Synthesis and biological activity of photostable and persistent abscisic acid analogs.

The plant hormone abscisic acid (ABA) plays a critical role in various environmental stress responses and has long been expected to be used in agriculture. However, the practical use of ABA has been limited, mainly because of its photoinstability and rapid biodegradation. We previously developed photostable ABA agonists, BP2A and Me 1',4'-trans-diol BP2A, in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. This finding validated our structure-based approach in designing photostable agonists and provided a basis for developing a more potent or long-lasting ABA agonist. In this study, we synthesized novel BP2A analogs in which the cyclohexenone ring was modified to avoid catabolism by the ABA metabolic enzyme, ABA 8'-hydroxylase. All synthesized analogs showed higher photostability than BP2A under sunlight. In an Arabidopsis seed germination assay, (+)-compounds 5 and 6 with a tetralone ring displayed significantly stronger ABA agonist activity than (+)-BP2A. In contrast, in the in vitro phosphatase assays, both compounds showed comparable or weaker ABA receptor (PYL1) agonistic activity than (+)-BP2A, suggesting that the stronger ABA-like activity of (+)-5 and (+)-6 may arise from their metabolic stability in vivo. This study provides data relevant to designing photostable and persistent ABA agonists.

1-Octen-3-ol is formed from its primeveroside after mechanical wounding of soybean leaves.

KEY MESSAGE: Hydrolysis of 1-octen-3-yl ß-primeveroside implemented by a system with high structure-specificity is accountable for the rapid formation of 1-octen-3-ol from soybean leaves after mechanical wounding. 1-Octen-3-ol is a volatile compound ubiquitous in fungi; however, a subset of plant species also has the ability to form 1-octen-3-ol. Owing to its volatile nature, it has been anticipated that 1-octen-3-ol is associated with the effort of the emitter to control the behavior of the surrounding organisms; however, its ecological significance and the enzymes involved in its biosynthesis have not been fully elucidated, particularly in plants. We previously found that soybean (Glycine max) seeds contain 1-octen-3-yl ß-primeveroside (pri). To elucidate the physiological significance and the biosynthesis of 1-octen-3-ol in plants, changes in the amount of 1-octen-3-yl pri during development of soybean plants was examined. A high 1-octen-3-yl pri level was found in young developing green organs, such as young leaves and sepals. Treatment of soybean leaves with methyl jasmonates resulted in a significant increase in the amount of 1-octen-3-yl pri; suggesting its involvement in defense responses. Although 1-octen-3-ol was below the detection limit in intact soybean leaves, mechanical damage to the leaves caused rapid hydrolysis of almost all 1-octen-3-yl pri to liberate volatile 1-octen-3-ol. Under the same conditions, the other glycosides, including isoflavone glycoside and linalool diglycoside, were hardly hydrolyzed. Therefore, the enzyme system to liberate aglycone from glycosides in soybean leaves should have strict substrate specificity. 1-Octen-3-yl pri might function as a storage form of volatile 1-octen-3-ol for immediate response against stresses accompanying tissue wounding.

Rhizotaxis Modulation in Arabidopsis Is Induced by Diffusible Compounds Produced during the Cocultivation of Arabidopsis and the Endophytic Fungus Serendipita indica.

Rhizotaxis is established under changing environmental conditions via periodic priming of lateral root (LR) initiation at the root tips and adaptive LR formation along the primary root (PR). In contrast to the adaptable LR formation in response to nutrient availability, there is little information on root development during interactions with beneficial microbes. The Arabidopsis root system is characteristically modified upon colonization by the root endophytic fungus Serendipita indica, accompanied by a marked stimulation of LR formation and the inhibition of PR growth. This root system modification has been attributed to endophyte-derived indole-3-acetic acid (IAA). However, it has yet to be clearly explained how fungal IAA affects the intrinsic LR formation process. In this study, we show that diffusible compounds (chemical signals) other than IAA are present in the coculture medium of Arabidopsis and S. indica and induce auxin-responsive DR5::GUS expression in specific sections within the pericycle layer. The DR5::GUS expression was independent of polar auxin transport and the major IAA biosynthetic pathways, implicating unidentified mechanisms responsible for the auxin response and LR formation. Detailed metabolite analysis revealed the presence of multiple compounds that induce local auxin responses and LR formation. We found that benzoic acid (BA) cooperatively acted with exogenous IAA to generate a local auxin response in the pericycle layer, suggesting that BA is one of the chemical signals involved in adaptable LR formation. Identification and characterization of the chemical signals will contribute to a greater understanding of the molecular mechanisms underlying adaptable root development and to unconventional technologies for sustainable agriculture.

Design of potent ABA receptor antagonists based on a conformational restriction approach.

The physiological functions of the plant hormone abscisic acid (ABA) are triggered by interactions between PYR/PYL/RCAR receptors (PYLs) and group-A protein phosphatases 2C (PP2Cs). PYL agonists/antagonists capable of inducing/disrupting these interactions would be valuable in investigating the regulatory mechanisms of ABA signaling. Previously, we developed (+)-PAO4, a high-affinity PYL antagonist, by conformationally restricting the S-hexyl chain of our first reported PYL antagonist, 3'-hexylsulfanyl-ABA. Although (+)-PAO4 shows a greater binding affinity for Arabidopsis PYL5 compared with 3'-hexylsulfanyl-ABA, it is not able to completely block the ABA responses both in vitro and in vivo. Therefore, we designed novel conformationally restricted PYL antagonists in which the O-butyl chain of (+)-PAO4 was replaced with a pentyl (PAC4), a pentyne (PAT3) or a pentadiyne (PATT1) chain. (+)-PAT3 and (+)-PATT1 suppressed the ABA-induced inhibition of Arabidopsis seed germination more strongly than (+)-PAO4, but contrary to expectations, the affinity of each compound for PYL5 was almost the same as that of (+)-PAO4. Subsequent biochemical analyses revealed that unlike (+)-PAO4, (+)-PAT3 and (+)-PATT1 completely abolished ABA-induced PYL-PP2C interactions without partial agonistic activities. The superior PYL antagonist functions of (+)-PAT3 and (+)-PATT1 over (+)-PAO4 may explain their potent antagonistic activities against exogenous ABA in vivo. Furthermore, (+)-PAT3 and (+)-PATT1 also suppressed ABA responses in rice, indicating that both compounds are useful chemical tools for ABA-signaling studies, not only in dicots but also in monocots.

Overexpression of geraniol synthase induces heat stress susceptibility in Nicotiana tabacum.

MAIN CONCLUSION: Transgenic tobacco plants overexpressing the monoterpene alcohol geraniol synthase exhibit hypersensitivity to thermal stress, possibly due to suppressed sugar metabolism and transcriptional regulation of genes involved in thermal stress tolerance. Monoterpene alcohols function in plant survival strategies, but they may cause self-toxicity to plants due to their hydrophobic and highly reactive properties. To explore the role of these compounds in plant stress responses, we assessed transgenic tobacco plants overexpressing the monoterpene alcohol geraniol synthase (GES plants). Growth, morphology and photosynthetic efficiency of GES plants were not significantly different from those of control plants (wild-type and GUS-transformed plants). While GES plants' direct defenses against herbivores or pathogens were similar to those of control plants, their indirect defense (i.e., attracting herbivore enemy Nesidiocoris tenuis) was stronger compared to that of control plants. However, GES plants were susceptible to cold stress and even more susceptible to extreme heat stress (50 °C), as shown by decreased levels of sugar metabolites, invertase activity and its products (Glc and Fru), and leaf starch granules. Moreover, GES plants showed decreased transcription levels of the WRKY33 transcription factor gene and an aquaporin gene (PIP2). The results of this study show that GES plants exhibit enhanced indirect defense ability against herbivores, but conversely, GES plants exhibit hypersensitivity to heat stress due to suppressed sugar metabolism and gene regulation for thermal stress tolerance.

Identification of a Hexenal Reductase That Modulates the Composition of Green Leaf Volatiles.

Green leaf volatiles (GLVs), including six-carbon (C6) aldehydes, alcohols, and esters, are formed when plant tissues are damaged. GLVs play roles in direct plant defense at wound sites, indirect plant defense via the attraction of herbivore predators, and plant-plant communication. GLV components provoke distinctive responses in their target recipients; therefore, the control of GLV composition is important for plants to appropriately manage stress responses. The reduction of C6-aldehydes into C6-alcohols is a key step in the control of GLV composition and also is important to avoid a toxic buildup of C6-aldehydes. However, the molecular mechanisms behind C6-aldehyde reduction remain poorly understood. In this study, we purified an Arabidopsis (Arabidopsis thaliana) NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980, named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR). CHR T-DNA knockout mutant plants displayed a normal growth phenotype; however, we observed significant suppression of C6-alcohol production following partial mechanical wounding or herbivore infestation. Our data also showed that the parasitic wasp Cotesia vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants compared with GLVs emitted from chr plants, which corresponded with reduced C6-alcohol levels in the mutant. Moreover, chr plants were more susceptible to exogenous high-dose exposure to (Z)-3-hexenal, as indicated by their markedly lowered photosystem II activity. Our study shows that reductases play significant roles in changing GLV composition and, thus, are important in avoiding toxicity from volatile carbonyls and in the attraction of herbivore predators.

Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis.

Strigolactones (SLs) are a class of plant hormones which regulate shoot branching and function as host recognition signals for symbionts and parasites in the rhizosphere. However, steps in SL biosynthesis after carlactone (CL) formation remain elusive. This study elucidated the common and diverse functions of MAX1 homologs which catalyze CL oxidation. We have reported previously that ArabidopsisMAX1 converts CL to carlactonoic acid (CLA), whereas a rice MAX1 homolog has been shown to catalyze the conversion of CL to 4-deoxyorobanchol (4DO). To determine which reaction is conserved in the plant kingdom, we investigated the enzymatic function of MAX1 homologs in Arabidopsis, rice, maize, tomato, poplar and Selaginella moellendorffii. The conversion of CL to CLA was found to be a common reaction catalyzed by MAX1 homologs, and MAX1s can be classified into three types: A1-type, converting CL to CLA; A2-type, converting CL to 4DO via CLA; and A3-type, converting CL to CLA and 4DO to orobanchol. CLA was detected in root exudates from poplar and Selaginella, but not ubiquitously in other plants examined in this study, suggesting its role as a species-specific signal in the rhizosphere. This study provides new insights into the roles of MAX1 in endogenous and rhizosphere signaling.

The effects of gene disruption of Kre6-like proteins on the phenotype of ß-glucan-producing Aureobasidium pullulans.

Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall ß-(1 → 6)-D-glucan in baker's yeast, Saccharomyces cerevisiae. The Δkre6Δskn1 mutant of S. cerevisiae and other fungi shows severe growth defects due to the failure to synthesize normal cell walls. In this study, two homologs of Kre6, namely, K6LP1 (Kre6-like protein 1) and K6LP2 (Kre6-like protein 2), were identified in Aureobasidium pullulans M-2 by draft genome analysis. The Δk6lp1, Δk6lp2, and Δk6lp1Δk6lp2 mutants were generated in order to confirm the functions of the Kre6-like proteins in A. pullulans M-2. The cell morphologies of Δk6lp1 and Δk6lp1Δk6lp2 appeared to be different from those of wild type and Δk6lp2 in both their yeast and hyphal forms. The productivity of the extracellular polysaccharides, mainly composed of ß-(1 → 3),(1 → 6)-D-glucan (ß-glucan), of the mutants was 5.1-17.3% less than that of wild type, and the degree of branching in the extracellular ß-glucan of mutants was 14.5-16.8% lower than that of wild type. This study showed that the gene disruption of Kre6-like proteins affected the cell morphology, the productivity of extracellular polysaccharides, and the structure of extracellular ß-glucan, but it did not have a definite effect on the cell viability even in Δk6lp1Δk6lp2, unlike in the Δkre6Δskn1 of S. cerevisiae.

'Hidden' Terpenoids in Plants: Their Biosynthesis, Localization and Ecological Roles.

Terpenoids are the largest group of plant specialized (secondary) metabolites. These naturally occurring chemical compounds are highly diverse in chemical structure. Although there have been many excellent studies of terpenoids, most have focused on compounds built solely of isoprene units. Plants, however, also contain many 'atypical' terpenoids, such as glycosylated volatile terpenes and composite-type terpenoids, the latter of which are synthesized by the coupling of isoprene units on aromatic compounds. This mini review describes these 'hidden' terpenoids, providing an overview of their biosynthesis, localization, and biological and ecological activities.

Generation of a novel artificial TrkB agonist, BM17d99, using T7 phage-displayed random peptide libraries.

Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors.

Fluid shear stress stimulates MATE2-K expression via Nrf2 pathway activation.

Accurate prediction of drug-induced renal toxicity is necessary for development of safer drugs for patients. Cellular assay systems that recapitulate physiologically relevant microenvironments have been proposed for correct estimation of drug responses in the human body. However, establishment of such assay systems for accurate prediction of renal toxicity is challenging because of the lack of readily available in vitro assay systems. In this study, we investigated the cellular response to fluid shear stress, which is a characteristic of the environment in the kidney proximal tubules, using microfluidic devices. The global gene expression profiles of human primary proximal tubule cells under the fluidic conditions revealed upregulation of MATE2-K and activation of Nrf2 signaling in response to fluid shear stress. Network and cell biological analysis additionally showed that expression of MATE2-K is regulated by Nrf2 signaling. These results strongly suggest that fluid shear stress is involved in the expression and maintenance of function of tissue-specific drug transporters in the proximal tubule, where the cells are exposed to continuous shear stress by primary urine. Furthermore, the microfluidic culture of human proximal tubules was demonstrated to be a useful system to analyze the regulatory mechanisms of gene expression in physiologically relevant cell conditions.

Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro.

Strigolactones (SLs) stimulate seed germination of root parasitic plants and induce hyphal branching of arbuscular mycorrhizal fungi in the rhizosphere. In addition, they have been classified as a new group of plant hormones essential for shoot branching inhibition. It has been demonstrated thus far that SLs are derived from carotenoid via a biosynthetic precursor carlactone (CL), which is produced by sequential reactions of DWARF27 (D27) enzyme and two carotenoid cleavage dioxygenases CCD7 and CCD8. We previously found an extreme accumulation of CL in the more axillary growth1 (max1) mutant of Arabidopsis, which exhibits increased lateral inflorescences due to SL deficiency, indicating that CL is a probable substrate for MAX1 (CYP711A1), a cytochrome P450 monooxygenase. To elucidate the enzymatic function of MAX1 in SL biosynthesis, we incubated CL with a recombinant MAX1 protein expressed in yeast microsomes. MAX1 catalyzed consecutive oxidations at C-19 of CL to convert the C-19 methyl group into carboxylic acid, 9-desmethyl-9-carboxy-CL [designated as carlactonoic acid (CLA)]. We also identified endogenous CLA and its methyl ester [methyl carlactonoate (MeCLA)] in Arabidopsis plants using LC-MS/MS. Although an exogenous application of either CLA or MeCLA suppressed the growth of lateral inflorescences of the max1 mutant, MeCLA, but not CLA, interacted with Arabidopsis thaliana DWARF14 (AtD14) protein, a putative SL receptor, as shown by differential scanning fluorimetry and hydrolysis activity tests. These results indicate that not only known SLs but also MeCLA are biologically active in inhibiting shoot branching in Arabidopsis.

Volatile Glycosylation in Tea Plants: Sequential Glycosylations for the Biosynthesis of Aroma ß-Primeverosides Are Catalyzed by Two Camellia sinensis Glycosyltransferases.

Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as ß-primeverosides (6-O-ß-D-xylopyranosyl-ß-D-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into ß-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 6'-hydroxy group of the sugar moiety of geranyl ß-D-glucopyranoside, producing geranyl ß-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with ß-primeverosidase encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma ß-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses.

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.

Biosynthesis of floral scent 2-phenylethanol in rose flowers.

Plants emit chemically diverse volatile compounds for attracting pollinators or putting up a chemical defense against herbivores. 2-Phenylethanol (2PE) is one of the abundantly emitted scent compounds in rose flowers. Feeding experiments with l-[(2)H8]phenylalanine into rose flowers and subsequent analysis using gas chromatography-mass spectrometry analysis revealed the hypothetical biosynthetic intermediates to [(2)H8]-2PE, and the biochemical and genetic analyses elucidated the principal pathway to [(2)H8]-2PE. We recently found season-specific 2PE pathway producing [(2)H7]-2PE from l-[(2)H8]phenylalanine. This is a unique example where the dominant pathway to a specific compound changes with the seasons. This review focuses on the biosynthesis of floral volatiles and their regulation to adapt to the changes in the environment.

CYP714B1 and CYP714B2 encode gibberellin 13-oxidases that reduce gibberellin activity in rice.

Bioactive gibberellins (GAs) control many aspects of growth and development in plants. GA(1) has been the most frequently found bioactive GA in various tissues of flowering plants, but the enzymes responsible for GA(1) biosynthesis have not been fully elucidated due to the enzymes catalyzing the 13-hydroxylation step not being identified. Because of the lack of mutants defective in this enzyme, biological significance of GA 13-hydroxylation has been unknown. Here, we report that two cytochrome P450 genes, CYP714B1 and CYP714B2, encode GA 13-oxidase in rice. Transgenic Arabidopsis plants that overexpress CYP714B1 or CYP714B2 show semidwarfism. There was a trend that the levels of 13-OH GAs including GA(1) were increased in these transgenic plants. Functional analysis using yeast or insect cells shows that recombinant CYP714B1 and CYP714B2 proteins can convert GA(12) into GA(53) (13-OH GA(12)) in vitro. Moreover, the levels of 13-OH GAs including GA(1) were decreased, whereas those of 13-H GAs including GA(4) (which is more active than GA(1)) were increased, in the rice cyp714b1 cyp714b2 double mutant. These results indicate that CYP714B1 and CYP714B2 play a predominant role in GA 13-hydroxylation in rice. The double mutant plants appear phenotypically normal until heading, but show elongated uppermost internode at the heading stage. Moreover, CYP714B1 and CYP714B2 expression was up-regulated by exogenous application of bioactive GAs. Our results suggest that GA 13-oxidases play a role in fine-tuning plant growth by decreasing GA bioactivity in rice and that they also participate in GA homeostasis.

The selectivity of 6-nor-ABA and 7'-nor-ABA for abscisic acid receptor subtypes.

Abscisic acid (ABA), a plant hormone, is involved in many plant development processes and environmental stress responses that are regulated by a Pyrabactin Resistant 1 (PYR)/Pyrabactin Resistant-Like (PYL)/Regulatory Component of ABA Receptor (RCAR) receptor protein-mediated signal transduction pathway. In Arabidopsis thaliana, PYL proteins constitute a 14-member family comprising two distinct subclasses: dimeric receptors (PYR1 and PYL1-PYL3) and monomeric receptors (PYL4-PYL13). The individual contributions of PYL subclasses/subtypes with specific physiological actions are still poorly understood; consequently, the development of PYL subclass/subtype-selective agonists should be useful to reveal the different functions of these receptors. In this study, we focused on the ABA analogs 6-nor-ABA and 7'-nor-ABA, which were expected to function as monomeric receptor-selective agonists on the basis of crystal structures of PYL-ABA complexes and sequence alignments of PYL subtypes. In a protein phosphatase 2C (PP2C) assay, the agonist activities of both analogs were lower than those of ABA toward all tested PYL proteins, regardless of subclass/subtype. Nevertheless, we found that 6-nor-ABA acts as a selective agonist at the physiological level: it induced stomatal closure but did not inhibit seed germination and root growth. On the basis of observed inhibitory activity against PP2C among different PYL subtypes, this biological effect of 6-nor-ABA may be attributed to the activity of that agonist on PYL5 and/or PYL6.

Conformationally restricted 3'-modified ABA analogs for controlling ABA receptors.

The physiological functions of abscisic acid (ABA) are regulated by a signal transduction pathway involving cytosolic ABA receptors, which include 14 PYR/PYL/RCAR (PYL) proteins in Arabidopsis. The development of a PYL antagonist could be a valuable tool to improve our understanding of the roles of ABA. We previously developed 3'-hexylsulfanyl-ABA (AS6), whose S-hexyl chain blocks protein phosphatase 2C (PP2C) binding by steric hindrance. This finding not only validated our structure-based approach to the design of a PYL antagonist, but also provided a basis for the development of a more potent or subclass/subtype selective PYL antagonist. In the present study, we synthesized a conformationally restricted analog of AS6, namely propenyl-ABA with an O-butyl chain (PAO4), to improve the affinity for PYL proteins by reducing the entropic penalty for binding to the receptors. In seed germination assays, (+)-PAO4 was a slightly stronger antagonist than AS6 in Arabidopsis and a significantly stronger antagonist in lettuce. Analysis of the thermodynamic parameters associated with the formation of the Arabidopsis PYL-(+)-PAO4 complex revealed that (+)-PAO4 binds more strongly to PYL5 than AS6 owing to an entropic advantage. In PP2C assays, this enhancement effect was observed only for the monomeric PYL subclass containing PYL5, suggesting that (+)-PAO4 is more effective than AS6 in physiological events involving monomeric PYL proteins as ABA receptors.

Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin.

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton ß-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding ß-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-ß-amyrin (a possible biosynthetic intermediate between ß-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-ß-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of ß-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.

The source of "fairy rings": 2-azahypoxanthine and its metabolite found in a novel purine metabolic pathway in plants.

Rings or arcs of fungus-stimulated plant growth occur worldwide; these are commonly referred to as "fairy rings". In 2010, we discovered 2-azahypoxanthine (AHX), a compound responsible for the fairy-ring phenomenon caused by fungus; AHX stimulated the growth of all the plants tested. Herein, we reveal the isolation and structure determination of a common metabolite of AHX in plants, 2-aza-8-oxohypoxanthine (AOH). AHX is chemically synthesized from 5-aminoimidazole-4-carboxamide (AICA), and AHX can be converted into AOH by xanthine oxidase. AICA is one of the members of the purine metabolic pathway in animals, plants, and microorganisms. However, further metabolism of AICA remains elusive. Based on these results and facts, we hypothesized that plants themselves produce AHX and AOH through a pathway similar to the chemical synthesis. Herein, we demonstrate the existence of endogenous AHX and AOH and a novel purine pathway to produce them in plants.