Binding of Plasma Membrane Lipids Recruits the Yeast Integral Membrane Protein Ist2 to the Cortical ER

Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein–protein and protein–membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein–lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P₂ at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.     

Adaptations in the Glucose Metabolism of Procyclic Trypanosoma brucei Isolates from Tsetse Flies and during Differentiation of Bloodstream Forms

Procyclic forms of Trypanosoma brucei isolated from the midguts of infected tsetse flies, or freshly transformed from a strain that is close to field isolates, do not use a complete Krebs cycle. Furthermore, short stumpy bloodstream forms produce acetate and are apparently metabolically preadapted to adequate functioning in the tsetse fly.African trypanosomatids comprise various pleomorphic trypanosome species that proliferate in the bloodstream of their mammalian hosts as long slender bloodstream form (BSF) trypanosomes, and at the peak of parasitemia they differentiate into nondividing short stumpy form trypanosomes (1). After being ingested during a bloodmeal by a tsetse fly (Glossina sp.), short stumpy form trypanosomes differentiate into procyclic form (PCF) trypanosomes, which actively multiply and colonize the midgut of the fly. Subsequently, PCF Trypanosoma brucei migrates to the salivary glands while undergoing a complex differentiation (22). Here, attached epimastigote forms start multiplying, after which nondividing metacyclic trypomastigotes develop. The life cycle of T. brucei is completed when these metacyclic trypomastigotes are injected into a mammal through the bite of an infected fly, after which they transform into long slender BSF trypanosomes. During this life cycle, trypanosomes encounter different environments to which they have adapted, resulting in distinct stages, characterized by morphological as well as metabolic changes. Long slender BSF trypanosomes degrade glucose by glycolysis and excrete pyruvate as the sole metabolic end product (12, 13, 23). On the other hand, PCF trypanosomes do not excrete pyruvate but degrade glucose to acetate and succinate as main end products (25). Krebs cycle activity was thought previously to be present in trypanosomatids, at least in insect stages of some African trypanosomatids (3, 9, 10, 12, 21). However, this presumed flux through the Krebs cycle is supported only poorly by direct experimental evidence and was based mainly on the presence of certain enzyme activities. Although genes for all enzymes of this cycle are indeed present in the genome and expressed in the insect stages, recent studies revealed that at least in T. brucei, the cycle is not used for the complete oxidation of acetyl-coenzyme A (CoA) to carbon dioxide (2, 26). Instead, parts of the cycle are most likely used in anabolic pathways, such as gluconeogenesis and fatty acid formation, and also for the final steps in the degradation of amino acids (26). It is possible that the reported discrepancies on the presence or absence of full-circle Krebs cycle activity are caused by differences in the number of passages through mice after the isolation of the strain from the field. Such passages may have been ongoing for many years, during which the parasites were continuously propagated as BSF trypanosomes. Furthermore, most insect form trypanosomes that were investigated up to now have been propagated for many years as PCF trypanosomes in rich culture media. Hence, the reported discrepancies could be due to differences between freshly differentiated PCF trypanosomes and those well adapted to in vitro culture, and the absence of an active Krebs cycle in PCF trypanosomes could be the result of an adaptation caused by the prolonged in vitro culturing. To investigate these possibilities, we analyzed the glucose metabolism of PCF T. brucei directly after isolation from the midguts of tsetse flies. We also studied freshly differentiated PCF trypanosomes from the AntAR 1 strain, a T. brucei strain that has had a minor history of animal passaging since its field isolation (15, 17).To investigate the cause of the conflicting reports on Krebs cycle activity in PCF trypanosomes, we first analyzed the effect of environmental factors by comparing the carbohydrate metabolism of PCF trypanosomes well adapted to in vitro culturing and PCF trypanosomes isolated from their natural environment, the midguts of tsetse flies. These experiments were performed with PCF TREU 927 T. brucei, a pleomorphic strain that has been thoroughly characterized and is still able to infect Glossina morsitans, performing a complete physiological life cycle (2). For the infection of tsetse flies, male G. morsitans flies originating from the colony maintained at the Institute of Tropical Medicine in Antwerp, Belgium, were infected with procyclic TREU 927 T. brucei by in vitro membrane feeding and subsequently maintained for 10 days by feeding on rabbit blood (15). Then, flies were dissected on a sterile glass slide and the infected midguts were isolated and incubated for at least 30 min at 28°C in SDM-79 medium that was gently rotated. After sedimentation of the midguts by gravity, insect gut debris was removed by centrifugation at 300 × g for 5 min. PCF trypanosomes were then isolated from the collected supernatant by centrifugation at 1,500 × g for 10 min. Since PCF trypanosomes could not be isolated from the midgut without minor amounts of contaminating insect gut material, such as gut cells and debris, we also investigated the glucose metabolism of this fraction. Analysis of metabolic end products produced from [6-¹⁴C]glucose in this control incubation of insect gut debris, which also contained minor amounts of trypanosome cells, showed the formation of ¹⁴C-labeled pyruvate, CO₂, acetate, and lactate (Fig. ​(Fig.1A).1A). Minor amounts of lactate were also produced in the incubations with PCF trypanosomes isolated from the midgut, which also contained minor amounts of insect gut debris. Since lactate is not an excreted end product of T. brucei, this labeled lactate is indicative for the glucose degradation activity of insect gut debris. Therefore, end product formation in the incubations with PCF trypanosomes isolated from the midgut was corrected for end products produced by the contaminating insect gut debris by subtracting all produced lactate and the calculated accompanying amounts of other end products produced in the insect gut debris incubation. The metabolic incubations with PCF trypanosomes directly after isolation from the tsetse midgut showed that these trypanosomes degrade glucose to the same metabolic end products, acetate, succinate, and pyruvate, as the in vitro culture-adapted PCF trypanosomes (Fig. ​(Fig.1A).1A). Furthermore, the ratio of acetate and succinate produced by PCF trypanosomes isolated from the midgut were similar to that of in vitro-cultured PCF trypanosomes (Fig. ​(Fig.1A).1A). On the other hand, a major difference was observed in the amount of glucose consumed since the PCF trypanosomes isolated from the midguts of tsetse flies consumed 16-fold less glucose than PCF trypanosomes that were derived from in vitro cultures. This difference in glucose consumption can probably be explained by our observation that both motility and especially growth of PCF trypanosomes isolated from the midgut were significantly reduced compared to the in vitro culture-derived PCF trypanosomes. Apparently, the environmental conditions in the midgut of the fly did affect the PCF trypanosomes, but they did not significantly alter the metabolic pathways used for energy metabolism. However, PCF trypanosomes isolated from the midgut of the fly excreted more pyruvate (Fig. ​(Fig.1A),1A), which suggests that pyruvate is a more important metabolic end product for PCF trypanosomes under physiological conditions than acknowledged thus far. Most importantly, however, just like continuously in vitro-cultured ones, PCF trypanosomes isolated from the midgut of the fly did not degrade [6-¹⁴C]glucose to labeled CO₂ (Fig. ​(Fig.1A),1A), which demonstrates the absence of a functional Krebs cycle in these tsetse fly-derived PCF trypanosomes.Open in a separate windowFIG. 1.Radioactive end products of [6-¹⁴C]glucose metabolism of procyclic TREU 927 T. brucei cells grown in vitro or isolated from the midguts of tsetse flies (A) and that of AntAR 1 T. brucei during differentiation of BSF to PCF trypanosomes (B). (A) The results of a single experiment for PCF trypanosomes isolated from the midgut and for insect gut debris and the mean + the standard deviation (SD) of three parallel incubations for in vitro-cultured PCF trypanosomes are shown. Total end product formation from [6-¹⁴C]glucose was 2.08 ± 0.19 μmol/h per 10⁸ cells and 0.23 μmol/h per 10⁸ cells for in vitro-cultured and midgut-isolated PCF trypanosomes, respectively, and was calculated using the number of trypanosome cells present at the beginning of the incubation. End product formation in the incubation with PCF trypanosomes isolated from the midgut was corrected for end products produced by contaminating insect gut debris (see text for details). (B) Metabolic incubations using 6-¹⁴C-labeled glucose were performed during differentiation from short stumpy BSF trypanosomes to insect stage PCF trypanosomes. Incubations with PCF trypanosomes were started at 24, 48, and 96 h after induction of differentiation (PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4, respectively); means + SDs of three parallel incubations are shown (for the short stumpy form, six incubations in two independent experiments). Total glucose consumption in incubations with long slender BSF trypanosomes, short stumpy BSF trypanosomes, PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4 was 4.8, 3.4, 1.5, 1.1, and 0.79 μmol/h per 10⁸ cells, respectively. Excreted labeled end products shown in panels A and B were analyzed as described previously (25) and are expressed as the percentage of the total amount of radioactive end products produced (in the incubation of gut debris, one other unidentified end product was produced, which explains why this total in the figure does not add up to 100%). The decrease in pyruvate production between long slender and short stumpy BSF trypanosomes as well as the increase in acetate production is significant as calculated using an unpaired t test (P < 0.01 for pyruvate and P < 0.001 for acetate).Although TREU 927 T. brucei is a pleomorphic trypanosome strain, it cannot be excluded that these trypanosomes have adapted their energy metabolism during the substantial period that this strain has been cultured in vitro. Therefore, we also studied the carbohydrate metabolism of freshly transformed PCF of the T. brucei AntAR 1 strain, a well-characterized pleomorphic strain that is close to the wild isolate (17). To investigate the energy metabolism of these freshly differentiated PCF trypanosomes, AntAR 1 BSF trypanosomes were harvested from the blood of infected immune-suppressed NMRI mice as described previously (16) and either directly incubated with [6-¹⁴C]glucose or differentiated to PCF trypanosomes, by addition of 6 mM cis-aconitate and incubation at 27°C (7). These trypanosomes were then incubated with [6-¹⁴C]glucose at different time points after the initiation of differentiation. Our experiments (Fig. ​(Fig.1B)1B) confirmed that differentiation of trypanosomes from BSF to PCF is accompanied by a metabolic shift in excreted end products from pyruvate to acetate and succinate (3, 14, 25). This metabolic shift during differentiation of BSF to PCF trypanosomes was complete after 1 to 2 days (Fig. ​(Fig.1B),1B), which is in agreement with previous observations (9). A subsequent switch in medium from HMI-9, a medium used to culture BSF T. brucei, to SDM-79, a medium used for the culture of PCF T. brucei, did not result in further changes in excreted end products (data not shown).Our experiments, however, did not show any significant production of labeled CO₂ and certainly not the massive increase in CO₂ formation upon differentiation of BSF into PCF trypanosomes that was reported in a comparable study by Durieux et al. (9). We cannot exclude that this difference in Krebs cycle activity between our study and that of Durieux et al. is caused by a strain difference, but since the AntAR 1 strain we used can be considered to be close to the field isolate, the results presented here are indicative of wild-type T. brucei metabolism and strongly suggest that a functional Krebs cycle is absent in PCF T. brucei cells in vivo.Next to the absence of carbon dioxide formation via Krebs cycle activity during differentiation of BSF to PCF trypanosomes, our metabolic experiments also demonstrated that acetate accounted for 30% of the glucose-derived excreted labeled end products in freshly isolated BSF AntAR 1 T. brucei cells (Fig. ​(Fig.1B).1B). This is a surprising observation since BSF trypanosomes are reported to rely on glycolysis only and to excrete pyruvate and minor amounts of glycerol (12, 13, 23). However, the BSF trypanosomes that we tested in our incubations were predominantly short stumpy BSF cells, whereas nearly all previously performed metabolic studies of BSF trypanosomes were performed with long slender BSF cells. In order to investigate whether differentiation from long slender to short stumpy form trypanosomes indeed shifts the metabolism toward acetate formation, we analyzed the energy metabolism of BSF trypanosomes harvested from mice at two different time points after infection. At day 4 after infection, predominantly long slender BSF trypanosomes were isolated (94% long slender versus 6% short stumpy), whereas at day 7 after infection, predominantly short stumpy BSF trypanosomes were isolated (92% short stumpy versus 8% long slender). Analysis of glucose-derived metabolic end products from incubations with BSF AntAR 1 trypanosomes isolated at day 4 or at day 7 after infection showed that short stumpy BSF trypanosomes indeed produce significant amounts of acetate as an end product of glucose metabolism (Fig. ​(Fig.1B).1B). In the incubations with predominantly long slender BSF AntAR 1 T. brucei cells, some acetate was also produced, but this relatively small amount of acetate formation can be explained by the presence of a certain amount of short stumpy cells. Although the incubations were started with nearly 95% long slender BSF cells, BSF cells from the AntAR 1 strain are highly pleomorphic and rapidly differentiate to short stumpy forms during in vitro culture conditions. Therefore, increasing amounts of short stumpy form T. brucei were formed during our incubations (up to 40 to 50% at the end of incubation), which accounts for the amount of acetate formed during these incubations.Since acetate production in Trypanosomatidae is catalyzed by the mitochondrial enzyme acetate:succinate CoA transferase (ASCT), which was previously shown not to be expressed in in vitro-cultured BSF T. brucei (20), we examined the ASCT enzyme activity in lysates derived from either over 92% short stumpy cells or 94% long slender cells. These experiments showed that the ASCT enzyme is present in short stumpy BSF trypanosomes in an amount equivalent to around 15% of that of PCF trypanosomes (Fig. ​(Fig.2).2). This is in agreement with the observation that acetate is a more prominent excreted end product in PCF trypanosomes than in short stumpy BSF cells. On the other hand, ASCT activity was nearly absent in long slender BSF trypanosomes (Fig. ​(Fig.2),2), which confirms the conclusion that in our incubations acetate is not produced by long slender BSF trypanosomes but by short stumpy BSF trypanosomes.Open in a separate windowFIG. 2.ASCT activity in total lysates of T. brucei AntAR 1. Enzymatic activity of ASCT was determined in total lysates derived from cultures containing predominantly long slender BSF trypanosomes (BSF LS; 94%), predominantly short stumpy BSF trypanosomes (BSF SS; 92%), or exclusively PCF trypanosomes (PCF). Shown are the means + standard deviations of three experiments.Hence, our experiments show that short stumpy BSF trypanosomes do not only degrade glucose by glycolysis but additionally produce acetate. Acetate formation in trypanosomes occurs via the mitochondrial enzyme ASCT and involves transfer of a CoA moiety from acetyl-CoA to succinate, yielding succinyl-CoA (24). This succinyl-CoA can then be converted back into succinate by succinyl-CoA synthetase, a reaction concomitantly converting ADP in ATP (6, 24). Therefore, our observations that short stumpy BSF trypanosomes produce acetate and express ASCT demonstrate that these stages in addition to glycolysis also use a mitochondrial pathway for the degradation of glucose and production of ATP.Multiple mitochondrial adaptations have been reported to occur during the transition from long slender BSF to short stumpy BSF T. brucei. Differential gene expression and the formation of cristea in the inner mitochondrial membrane have been shown to occur during this transition (8, 11, 19). Furthermore, the trypanosomal homologue of complex I of the respiratory chain is expressed in short stumpy BSF trypanosomes (4, 5, 18). Our experiments show that this more elaborate composition of the electron transport chain is also used by this stage, as the production of acetate implies that acetyl-CoA is formed, which is catalyzed by the pyruvate dehydrogenase complex and results in the production of NADH inside the mitochondrion. This means that either complex I or the alternative NADH dehydrogenase is active in this stage (18). Moreover, our experiments show that the previously reported mitochondrial adaptations in short stumpy BSF trypanosomes are not restricted to morphological changes and to changes in the composition of the electron transport chain but also result in a functionally altered energy metabolism.In conclusion, the data described in this paper demonstrate the absence of a functional Krebs cycle in the mitochondria of PCF T. brucei, isolated from the tsetse midgut or freshly differentiated from BSF trypanosomes. Furthermore, we show that short stumpy BSF T. brucei cells produce large amounts of acetate. Therefore, the mitochondria of short stumpy trypanosomes are metabolically divergent from the mitochondria in long slender BSF T. brucei cells. These results are consistent with prior work (4, 5, 8, 11). The functional changes might be a preadaptation that allows short stumpy BSF T. brucei to function in the intestines of infected tsetse flies and enables them to differentiate further into PCF trypanosomes.     

In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.Nitration of tyrosine to 3-nitrotyrosine is one of several protein modifications occurring during oxidative stress (1, 2). This modification is considered as a two-step process in which a tyrosine radical is first formed followed by the addition of ^•NO₂ yielding 3-nitrotyrosine. One of the mechanisms through which tyrosine can be nitrated is via the peroxynitrite radical (ONOO⁻); however, alternative pathways exist such as nitration by hemoperoxidases in the presence of hydrogen peroxide and nitrite (3) and reaction of the tyrosyl radical with nitric oxide yielding 3-nitrosotyrosine, which can be further oxidized to 3-nitrotyrosine.Nitration of protein-bound tyrosines can have important implications on the structure and activity of proteins (4–6) and is linked to a variety of pathological conditions such as Alzheimer disease (7) and atherosclerosis (8). Proteins containing 3-nitrotyrosine residues have mainly been identified by one- or two-dimensional PAGE followed by Western blotting using 3-nitrotyrosine-specific antibodies (9) or following affinity enrichment (10, 11). However, rather few in vivo tyrosine nitration sites have thus far been mapped onto proteins, and hence, the exact sites of in vivo nitration remain elusive. This is highly likely due to the overall low abundance of this modification as was recently illustrated by the identification of only 31 nitration sites in 29 proteins in a comprehensive analysis of mouse brain tissue covering 7,792 proteins (12). Furthermore, it was estimated that in diseased cells or organs the number of nitrated tyrosines should be as low as 0.00001–0.001% of all tyrosines (5), numbers that clearly indicate the need to enrich for 3-nitrotyrosine peptides prior to mass spectrometric analysis. In addition, several MS and MS/MS detection problems for 3-nitrotyrosine peptides were reported (13, 14) that also influence identification of such peptides.Recently, a procedure for enriching 3-nitrotyrosine peptides was described (10). In brief, reduced and alkylated proteins were digested after which primary amino groups were blocked by acetylation. Nitrotyrosines were then reduced to aminotyrosine using dithionite (15), unveiling novel primary amino groups on which sulfhydryl groups were coupled that allowed binding peptides to thiopropyl-Sepharose beads. In contrast to an earlier affinity-based isolation protocol (16), this improved enrichment procedure was more effective and led to the characterization of 150 tyrosine nitration sites in 102 proteins using a total of 3.1 mg of a mouse brain homogenate sample that was in vitro nitrated (10). However, this procedure requires many modification steps, which, when incomplete, will introduce artifacts (see “Results”). Among others, these explain the rather low number of identified nitrated tyrosines peptides using the high amount of starting material that was in vitro nitrated.Our laboratory adapted diagonal chromatography (17) for contemporary mass spectrometry-driven proteomics. Central in our combined fractional diagonal chromatography (COFRADIC1 (18)) approach is a chemical or enzymatic step that changes the reverse-phase column retention properties of a set of peptides such that these can be isolated. Among others, we developed COFRADIC protocols for isolating peptides carrying amino acid modifications such as phosphorylation (19), N-glycosylation (20), and sialylation (21) or peptides that are the result of protein processing (22–24). Here we describe a COFRADIC procedure for sorting peptides carrying nitrated tyrosines. Peptide sorting is based on a hydrophilic shift after reducing the nitro group to its amino counterpart. The applicability of COFRADIC for analyzing this modification is illustrated by characterization of four 3-nitrotyrosines in BSA treated with tetranitromethane, the mapping of 335 different nitration sites in 267 different proteins starting from 300 μg of an in vitro peroxynitrite (ONOO⁻)-treated Jurkat lysate, and the identification of six unique nitrated tyrosine residues in four serum proteins in a mouse sepsis model.     

The Effect of Map Boundary on Estimates of Landscape Resistance to Animal Movement

Background

Artificial boundaries on a map occur when the map extent does not cover the entire area of study; edges on the map do not exist on the ground. These artificial boundaries might bias the results of animal dispersal models by creating artificial barriers to movement for model organisms where there are no barriers for real organisms. Here, we characterize the effects of artificial boundaries on calculations of landscape resistance to movement using circuit theory. We then propose and test a solution to artificially inflated resistance values whereby we place a buffer around the artificial boundary as a substitute for the true, but unknown, habitat.

Methodology/Principal Findings

We randomly assigned landscape resistance values to map cells in the buffer in proportion to their occurrence in the known map area. We used circuit theory to estimate landscape resistance to organism movement and gene flow, and compared the output across several scenarios: a habitat-quality map with artificial boundaries and no buffer, a map with a buffer composed of randomized habitat quality data, and a map with a buffer composed of the true habitat quality data. We tested the sensitivity of the randomized buffer to the possibility that the composition of the real but unknown buffer is biased toward high or low quality. We found that artificial boundaries result in an overestimate of landscape resistance.

Conclusions/Significance

Artificial map boundaries overestimate resistance values. We recommend the use of a buffer composed of randomized habitat data as a solution to this problem. We found that resistance estimated using the randomized buffer did not differ from estimates using the real data, even when the composition of the real data was varied. Our results may be relevant to those interested in employing Circuitscape software in landscape connectivity and landscape genetics studies.     

Bottlenecks for High Coverage of Intermittent Preventive Treatment in Pregnancy: The Case of Adolescent Pregnancies in Rural Burkina Faso

Background

While IPTp-SP is currently being scaled up in sub-Saharan Africa (SSA), the coverage with the required ≥2 doses of SP remains considerably short of the Roll Back Malaria (RBM) goal of 80%, not to mention of the recently advocated universal coverage.

Methods

The study triangulates quantitative data from a health center randomized community-based trial on IPTp-SP effectiveness and the additional benefit of a promotional campaign with qualitative data from focused ethnography.

Findings

In rural Burkina Faso, despite the significantly higher risk of malaria infection among adolescent primigravidae (PG) (OR 2.44 95%CI 1.81–3.28, p<0.001), making them primary target beneficiaries of IPTp-SP, adolescents adhered to the required three or more ANC visits significantly less (PG: 46.6%; SG 43.7%) than adults (PG: 61.9%; SG 54.9%) and had lower SP uptake during the malaria transmission season, further showing the difficulty of reaching this age group. Adolescents'' structural constraints (such as their social position and household labor requirements) and needs (such as anonymity in the health encounter) leave them highly vulnerable during their pregnancies and, especially, during the high malaria transmission season.

Conclusion

Our study shows that adolescents need to be targeted specifically, prior to their first pregnancy and with measures adapted to their social context, addressing their structural constraints and needs and going beyond standard health promotion campaigns. Unless such specific measures are taken, adolescents'' social vulnerability will present a serious bottleneck for the effectiveness of IPTi-SP.     

Litterfall and associated nutrient pools extend beyond the canopy of scattered eucalypt trees in temperate pastures

Scattered paddock trees are a keystone feature of temperate grazing landscapes of Australia. However, our understanding of their influence on their immediate environment, and specifically the spatial distribution and characteristics of litter, is still limited. Here, we quantified the spatial pattern of litter around 4 Eucalyptus species (Eucalyptus melliodora A. Cunn. Ex Schauer, E. viminalis Labill., E. blakelyi Maiden and E. michaeliana Blakely) in grazing landscapes on the Northern Tablelands of NSW, Australia. We examined the effect of species and soil parent material (basalt, granite and meta-sediments) on litter chemistry and chemical pools. Between 54–145 kg of litter was found around individual trees and litter density consistently declined with distance from the tree (330 g.m^?2 in the inner canopy to 4 g.m^?2 in the open paddock). However, an equivalent quantity of litter was found beneath and beyond the canopy indicating that a large quantity of the litter and nutrients fell beyond the edge of the canopy. Overall, leaf litter accounted for 23 to 34% of litterfall and had larger nutrient concentrations and pools than bark or stick litter. Most litter nutrients concentrations were independent of tree species or parent material but our results suggest that P, K and S were removed in foliage prior to abscission whilst Ca and Fe concentrations increased. The spatial patterns of litter distribution around scattered trees coincide with spatial patterns in soil properties that are frequently observed in these environments, and provide strong evidence of a significant link between these factors. Our results suggest that the removal of scattered trees from pastoral landscapes in this region of Australia will result in the loss of a significant litter input to the soil surface and will diminish this potentially important source of soil nutrients.     

Production of complex multiantennary N-glycans in Nicotiana benthamiana plants

In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions.