Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.     

Wide ranging stopover movements and substantial fuelling in first year garden warblers at a northern stopover site

Migratory birds use stopovers to replenish their fuel reserves and they generally spend more time at stopover sites than they do in actual flight. When arriving at a new stopover site birds may need to search extensively to find a suitable feeding area and this search and settling period may affect the duration of stopover. Stopover behaviour can thus have profound effects on the migratory programme and studies on stopover behaviour are important to understand migratory strategies. We followed 51 first‐year garden warblers Sylvia borin with radio‐transmitters at an autumn stopover site on the island of Gotland in southern Sweden. Our aim was to determine the distance birds relocated from the coastal capture site when searching for an area to settle in, and also to establish the duration of stopover and put it in relation to refuelling rate by recapturing a subset of the radio‐tracked individuals. Sixteen birds made an extended stopover (> 2 d), relocated inland from the capture site and settled on average 5.6 km from the capture site, with the longest recorded relocation being fourteen kilometres. Birds that relocated nocturnally settled in areas further away than birds that relocated diurnally. Thirteen birds that continued migration after a short stop carried larger fuel stores than birds that stopped over longer and they remained close to the capture site until departure. Three birds were re‐trapped and showed high fuelling rates, between 0.3 and 1.1 g d^–1. They left the stopover site with fuel loads between 40–56 percent of lean body mass, which possibly would have allowed them to reach the Mediterranean area without additional refuelling stops.     

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.     

Interpreting an Archaean paleoenvironment through 3D imagery of microbialites

While stromatolites, and to a lesser extent thrombolites, have been extensively studied in order to unravel Precambrian (>539 Ma) biological evolution, studies of clastic-dominated microbially induced sedimentary structures (MISS) are relatively scarce. The lack of a consolidated record of clastic microbialites creates questions about how much (and what) information on depositional and taphonomic settings can be gleaned from these fossils. We used μCT scanning, a non-destructive X-ray-based 3D imaging method, to reconstruct morphologies of ancient MISS and mat textures in two previously described coastal Archaean samples from the ~3.48 Ga Dresser Formation, Pilbara, Western Australia. The aim of this study was to test the ability of μCT scanning to visualize and make 3D measurements that can be used to interpret the biotic–environmental interactions. Fossil MISS including mat laminae with carpet-like textures in one sample and mat rip-up chips in the second sample were investigated. Compiled δ¹³C and δ³⁴S analyses of specimens from the Dresser Fm. are consistent with a taxonomically diverse community that could be capable of forming such MISS. 3D measurements of fossil microbial mat chips indicate significant biostabilization and suggest formation in flow velocities >25 cm s⁻¹. Given the stratigraphic location of these chips in a low-flow lagoonal layer, we conclude that these chips formed due to tidal influence, as these assumed velocities are consistent with recent modeling of Archaean tides. The success of μCT scanning in documenting these microbialite features validates this technique both as a first step analysis for rare samples prior to the use of more destructive techniques and as a valuable tool for gaining insight into microbialite taphonomy.     

Increase in protandry over time in a long‐distance migratory bird

Protandry is a widespread life‐history phenomenon describing how males precede females at the site or state of reproduction. In migratory birds, protandry has an important influence on individual fitness, the migratory syndrome, and phenological response to climate change. Despite its significance, accurate analyses on the dynamics of protandry using data sets collected at the breeding site, are lacking. Basing our study on records collected during two time periods, 1979 to 1988 and 2006 to 2016, we aim to investigate protandry dynamics over 38 years in a breeding population of willow warblers (Phylloscopus trochilus). Change in the timing of arrival was analyzed in males and females, and protandry (number of days between male and female arrival) was investigated both at population level and within breeding pairs. Our results show advancement in the arrival time at the breeding site in both sexes, but male arrival has advanced to a greater extent, leading to an increase in protandry both at the population level and within breeding pairs. We did not observe any change in sex ratio that could explain the protandry increase, but pronounced temperature change has occurred and been reported in the breeding area and along the migratory route. Typically, natural selection opposes too early arrival in males, but given warmer springs, this counteracting force may be relaxing, enabling an increase in protandry. We discuss whether our results suggest that climate change has induced sex‐specific effects, if these could be evolutionary and whether the timing of important life‐history stages such as arrival at the breeding site may change at different rates in males and females following environmental shifts.     

Different root strategies of perennial native grasses under two contrasting water availability conditions: implications for their spatial distribution in desert dunes

Plant Ecology - The distribution pattern of perennial native grasses in the dune systems of the Monte desert might be determined by the ability of plant roots to acquire water under drought...     

Effect of competition intensity on recruitment of palatable and unpalatable grasses

In this study, we made an attempt to reveal how competition intensity from established plants impacts on palatable and unpalatable grass seedlings recruitment, in a natural mesic grassland of central Argentina. Our objective was to assess the seedling recruitment of a palatable species (Chascolytrum subaristatum) and an unpalatable species (Nassella trichotoma) in microsites differing in competition intensity from established plants. Identity (C. subaristatum and N. trichotoma) and defoliation severity were used as surrogate for competition intensity. In March 2017, we permanently marked established individuals of N. trichotoma and C. subaristatum and placed two circular plots adjacent to each individual. In one plot we added seeds of N. trichotoma and in the other seeds of C. subaristatum. After seeding, established plants were randomly assigned to one of three level of defoliation: without defoliation, low defoliation severity and high defoliation severity. From April to November 2017 (i.e. over a complete annual growing cycle), we measured seedling density, recruitment and growth. Our results supported the hypothesis that seedlings of palatable grasses are more competitive than seedlings of unpalatable grasses. Seedling of the palatable grass C. subaristatum recruited successfully regardless the intensity of competition from established plants, whereas seedlings of the unpalatable grass N. trichotoma recruited better under low competitive pressure from established plants. Our results suggest that the availability of microsites with low competitive pressure from the established vegetation, created by selective grazing of palatable grasses, promotes the recruitment of unpalatable grass seedlings. This mechanism may contribute to the species replacement process commonly observed in heavy grazed grasslands.