Impact of atmospheric particulate matter retention on physiological characters of five plant species under different pollution levels in Zhengzhou.

Atmospheric particulate matter (PM) pollution has become a major environmental risk, and green plants can mitigate air pollution by regulating their enzymatic activity, osmoregulatory substances, photosynthetic pigments, and other biochemical characteristics. The present investigation aims to evaluate the mitigation potential of five common evergreen tree species (Photinia serrulata, Ligustrum lucidum, Eriobotrya japonica, Euonymus japonicus, Pittosporum tobira) against air pollution and to assess the effect of dust retention on plant physiological functions exposed to three different pollution levels (road, campus, and park). The results found that the amount of dust retained per unit leaf area of the plants was proportional to the mass concentration of atmospheric particulate matter in the environment, and that dust accumulation was higher on the road and campus than in the park. There were significant differences in dust retention among the five tree species, with the highest leaf dust accumulation observed for E. japonica (5.45 g·m-2), and the lowest for P. tobira (1.53 g·m-2). In addition, the increase in PM adsorption by different plants was uneven with increasing pollution levels, with significant decreases in chlorophyll content, photosynthetic and transpiration rate. From a physiological perspective, P. tobira exhibited greater potential to respond to PM pollution. Biochemical indicators suggested that PM pollution caused changes in plant protective enzyme activities, with a decrease in superoxide dismutase (SOD) and peroxidase (POD) activities, as well as promoting membrane lipid peroxidation, and appropriate stress also enables plants to counteract oxidative damage. In particular, PM exposure also induced stomatal constriction. Overall, PM retention was significantly associated with physiological and photosynthetic traits. In conclusion, our study contributes to the understanding of the effects of PM on plant physiology. Furthermore, it also provides insights into the selection of plants that are tolerant to PM pollution.

Mechanism of Rhodiola rosea-Euonymus alatus drug pair against rheumatoid arthritis: Network pharmacology and experimental validation.

PURPOSE: The present study aimed to explore the potential components and mechanisms of Rhodiola rosea-Euonymus alatus drug pair (TY) that ameliorate rheumatoid arthritis (RA). METHODS: The main active components, core targets, and important pathways of TY against RA were predicted by network pharmacology analysis. The binding activity between the main active components and the core targets was verified by the molecular docking technique. Collagen-induced arthritis (CIA) rat model and tumor necrosis factor (TNF)-α-induced fibroblast-like synovial cells in human RA (HFLS-RA) model were established, respectively. The core targets were verified by cell counting kit-8 assay, hematoxylin eosin, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis, and the therapeutic effect of TY was evaluated. RESULTS: A total of 18 possible components and 34 core targets were obtained by network pharmacology, among which inflammatory response, phosphatidylinositide 3-kinases (PI3K)-AKT and MAPK pathways were involved in the therapeutic effect of TY on RA. The results of molecular docking showed that kaempferol and quercetin had high binding affinity to interleukin (IL)-1ß, IL-6, matrix metalloproteinase (MMP)9, and TNF-α. In vivo and in vitro experiments showed that TY dose-dependently inhibited the proliferation of HFLS-RA cells induced by TNF-α, and significantly reduced the paw swelling and arthritis scores in CIA rats. At the same time, TY inhibited the production of inflammatory factors in CIA rat serum and TNF-α-induced HFLS-RA cells. It also decreased the expression of PI3K, phospho-protein kinase B, MMP1, MMP3, MMP9, and increased the protein and mRNA levels of tissue inhibitors of MMPs (TIMP)1 in synovial tissue. CONCLUSION: TY can inhibit the PI3K/AKT signaling pathway and regulate the balance between MMPs and TIMP, thus playing a therapeutic role in RA.

Euonymus alatus Twig Extract Protects against Scopolamine-Induced Changes in Brain and Brain-Derived Cells via Cholinergic and BDNF Pathways.

In the current study, the therapeutic and preventive effects of Euonymus alatus (EA) twig extract were investigated in a mouse model of cognitive deficit and B35 cells. Twig extract 1 was extracted with 70% ethanol and later twig extract 2 was extracted through liquid-liquid extraction with 70% ethanol and hexane. EA twig 2 (300 mg/kg) along with the standard drug donepezil (5 mg/kg) were orally administered to the mice for 34 days. Scopolamine was given intraperitoneally for 7 days. Administration of EA twig extract 2 significantly improved the passive avoidance test (PAT) in mice. EA twigs extract also restored the scopolamine-reduced brain-derived neurotrophic factor (BDNF)/extracellular regulated kinase (ERK)/cyclic AMP responsive element binding protein (CREB) signaling in B35 cells and the mouse hippocampus. In addition, EA twig extract significantly inhibited the acetylcholine esterase (AChE) activity in B35 cells in a dose-dependent manner. Chromatography and ESI MS analysis of EA twig extract revealed the presence of flavonoids; epicatechin, taxifolin, aromadendrin, and naringenin with catechin being the most abundant. These flavonoids exerted protective effects alone and had the possibility of synergistic effects in combination. Our work unmasks the ameliorating effect of EA twig extract 2 on scopolamine-associated cognitive impairments through the restoration of cholinergic systems and the BDNF/ERK/CREB pathway.

Effects of arbuscular mycorrhizal inoculation on the growth, photosynthesis and antioxidant enzymatic activity of Euonymus maackii Rupr. under gradient water deficit levels.

The role of arbuscular mycorrhizal (AM) fungus (Rhizophagus intraradices) in the amelioration of the water deficit-mediated negative influence on the growth, photosynthesis, and antioxidant system in Euonymus maackii Rupr. was examined. E. maackii seedlings were subjected to 5 water deficit levels, soil water contents of 20%, 40%, 60%, 80% and 100% field capacity (FC), and 2 inoculation treatments, with and without AM inoculation. The water deficit increasingly limited the seedling height, biomass accumulation in shoots and roots, chlorophyll content, gas exchange and chlorophyll fluorescence parameters with an increasing water deficit level. In addition, water deficit stimulated the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), in both shoots and roots, except under 20% FC conditions. E. maackii seedlings under all water deficit conditions formed symbiosis well with AM fungi, which significantly ameliorated the drought-mediated negative effect, especially under 40% and 60% FC conditions. Under 40% to 80% FC conditions, AM formation improved seedling growth and photosynthesis by significantly enhancing the biomass accumulation, chlorophyll content and assimilation. Mycorrhizal seedlings showed better tolerance and less sensitivity to a water deficit, reflected in the lower SOD activities of shoots and roots and CAT activity of shoots under 40% and 60% FC conditions. Downregulation of the antioxidant system in mycorrhizal seedlings suggested better maintenance of redox homeostasis and protection of metabolism, including biomass accumulation and assimilation. All the results advocated the positive role of R. intraradices inoculation in E. maackii against a water deficit, especially under 40% FC, which suggested the distinct AM performance in drought tolerance and the potential role of the combination of E. maackii-AM fungi in ecological restoration in arid regions.

Arbuscular mycorrhizal fungi (AMF) enhance the tolerance of Euonymus maackii Rupr. at a moderate level of salinity.

Salt stress is one of the major environmental constraints for plant growth. Although the ways in which mycorrhizal plants deal with salt stress have been well documented, it still is blank for Euonymus maackii, an important local ecological restoration tree, to arbuscular mycorrhizal fungi (AMF) inoculation and salt stress. In this study, we tested the effect of different salt levels (0, 50, 100,150 and 200 mM) and AMF inoculation on E. maackii growth rate, photosynthesis, antioxidant enzymes, nutrient absorption and salt ion distribution. The results indicated negative effect of salt on height, photosynthesis capacity, nutrition accumulation, while salt stimulated the antioxidant defense system and salt ions accumulation. The toxic symptom by excessive accumulation of salt ions worsen with salt level increased gradually (except for the 50 mM NaCl treatment). AMF inoculation alleviated the toxic symptom under moderate salt levels (100 and 150 mM) by increasing photosynthesis capacity, accelerating nutrient absorption and activating antioxidant enzyme activities under salt stress. Meanwhile, effect of AMF was not detected on seedlings under slight (0 and 50 mM) and high (200 mM) NaCl concentration. Our study indicated AMF had positive impact on E. maackii subjected to salt, which suggested potential application of AMF- E. maackii on restoration of salt ecosystems.

Electrophysiological and Behavioral Responses of Adult Vine Weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae), to Host Plant Odors.

Vine weevil, Otiorhynchus sulcatus F. (Coleoptera: Curculionidae), is an economically important pest species in many soft-fruit and ornamental crops. Economic losses arise from damage to the roots, caused by larvae, and to the leaves, caused by adults. As adults are nocturnal and larvae feed below ground, infestations can be missed initially, with controls applied too late. In the absence of a vine weevil sex or aggregation pheromone, the development of an effective semiochemical lure for better management of this pest is likely to focus on host-plant volatiles. Here, we investigate the electrophysiological and behavioral responses of adult vine weevils to volatile organic compounds (VOCs) originating from their preferred host plant Euonymus fortunei, and synthetic VOCs associated with this host when presented individually or as blends. Consistent electroantennographic responses were observed to a range of generalist VOCs. Behavioral responses of weevils to VOCs, when presented individually, were influenced by concentration. Vine weevil adults showed directional movement toward a mixture of seven plant volatiles, methyl salicylate, 1-octen-3-ol, (E)-2-hexenol, (Z)-3-hexenol, 1-hexanol, (E)-2-pentenol, and linalool, even though no, or negative, responses were recorded to each of these compounds presented individually. Similarly, vine weevils showed directional movement toward a 1:1 ratio mixture of (Z)-2-pentenol and methyl eugenol. Results presented here point to the importance of blends of generalist compounds and their concentrations in the optimization of a lure.

Slower development of PSI activity limits photosynthesis during Euonymus japonicus leaf development.

This study primarily explored the limiting factor for photosynthesis during the development of Euonymus japonicus leaves. The analysis of the chlorophyll fluorescence transient, pulse-modulated fluorescence, 820-nm reflection, and expression of core proteins for photosystems demonstrated that photosystem II (PSII) activity developed more rapidly than did photosystem I (PSI) activity. The slower development of the PSI activity restricted linear and cyclic electron transport and thus inhibited the production of ATP and NADPH, which inhibits the activation of Rubisco, resulting in low activity of carboxylation efficiency. The application of exogenous NADPH (50 µM) and ATP (100 µM) to leaves remarkably increased the Pn and CE in the youngest leaf but not in the fully expanded leaf, which indicated that an inadequate supply of the assimilatory power significantly inhibited CE and Pn. We concluded that the slower development of the PSI activity was one of the most important limiting factors for photosynthesis during the development of E. japonicus leaves.

Evaluation of the Possibility of Using Hydroponic Cultivations for the Removal of Pharmaceuticals and Endocrine Disrupting Compounds in Municipal Sewage Treatment Plants.

The problem of the presence of pharmaceuticals and endocrine disrupting compounds (EDCs) in the environment is closely related to municipal wastewater and in consequence to municipal wastewater treatment plants (MWWTPs) because wastewater is the main way in which these compounds are transferred to the ecosystem. For this reason, the development of cheap, simple but very effective techniques for the removal of such residues from wastewater is very important. In this study, the analysis of the potential of using three new plants: Cyperus papyrus (Papyrus), Lysimachia nemorum (Yellow pimpernel), and Euonymus europaeus (European spindle) by hydroponic cultivation for the removal of 15 selected pharmaceuticals and endocrine disrupting compounds (EDCs) in an MWWTP is presented. In order to obtain the most reliable data, this study was performed using real WWTP conditions and with the determination of the selected analytes in untreated sewage, treated sewage, and in plant materials. For determining the target compounds in plant materials, an Accelerated Solvent Extraction (ASE)-Solid-Phase Extraction (SPE)-GC-MS(SIM) method was developed and validated. The obtained data proved that the elimination efficiency of the investigated substances from wastewater was in the range of 35.8% for diflunisal to above 99.9% for paracetamol, terbutaline, and flurbiprofen. Lysimachia nemorum was the most effective for the uptake of target compounds among the tested plant species. Thus, the application of constructed wetlands for supporting conventional MWWTPs allowed a significant increase in their removal from the wastewater stream.

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value.

Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild-type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl-TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl-triacylglycerols (acetyl-TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl-TAG levels to up to 85 mol% in field-grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl-TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn-3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl-TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl-TAG oils were reduced, enabling use of this oil in several nonfood and food applications.

An endoparasitoid avoids hyperparasitism by manipulating immobile host herbivore to modify host plant morphology.

Many parasitic organisms have an ability to manipulate their hosts to increase their own fitness. In parasitoids, behavioral changes of mobile hosts to avoid or protect against predation and hyperparasitism have been intensively studied, but host manipulation by parasitoids associated with endophytic or immobile hosts has seldom been investigated. We examined the interactions between a gall inducer Masakimyia pustulae (Diptera: Cecidomyiidae) and its parasitoids. This gall midge induces dimorphic leaf galls, thick and thin types, on Euonymus japonicus (Celastraceae). Platygaster sp. was the most common primary parasitoid of M. pustulae. In galls attacked by Platygaster sp., whole gall thickness as well as thicknesses of upper and lower gall wall was significantly larger than unparasitized galls, regardless of the gall types, in many localities. In addition, localities and tree individuals significantly affected the thickness of gall. Galls attacked by Platygaster sp. were seldom hyperparasitized in the two gall types. These results strongly suggest that Platygaster sp. manipulates the host plant's development to avoid hyperparasitism by thickening galls.

Transcriptional behavior of EUL-related rice lectins toward important abiotic and biotic stresses.

The rice genome encodes several genes for putative carbohydrate-binding proteins belonging to the family of Euonymus related lectins (EULs). This lectin family was discovered recently and evidence shows that the expression of these proteins is subject to multiple environmental stresses. In this study, quantitative reverse transcription PCR (qRT-PCR) was conducted on rice seedlings exposed to various abiotic (150mM NaCl, 100mM mannitol, and 100µM abscisic acid (ABA)) and biotic (Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae) stresses to compare the transcriptional behavior of the EULs and a known stress related lectin Orysata belonging to the family of jacalin-related lectins. All EUL transcripts were strongly up-regulated after ABA and NaCl treatments in the roots whereas the overall expression level was generally lower and more variable in the shoots. Moreover, all abiotic stresses induced Orysata in both tissues except for mannitol treatment which failed to show an effect in the roots. Orysata also strongly accumulated after X. oryzae pv. oryzae infection, as were various D-type EUL lectins. In contrast, some of the EUL proteins, including OrysaEULS3, OrysaEULD1A and OrysaEULD2, as well as Orysata were significantly down-regulated upon M. oryzae attack, suggesting fungal manipulation of these genes. Collectively, our results clearly show that rice expresses multiple carbohydrate-binding proteins in response to a wide variety of abiotic and biotic stress conditions. We hypothesize that the Euonymus related proteins fulfill a prominent role in sensing and responding to multiple environmental cues.

Water relations, nutrient content and developmental responses of Euonymus plants irrigated with water of different degrees of salinity and quality.

For 20 weeks, the physiological responses of Euonymus japonica plants to different irrigation sources were studied. Four irrigation treatments were applied at 100 % water holding capacity: control (electrical conductivity (EC) <0.9 dS m(-1)); irrigation water normally used in the area (irrigator's water) IW (EC: 1.7 dS m(-1)); NaCl solution, NaCl (EC: 4 dS m(-1)); and wastewater, WW (EC: 4 dS m(-1)). This was followed by a recovery period of 13 weeks, when all the plants were rewatered with the same amount and quality of irrigation water as the control plants. Despite the differences in the chemical properties of the water used, the plants irrigated with NaCl and WW showed similar alterations in growth and size compared with the control even at the end of the recovery period. Leaf number was affected even when the EC of the irrigation water was of 1.7 dS m(-1) (IW), indicating the salt sensitivity of this parameter. Stomatal conductance (gs) and photosynthesis (Pn), as well as stem water potential (Ψstem), were most affected in plants irrigated with the most saline waters (NaCl and WW). At the end of the experiment the above parameters recovered, while IW plants showed similar values to the control. The higher Na(+) and Cl(+) uptake by NaCl and WW plants led them to show osmotic adjustment throughout the experiment. The highest amount of boron found in WW plants did not affect root growth. Wastewater can be used as a water management strategy for ornamental plant production, as long as the water quality is not too saline, since the negative effect of salt on the aesthetic value of plants need to be taken into consideration.

A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds.

Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications.

[Environmental responses of four urban tree species transpiration in northern China].

By using thermal dissipation probes (TDP), this paper monitored the sap flow of four tree species (Cedrus deodara, Zelkova schneideriana, Euonymus bungeanus, and Metasequoia glyptostroboides) at the Laodong Park in Dalian City from June to August 2008, and the soil moisture content and micrometeorological variables were mehsured simultaneously. Due to the absence of water-stress in the habitat, the sap flow of all sampled trees had no significant correlation with soil moisture content (R2 < 0.050, P > 0.211, n=1296). The correlation coefficient between solar radiation and sap flow reached 0.624-0.773 (P = 0.00, n=1296) despite the existing hysteresis. Solar radiation had major effect (R2 > 0.700, P < 0.05) during early morning (5:00-8:00) and late afternoon (18:00-20:00) when undergoing dramatic changes. As the main factor determining nighttime sap flow (R2 > 0.660, P < 0.05, n=1872), vapor pressure deficit (VPD) had a correlation coefficient as high as 0.650-0.823 (P = 0.00, n=1296) with the sap flow in whole-day scale. Meanwhile, the models constructed on the basis of VPD were able to explain 90% of daily sap flow change (P = 0.00). The correlation coefficient between sap flow and wind speed was relatively smaller than the previous two (R2 < 0.380, P = 0.00, n=1296), though showing significant correlation in affecting sap flow. Observations also detected the saturation phenomenon of sap flow to the environmental demands.

The "old" Euonymus europaeus agglutinin represents a novel family of ubiquitous plant proteins.

Molecular cloning of the "old" but still unclassified Euonymus europaeus agglutinin (EEA) demonstrated that the lectin is a homodimeric protein composed of 152 residue subunits. Analysis of the deduced sequence indicated that EEA is synthesized without a signal peptide and undergoes no posttranslational processing apart from the removal of a six-residue N-terminal peptide. Glycan array screening confirmed the previously reported high reactivity of EEA toward blood group B oligosaccharides but also revealed binding to high mannose N-glycans, providing firm evidence for the occurrence of a plant carbohydrate-binding domain that can interact with structurally different glycans. Basic Local Alignment Search Tool searches indicated that EEA shares no detectable sequence similarity with any other lectin but is closely related evolutionarily to a domain that was first identified in some abscisic acid- and salt stress-responsive rice (Oryza sativa) proteins, and, according to the available sequence data, might be ubiquitous in Spermatophyta. Hence, EEA can be considered the prototype of a novel family of presumably cytoplasmic/nuclear proteins that are apparently ubiquitous in plants. Taking into account that some of these proteins are definitely stress related, the present identification of the EEA lectin domain might be a first step in the recognition of the involvement and importance of protein-glycoconjugate interactions in some essential cellular processes in Embryophyta.

Kaempferol and quercetin isolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activity.

Euonymus alatus as a folk medicine in China has been clinically used to treat type 2 diabetes for many years, and also exerts beneficial effects on hyperglycemia of diabetic animals. Our previous studies have isolated kaempferol and quercetin from the extract of E. alatus. In the present study, we investigated the possible mechanism of antidiabetic activity of these compounds. Kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. In addition, further experiments showed that kaempferol and quercetin served as weak partial agonists in the peroxisome proliferator-agonist receptor gamma (PPARgamma) reporter gene assay. Kaempferol and quercetin could not induce differentiation of 3T3-L1 preadipocytes as traditional PPARgamma agonist. When added together with the PPARgamma agonist rosiglitazone to 3T3-L1 preadipocytes, they could inhibit 3T3-L1 differentiation in a dose-dependent manner. Competitive ligand-binding assay confirmed that kaempferol and quercetin could compete with rosiglitazone at the same binding pocket site as PPARgamma. Kaempferol and quercetin showed significant inhibitory effects on NO production in response to lipopolysaccharide treatment in macrophage cells in which the PPARgamma was overexpressed; rosiglitazone was less potent than kaempferol and quercetin. These observations suggest that kaempferol and quercetin potentially act at multiple targets to ameliorate hyperglycemia, including by acting as partial agonists of PPARgamma.

Chemical shift assignments of two oleanane triterpenes from Euonymus hederaceus.

(1)H-NMR and (13)C-NMR assignments of 12-oleanene-3,11-dione (compound 1) were completely described for the first time through conventional 1D NMR and 2D shift-correlated NMR experiments using (1)H-(1)HCOSY, HMQC, HMBC techniques. Based on its NMR data, the assignments of 28-hydroxyolean-12-ene-3,11-dione (compound 2) were partially revised.

Synergistic antifungal activity of two chitin-binding proteins from spindle tree (Euonymus europaeus L.).

Two structurally different chitin-binding proteins were isolated from bark and leaves of the spindle tree (Euonymus europaeus L.). Both the small hevein-like chitin-binding protein (Ee-CBP) and the classical class-I chitinase (Ee-chitinase) possess antifungal properties, Ee-CBP being far more potent than Ee-chitinase. In addition, Ee-CBP and Ee-chitinase display a pronounced synergistic effect when added together in the test medium. Determination of the biological activities indicates that the synergism between Ee-CBP and Ee-chitinase relies on a different mode of action. Cloning and sequencing of the corresponding genes further revealed that Ee-CBP and Ee-chitinase are simultaneously expressed in bark and leaf tissues, and hence can act synergistically in planta. Moreover, analysis of the deduced sequences allowed the exact relationship between the structurally different Ee-CBP and Ee-chitinase to be corroborated. Both proteins are synthesized as similar chimeric precursors consisting of an N-terminal hevein domain linked to a C-terminal chitinase-like domain by a hinge region. However, whereas in the case of Ee-chitinase the C-terminal chitinase domain remains linked to the N-terminal hevein domain, the corresponding domain is cleaved from the Ee-CBP-precursor resulting in the formation of the hevein-type Ee-CBP. Since both precursors are--apart from the hinge region between the hevein and chitinase domains--very similar, the Ee-CBP/Ee-chitinase system offers a unique opportunity to study the importance of sequence and/or structural information comprised in the hinge region for the posttranslational processing of the respective precursor proteins.