Inorganic Nitrogen Assimilation Research Articles

Amoebocytes facilitate efficient carbon and nitrogen assimilation in the Cassiopea-Symbiodiniaceae symbiosis.

The upside-down jellyfish Cassiopea engages in symbiosis with photosynthetic microalgae that facilitate uptake and recycling of inorganic nutrients. By contrast to most other symbiotic cnidarians, algal endosymbionts in Cassiopea are not restricted to the gastroderm but are found in amoebocyte cells within the mesoglea. While symbiont-bearing amoebocytes are highly abundant, their role in nutrient uptake and cycling in Cassiopea remains unknown. By combining isotopic labelling experiments with correlated scanning electron microscopy, and Nano-scale secondary ion mass spectrometry (NanoSIMS) imaging, we quantified the anabolic assimilation of inorganic carbon and nitrogen at the subcellular level in juvenile Cassiopea medusae bell tissue. Amoebocytes were clustered near the sub-umbrella epidermis and facilitated efficient assimilation of inorganic nutrients. Photosynthetically fixed carbon was efficiently translocated between endosymbionts, amoebocytes and host epidermis at rates similar to or exceeding those observed in corals. The Cassiopea holobionts efficiently assimilated ammonium, while no nitrate assimilation was detected, possibly reflecting adaptation to highly dynamic environmental conditions of their natural habitat. The motile amoebocytes allow Cassiopea medusae to distribute their endosymbiont population to optimize access to light and nutrients, and transport nutrition between tissue areas. Amoebocytes thus play a vital role for the assimilation and translocation of nutrients in Cassiopea, providing an interesting new model for studies of metabolic interactions in photosymbiotic marine organisms.

Heterotrophic Foraminifera Capable of Inorganic Nitrogen Assimilation.

Nitrogen availability often limits biological productivity in marine systems, where inorganic nitrogen, such as ammonium is assimilated into the food web by bacteria and photoautotrophic eukaryotes. Recently, ammonium assimilation was observed in kleptoplast-containing protists of the phylum foraminifera, possibly via the glutamine synthetase/glutamate synthase (GS/GOGAT) assimilation pathway imported with the kleptoplasts. However, it is not known if the ubiquitous and diverse heterotrophic protists have an innate ability for ammonium assimilation. Using stable isotope incubations (15N-ammonium and 13C-bicarbonate) and combining transmission electron microscopy (TEM) with quantitative nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, we investigated the uptake and assimilation of dissolved inorganic ammonium by two heterotrophic foraminifera; a non-kleptoplastic benthic species, Ammonia sp., and a planktonic species, Globigerina bulloides. These species are heterotrophic and not capable of photosynthesis. Accordingly, they did not assimilate 13C-bicarbonate. However, both species assimilated dissolved 15N-ammonium and incorporated it into organelles of direct importance for ontogenetic growth and development of the cell. These observations demonstrate that at least some heterotrophic protists have an innate cellular mechanism for inorganic ammonium assimilation, highlighting a newly discovered pathway for dissolved inorganic nitrogen (DIN) assimilation within the marine microbial loop.

The Contribution of Small Phytoplankton Communities to the Total Dissolved Inorganic Nitrogen Assimilation Rates in the East/Japan Sea: An Experimental Evaluation

As a part of Korean-Russian joint expeditions in the East/Japan Sea during 2012 and 2015, a set of total and small (<2 μm) phytoplankton NO3− and NH4+ uptake rate estimations were carried out. The study aimed to assess the spatio-temporal variations in dissolved inorganic nitrogen (DIN) assimilation by the total and small phytoplankton. The results show that the total NO3− uptake rates during 2012 varied between 0.001 and 0.150 μmol NL−1h−1 (mean ± SD = 0.034 ± 0.033) and that the total NH4+ uptake rates ranged between 0.002 and 0.707 μmol NL−1h−1 (mean ± SD = 0.200 ± 0.158). The total uptake rates during 2015 were ranged from 0.003 to 0.530 (mean ± S.D. = 0.117 ± 0.120 μmol NL−1h−1) for NO3− and from 0.008 to 1.17 (mean ± S.D. = 0.199 ± 0.266 NL−1h−1) for NH4+. The small phytoplankton NO3− and NH4+ uptake rates during 2015 ranged between 0.001 and 0.164 (mean ± S.D. = 0.033 ± 0.036) μmol NL−1h−1 and 0.010–0.304 (mean ± S.D. = 0.101 ± 0.073) μmol NL−1h−1, respectively. Small phytoplankton’s contribution to the total depth-integrated NO3− and NH4+ uptake rates ranged from 10.24 to 59.36% and from 30.21 to 68.55%, respectively. The significant negative relationship observed between the depth-integrated total NO3− and NH4+ uptake rates and small phytoplankton contributions indicates a possible decline in the DIN assimilation rates under small phytoplankton dominance. The results from the present study highlight the possibility of a reduction in the total DIN assimilation process in the East/Japan Sea when small phytoplankton dominate under strong thermal stratification due to sea surface warming. The present study’s findings agree with the model projections, which suggested a decline in primary production in the global warming scenario.

Utilization of biofloc pond water as a substitute for nutrition in the pak choi hydroponic system

Increased fish consumption needs can be overcome by high stocking density in fish farming. Stocking density and high feeding rise the problem of increasing organic waste which can reduce water quality. Biofloc technology is one alternative that can overcome this problem by adding the amount of organic carbon in the pond, thereby increasing the C / N ratio of water that will grow heterotrophic bacteria. The basic principle of biofloc technology is the assimilation of inorganic nitrogen by heterotrophic microbial communities in cultivation media which can then be utilized by aquatic organisms as a food source. The government through the Ministry of Marine Affairs and Fisheries promotes freshwater fish farming with biofloc technology. The objective of this research was to assessing the effect of biofloc pond water on pak choi growth. The research was conducted for two months, planting the pak choi in pipe line using rockwool as its growth media. The cultivation used two treatments, biofloc pond water and ab mix as its nutrition. The result showed that pak choi growth with biofloc pond water was lower than ab mix nutrition.

Differential contributions of NO3\u2212/NH4+ to nitrogen use in response to a variable inorganic nitrogen supply in plantlets of two Brassicaceae species in vitro

BackgroundThe primary sources of nitrogen for plants have been suggested to be nitrate (NO3−) and ammonium (NH4+). However, when both nitrate and ammonium are simultaneously available to plants, it is very difficult to differentially quantify NO3−/NH4+ utilization in culture media or soil. Consequently, the contribution of NO3−/NH4+ to total inorganic nitrogen assimilation cannot be determined.ResultsWe developed a method called the bidirectional stable nitrogen isotope tracer to differentially quantify the nitrate and ammonium utilization by Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets in vitro. The utilization efficiency of nitrate was markedly lower than the utilization efficiency of ammonium for plantlets of both Ov and Bn. In both Ov and Bn, the proportion of NO3−/NH4+ utilization did not show a linear relationship with inorganic nitrogen supply. The Ov plantlets assimilated more nitrate than the Bn plantlets at the lowest inorganic nitrogen concentration.ConclusionsQuantifying the utilization of nitrate and ammonium can reveal the differences in nitrate and ammonium assimilation among plants at different inorganic nitrogen supply levels and provide an alternate way to conveniently optimize the supply of inorganic nitrogen in culture media.

Quantifying Inorganic Nitrogen Assimilation by Synechococcus Using Bulk and Single-Cell Mass Spectrometry: A Comparative Study.

Microorganisms drive most of the major biogeochemical cycles in the ocean, but the rates at which individual species assimilate and transform key elements is generally poorly quantified. One of these important elements is nitrogen, with its availability limiting primary production across a large proportion of the ocean. Nitrogen uptake by marine microbes is typically quantified using bulk-scale approaches, such as Elemental Analyzer-Isotope Ratio Mass Spectrometry (EA-IRMS), which averages uptake over entire communities, masking microbial heterogeneity. However, more recent techniques, such as secondary ion mass spectrometry (SIMS), allow for elucidation of assimilation rates at the scale at which they occur: the single-cell level. Here, we combine and compare the application of bulk (EA-IRMS) and single-cell approaches (NanoSIMS and Time-of-Flight-SIMS) for quantifying the assimilation of inorganic nitrogen by the ubiquitous marine primary producer Synechococcus. We aimed to contrast the advantages and disadvantages of these techniques and showcase their complementarity. Our results show that the average assimilation of 15N by Synechococcus differed based on the technique used: values derived from EA-IRMS were consistently higher than those derived from SIMS, likely due to a combination of previously reported systematic depletion as well as differences in sample preparation. However, single-cell approaches offered additional layers of information, whereby NanoSIMS allowed for the quantification of the metabolic heterogeneity among individual cells and ToF-SIMS enabled identification of nitrogen assimilation into peptides. We suggest that this coupling of stable isotope-based approaches has great potential to elucidate the metabolic capacity and heterogeneity of microbial cells in natural environments.

Kleptoplastidic benthic foraminifera from aphotic habitats: insights into assimilation of inorganic C, N and S studied with sub-cellular resolution.

The assimilation of inorganic compounds in foraminiferal metabolism compared to predation or organic matter assimilation is unknown. Here, we investigate possible inorganic-compound assimilation in Nonionellina labradorica, a common kleptoplastidic benthic foraminifer from Arctic and North Atlantic sublittoral regions. The objectives were to identify the source of the foraminiferal kleptoplasts, assess their photosynthetic functionality in light and darkness and investigate inorganic nitrogen and sulfate assimilation. We used DNA barcoding of a ~ 830 bp fragment from the SSU rDNA to identify the kleptoplasts and correlated transmission electron microscopy and nanometre-scale secondary ion mass spectrometry (TEM-NanoSIMS) isotopic imaging to study 13 C-bicarbonate, 15 N-ammonium and 34 S-sulfate uptake. In addition, respiration rate measurements were determined to assess the response of N. labradorica to light. The DNA sequences established that over 80% of the kleptoplasts belonged to Thalassiosira (with 96%-99% identity), a cosmopolitan planktonic diatom. TEM-NanoSIMS imaging revealed degraded cytoplasm and an absence of 13 C assimilation in foraminifera exposed to light. Oxygen measurements showed higher respiration rates under light than dark conditions, and no O2 production was detected. These results indicate that the photosynthetic pathways in N. labradorica are not functional. Furthermore, N. labradorica assimilated both 15 N-ammonium and 34 S-sulfate into its cytoplasm, which suggests that foraminifera might have several ammonium or sulfate assimilation pathways, involving either the kleptoplasts or bona fide foraminiferal pathway(s) not yet identified.

Does bicarbonate affect the nitrate utilization and photosynthesis of Orychophragmus violaceus?

The effect of bicarbonate (HCO3−) on the growth and development of plants varies by species. To better understand inorganic carbon and nitrogen assimilation changes of karst-adaptable plants under different HCO3− treatments, we conducted experiments on seedlings and in vitro plantlets of Orychophragmus violaceus (Ov). We found that the vital photosynthesis potential (as measured by net photosynthetic rate, actual photochemical efficiency of photosystem-II, photochemical quenching coefficient, and the instantaneous carbon isotope ratio of 3-phosphoglycerate) was consistent under different HCO3− treatments of Ov. Bicarbonate’s lack of effect on carbon assimilation of Ov may be related to carbonic anhydrase in Ov converting HCO3− to H2O and CO2. In this way, Ov could prevent HCO3− ion toxicity and high pH from harming its growth and development under HCO3− stress. This study also found that high HCO3− concentrations could promote nitrogen assimilation and utilization of Ov through changes in related indexes (foliar nitrogen isotope fractionation ratio, stable nitrogen isotope assimilation ratio, foliar stable nitrogen isotope fractionation, nitrate nitrogen utilization efficiency, and nitrate utilization share) under different HCO3− treatments. Bicarbonate has different effects on photosynthesis and on inorganic nitrogen assimilation of Ov, which may be connected to photosynthesis providing electrons for nitrate/nitrite reduction through the photosynthetic chain.

Inorganic carbon and nitrogen assimilation in cellular compartments of a benthic kleptoplastic foraminifer

Haynesina germanica, an ubiquitous benthic foraminifer in intertidal mudflats, has the remarkable ability to isolate, sequester, and use chloroplasts from microalgae. The photosynthetic functionality of these kleptoplasts has been demonstrated by measuring photosystem II quantum efficiency and O2 production rates, but the precise role of the kleptoplasts in foraminiferal metabolism is poorly understood. Thus, the mechanism and dynamics of C and N assimilation and translocation from the kleptoplasts to the foraminiferal host requires study. The objective of this study was to investigate, using correlated TEM and NanoSIMS imaging, the assimilation of inorganic C and N (here ammonium, NH4+) in individuals of a kleptoplastic benthic foraminiferal species. H. germanica specimens were incubated for 20 h in artificial seawater enriched with H13CO3− and 15NH4+ during a light/dark cycle. All specimens (n = 12) incorporated 13C into their endoplasm stored primarily in the form of lipid droplets. A control incubation in darkness resulted in no 13C-uptake, strongly suggesting that photosynthesis is the process dominating inorganic C assimilation. Ammonium assimilation was observed both with and without light, with diffuse 15N-enrichment throughout the cytoplasm and distinct 15N-hotspots in fibrillar vesicles, electron-opaque bodies, tubulin paracrystals, bacterial associates, and, rarely and at moderate levels, in kleptoplasts. The latter observation might indicate that the kleptoplasts are involved in N assimilation. However, the higher N assimilation observed in the foraminiferal endoplasm incubated without light suggests that another cytoplasmic pathway is dominant, at least in darkness. This study clearly shows the advantage provided by the kleptoplasts as an additional source of carbon and provides observations of ammonium uptake by the foraminiferal cell.

The Importance of Mesodinium rubrum at Post-Spring Bloom Nutrient and Phytoplankton Dynamics in the Vertically Stratified Baltic Sea

The inter-annual dynamics of the photosynthetic ciliate Mesodinium rubrum in the central Gulf of Finland in spring-summer continuum during five years were followed. The analysis was mainly based on high-resolution measurements and sampling in the surface layer along the ferry route Tallinn-Helsinki. The main purpose was to analyze the dynamics of M. rubrum biomass, its contribution to the photosynthetic plankton biomass, and the influence of water temperature and variations of inorganic nutrients in the surface and sub-surface layer on its dynamics. The analysis revealed that the outcome of the M. rubrum bloom in spring was largely related to the surface layer water temperature – in the years of earlier warming, the higher biomass of this species was formed. The photosynthetic ciliate was an important primary producer in all studied years during the late phase or post-spring bloom period in the Gulf of Finland. The maximum proportion of M. rubrum in the photosynthetic plankton community was estimated up to 88% in May and up to 91% in June. We relate the observed post-spring bloom decrease of phosphate concentrations in the surface layer to the dominance and growth of M. rubrum. We suggest that this link can be explained by the vertical migration behaviour of M. rubrum and phosphate utilization in the surface layer coupled with inorganic nitrogen assimilation in the sub-surface layer. Thus, the dynamics of M. rubrum could strongly influence the amount of post-spring bloom excess PO43- in the euphotic layer and the depth of nitracline in the Gulf of Finland.

Carbon and Nitrogen Acquisition in Shallow and Deep Holobionts of the Scleractinian Coral S. pistillata

Reef building corals can host different symbiont genotypes (clades), and form distinct holobionts in response to environmental changes. Studies on the functional significance of genetically different symbionts have focused on the thermal tolerance rather than on the nutritional significance. Here, we characterized the nitrogen and carbon assimilation rates, the allocation patterns of these nutrients within the symbiosis, and the trophic condition of two distinct holobionts of Stylophora pistillata: one associated with Symbiodinium clade A in shallow reefs and the other one associated with clade C in mesophotic reefs. The main findings are that: (1) clade C-symbionts have a competitive advantage for the acquisition of carbon at low irradiance compared to clade A-symbionts; (2) light is however the primary factor that determines the positive relationship between the amount of carbon fixed in photosynthesis by the symbionts and the amount of carbon translocated to the host; and (3) by contrast, the dominant Symbiodinium type preferentially determines a negative relationship between rates of coral feeding and nitrogen assimilation, although light still plays a relevant role in this relationship. Clade C-holobionts had indeed higher heterotrophic capacities, but lower inorganic nitrogen assimilation rates than clade A-holobionts, at all light levels. Broadly, our results show that the assimilation and translocation rates of inorganic carbon and nitrogen are clade and light-dependent. In addition, the capacity of S. pistillata to form mesophotic reefs in the Red Sea relies on its ability to select Symbiodinium clade C, as this symbiont type is more efficient to fix carbon at low light.

The δ15N response and nitrate assimilation of Orychophragmus violaceus and Brassica napus plantlets in vitro during the multiplication stage cultured under different nitrate concentrations

Natural nitrogen isotope composition (δ15N) is an indicator of nitrogen sources and is useful in the investigation of nitrogen cycling in organisms and ecosystems. δ15N is also used to study assimilation of inorganic nitrogen. However, the foliar δ15N of intact plants, which is a consequence of nitrate assimilation occurring in the roots and shoots, is not suited for studying nitrate assimilation in cases where nitrate is the sole nitrogen source. In this study, Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets, in which nitrate assimilation occurred in the leaves, were used to study the relationship between foliar δ15N and nitrate assimilation. The plantlets were grown in vitro in culture media with different nitrate concentrations, and no root formation occurred for the plantlets during the multiplication stage. Nitrogen isotope fractionation occurred in both the Ov and the Bn plantlets under all treatments. Furthermore, the foliar nitrogen content of both the Ov and Bn plantlets increased with increasing nitrate concentration. Foliar nitrogen isotope fractionation was negatively correlated with foliar nitrogen content for both the Ov and Bn plantlets. Our results suggest that the foliar nitrogen isotope fractionation value could be employed to evaluate nitrate assimilation ability and leaf nitrate reductase activity. Moreover, high external nitrate concentrations could contribute to improved foliar nitrogen content and enhanced nitrate assimilation ability.

Production of transgenic tobacco plants expressing GS1 gene for the increase ofnitrogen use efficiency

Glutamine synthetase (GS, CE 6.3.1.2) is an enzyme that catalyzes the ATP-dependent condensation of glutamic acid with ammonia to yield glutamine. Glutamine synthetase is a key enzyme involved in the assimilation of inorganic nitrogen into organic forms. In plant cells, GS is present in both chloroplasts (GS2) and cytoplasm (GS1), in which GS1 can assimilate nitrogen source. In this study, one transgenic vector pBI121 carrying GS1 gene under the control of promoter 35S (pBI121::GS1) were successfully constructed. This vector containing G1S gene was transformed into tobacco leaves pieces via Agrobacterium tumefaciens strain C58. Five weeks after cultivation, there were 28 tobacco lines which had roots on the medium added kanamycin 50 mg/l. Then, the presence of G1S gene in these tobacco lines was tested from leaves in the next experiments. PCR and Southern blot confirmed that there are five tobacco lines carrying transferred GS1 gene. The effectiveness of nitrogen using in GS1 transgenic tobacco plants in vitro was evaluated. The tissue fresh weight, number and height of shoots forming buds and rooting ability of GS1 transgenic tobacco plants were greater than those of non-GM plants in the medium of low nitrogen concentration (0.1X - 0.2X). Assessment of crop growing in a greenhouse demonstrated that GS1 transgenic tobacco plants grow faster than non-transgenic ones. In detail, the increment of plant height after planting 03 months and 05 months in greenhouse is 43.55% and 33.29%, respectively. These results provide a scientific basis for the development of other genetically modified plants which enhanced nitrogen-use efficiency.

Stable Isotope Resolved Metabolomics Reveals the Role of Anabolic and Catabolic Processes in Glyphosate-Induced Amino Acid Accumulation in Amaranthus palmeri Biotypes.

Biotic and abiotic stressors often result in the buildup of amino acid pools in plants, which serve as potential stress mitigators. However, the role of anabolic (de novo amino acid synthesis) versus catabolic (proteolytic) processes in contributing to free amino acid pools is less understood. Using stable isotope-resolved metabolomics (SIRM), we measured the de novo amino acid synthesis in glyphosate susceptible (S-) and resistant (R-) Amaranthus palmeri biotypes. In the S-biotype, glyphosate treatment at 0.4 kg ae/ha resulted in an increase in total amino acids, a proportional increase in both (14)N and (15)N amino acids, and a decrease in soluble proteins. This indicates a potential increase in de novo amino acid synthesis, coupled with a lower protein synthesis and a higher protein catabolism following glyphosate treatment in the S-biotype. Furthermore, the ratio of glutamine/glutamic acid (Gln/Glu) in the glyphosate-treated S- and R-biotypes indicated that the initial assimilation of inorganic nitrogen to organic forms is less affected by glyphosate. However, amino acid biosynthesis downstream of glutamine is disproportionately disrupted in the glyphosate treated S-biotype. It is thus concluded that the herbicide-induced amino acid abundance in the S-biotype is contributed by both protein catabolism and de novo synthesis of amino acids such as glutamine and asparagine.

Metabolic changes and associated cytokinin signals in response to nitrate assimilation in roots and shoots of Lolium perenne

The efficiency of inorganic nitrogen (N) assimilation is a critical component of fertilizer use by plants and of forage production in Lolium perenne, an important pasture species worldwide. We present a spatiotemporal description of nitrate use efficiency in terms of metabolic responses and carbohydrate remobilization, together with components of cytokinin signal transduction following nitrate addition to N-impoverished plants. Perennial ryegrass (L. perenne cv. Grasslands Nui) plants were grown for 10 weeks in unfertilized soil and then treated with nitrate (5 mM) hydroponically. Metabolomic analysis by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry revealed a dynamic interaction between N and carbon metabolism over a week-long time course represented by the relative abundance of amino acids, tricarboxylic acid intermediates and stored water-soluble carbohydrates (WSCs). The initial response to N addition was characterized by a rapid remobilization of carbon stores from the low-molecular weight WSC, along with an increase in N content and assimilation into free amino acids. Subsequently, the shoot became the main source of carbon through remobilization of a large pool of high-molecular weight WSC. Associated quantification of cytokinin levels and expression profiling of putative cytokinin response regulator genes by quantitative reverse transcription polymerase chain reaction support a role for cytokinin in the mediation of the response to N addition in perennial ryegrass. The presence of high levels of cis-zeatin-type cytokinins is discussed in the context of hormonal homeostasis under the stress of steady-state N deficiency.

An RNA interference knock-down of nitrate reductase enhances lipid biosynthesis in the diatom Phaeodactylum tricornutum.

When diatoms are stressed for inorganic nitrogen they remodel their intermediate metabolism and redirect carbon towards lipid biosynthesis. However, this response comes at a significant cost reflected in decreased photosynthetic energy conversion efficiency and growth. Here we explore a molecular genetics approach to restrict the assimilation of inorganic nitrogen by knocking down nitrate reductase (NR). The transformant strain, NR21, exhibited about 50% lower expression and activity of the enzyme but simultaneously accumulated over 40% more fatty acids. However, in contrast to nitrogen-stressed wild-type (WT) cells, which grow at about 20% of the rate of nitrogen-replete cells, growth of NR21 was only reduced by about 30%. Biophysical analyses revealed that the photosynthetic energy conversion efficiency of photosystem II was unaffected in NR21; nevertheless, the plastoquinone pool was reduced by 50% at the optimal growth irradiance while in the WT it was over 90% oxidized. Further analyses reveal a 12-fold increase in the glutamate/glutamine ratio and an increase NADPH and malonyl-CoA pool size. Transcriptomic analyses indicate that the knock down resulted in changes in the expression of genes for lipid biosynthesis, as well as the expression of specific transcription factors. Based on these observations, we hypothesize that the allocation of carbon and reductants in diatoms is controlled by a feedback mechanism between intermediate metabolites, the redox state of the plastid and the expression and binding of transcription factors related to stress responses.

Ameliorating Effects of Fungus Chaff and Its Biochar on Soil Acidity

A biochar was generated from fungus chaff at 300 °C, and the ameliorating effects of fungus chaff and its biochar on an acidic Ultisol were compared using incubation experiments. Incorporation of fungus chaff and its biochar significantly increased soil pH and soil exchangeable base cations but decreased soil exchangeable acidity. The ameliorating effect was greater for the biochar than the fungus chaff, and thus the biochar was a better amendment for acidic soil than its feedstock of fungus chaff. The biochar ameliorated soil acidity mainly through the release of its contained alkaline substances, while fungus chaff increased pH of acidic soils through two mechanisms: release of alkaline substances and inhibition of soil nitrification. The incorporation of fungus chaff increased soil-available organic carbon and thus accelerated the microbial assimilation of inorganic nitrogen, while incorporation of fungus chaff biochar enhanced nitrification due to increased soil pH.

Interspecific variation in leaf–root differences in δ15N among three tree species grown with either nitrate or ammonium

Interspecific variation in nitrogen isotope composition of roots and leaves of tree seedlings grown in a steady-state nitrogen environment reflects known variation in sites of assimilation and nitrogen source preference in three tree species. As a time-integrated measure of nitrogen use, discrimination against the heavier stable isotope (15N) during the uptake and assimilation of inorganic nitrogen has the potential to provide information on interspecific differences in inorganic nitrogen source preference. Here, nitrogen isotope composition (δ15N) at natural abundance was measured for the roots and shoots from seedlings of three forest tree species: Populus tremuloides (aspen), Pinus contorta var. latifolia (pine) and Picea glauca (spruce). The seedlings were grown hydroponically with low (0.1 mM) or high (1.5 mM) concentrations of NO3 − or NH4 +, or in sand with NO3 −, NH4 + or an equal mix of NO3 − and NH4 + (0.1 mM). Whole-plant nitrogen isotope discrimination was observed in hydroponically grown seedlings but not in sand culture. Differences in δ15N between shoots and roots were greater in aspen when grown with NO3 − (3.02 ‰) than with NH4 + (1.27 ‰). There were no significant differences between the δ15N of leaves and roots for pine and spruce on either source. Although whole-plant nitrogen isotope discrimination was not observed in sand culture, shoot δ15N was, again, greater than root δ15N for NO3 −-grown aspen. Interspecific variation in nitrogen isotope discrimination was observed in both hydroponics and sand culture. The differences in nitrogen isotope composition under steady-state conditions indicate that interspecific differences in nitrogen source preference are consistent with previous experiments using alternative methods to identify differences in nitrogen uptake and assimilation in the same tree species.

Comparative Genomics Suggests That the Human Pathogenic Fungus Pneumocystis jirovecii Acquired Obligate Biotrophy through Gene Loss

Pneumocystis jirovecii is a fungal parasite that colonizes specifically humans and turns into an opportunistic pathogen in immunodeficient individuals. The fungus is able to reproduce extracellularly in host lungs without eliciting massive cellular death. The molecular mechanisms that govern this process are poorly understood, in part because of the lack of an in vitro culture system for Pneumocystis spp. In this study, we explored the origin and evolution of the putative biotrophy of P. jirovecii through comparative genomics and reconstruction of ancestral gene repertoires. We used the maximum parsimony method and genomes of related fungi of the Taphrinomycotina subphylum. Our results suggest that the last common ancestor of Pneumocystis spp. lost 2,324 genes in relation to the acquisition of obligate biotrophy. These losses may result from neutral drift and affect the biosyntheses of amino acids and thiamine, the assimilation of inorganic nitrogen and sulfur, and the catabolism of purines. In addition, P. jirovecii shows a reduced panel of lytic proteases and has lost the RNA interference machinery, which might contribute to its genome plasticity. Together with other characteristics, that is, a sex life cycle within the host, the absence of massive destruction of host cells, difficult culturing, and the lack of virulence factors, these gene losses constitute a unique combination of characteristics which are hallmarks of both obligate biotrophs and animal parasites. These findings suggest that Pneumocystis spp. should be considered as the first described obligate biotrophs of animals, whose evolution has been marked by gene losses.

Nitrogen isotope discrimination as an integrated measure of nitrogen fluxes, assimilation and allocation in plants

Fractionation of nitrogen isotopes between a plant and its environment occurs during uptake and assimilation of inorganic nitrogen. Fractionation can also occur between roots and the shoot. Under controlled nitrogen conditions, whole-plant and organ-level nitrogen isotope discrimination (Δ(15) N) is suggested to primarily be a function of three factors: nitrogen efflux back to the substrate relative to gross influx at the root (efflux/influx), the proportion of net influx assimilated in the roots and the export of remaining inorganic nitrogen for assimilation in the leaves. Here, an isotope discrimination model combining measurements of δ(15) N and nitrogen content is proposed to explain whole-plant and organ-level variation in δ(15) N under steady-state conditions and prior to any significant retranslocation. We show evidence that nitrogen isotope discrimination varies in accordance with changes to nitrogen supply or demand. Increased whole-plant discrimination (greater Δ(15) N or more negative δ(15) N relative to the source nitrogen δ(15) N) indicates increased turnover of the cytosolic inorganic nitrogen pool and a greater efflux/influx ratio. A greater difference between shoot and root δ(15) N indicates a greater proportion of inorganic nitrogen being assimilated in the leaves. In addition to calculations of integrated nitrogen-use traits, knowledge of biomass partitioning and nitrogen concentrations in different plant organs provides a spatially and temporally integrated, whole-plant phenotyping approach for measuring nitrogen-use in plants. This approach can be used to complement instantaneous cell- and tissue-specific measures of nitrogen use currently used in nitrogen uptake and assimilation studies.