RNAi activity of siRNAs modified with 2'-aminoalkyl-substituted fluorinated nucleobases

We recently reported that a 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-d-ribofuranose nucleoside appears to be a universal nucleoside which does not differentiate between the four natural nucleosides A, C, G, and U in duplexes. Moreover, ribozymes modified with this nucleoside analog showed a better or at least equal catalytic activity relative to Watson-Crick mismatches.[1] Due to these data, we investigated the ability of this compound to tolerate Watson-Crick mismatches in order to avoid HIV escape mutations in RNA interference. The influence of this nucleoside analog on siRNA efficiency was analyzed with a proven siRNA targeting GFP.     

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Spatio-temporal variation in post-recovery dynamics in a large Peregrine Falcon (Falco peregrinus) population in the Jura mountains 2000–2020†

After spectacular population crashes in the 1960 and 1970s, Peregrine Falcon Falco peregrinus populations recovered worldwide, and in the 2000s many study populations were back to normal. However, post-recovery trends have been documented less well. We combined three long-term population studies covering the entire Jura mountain range (16 304 km²) to examine spatio-temporal variation in population dynamics during 2000–2020 in 420 known nesting sites in five regions: Ain, Jura and Doubs in France, and Jura Sud and Jura Nord in Switzerland. Every year about 60% of all sites were surveyed at least once, and this proportion increased over time. We used Bayesian occupancy modelling to investigate site persistence and colonization probabilities and correct status and trend estimates for bias due to non-random site coverage. Based on the average of two models correcting for preferential sampling, we estimated that population size peaked in 2002 (Jura), 2005 (Doubs), 2007 (Ain, Jura Nord) and in 2007 for the Jura as a whole. Thereafter, the population declined significantly in four regions and in the Jura as a whole, with the strongest decline found in the Jura Nord ( –38%, 95% credible interval –34%/–41%) during 2007–2020. Thus, we found considerable spatial heterogeneity in post-recovery trends and widespread post-recovery declines. Reasons for the latter may include Eagle Owl Bubo bubo predation, illegal persecution and human disturbance. Our results illustrate how quickly positive population trends may be reversed, such that continuous long-term monitoring even for species seemingly ‘out of danger’ remains invaluable. Our study also emphasizes the importance of correcting for both coverage bias in general and preferential sampling in particular when assessing population trends in studies where not every territory can be surveyed in all years.     

The regulatory role of CO2 on nutrient releases from ashed rice straw phytoliths

Biogeochemistry - Phytolith is widely known as a silica structure in numerous silicon (Si) accumulator plants, e.g., rice, and it contains various nutrients and other beneficial elements. When rice...     

Dwarf shrub hydraulics: two Vaccinium species (Vaccinium myrtillus,Vaccinium vitis‐idaea) of the European Alps compared

Vaccinium myrtillus and Vaccinium vitis‐idaea are two dwarf shrubs widespread in the European Alps. We studied the hydraulics of these species hypothesizing that (1) the hydraulic architecture of dwarf shrubs differs from trees, (2) hydraulic properties reflect the species' ecological amplitude and (3) hydraulic properties vary spatially and seasonally. Key hydraulic parameters (osmotic potential, turgor loss point, xylem hydraulic conductivity, vulnerability to drought‐induced embolism, stomata closure, drought‐induced cell damage and embolism repair) and related wood anatomical traits (conduit diameter and conduit wall reinforcement) were analyzed at four sites in Tyrol, Austria. Both species exhibited low hydraulic safety as well as low hydraulic efficiency. Fifty percentage embolism accumulated at ?2.08 (V. myrtillus) and ?1.97 MPa (V. vitis‐idaea), 88% stomata closure was at ?2.19 and ?2.35 MPa, respectively. After drought, both species showed embolism repair on re‐watering. Site‐specific variation within species was low, while seasonal changes in embolism resistance and turgor loss point were observed. Results indicate that studied Vaccinium species have a high risk for embolism formation. This is balanced by refilling capacities, which are probably based on the small growth height of dwarf shrubs. V. vitis‐idaea, which occurs on drier sites, showed more efficient repair and a lower turgor loss point than V. myrtillus.     

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.Plants have evolved both constitutive and inducible defense mechanisms against herbivores. Constitutive mechanisms include structural defenses (e.g. spines and trichomes) and specific chemical compounds. Constitutive defense mechanisms provide immediate protection against herbivore attacks, although they represent an energy investment by the plant regardless of whether herbivory occurs or not (Mauricio, 1998; Bekaert et al., 2012). By contrast, inducible defense mechanisms do not require an up-front energy cost, although such mechanisms may not be as immediate as constitutive ones when herbivore feeding occurs (Windram et al., 2012). Accordingly, plants exhibit both constitutive and inducible defense mechanisms against herbivory to balance the speed and cost of response. In this regard, it is plausible that the recruitment of abundant primary metabolites for defensive purposes might represent a substantial benefit to plants, providing both a swift and economical defense function.Toxic chemical compounds form an essential part in both constitutive and inducible defense mechanisms. However, these compounds are potentially a double-edged sword for plants, in a sense that they might pose toxic effects for both plants and herbivores. Plants have evolved an intricate binary system that prevents autointoxication by their own chemical compounds. Specifically, a toxic substance is stored in its inactive form and is spatially isolated from specific activating enzymes. These enzymes activate the substance when cells are disrupted by chewing herbivores (Saunders and Conn, 1978; Thayer and Conn, 1981; Morant et al., 2008). One of the most extensively studied binary defense systems is the glucosinolate/myrosinase system, in which the glucosinolate substrate and their hydrolyzing enzyme, a thioglucosidase myrosinase, are compartmentalized. Upon tissue damage, both the substrate and the enzyme come into contact to produce unstable aglycones, and various toxic compounds are then spontaneously produced (Bones and Rossiter, 1996). Another well-known example of the binary system is comprised of cyanogenic glucosides and β-glucosidase (Vetter, 2000; Mithöfer and Boland, 2012). In this system, nontoxic cyanogenic glycoside compounds are stored in the vacuole, whereas, the related glycosidase is localized in the cytoplasm. Upon cell destruction by chewing herbivores, the cyanogenic glycosides are hydrolyzed by glycosidase to yield unstable cyanohydrin that is either spontaneously or enzymatically converted into toxic hydrogen cyanide and a ketone or an aldehyde. Because the binary defense system is efficient and effective, a use of ubiquitous compounds for such systems would provide further benefits for plants.Tetrapyrrole compounds, in particular heme and chlorophyll, are abundant in plant cells. Despite their significant roles in various biological processes including photosynthesis and respiration, many tetrapyrroles are highly toxic to plant and animal cells, if present in excess amounts (Kruse et al., 1995; Meskauskiene et al., 2001). Their photodynamic properties can cause the generation of reactive oxygen species upon illumination, resulting in cell injury or direct cell death. For example, Tapper et al. (1975) showed that a tetrapyrrole compound (pheophorbide a), which is readily converted from dietary chlorophyll through the loss of magnesium and phytol, reduces the growth and survival rates of young albino rats through its photodynamic property. More recently, Jonker et al. (2002) demonstrated that dietary-derived pheophorbide a causes severe damages on the skin of mutant mice that lack a transporter to excrete pheophorbide a from cells. These studies indicate that incorporation of an excessive amount of tetrapyrrole compounds can induce photosensitization in animals. Previous studies also showed that tetrapyrroles have illumination-independent deleterious effects on insects. For example, pheophorbide a affected the assimilation of the plant sterols to synthesize developmental hormones of insects by inhibiting the activity of a key enzyme, cholesterol acyltransferase (Song et al., 2002). Moreover, some tetrapyrroles, including pheophorbide a, have been suggested to induce illumination-independent cell death in plants as well by an unknown mechanism (Hirashima et al., 2009). It is proposed that organisms use the toxicity of tetrapyrroles for their defense systems. The larvae of tortoise beetle (Chelymorpha alternans) even utilize pheophorbide a as a powerful deterrent in the fecal shield to protect themselves from their predators (Vencl et al., 2009). Kariola et al. (2005) suggested that a chlorophyll derivative, chlorophyllide, is involved in the defense against fungi, based on their observations that down-regulation of a chlorophyll-hydrolyzing enzyme, chlorophyllase (CLH), results in increased susceptibility of Arabidopsis (Arabidopsis thaliana) plants to the necrotrophic fungus Alternaria brassicicola.In this study, we examined the possibility that plants use tetrapyrroles for defense against herbivores by analyzing CLH, a well-known hydrolase common in plants. Chlorophyll consists of a tetrapyrrolic macrocycle and a hydrophobic phytol side chain (Fig. 1). Phytol hydrolysis results in the formation of chlorophyllide (Fig. 1), a less hydrophobic chlorophyll derivative, which has photochemical properties similar to chlorophyll. Two different plant enzymes are known to catalyze the cleavage of phytol, pheophytinase (PPH) and CLH. PPH is a chloroplast-located enzyme that specifically catalyzes the removal of phytol from Mg-free chlorophyll catabolites (Schelbert et al., 2009). This enzyme was only recently discovered and has been shown to be responsible for chlorophyll degradation during leaf senescence. By contrast, CLH has a broader substrate specificity and removes the side chain from chlorophyll or other chlorophyll derivatives (McFeeters et al., 1971). CLH activity was first reported in leaf extracts in 1913 (Willstätter and Stoll, 1913), but despite a century of research, in vivo function and intracellular localization of this enzyme remained controversial. Some reports have indicated CLH to localize to chloroplasts (Azoulay Shemer et al., 2008; Azoulay-Shemer et al., 2011), while Schenk et al. (2007), by examining the intracellular localization of transiently expressed CLH-GFP fusions, proposed Arabidopsis CLH to localize outside the chloroplast. Schenk et al. (2007) also reported that the lack of CLH does not affect chlorophyll degradation during leaf senescence. However, it remains possible that CLH is specifically involved in chlorophyll degradation in response to stresses that activate jasmonate signaling, such as wounding or pathogen attack. This hypothesis is based on the observation that the expression of a CLH gene was highest when methyl-jasmonate (MeJA; a derivative of jasmonic acid) was applied to Arabidopsis plants (Tsuchiya et al., 1999).Open in a separate windowFigure 1.Early steps of proposed chlorophyll breakdown pathways. MCS, Magnesium-dechelating substance.Here, we report that CLH is not involved in endogenous chlorophyll breakdown even when leaf senescence was promoted by jasmonate signaling. CLH is shown to localize to the chlorophyll-free endoplasmic reticulum (ER) and the tonoplast of intact plant cells. We found that CLH promotes the conversion of chlorophyll into chlorophyllide when leaf cells are disrupted or when CLH is genetically mislocalized to chloroplasts. To examine the possibility that plants use chlorophyll and CLH to form a binary defense system against herbivores, a generalist herbivore, Spodoptera litura larvae, was employed to investigate the toxicity of Arabidopsis leaves with genetically enhanced CLH activity and purified chlorophyllide. The results support our hypothesis, indicating plants to deploy an abundant photosynthetic pigment for defense against herbivores, which would be economic and provide adaptation benefits to plants. A potential mechanism of chlorophyllide action as part of the plant defense system is discussed based on the examination of chlorophyllide binding to the insect gut.