Requirement of catalytic-triad and related amino acids for the acyltransferase activity of Tanacetum cinerariifolium GDSL lipase/esterase TcGLIP for ester-bond formation in pyrethrin biosynthesis.

We have recently discovered that a GDSL lipase/esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltose-binding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.

Identification and characterization of a GDSL lipase-like protein that catalyzes the ester-forming reaction for pyrethrin biosynthesis in Tanacetum cinerariifolium- a new target for plant protection.

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.

Specific regulation of pyrethrin biosynthesis in Chrysanthemum cinerariaefolium by a blend of volatiles emitted from artificially damaged conspecific plants.

Plants emit specific blends of volatile organic compounds (VOCs) in response to mechanical wounding. Such induced VOCs have been shown to mediate in plant and interplant communication, yet little is known about the time- and dose-response relationships in VOC-mediated communications. Here, we employed young seedlings of Chrysanthemum cinerariaefolium to examine the effects of volatiles emitted by artificially damaged seedlings on the biosynthesis of the natural insecticides pyrethrins in intact conspecific plants. Wounded leaves emitted (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate and (E)-ß-farnesene as dominant wound-induced VOCs. Exposing intact seedlings to a mixture of these VOCs at concentrations mimicking those emitted from wounded seedlings, as well as placing the intact seedlings next to the wounded seedlings, resulted in enhanced pyrethrin contents in the intact seedlings. Thus we quantified mRNA transcripts of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), chrysanthemyl diphosphate synthase (CPPase), 13-lipoxygenase (13-LOX) and allene oxide synthase (AOS) genes in intact seedlings exposed to the VOC mixture to show that DXS and 13-LOX gene expression reached a maximum at 3 h, whereas CPPase and AOS reached it at 6 h. Interestingly, both increasing and decreasing the VOC mixture concentrations from those observed on injury reduced the expression of DXS, CPPase and AOS genes to the control level. Also, separating the VOC mixture into individual components eliminated the ability to enhance the expression of all the biosynthetic genes examined. This is the first study showing that the wound-induced VOCs function as a blend to control the biosynthesis of second metabolites at specific concentrations.

Green odor attenuates a cold pressor test-induced cardiovascular response in healthy adults.

BACKGROUND: Green odor, a mixture of equal amounts of 2E-hexenal (leaf aldehyde) and 3Z-hexenol (leaf alcohol) has been demonstrated to have an anti-stress effect in rats. This study investigated whether or not green odor also has an anti-stress effect in humans. METHODS: Changes in blood pressure, heart rate, and the skin temperature of a fingertip were observed after presenting green odor at a concentration of 0.03% or vehicle via inhalation through the nose for 10 min to eight healthy normotensive adults. We also assessed the pleasantness of green odor and its effect on mood states via assessment with the Profile of Mood States (POMS) questionnaire. Cardiovascular response to green odor and the vehicle were compared among 11 additional healthy adults by use of the cold pressor test. RESULTS: Of 19 subjects, 15 (79%) reported that the green odor was pleasant. Green odor had no effect on blood pressure, heart rate, skin temperature, or POMS score under non-stressful conditions. In the second experiment, green odor attenuated cold pressor test-induced increases in systolic and diastolic blood pressure and facilitated the recovery of skin temperature. CONCLUSION: These findings suggest that green odor has an anti-stress effect in healthy humans.

Biosynthesis of pyrethrin I in seedlings of Chrysanthemum cinerariaefolium.

The biosynthetic pathway to natural pyrethrins in Chrysanthemum cinerariaefolium seedlings was studied using [1-13C]d-glucose as a precursor, with pyrethrin I isolated using HPLC from a leaf extract. The 13C NMR spectrum of pyrethrin I from the precursor-administered seedlings indicated that the acid moiety was biosynthesized from d-glucose via 2-C-methyl-d-erythritol 4-phosphate, whereas the alcohol moiety was possibly biosynthesized from linolenic acid.

Attenuation of stress-induced elevations in plasma ACTH level and body temperature in rats by green odor.

To investigate the effect of green odor on the elevation of the plasma adrenocorticotropic hormone (ACTH) levels and body temperature (T(b)) induced by stress, adult male rats were subjected to a 2-h immobilization stress and exposed to green odor or its vehicle only. In comparison with the vehicle group, animals in the green odor group showed a significant reduction in plasma ACTH levels at the end of the stress when green odor was applied during the stress. The elevated plasma ACTH levels 2 days after the stress were reduced by green odor applied 0, 1, 2, 4 or 6 h after the beginning of the stress. Neither the immediate nor the long-lasting plasma ACTH response was affected by a prestress treatment of green odor. T(b) elevation was evident following the end of the stress and during the light phase the day after the stress. Both the immediate and the long-lasting elevations in T(b) were attenuated by green odor. These data suggest that green odor extracted from green leaves has a relieving effect on plasma ACTH and T(b) levels induced by an immobilization stress for not only immediate but also long-lasting periods.

Activation of the anterior cingulate gyrus by 'Green Odor': a positron emission tomography study in the monkey.

The equivalent mixture of cis-3-hexenol and trans-2-hexenal (hexenol/hexenal), 'green odor', is known to have a healing effect on the psychological damage caused by stress. Behavioral studies in humans and monkeys have revealed that hexenol/hexenal prevents the prolongation of reaction time caused by fatigue. In the present study, we investigated which brain regions are activated by the odor of hexenol/hexenal using positron emission tomography with alert monkeys. Regional cerebral blood flow (rCBF) in the prepyriform area (the primary olfactory cortex) was commonly increased by the passive application of odor: acetic acid, isoamylacetate or hexenol/hexenal. We observed rCBF increases in the orbitofrontal cortex (the secondary olfactory cortex) by these olfactory stimuli in two of three monkeys, and found no predominance of laterality of the activated hemisphere. Furthermore, rCBF increase in the cerebellum was observed in two of three monkeys, and the odor of acetic acid increased rCBF in the substantia innominata in all monkeys. In addition to these olfactory related regions, the anterior cingulate gyrus was activated by the odor of hexenol/hexenal. These findings suggest that the increase of rCBF in the anterior cingulate gyrus by the odor of hexenol/hexenal may contribute the healing effects of this mixture observed in the monkey.

Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacteria.

All eight C6-aliphatic alcohol and aldehyde compounds in naturally occurring green leaves showed bacteriostatic effects against Staphylococcus aureus IFO 12732, methicillin-resistant S. aureus, Escherichia coli IFO 3301, E. coli O157:H7, and Salmonella enteritidis, with bacteriostatic activities of less than 12.5 microg mL(-1). In this study, the susceptibility of Gram-positive bacteria tested was observed to be greater than that of Gram-negative bacteria. The bactericidal action of the aldehyde compounds was found to be much stronger than that of the alcohol compounds under both liquid and gaseous conditions. The most effective compound was (3E)-hexenal at concentrations of 0.1 and 1 microg mL(-1), which killed 2.1 x 10(5) cfu mL(-1) of S. aureus IFO 12732 and 1.4 x 10(5) cfu mL(-1) of E. coli IFO 3301, respectively, by direct contact with the compound. Lethality of (3E)-hexenal against S. aureus IFO 12732 and E. coli IFO 3301 was also observed as a result of gaseous contact at concentrations of 3 and 30 microg mL(-1), respectively. The bactericidal effects of 30 microg mL(-1) (3E)-hexenal were thoroughly maintained throughout periods of 2 days and 1 day against S. aureus IFO 12732 and E. coli IFO 3301, respectively, by a complex formation with alpha-cyclodextrin.

Alleviating effects of plant-derived fragrances on stress-induced hyperthermia in rats.

In the present study, the effects of exposure to plant-derived odors on the autonomic and behavioral responses to novel environment were examined in rats. Male rats (n=42) carrying a telemetry transmitter were individually housed, and on the test day each rat was transferred to a new cage containing bedding that had been sprayed immediately before testing with 200 microl of 0.03% dilution of either lavender essential oil, green leaf odor (a mixture of hexenol and hexenal), alpha-pinene, or solvent (triethyl citrate) as a control. Following the transfer to this novel environment, the body temperature of the rats increased by nearly 1 degrees C, showing a stress-induced hyperthermia. Stress-induced hyperthermia was attenuated by the green odor and the alpha-pinene, but not by the lavender or solvent. There was no clear effect of fragrances on heart rate or behavioral responses. These results suggest that plant-derived fragrances, such as green odor and alpha-pinene, have calming effects on autonomic stress response to novel environments.

Concentration effects of green odor on event-related potential (P300) and pleasantness.

The effects of eight compounds, constituting the so-called "natural green odor", including leaf alcohol, on the event-related potential (P300) were investigated. In experiments with a series of single compounds, each of these eight compounds could be characterized by an overall change consisting predominantly of an increase, a decrease or no change in the amplitude of P300, whereas in experiments with a series of two-component mixtures, noticeable synergism could not be demonstrated, contrary to our expectation. Experiments with leaf alcohol (3Z-hexenol) performed at two concentrations showed a significantly different degree of pleasantness and an increase or decrease in the amplitude of P300 depending on their concentration, suggesting that concentration is important in odorant-presentation studies.