eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM)

The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H⁺ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Does a sap feeding marsupial choose trees with specific chemical characteristics?

Only about 20 vertebrate species are known to feed regularly on tree sap. One of these is the yellow‐bellied glider (Petaurus australis), a marsupial that obtains between 16% and 80% of its diet from eucalypt sap. We reviewed the literature on sap feeding by the yellow‐bellied glider and identified 10 species from which it most frequently obtains sap. These species come mainly from a few sections of the subgenus Symphyomyrtus, which accounts for two‐thirds of over 2000 sap trees reported, but more specifically sections Latoangulatae and Maidenii within this subgenus. Some of these key species contain relatively high concentrations of foliar nutrients and each is an important food tree for at least one of the arboreal folivores – koala (Phascolarctos cinereus), greater glider (Petauroides volans), and common ringtail (Pseudocheirus peregrinus) and brushtail possums (Trichosurus vulpecula). Because yellow‐bellied gliders, like the arboreal folivores, prefer to feed from certain individuals within a species, we hypothesized that these trees possess a unique chemical signature that links sap‐feeding with the concentrations of available nitrogen and formylated phloroglucinol compounds in leaves – the nutritive and defensive chemicals that influence feeding on these species by marsupial folivores. We tested this hypothesis in samples of leaves and bark collected from E. punctata and E. viminalis but found no link between chemistry and sap feeding and conclude that other aspects of an individual tree, such as sap flow or sap chemistry, determine whether gliders will target it for sap.     

Invasive aphids of the tribe Siphini: a model of potentially suitable ecological niches

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Distribution of Norway spruce bark and wood‐boring beetles along Alpine elevational gradients

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Change in diel catchability of young-of-year yellow perch associated with establishment of dreissenid mussels

1. Non‐native mussels have increased water clarity in many lakes and streams in North America and Europe. Diel variation in catchability of some fish species has been linked to visibility during survey trawls (used to measure escapement). 2. Water clarity increased in nearshore areas of western Lake Erie by the early 1990s, following passage of legislation in 1972 to improve water quality (e.g. reduce phosphorus loading) and the invasion of dreissenid mussels (Dreissena spp.) beginning in 1987. 3. We hypothesised that increased water clarity in Lake Erie resulted in decreased catchability of young‐of‐year (age‐0) yellow perch (Perca flavescens Mitchill) during daylight compared to during night. We used a two‐tiered modelling approach to test this hypothesis on the ratio (R) of catch per hour (CPH) during night to CPH during daylight in bottom trawl surveys conducted during 1961–2005. 4. First, we examined seven a priori models. The first model, the ‘null’ model, represented no change in R over time. Three more models tested whether the timing of the change in R was associated with passage of water quality legislation only, dreissenids only (two‐period models) and both legislation and dreissenids (three‐period models). Three additional models included a 3‐year lag before the effects of legislation, dreissenids or both occurred. Secondly, all possible two‐ and three‐period models with a minimum of 2 years per time period were explored a posteriori. The a posteriori procedure determined the temporal transitions to higher R that were best supported by the data, without regard to a priori hypotheses. 5. Night CPH was greater than daylight CPH in 3 of 11 years during 1961–72, in 10 of 15 years during 1973–87, and in 14 of 18 years during 1988–2005. During 1991–2005 night CPH exceeded daylight CPH in all years except one, and night CPH was more than twice daylight CPH in 10 years during this period. 6. The best a priori model had two periods, with a break between 1990 and 1991, corresponding to 3 years after the dreissenid invasion. Similarly, the best two‐ and three‐period a posteriori models both had breaks between 1990 and 1991. The results supported our hypothesis that age‐0 yellow perch exhibited a transition to lower catchability during daylight compared to night, and the timing of the transition coincided with the establishment of dreissenid mussels. 7. The most plausible mechanism for our results was increased visibility of the trawl during daylight, resulting in increased avoidance of the trawl. These results have potential applications wherever non‐native mussels have increased water clarity.     

Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters

Background: Since the identification of the genes controlling the root acquisitionof iron (Fe), the control of inter- and intracellular distributionhas become an important challenge in understanding metal homeostasis.The identification of the yellow stripe-like (YSL) transporterfamily has paved the way to decipher the mechanisms of long-distancetransport of Fe. Scope: Once in the plant, Fe will systematically react with organicligands whose identity is poorly known so far. Among potentialligands, nicotianamine has been identified as an important moleculefor the circulation and delivery of metals since it participatesin the loading of copper (Cu) and nickel in xylem and preventsFe precipitation in leaves. Nicotianamine is a precursor ofphytosiderophores, which are high-affinity Fe ligands exclusivelysynthesized by Poaceae species and excreted by roots for thechelation and acquisition of Fe. Maize YS1 is the founding memberof a family of membrane transporters called YS1-like (YSL),which functions in root Fe–phytosiderophore uptake fromthe soil. Next to this well-known Fe acquisition role, mostof the other YSL family members are likely to function in plant-widedistribution of metals since (a) they are produced in vasculartissues throughout the plant and (b) they are found in non-Poaceaespecies that do not synthesize phytosiderophores. The hypothesizedactivity as Fe–nicotianamine transporters of several YSLmembers has been demonstrated experimentally by heterologousexpression in yeast or by electrophysiology in Xenopus oocytesbut, despite numerous attempts, proof of the arabidopsis YSLsubstrate specificity is still lacking. Reverse genetics, however,has revealed a role for AtYSL members in the remobilizationof Cu and zinc from senescing leaves, in the formation of pollenand in the Fe, zinc and Cu loading of seeds. Conclusions: Preliminary data on the YSL family of transporters clearly arguesin favour of its role in the long-distance transport of metalsthrough and between vascular tissues to eventually support gametogenesisand embryo development.     

High-resolution spatial and temporal analysis of phytoalexin production in oats

The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of AFLP fragments linked to hydroxysafflor yellow A in Flos Carthami and conversion to a SCAR marker for rapid selection

Hydroxysafflor yellow A (HSYA), an important active compound in treating focal cardiac and cerebral ischemia, is uniquely present in flower petals of Carthamus tinctorius. In this study, inheritance and molecular marker analyses for HSYA trait in safflower were carried out. HSYA contents in parents, cross hybridized F₁ and F₂ individuals were analyzed by high performance liquid chromatography. Results revealed that the presence/absence of HSYA was controlled by one major nuclear gene termed HSya. A total of 48 AFLP primer combinations were screened, and bulked segregant analysis was performed by preparing two pools of 10 present-HSYA and ten absent-HSYA plants selected from the 498 individuals of the F₂ segregating population. Four AFLP markers, AFLP-5, AFLP-7, AFLP-15 and AFLP-16, were identified to be closely associated with HSya. Of those, AFLP-16 was the closest to HSya, estimated at about 9.4 cM in genetic distance. The dominant AFLP-16 marker was converted into a simple sequence characterized amplified region marker based on the sequence information of the cloned flanking regions of the AFLP fragment and was designated as SCM16. Our result has direct application for marker-assisted selection of quality breeding in safflower.