Nitrogen Stress Affects the Turnover and Size of Nitrogen Pools Supplying Leaf Growth in a Grass

The effect of nitrogen (N) stress on the pool system supplying currently assimilated and (re)mobilized N for leaf growth of a grass was explored by dynamic ¹⁵N labeling, assessment of total and labeled N import into leaf growth zones, and compartmental analysis of the label import data. Perennial ryegrass (Lolium perenne) plants, grown with low or high levels of N fertilization, were labeled with ¹⁵NO₃⁻/¹⁴NO₃⁻ from 2 h to more than 20 d. In both treatments, the tracer time course in N imported into the growth zones fitted a two-pool model (r² > 0.99). This consisted of a “substrate pool,” which received N from current uptake and supplied the growth zone, and a recycling/mobilizing “store,” which exchanged with the substrate pool. N deficiency halved the leaf elongation rate, decreased N import into the growth zone, lengthened the delay between tracer uptake and its arrival in the growth zone (2.2 h versus 0.9 h), slowed the turnover of the substrate pool (half-life of 3.2 h versus 0.6 h), and increased its size (12.4 μg versus 5.9 μg). The store contained the equivalent of approximately 10 times (low N) and approximately five times (high N) the total daily N import into the growth zone. Its turnover agreed with that of protein turnover. Remarkably, the relative contribution of mobilization to leaf growth was large and similar (approximately 45%) in both treatments. We conclude that turnover and size of the substrate pool are related to the sink strength of the growth zone, whereas the contribution of the store is influenced by partitioning between sinks.This article examines the nitrogen (N) supply system of growing grass leaves, and it investigates how functional and kinetic properties of this system are affected by N stress. The N supply of growing leaves is a dominant target of whole-plant N metabolism. This is primarily related to the high N demand of the photosynthetic apparatus and the related metabolic machinery of new leaves (Evans, 1989; Makino and Osmond, 1991; Grindlay, 1997; Lemaire, 1997; Wright et al., 2004; Johnson et al., 2010; Maire et al., 2012). The N supply system, as defined here, is an integral part of the whole plant: it includes all N compounds that supply leaf growth. Hence, it integrates all events between the uptake of N from the environment (source), intermediate uses in other processes of plant N metabolism, and the eventual delivery to the leaf growth zone (sink; Fig. 1). N that does not ultimately serve leaf growth is not included in this system; all N that serves leaf growth is included, irrespective of its localization in the plant. Conceptually, two distinct sources supply N for leaf growth: N from current uptake and assimilation that is directly transferred to the growing leaf (“directly transferred N”) and N from turnover/redistribution of organic compounds (“mobilized N”).Open in a separate windowFigure 1.Schematic representation of N fluxes in the leaf growth zone and in the N supply system of leaf growth in a grass plant. A, Scheme of a growing leaf, with its growth zone (including zones of cell division, expansion, and maturation) and recently produced tissue (RPT). N import (I; μg h⁻¹) into the growth zone is mostly in the form of amino acids. Inside the growth zone, the nitrogenous substrate is used in new tissue construction. Then, N export (E; μg h⁻¹) is in the form of newly formed, fully expanded nitrogenous tissue (tissue-bound export with RPT) and is calculated as leaf elongation rate (L_ER; mm h⁻¹) times the lineal density of N in RPT (ρ; μg mm⁻¹): E = L_ER × ρ (Lattanzi et al., 2004). In a physiological steady state, import equals export (I = E) and the N content of the growth zone (G; μg [not shown]) is constant. Labeled N import into the growth zone (I_lab) commences shortly after labeling of the nutrient solution with ¹⁵N. The labeled N content of the growth zone (G_lab; μg) increases over time (dG_lab/dt) until it eventually reaches isotopic saturation (Fig. 2B). Similarly, the lineal density of labeled N in RPT (ρ_lab) increases until it approaches ρ. At any time, the export of labeled N in RPT (E_lab) equals the concurrent ρ_lab × L_ER. The import of labeled N is obtained as I_lab = E_lab + dG_lab/dt (Lattanzi et al., 2005) and considers the increasing label content in the growth zone during labeling. The fraction of labeled N in the import flux (f_{lab I}) is calculated as f_{lab I} = I_lab/I. The time course of f_{lab I} (Fig. 3) reflects the kinetic properties of the N supply system of leaf growth (C). B, Scheme of a vegetative grass plant (reduced to a rooted tiller with three leaves) with leaf growth zone. N import into the growth zone (I) originates from (1) N taken up from the nutrient solution that is transferred directly to the growth zone following assimilation (directly transferred N) and (2) N derived from turnover/redistribution of stores (mobilized N). The store potentially includes proteins in all mature and senescing tissue in the shoot and root of the entire plant. As xylem, phloem, and associated transfer cells/tissue provide for a vascular network that connects all parts of the plant, the mobilized N may principally originate from any plant tissue that exhibits N turnover/mobilization. The fraction of total N uptake that is allocated to the N supply system of the growth zone equals U (see model in C). The fraction of total mobilized N allocated to the growth zone equals M (see model in C). C, Compartmental model of the source-sink system supplying N to the leaf growth zone, as shown by Lattanzi et al. (2005) and used here. Newly absorbed N (U; μg h⁻¹) enters a substrate pool (Q₁); from there, the N is either imported directly into the growth zone (I) or exchanged with a store (Q₂). Q₁ integrates the steps of transport and assimilation that precede the translocation to the growth zone. Q₂ includes all proteins that supply N for leaf growth during their turnover and mobilization. The parameters of the model, including the (relative) size and turnover of pools Q₁ and Q₂, the deposition into the store (D; μg h⁻¹), and the mobilization from the store (M; μg h⁻¹), and the contribution of direct transfer relative to mobilization to the N supply of the growth zone are obtained by fitting the compartmental model to the f_{lab I} data (A) obtained in dynamic ¹⁵N labeling experiments (for details, see “Materials and Methods”). During physiological steady state, the sizes of Q₁ and Q₂ are constant, I = U, and M = D. [See online article for color version of this figure.]Amino acids are the predominant form in which N is supplied for leaf growth in grasses, and incorporation in new leaf tissue occurs mainly in the leaf growth zone (Gastal and Nelson, 1994; Amiard et al., 2004). This is a heterotrophic piece of tissue that includes the zones of cell division and elongation, is located at the base of the leaf, and is encircled by the sheath of the next older leaf (Volenec and Nelson, 1981; MacAdam et al., 1989; Schnyder et al., 1990; Kavanová et al., 2008). As most N is taken up in the form of nitrate but supplied to the growth zone in the form of amino acids, the path of directly transferred N includes a series of metabolic and transport steps. These include transfer to and loading into the xylem, xylem transport and unloading, reduction and ammonium assimilation, cycling through photorespiratory N pools, amino acid synthesis, loading into the phloem, and transport to the growth zone (Hirel and Lea, 2001; Novitskaya et al., 2002; Stitt et al., 2002; Lalonde et al., 2003; Dechorgnat et al., 2011). The time taken to pass through this sequence is unknown at present, as is the effect of N deficiency on that time. Also, it is not known how much N is contained in, and moving through, the different compartments that supply leaf growth with currently assimilated N.At the level of mature organs, mainly leaves, there is considerable knowledge about N turnover and redistribution. Much less is known about the fate of the mobilized N and its actual use in sink tissues like the leaf growth zone. The processes in mature organs are associated with the maintenance metabolism of proteins, organ senescence, and adjustments in leaf protein levels to decreasing irradiance inside growing canopies when leaves become shaded by overtopping newer ones (Evans, 1993; Vierstra, 1993; Hikosaka et al., 1994; Anten et al., 1995; Hirel et al., 2007; Jansson and Thomas, 2008; Moreau et al., 2012). N mobilization in shaded leaves supports the optimization of photosynthetic N use efficiency at plant and canopy scale (Field, 1983; Evans, 1993; Anten et al., 1995), it reduces the respiratory burden of protein maintenance costs (Dewar et al., 1998; Amthor, 2000; Cannell and Thornley, 2000), and it provides a mechanism for the conservation of the most frequently growth-limiting nutrient (Aerts, 1996). Mobilization of N involves protein turnover and net degradation (Huffaker and Peterson, 1974), redistribution in the form of amino acids (Simpson and Dalling, 1981; Simpson et al., 1983; Hörtensteiner and Feller, 2002), and (at least) some of the mobilized N is supplied to new leaf growth (Lattanzi et al., 2005).N fertilizer supply has multiple direct and indirect effects on plant N metabolism (Stitt et al., 2002; Schlüter et al., 2012). In particular, it modifies the N content of newly produced leaves, leaf longevity/senescence, and the dynamics of light distribution inside expanding canopies (Evans, 1983, 1989; Lötscher et al., 2003; Moreau et al., 2012). Thus, N fertilization influences the availability of recyclable N. At the same time, it augments the availability of directly transferable N to leaf growth. The net effect of these factors on the importance of mobilized versus directly transferred N substrate for leaf growth is not known. Also, it is unknown how N fertilization influences the functional characteristics of the N supply system, such as the size and turnover of its component pools.The assessment of the importance of directly transferred versus mobilized N for leaf growth requires studies at the sink end of the system (i.e. investigations of the N import flux into the leaf growth zone). Directly transferred N and mobilized N can be distinguished on the basis of their residence time in the plant, the time between uptake from the environment and import into the leaf growth zone: direct transfer involves a short residence time (fast transfer), whereas mobilized N resides much longer in the plant before it is delivered to the growth zone (slow transfer; De Visser et al., 1997; Lattanzi et al., 2005). Such studies require dynamic labeling of the N taken up by the plant (Schnyder and de Visser, 1999) and monitoring of the rate and isotopic composition/label content of N import into the leaf growth zone (Lattanzi et al., 2005). For grass plants in a physiological steady state, N import and the isotopic composition of the imported N are calculated from the leaf elongation rate and the lineal density of N in newly formed tissue (Fig. 1A; Lattanzi et al., 2004) and the change of tracer content in the leaf growth zone and recently produced leaf tissue over time (Lattanzi et al., 2005). Such data reveal the temporal change of the fraction of labeled N in the N import flux (f_{lab I}), which then can be used to characterize the N supply system of leaf growth via compartmental modeling. So far, there is only one study that has partially characterized this system (Lattanzi et al., 2005): this work was conducted with a C₃ grass, perennial ryegrass (Lolium perenne), and a C₄ grass, Paspalum dilatatum, growing in mixed stands and indicated that two interconnected N pools supplied the leaf growth zone in both species: a “substrate pool” (Q₁), which provided a direct route for newly absorbed and assimilated N import into the leaf growth zone (directly transferred N), and a mobilizing “store” (Q₂), which supplied N to the leaf growth zone via the substrate pool (Fig. 1C). The relative contribution of mobilization from the store was least important in the fast-growing, dominant individuals and most important in subordinate, shaded individuals. That work did not address the role of N deficiency, and the limited short-term resolution of the study (labeling intervals of 24 h or greater) precluded an analysis of the fast-moving parts of the system.Accordingly, this work addresses the following questions. How does N deficiency influence the substrate supply system of the leaf growth sink in terms of the number, size, and turnover (half-life) of its kinetically distinct pools? How does N deficiency affect the relationship between directly transferred and mobilized N for leaf growth? And what additional insight on the compartmental structure of the supply system is obtained when the short-term resolution of the analysis is increased by 1 order of magnitude? The work was performed with vegetative plants of perennial ryegrass grown in constant conditions with either a low (1.0 mm; termed low N) or high (7.5 mm; high N) nitrate concentration in the nutrient solution. In both treatments, a large number of plants were dynamically labeled with ¹⁵N over a wide range of time intervals (2 h to more than 20 d). The import of total N and ¹⁵N tracer into growth zones was estimated at the end of each labeling interval. Tracer data were analyzed with compartmental models following principles detailed by Lattanzi et al. (2005, 2012) and Lehmeier et al. (2008) to address the specific questions. Previous articles reported on root and shoot respiration (Lehmeier et al., 2010) and cell division and expansion in leaf growth zones (Kavanová et al., 2008) in the same experiment.     

A comparison of the strength of biodiversity effects across multiple functions

In order to predict which ecosystem functions are most at risk from biodiversity loss, meta-analyses have generalised results from biodiversity experiments over different sites and ecosystem types. In contrast, comparing the strength of biodiversity effects across a large number of ecosystem processes measured in a single experiment permits more direct comparisons. Here, we present an analysis of 418 separate measures of 38 ecosystem processes. Overall, 45 % of processes were significantly affected by plant species richness, suggesting that, while diversity affects a large number of processes not all respond to biodiversity. We therefore compared the strength of plant diversity effects between different categories of ecosystem processes, grouping processes according to the year of measurement, their biogeochemical cycle, trophic level and compartment (above- or belowground) and according to whether they were measures of biodiversity or other ecosystem processes, biotic or abiotic and static or dynamic. Overall, and for several individual processes, we found that biodiversity effects became stronger over time. Measures of the carbon cycle were also affected more strongly by plant species richness than were the measures associated with the nitrogen cycle. Further, we found greater plant species richness effects on measures of biodiversity than on other processes. The differential effects of plant diversity on the various types of ecosystem processes indicate that future research and political effort should shift from a general debate about whether biodiversity loss impairs ecosystem functions to focussing on the specific functions of interest and ways to preserve them individually or in combination.     

Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C₁ compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.     

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N₃-ATP) and 8-azidoadenosine 5′-monophosphate (8-N₃-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N₃-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N₃-ATP at ATP-binding site 2. The adenylate kinase active center probe P¹,P⁵-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

DEAD-Box RNA Helicases in Bacillus subtilis Have Multiple Functions and Act Independently from Each Other

DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the importance of RNA, our knowledge about RNA helicases is limited. In this study, we investigated the role of the four DEAD-box RNA helicases in the Gram-positive model organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. The deletion of cshA, cshB, or yfmL in particular leads to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis, suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype, and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis, we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis.