stressed cells from nitrate to ammonium results in a rapid increase in growth rate and iron use efficiency with an increase in pigment content as NNiR content declines. When nitrate replaces ammonium, acclimation is slower because of the redirection of Fe formerly associated in photosystems to NNiR. Manipulation of the model may prove useful as an indicator for new research, by revealing elements of physiology that may be most significantin determining competitive advantage between species.

Changes in fatty acids, amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grown Isochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.

Comparison of Genetic Diversities in Native and Alien Populations of Hoary Mustard (Hirschfeldia incana [L.] Lagreze‐Fossat)

Increased selfing and inbreeding and, consequently, depauperate genetic diversities are commonly expected for alien colonies. We compared RAPDs data for native (southern Europe) and alien (British Isles) populations of hoary mustard (Hirschfeldia incana). This species is normally out‐breeding, but it is capable of self‐fertilization. Contrary to the common expectations, genetic diversities in native and alien populations were similar, without any strong evidence of decreased levels of genetic diversities in alien populations. A variety of factors may have contributed to this observation, including high variation in founding groups, founders originating from multiple H. incana source populations, and high rates of past and/or current gene flow. A review of other studies showed that this pattern of similar genetic diversities in native and alien populations was not unusual but has been regularly observed in other invasive plant species.

Nitrification by plants that also fix nitrogen

Nitrification is a key stage in the nitrogen cycle; it enables the transformation of nitrogen into an oxidized, inorganic state. The availability of nitrates produced by this process often limits primary productivity and is an important determinant in plant community ecology and biodiversity. Chemoautotrophic prokaryotes are recognized as the main facilitators of this process, although heterotrophic nitrification by fungi may be significant under certain conditions. However, there has been neither biochemical nor ecological evidence to support nitrification by photoautotrophic plants. Here we show how certain legumes that accumulate the toxin, 3-nitropropionic acid, generate oxidized inorganic nitrogen in their shoots, which is returned to the soil in their litter. In nitrogen-fixing populations this ‘new’ nitrate and nitrite can be derived from the assimilation of nitrogen gas. Normally, the transformation of elemental nitrogen from the atmosphere into a fixed oxidized form (as nitrate) is represented in the nitrogen cycle as a multiphasic process involving several different organisms. We show how this can occur in a single photoautotrophic organism, representing a previously undescribed feature of this biogeochemical cycle.

Changes in carbon and nitrogen physiology during ammonium and nitrate nutrition and nitrogen starvation in Isochrysis galbana

Isochrysis galbana was growth in a 12/12 h light/dark cycle with either nitrate or ammonium as the N-source, and then resuspended in N-free medium. During exponential growth ammonium-growing cells contained half the fatty acid content, higher concentrations of intracellular free glutamine and asparagine, and little nitrate reductase activity in comparison with nitrate-growing cells. Differences in the glutamine/glutamate and asparagine/aspartate ratios suggest that nitrate-growing cells were relatively more N-stressed than ammonium-growing cells. During N-starvation the fatty acid content of ammonium-grown cells increased to levels similar to those in nitrate-grown cells and the proportion of oleic acid increased in both cultures. Possible reasons for the increased synthesis of fatty acids, with its additional demand for reductant, in cells growing on nitrate-N are discussed. Nitrate reductase activity remained low in the ammonium-grown cells and decreased rapidly in the nitrate-grown cells during N-starv...

Interactions between nitrate and ammonium in Emiliania huxleyi

Abstract The coccolithophorid Emiliania huxleyi was grown with ammonium and nitrate in “stretched-batch” culture. This provided a continuous input of fresh media at a dilution rate of 0.1 d −1 and maintained the derepression of N-assimilatory systems during increasing N-stress, while exhibiting batch culture growth dynamics. Estimates of Kt (half-saturation constant for transport) for both N nutrients were less than 0.4 μM, with a median of 0.2 μM. During the development of N-stress, the maximum rate of N-specific transport ( Vt max ) for ammonium increased while that for nitrate more closely matched the concurrent rate of N-specific growth as N-stress developed. When both N nutrients were added together, respective Kt values showed no significant change, suggesting the operation of discrete nitrate and ammonium porters. In contrast, there were changes in Vt max when both nutrients were assimilated concurrently. However, higher concentrations of nitrate (4 μM) were required to achieve a 50% depression of ammonium Vt max compared to the concentration of ammonium (0.5 μM) required to bring about a similar depression of nitrate Vt max . This is consistent with the regulation of transport of both nutrients by an organic product of N-assimilation, which accumulates more rapidly following ammonium addition to N-stressed cells. Ammonium-grown cells contained higher concentrations of internal free amino acids, such as glutamine, than nitrate-grown cells. Interactions between ammonium and nitrate transport and assimilations could be simulated using the Ammonium–Nitrate Interaction Model, which contains constants for Kt and transports regulated by the level of the early product of N-assimilation, glutamine. There were no indications of a significant loss of N from the [dissolved inorganic N plus particulate organic N] fraction for exponentially growing Emiliania huxleyi in experiments, and there was no need to include such a process in the model for it to simulate the experiments.

3-nitropropionic acid oxidase from horseshoe vetch (Hippocrepis comosa): a novel plant enzyme.

A novel enzyme that catalyses the oxygen-dependent oxidation of 3-nitropropionic acid (3NPA) to malonate semialdehyde, nitrate, nitrite and H2O2 has been purified from leaf extracts of the horseshoe vetch, Hippocrepis comosa, and named 3NPA oxidase. The enzyme is a flavoprotein with a subunit molecular mass of 36 kDa containing 1 molecule of FMN and exhibits little specificity for all nitroalkanes tested other than 3NPA (apparent Km 620 microM). The maximum enzyme activity in vitro was expressed at pH4.8 and was inhibited strongly by the products nitrate and nitrite. 3NPA oxidase activity was detected in green shoots, which also contain high concentrations of 3NPA, from plants grown with nitrate, ammonium or N2 as sources of nitrogen. Enzyme activity was absent from roots and cell cultures, neither of which accumulate high levels of 3NPA.

Nitroaliphatic compounds in Hippocrepis comosa and other legumes in the European flora

Abstract In a survey of 148, mostly European, angiosperm species, 3-nitropropionate was found only in extracts of the legumes, Hippocrepis balearica, H. comosa, Lotus uliginosus and Scorpiurus muricatus . Three separate nitropropanoyl esters of glucose, 6-(3-nitropropanoyl)-αβ- d -glucopyranose, 1,6- di -O-(3- nitropropanoyl -β- d - glucopyranose (cibarian) and 1,2,6- tri -O-(3- nitropropanoyl )-β- d - glucopyranose (karakin) were identified in shoot extracts of H . comosa . Evidence for the presence of the 3-nitropropanoyl ester of 4-hydroxybutanoic acid in extracts of H . comosa is also presented.

Conservation of genetic diversity in British populations of the diploid endemic Coincya monensis ssp monensis (Isle of Man Cabbage): the risk of hybridisation with the tetraploid alien, Coincya monensis ssp cheiranthos

Coincya monensis is represented in the British flora by two, cytologically distinct subspecies. Coincya monensis ssp monensis is an endemic diploid with a coastal sand dune distribution that includes a number of isolated populations. Coincya monensis ssp cheiranthos is a tetraploid alien, well established in South Wales in early successional habitats. Both subspecies share similar life form traits, flowering times and pollinators. Cluster analysis and phylogenetic reconstruction based on sequences of the mitochondrial nad4 gene confirmed the distinction between alien and endemic taxa. Tetraploid populations carry more polymorphic RAPDs loci and their genetic diversity is partitioned more within than among populations. In contrast, C. monensis ssp monensis has a distinct population genetic structure. Analysis of the multilocus genetic data confirmed a structure of genetically isolated, endemic population clusters in Scotland, Arran, the Isle of Man and South Wales. Experimental hybridisation showed the two subspecies are interfertile. Multivariate analysis of RAPDs data resolved hybrids between alien and endemic clusters and hybrids contained a proportion of alien-specific polymorphic loci. Hybrids of alien maternal parentage contained the mitochondrial nad4 sequence characteristic of the alien subspecies. Since the alien subspecies can invade mobile sand dune communities from urban sites and compete for pollinators, there is a risk that alien and endemic populations will mix and introgress. Conservation of endemic genetic diversity in Britain will require protection for all C. monensis ssp monensis populations. Currently, the most disjunct endemic population in South Wales is most at risk from introgression.

Presence of 3-nitropropionic acid, in widely distributed pasture legumes in Britain

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