Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.

Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily.

BACKGROUND: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects. RESULTS: A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ. CONCLUSION: Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

Quantification of canavanine, 2-aminoethanol, and cyanamide in Aphis craccivora and its host plants, Robinia pseudoacacia and Vicia angustifolia: effects of these compounds on larval survivorship of Harmonia axyridis.

The cowpea aphid Aphis craccivora that infests the black locust Robinia pseudoacacia shows toxicity to its predator, the multicolored Asian ladybird beetle, Harmonia axyridis. In contrast, the same aphid species that infests the common vetch, Vicia angustifolia, is suitable prey for H. axyridis larvae. Previously, it was reported that the toxicity of A. craccivora infesting R. pseudoacacia was due to canavanine and 2-aminoethanol, but there was some doubt about the toxicity of these compounds and their concentrations in the aphids. In the present study, we determined the concentrations of cyanamide, canavanine, and 2-aminoethanol in A. craccivora infesting the two host plants. In the extracts of A. craccivora that infested either of the host plants, canavanine was undetectable, and 2-aminoethanol was detected at the concentration of 3.0-4.0 µg/g fresh weight. Cyanamide was detected in the extract of A. craccivora that infested R. pseudoacacia (7.7 µg/g fresh weight) but not in that infesting V. angustifolia. The toxicity of canavanine, 2-aminoethanol, and cyanamide was evaluated against H. axyridis larvae in a bioassay by using an artificial diet containing these compounds at various concentrations. Cyanamide exhibited 10-100 times stronger toxicity than canavanine and 2-aminoethanol. These results indicate that the toxicity is at least partly due to cyanamide, which is present in the toxic A. craccivora that infests R. pseudoacacia but absent from the non-toxic A. craccivora that infests V. angustifolia.

Quantification of cyanamide in young seedlings of Vicia species, Lens culinaris, and Robinia pseudo-acacia by gas chromatography-mass spectrometry.

We quantified the cyanamide content of young leaves of nine Vicia species, Lens culinaris, and Robinia pseudo-acacia using a modified analytical procedure that made it possible to measure the cyanamide content of a single leaf. Recent molecular phylogenetic analysis suggests that cyanamide is present in V. benghalensis, which is placed in a monophyletic group with cyanamide-biosynthesizing plants, V. villosa and V. cracca; this suggestion was verified.

Synthesis of (-)-epicatechin 3-(3-O-methylgallate) and (+)-catechin 3-(3-O-methylgallate), and their anti-inflammatory activity.

A concise synthesis of (-)-epicatechin 3-(3-O-methylgallate) (1; ECG3''Me), which is a minor constituent of tea, and (+)-catechin 3-(3-O-methylgallate) (2; CG3''Me) via condensation of equimolar amount of catechin and gallate derivatives has been achieved. The anti-inflammatory effect of the synthetic compounds on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation of mouse ears was examined. Compounds 1 and 2 suppressed the TPA-induced inflammation of mouse ears by 50 and 43%, respectively, at a dose of 200 microg. Their activities are stronger than those of indomethacin and glycyrrhetinic acid, the normally used anti-inflammatory agents.

Limited distribution of natural cyanamide in higher plants: occurrence in Vicia villosa subsp. varia, V. cracca, and Robinia pseudo-acacia.

Cyanamide (NH2CN) has recently been proven to be a natural product, although it has been synthesized for over 100 years for agricultural and industrial purposes. The distribution of natural cyanamide appears to be limited, as indicated by our previous investigation of 101 weed species. In the present study, to investigate the distribution of natural cyanamide in Vicia species, we monitored the cyanamide contents in V. villosa subsp. varia, V. cracca, and V. amoena during their pre-flowering and flowering seasons. It was confirmed that V. cracca was superior to V. villosa subsp. varia in accumulating natural cyanamide, and that V. amoena was unable to biosynthesize this compound under laboratory condition examined. The localization of cyanamide in the leaves of V. villosa subsp. varia seedlings was also clarified. In a screening study to find cyanamide-biosynthesizing plants, only Robinia pseudo-acacia was found to contain cyanamide among 452 species of higher plants. We have investigated 553 species to date, but have so far found the ability to biosynthesize cyanamide in only three species, V. villosa subsp. varia, V. cracca and R. pseudo-acacia.

A DNA barcoding method for identifying and quantifying the composition of pollen species collected by European honeybees, Apis mellifera (Hymenoptera: Apidae).

The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), is the most important crop pollinator, and there is an urgent need for a sustained supply of honeybee colonies. Understanding the availability of pollen resources around apiaries throughout the brood-rearing season is crucial to increasing the number of colonies. However, detailed information on the floral resources used by honeybees is limited due to a scarcity of efficient methods for identifying pollen species composition. Therefore, we developed a DNA barcoding method for identifying the species of each pollen pellet and for quantifying the species composition by summing the weights of the pellets for each species. To establish the molecular biological protocol, we analyzed 1008 pellets collected between late July and early September 2016 from five hives placed in a forest/agricultural landscape of Hokkaido, northern Japan. Pollen was classified into 31 plant taxa, of which 29 were identified with satisfactory discrimination (25 species and 4 genera) using trnL-trnF and ITS2 as DNA barcoding regions together with available floral and phenological information. The remaining two taxa were classified to the species level using other DNA barcoding regions. Of the 1008 pollen pellets tested, 1005 (99.7%) were successfully identified. As an example of the use of this method, we demonstrated the change in species composition of pollen pellets collected each week for 9 weeks from the same hive.

A tyrosinase inhibitor, Daedalin A, from mycelial culture of Daedalea dickinsii.

A chromene-type compound, daedalin A (1), was isolated from mycelial culture broth of Daedalea dickinsii. Based on spectroscopic data, the structure of 1 was found to be (2R)-6-hydroxy-2-hydroxymethyl-2-methyl-2H-chromene. Daedalin A (1) strongly inhibited the activity of tyrosinase (IC(50): 194 muM). In addition, 1 also showed 1,1-diphenyl-2-picrylhydrazyl scavenging activity (IC(50): 16.9 microM) and superoxide anion scavenging activity (IC(50): 28.5 microM).

Phyllosphere Methylobacterium bacteria contain UVA-absorbing compounds.

Microbes inhabiting the phyllosphere encounter harmful ultraviolet rays, and must develop adaptive strategies against this irradiation. In this study, we screened bacterial isolates originating from the phyllosphere of various plants which harbored absorbers of ultraviolet A (UVA), a wavelength range which is recognized as harmful to human skin. Of the 200 phyllosphere bacterial isolates we screened, methanol extracts from bacterial cells of seventeen isolates absorbed wavelengths in the range of 315-400nm. All of the UVA-absorbing strains belonged to Methylobacterium species based on 16S ribosomal RNA gene sequences, suggesting that cells of this bacterial genus contain specific UVA-absorbing compounds. When cells of a representative Methylobacterium strain were extracted using various solvents, UVA absorption was observed in the extracts obtained using several aqueous solvents, indicating that the UVA-absorbing compounds were highly polar. A compound was purified using solid columns and HPLC separation, and comparative analysis revealed that the absorption strength and spectrum of the compound were similar to those of the known UVA filter, avobenzone. The compound was also verified to be stable under UVA exposure for at least 480min. Based on these results, the UVA-absorbing compound harbored by Methylobacterium has potential to be used as a novel sunscreen ingredient.

Evidence of cyanamide production in hairy vetch Vicia villosa.

Cyanamide (NH(2)CN) has recently been isolated as a plant growth inhibitor from Vicia villosa, which is the first discovery of cyanamide from natural sources. To reveal the presence of the biosynthesized cyanamide in plants, 3.4 mM potassium ((15)N)nitrate was administered to 15- to 35-day-old plants of V. villosa, from which the cyanamide was purified and subjected to GC/MS analysis. The isotopic ratio (15)N/((14)N + (15)N) of the cyanamide was calculated to be 0.143, while that of the cyanamide extracted from V. villosa grown in the presence of a natural N source was 0.0065. The (15)N-enrichment proved de novo biosynthesis of cyanamide.

Plant growth inhibitory activity of azo- and azoxyformamides from Calvatia craniiformis and Lycoperdon hiemale.

4-Methoxybenzene-1-ONN-azoxyformamide (1), 4-methoxybenzene-1-azoformamide (2), 4-hydroxyben-zene-1-azoformamide (3) and 4-hydroxybenzene-1-ONN-azoxyformamide (4) were isolated from the fruiting bodies of Calvatia craniiformis and Lycoperdon hiemale, respectively. Compounds 1 and 4 showed radicle growth inhibitory activities against the lettuce seedlings by more than 50% at 5.0 x 10(-1) mM, suggesting that the azoxy moiety contributes to the inhibitory activity. The plant growth inhibitory activities of 1, 2 and 4 against the barnyard millet seedlings were also measured.

Direct quantitative determination of cyanamide by stable isotope dilution gas chromatography-mass spectrometry.

Cyanamide is a multifunctional agrochemical used, for example, as a pesticide, herbicide, and fertilizer. Recent research has revealed that cyanamide is a natural product biosynthesized in a leguminous plant, hairy vetch (Vicia villosa). In the present study, gas chromatography-mass spectrometry (GC-MS) equipped with a capillary column for amines was used for direct quantitative determination of cyanamide. Quantitative signals for ((14)N(2))cyanamide, ((15)N(2))cyanamide (internal standard for stable isotope dilution method), and m-(trifluoromethyl)benzonitrile (internal standard for correcting errors in GC-MS analysis) were recorded as peak areas on mass chromatograms at m/z 42 (A(42)), 44 (A(44)), and 171 (A(IS)), respectively. Total cyanamide content, ((14)N(2))cyanamide plus ((15)N(2))cyanamide, was determined as a function of (A(42)+A(44))/A(IS). Contents of ((14)N(2))cyanamide and ((15)N(2))cyanamide were then calculated by multiplying the total cyanamide content by A(42)/(A(42)+A(44)) and A(44)/(A(42)+A(44)), respectively. The limit of detection for the total cyanamide content by the GC-MS analysis was around 1ng. The molar ratio of ((14)N(2))cyanamide to ((15)N(2))cyanamide in the injected sample was equal to the observed A(42)/A(44) value in the range from 0.1 to 5. It was, therefore, possible to use the stable isotope dilution method to quantify the natural cyanamide content in samples; i.e., the natural cyanamide content was derived by subtracting the A(42)/A(44) ratio of the internal standard from the A(42)/A(44) ratio of sample spiked with internal standard, and then multiplying the resulting difference by the amount of added ((15)N(2))cyanamide (SID-GC-MS method). This method successfully gave a reasonable value for the natural cyanamide content in hairy vetch, concurring with the value obtained by a conventional method in which cyanamide was derivatized to a photometrically active compound 4-cyanimido-1,2-naphthoquinone and analyzed with reversed-phase HPLC (CNQ-HPLC method). The determination range of cyanamide in the SID-GC-MS method was almost the same as that in the CNQ-HPLC method; however, the SID-GC-MS method was much simpler than the CNQ-HPLC method.

Cyanamide is biosynthesized from L-canavanine in plants.

Cyanamide had long been recognized as a synthetic compound but more recently has been found as a natural product from several leguminous plants. This compound's biosynthetic pathway, as yet unelaborated, has attracted attention because of its utility in many domains, such as agriculture, chemistry, and medicine. We noticed that the distribution of L-canavanine in the plant kingdom appeared to include that of cyanamide and that the guanidino group structure in L-canavanine contained the cyanamide skeleton. Here, quantification of these compounds in Vicia species suggested that cyanamide was biosynthesized from L-canavanine. Subsequent experiments involving L-[guanidineimino-(15)N2]canavanine addition to young Vicia villosa seedlings resulted in significant incorporation of (15)N-label into cyanamide, verifying its presumed biosynthetic pathway.

Novel C-xylosylflavones from the leaves and flowers of Iris gracilipes.

Two new C-glycosylflavones, apigenin 7,4'-dimethyl ether 6-C-ß-[(4"'-acetyl-L-rhamnopyranosyl)-(1-->2)-xylopyranoside] (1) and apigenin 7,4'-dimethyl ether 6-C-ß-L-rhamnopyranosyl-(1-->2)-xylopyranoside (2) were isolated from the leaves of Iris gracilipes (Iridaceae), along with two known flavonoids, swertiajaponin (3) and swertisin (4). C-Xylosylflavones 1 and 2 were elucidated by UV and NMR spectroscopy, mass spectrometry, and acid and alkaline hydrolyses. These novel compounds were also presented in the flowers.

Revised chirality of the acyl group of 8'-O-(3-hydroxy-3-methylglutaryl)-8'-hydroxyabscisic acid.

8'-O-(3-Hydroxy-3-methylglutaryl)-8'-hydroxyabscisic acid is a stable conjugate of the first metabolite of abscisic acid, 8'-hydroxyabscisic acid, that is spontaneously isomerized to phaseic acid. The chirality of the 3-hydroxy-3-methylglutaryl group of the conjugate was revised to S based on an HPLC analysis of the diastereomer derived from mevalonolactone obtained by reduction of the conjugate with lithium borohydride.

Plant growth inhibitor from the Malaysian medicinal plant Goniothalamus andersonii and related species.

A crude methanol extract of Goniothalamus andersonii J. Sinclair strongly inhibited elongation of lettuce (Lactuca sativa L.) radicles. We conducted bioassay-guided purification of G. andersonii bark extract and obtained goniothalamin as the major bioactive compound. Its EC50 values against elongation of lettuce radicles and hypocotyls were 50 and 125 micromol L(-1), respectively. Among the six species tested, timothy was the most sensitive to goniothalamin. Quantification of this compound in other Goniothalamus species suggested that the plant inhibitory activity of this genus is explainable by goniothalamin, with G. calcareus as an exception.

Carbon sources of natural cyanamide in Vicia villosa subsp. varia.

The ¹³C labels of [¹³C]carbon dioxide and D-[¹³C6]glucose were incorporated into cyanamide (NH2CN) when they were administered to Vicia villosa subsp. varia shoots. In contrast, the administration of sodium [2,3-¹³C2]pyruvate did not affect the relative area of the [M + 1]+ ion of cyanamide in the gas chromatography-mass spectrometry analysis. [2,3-¹³C2]pyruvate was incorporated into organic acids that are part of the citric acid cycle, such as succinate and fumarate, confirming that the shoots absorbed and metabolised it. These observations demonstrated that the carbon atom of cyanamide is derived from any of the carbohydrates that are present upstream of pyruvate in the metabolic pathway.

1,2-Dehydrolactarolide A, a new plant growth regulatory lactarane sesquiterpene from Lactarius vellereus.

A new lactarane sesquiterpene, 1,2-dehydrolactarolide A (1), together with lactarorufin A (2), 3-O-ethyllactarolide A (3) and 3-O-ethyllactarolide B (4), was isolated from a mushroom of the Russulaceae family, Lactarius vellereus. Additionally, two lactarane sesquiterpenes, lactarorufin A (2) and lactarolide A (5) were isolated from L. subpiperatus. 1,2-Dehydrolactarolide A (1) showed promotional activity (152% at 3.6 x 10(1) microM) toward radicle elongation in lettuce seedlings.

Repraesentins D, E and F, new plant growth promoters from Lactarius repraesentaneus.

Three new plant growth regulatory sesquiterpenes were isolated from the Lactarius repraesentaneus fungus. Their structures were elucidated to be lactarane sesquiterpenes, namely repraesentins D (1) and E (2), and a protoilludane-related sesquiterpene, namely repraesentin F (3). Repraesentin E (2) showed the strongest promotion activity, 164% at 3.6 microM, of the three compounds toward the radicle elongation of lettuce seedlings.

Quantification of cyanamide contents in herbaceous plants.

Cyanamide (NH2CN) is found in nature, although it has long been recognized as an industrial product. Distribution of cyanamide in the plant kingdom was investigated using a direct quantitative determination method to detect and measure cyanamide by stable isotope dilution gas chromatography-mass spectrometry (the SID-GC-MS method). The SID-GC-MS method proved to be a robust way to quantify cyanamide contents in the extracts of 101 species of herbaceous plants. The average recovery of cyanamide from all plants tested was 55.6+/-20.3%. Vicia villosa and V. cracca contained cyanamide at 369-498 microg/gFW and 3,460-3,579 microg/gFW respectively, while the other 99 species contained no detectable cyanamide (<1 microg/gFW). This result suggests that distribution of cyanamide in the plant kingdom is limited and uneven.