The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.      

Boosting protein stability with the computational design of β‐sheet surfaces

β‐sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent‐facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β‐sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β‐sheet proteins. Two design variants of the β‐sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β‐sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β‐sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded.     

Animal behaviour shapes the ecological effects of ocean acidification and warming: moving from individual to community‐level responses

Biological communities are shaped by complex interactions between organisms and their environment as well as interactions with other species. Humans are rapidly changing the marine environment through increasing greenhouse gas emissions, resulting in ocean warming and acidification. The first response by animals to environmental change is predominantly through modification of their behaviour, which in turn affects species interactions and ecological processes. Yet, many climate change studies ignore animal behaviour. Furthermore, our current knowledge of how global change alters animal behaviour is mostly restricted to single species, life phases and stressors, leading to an incomplete view of how coinciding climate stressors can affect the ecological interactions that structure biological communities. Here, we first review studies on the effects of warming and acidification on the behaviour of marine animals. We demonstrate how pervasive the effects of global change are on a wide range of critical behaviours that determine the persistence of species and their success in ecological communities. We then evaluate several approaches to studying the ecological effects of warming and acidification, and identify knowledge gaps that need to be filled, to better understand how global change will affect marine populations and communities through altered animal behaviours. Our review provides a synthesis of the far‐reaching consequences that behavioural changes could have for marine ecosystems in a rapidly changing environment. Without considering the pervasive effects of climate change on animal behaviour we will limit our ability to forecast the impacts of ocean change and provide insights that can aid management strategies.     

Photosynthetic activity and proteomic analysis highlights the utilization of atmospheric CO2 by Ulva prolifera (Chlorophyta) for rapid growth

Free‐floating Ulva prolifera is one of the causative species of green tides. When green tides occur, massive mats of floating U. prolifera thalli accumulate rapidly in surface waters with daily growth rates as high as 56%. The upper thalli of the mats experience environmental changes such as the change in carbon source, high salinity, and desiccation. In this study, the photosynthetic performances of PSI and PSII in U. prolifera thalli exposed to different atmospheric carbon dioxide (CO₂) levels were measured. Changes in photosynthesis within salinity treatments and dehydration under different CO₂ concentrations were also analyzed. The results showed that PSII activity was enhanced as CO₂ increased, suggesting that CO₂ assimilation was enhanced and U. prolifera thalli can utilize CO₂ in the atmosphere directly, even when under moderate stress. In addition, changes in the proteome of U. prolifera in response to salt stress were investigated. Stress‐tolerance proteins appeared to have an important role in the response to salinity stress, whereas the abundance of proteins related to metabolism showed no significant change under low salinity treatments. These findings may be one of the main reasons for the extremely high growth rate of free‐floating U. prolifera when green tides occur.     

Tricin‐lignins: occurrence and quantitation of tricin in relation to phylogeny

Tricin [5,7‐dihydroxy‐2‐(4‐hydroxy‐3,5‐dimethoxyphenyl)‐4H‐chromen‐4‐one], a flavone, was recently established as an authentic monomer in grass lignification that likely functions as a nucleation site. It is linked onto lignin as an aryl alkyl ether by radical coupling with monolignols or their acylated analogs. However, the level of tricin that incorporates into lignin remains unclear. Herein, three lignin characterization methods: acidolysis; thioacidolysis; and derivatization followed by reductive cleavage; were applied to quantitatively assess the amount of lignin‐integrated tricin. Their efficiencies at cleaving the tricin‐(4′–O–β)‐ether bonds and the degradation of tricin under the corresponding reaction conditions were evaluated. A hexadeuterated tricin analog was synthesized as an internal standard for accurate quantitation purposes. Thioacidolysis proved to be the most efficient method, liberating more than 91% of the tricin with little degradation. A survey of different seed‐plant species for the occurrence and content of tricin showed that it is widely distributed in the lignin from species in the family Poaceae (order Poales). Tricin occurs at low levels in some commelinid monocotyledon families outside the Poaceae, such as the Arecaceae (the palms, order Arecales) and Bromeliaceae (Poales), and the non‐commelinid monocotyledon family Orchidaceae (Orchidales). One eudicotyledon was found to have tricin (Medicago sativa, Fabaceae). The content of lignin‐integrated tricin is much higher than the extractable tricin level in all cases. Lignins, including waste lignin streams from biomass processing, could therefore provide a large and alternative source of this valuable flavone, reducing the costs, and encouraging studies into its application beyond its current roles.     

昆明山海棠对中国仓鼠V79细胞二酰基甘油含量的影响

本研究以昆明山海棠根部水抽提物(Tripterygium Hypoglaucum(Level)Hutch,THH)处理中国仓鼠V79细胞,通过检测V79细胞C-M细胞频率以及二酰基甘油(1,2-diacylgcerol,DAG)的含量测定,分析了THH诱发非整倍体与细胞醇磷酯信号通路的关系.结果指出THH能在1mg/ml、2mg/ml两个剂量上使V79细胞的DAG含量显著升高(P＜0.001),并明显的提高C-M细胞频率(P＜0.05),提示肌醇酯信号通路是介导THH诱发非整倍体的途径之一.     

Antioxidative Activity of 5,6,7,8-Tetrahydrobiopterin and its Inhibitory Effect on Paraquat-Induced Cell Toxicity in Cultured Rat Hepatocytes

The in vitro antioxidative activity of 5,6,7,8-tetrahydrobiopterin (BPH4) was measured and the ability of BPH4 to prevent paraquat-induced cell damage was examined in cultured hepatocytes. The scavenging activity of BPH4 against superoxide anion radicals was assayed in two systems, i.e., xanthine/xanthine oxidase (X/XOD) and rat macrophage/phorbol myristate acetate (MξPMA) radical-generating systems. BPH4 showed an extremely strong superoxide anion radical-scavenging activity in both assay systems. Biopterin (BP) itself did not show any activity in the X/XOD system, but was effective in the MξPMA system. The antioxidative activities of BPH4 against both superoxide anion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. BPH4 also protected rat brain homogenate against auto-oxidation. We further examined the effect of BPH4 on paraquat-induced cell toxicity in cultured rat hepatocytes. The paraquat-induced elevation of the release of lactate dehydrogenase (LDH), a marker enzyme for cytotoxicity from cultured hepatocytes was suppressed by BPH4 in a dose-dependent manner. The elevation of lipid peroxides simultaneously induced by paraquat was also inhibited by BPH4 in the same manner. These results suggest that BPH4 might be useful in the treatment of various diseases whose pathogenesis is active oxygen-related.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate