Insights into stereoselective ring formation in canonical strigolactone: Identification of a dirigent domain-containing enzyme catalyzing orobanchol synthesis.

Strigolactones (SLs) are plant apocarotenoids with diverse roles and structures. Canonical SLs, widespread and characterized by structural variations in their tricyclic lactone (ABC-ring), are classified into two types based on C-ring configurations. The steric C-ring configuration emerges during the BC-ring closure, downstream of the biosynthetic intermediate, carlactonoic acid (CLA). Most plants produce either type of canonical SLs stereoselectively, e.g., tomato (Solanum lycopersicum) yields orobanchol with an α-oriented C-ring. The mechanisms driving SL structural diversification are partially understood, with limited insight into functional implications. Furthermore, the exact molecular mechanism for the stereoselective BC-ring closure reaction is yet to be known. We identified an enzyme, the stereoselective BC-ring-forming factor (SRF), from the dirigent protein (DIR) family, specifically the DIR-f subfamily, whose biochemical function had not been characterized, making it a key enzyme in stereoselective canonical SL biosynthesis with the α-oriented C-ring. We first confirm the precise catalytic function of the tomato cytochrome P450 SlCYP722C, previously shown to be involved in orobanchol biosynthesis [T. Wakabayashi et al., Sci. Adv. 5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. We then show that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid, forming orobanchol. Our methodology combines experimental and computational techniques, including SRF structure prediction and conducting molecular dynamics simulations, suggesting a catalytic mechanism based on the conrotatory 4π-electrocyclic reaction for the stereoselective BC-ring formation in orobanchol. This study sheds light on the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.

Mediation analysis unveils a carcinogenic effect of ADH1B rs1229984 through mechanisms other than change in drinking intensity: oesophageal cancer case-control study.

BACKGROUND: Ingested alcohol is predominantly oxidized to acetaldehyde by alcohol dehydrogenase 1B (ADH1B), and acetaldehyde is further oxidized to acetate mainly by aldehyde dehydrogenase 2 (ALDH2). Although alcohol consumption is a convincing risk factor for oesophageal cancer, the role of ADH1B rs1229984 (His48Arg), the single-nucleotide polymorphism associated with slow alcohol metabolism, in oesophageal cancer development is unclear. Because this single-nucleotide polymorphism is associated with both increased risk of oesophageal cancer and drinking intensity, its association with oesophageal cancer might operate either through a direct pathway independently of drinking intensity, via an indirect pathway mediated by drinking intensity, or both. METHODS: To disentangle these different pathways, we applied a mediation analysis to an oesophageal cancer case-control study (600 cases and 865 controls) by defining the ADH1B Arg allele and alcohol consumption as exposure and mediator, respectively, and decomposed the total-effect odds ratio of the ADH1B Arg allele into direct- and indirect-effect odds ratio. RESULTS: The ADH1B Arg allele was associated with oesophageal cancer risk through pathways other than change in drinking intensity (direct-effect odds ratio, 2.03; 95% confidence interval, 1.41-2.92), in addition to the indirect pathway mediated by drinking intensity (indirect-effect odds ratio, 1.27; 95% confidence interval, 1.05-1.53). Further analyses by stratifying genotypes of ALDH2 rs671 (Glu504Lys), the functional single-nucleotide polymorphism that strongly attenuates the enzymatic activity, showed significant direct-effect odds ratio within each stratum. CONCLUSIONS: These results indicate that ADH1B Arg allele contributes to oesophageal cancer risk by slowing alcohol breakdown, in addition to its effect on the amount of alcohol consumed.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Tandem Gene Duplication of Dioxygenases Drives the Structural Diversity of Steroidal Glycoalkaloids in the Tomato Clade.

Cultivated tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid (SGA), which functions as a defense compound to protect against pathogens and herbivores; interestingly, wild species in the tomato clade biosynthesize a variety of SGAs. In cultivated tomato, the metabolic detoxification of α-tomatine during tomato fruit ripening is an important trait that aided in its domestication, and two distinct 2-oxoglutarate-dependent dioxygenases (DOXs), a C-23 hydroxylase of α-tomatine (Sl23DOX) and a C-27 hydroxylase of lycoperoside C (Sl27DOX), are key to this process. There are tandemly duplicated DOX genes on tomato chromosome 1, with high levels of similarity to Sl23DOX. While these DOX genes are rarely expressed in cultivated tomato tissues, the recombinant enzymes of Solyc01g006580 and Solyc01g006610 metabolized α-tomatine to habrochaitoside A and (20R)-20-hydroxytomatine and were therefore named as habrochaitoside A synthase (HAS) and α-tomatine 20-hydroxylase (20DOX), respectively. Furthermore, 20DOX and HAS exist in the genome of wild tomato S. habrochaites accession LA1777, which accumulates habrochaitoside A in its fruits, and their expression patterns were in agreement with the SGA profiles in LA1777. These results indicate that the functional divergence of α-tomatine-metabolizing DOX enzymes results from gene duplication and the neofunctionalization of catalytic activity and gene expression, and this contributes to the structural diversity of SGAs in the tomato clade.

Identification and characterization of sorgomol synthase in sorghum strigolactone biosynthesis.

Strigolactones (SLs), first identified as germination stimulants for root parasitic weeds, act as endogenous phytohormones regulating shoot branching and as root-derived signal molecules mediating symbiotic communications in the rhizosphere. Canonical SLs typically have an ABCD ring system and can be classified into orobanchol- and strigol-type based on the C-ring stereochemistry. Their simplest structures are 4-deoxyorobanchol (4DO) and 5-deoxystrigol (5DS), respectively. Diverse canonical SLs are chemically modified with one or more hydroxy or acetoxy groups introduced into the A- and/or B-ring of these simplest structures, but the biochemical mechanisms behind this structural diversity remain largely unexplored. Sorgomol in sorghum (Sorghum bicolor [L.] Moench) is a strigol-type SL with a hydroxy group at C-9 of 5DS. In this study, we characterized sorgomol synthase. Microsomal fractions prepared from a high-sorgomol-producing cultivar of sorghum, Sudax, were shown to convert 5DS to sorgomol. A comparative transcriptome analysis identified SbCYP728B subfamily as candidate genes encoding sorgomol synthase. Recombinant SbCYP728B35 catalyzed the conversion of 5DS to sorgomol in vitro. Substrate specificity revealed that the C-8bS configuration in the C-ring of 5DS stereoisomers was essential for this reaction. The overexpression of SbCYP728B35 in Lotus japonicus hairy roots, which produce 5DS as an endogenous SL, also resulted in the conversion of 5DS to sorgomol. Furthermore, SbCYP728B35 expression was not detected in nonsorgomol-producing cultivar, Abu70, suggesting that this gene is responsible for sorgomol production in sorghum. Identification of the mechanism modifying parental 5DS of strigol-type SLs provides insights on how plants biosynthesize diverse SLs.

Involvement of α-galactosidase OmAGAL2 in planteose hydrolysis during seed germination of Orobanche minor.

Root parasitic weeds of the Orobanchaceae, such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.), cause serious losses in agriculture worldwide, and efforts have been made to control these parasitic weeds. Understanding the characteristic physiological processes in the life cycle of root parasitic weeds is particularly important to identify specific targets for growth modulators. In our previous study, planteose metabolism was revealed to be activated soon after the perception of strigolactones in germinating seeds of O. minor. Nojirimycin inhibited planteose metabolism and impeded seed germination of O. minor, indicating a possible target for root parasitic weed control. In the present study, we investigated the distribution of planteose in dry seeds of O. minor by matrix-assisted laser desorption/ionization-mass spectrometry imaging. Planteose was detected in tissues surrounding-but not within-the embryo, supporting its suggested role as a storage carbohydrate. Biochemical assays and molecular characterization of an α-galactosidase family member, OmAGAL2, indicated that the enzyme is involved in planteose hydrolysis in the apoplast around the embryo after the perception of strigolactones, to provide the embryo with essential hexoses for germination. These results indicate that OmAGAL2 is a potential molecular target for root parasitic weed control.

Identification of 6-epi-heliolactone as a biosynthetic precursor of avenaol in Avena strigosa.

Strigolactones (SLs), which are known as rhizosphere signaling molecules and plant hormones regulating shoot architecture, are classified into 2 distinct groups, canonical and noncanonical SLs, based on their structures. Avenaol, a noncanonical SL found in the root exudates of black oat (Avena strigosa), has a characteristic bicyclo[4.1.0]heptane skeleton. Elucidating the biosynthetic mechanism of this peculiar structure is a challenge for further understanding of the structural diversification of noncanonical SLs. In this study, a novel noncanonical SL, 6-epi-heliolactone in black oat root exudates was identified. Feeding experiments showed that 6-epi-heliolactone was a biosynthetic intermediate between methyl carlactonoate and avenaol. Inhibitor experiments proposed the involvement of 2-oxoglutarate-dependent dioxygenase in converting 6-epi-heliolactone to avenaol. These results provide new insights into the stereochemistry diversity of noncanonical SLs and a basis to explore the biosynthetic pathway causing avenaol.

Tomato E8 Encodes a C-27 Hydroxylase in Metabolic Detoxification of α-Tomatine during Fruit Ripening.

Tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid that contributes to the plant defense against pathogens and herbivores through its bitter taste and toxicity. It accumulates at high levels in all the plant tissues, especially in leaves and immature green fruits, whereas it decreases during fruit ripening through metabolic conversion to the nontoxic esculeoside A, which accumulates in the mature red fruit. This study aimed to identify the gene encoding a C-27 hydroxylase that is a key enzyme in the metabolic conversion of α-tomatine to esculeoside A. The E8 gene, encoding a 2-oxoglutalate-dependent dioxygenase, is well known as an inducible gene in response to ethylene during fruit ripening. The recombinant E8 was found to catalyze the C-27 hydroxylation of lycoperoside C to produce prosapogenin A and is designated as Sl27DOX. The ripe fruit of E8/Sl27DOX-silenced transgenic tomato plants accumulated lycoperoside C and exhibited decreased esculeoside A levels compared with the wild-type (WT) plants. Furthermore, E8/Sl27DOX deletion in tomato accessions resulted in higher lycoperoside C levels in ripe fruits than in WT plants. Thus, E8/Sl27DOX functions as a C-27 hydroxylase of lycoperoside C in the metabolic detoxification of α-tomatine during tomato fruit ripening, and the efficient detoxification by E8/27DOX may provide an advantage in the domestication of cultivated tomatoes.

Specific methylation of (11R)-carlactonoic acid by an Arabidopsis SABATH methyltransferase.

MAIN CONCLUSION: An Arabidopsis S-adenosyl-L-methionine-dependent methyltransferase belonging to the SABATH family catalyzes the specific carboxymethylation of (11R)-carlactonoic acid. Methyl carlactonoate (MeCLA), found in Arabidopsis (Arabidopsis thaliana) as a non-canonical strigolactone (SL), may be a biosynthetic intermediate of various non-canonical SLs and biologically active as a plant hormone. MeCLA is formed from carlactonoic acid (CLA), but the methyltransferases (MTs) converting CLA to MeCLA remain unclear. Previous studies have demonstrated that the carboxymethylation of acidic plant hormones is catalyzed by the same protein family, the SABATH family (Wang et al. in Evol Bioinform 15:117693431986086. https://doi.org/10.1177/1176934319860864 , 2019). In the present study, we focused on the At4g36470 gene, an Arabidopsis SABATH MT gene co-expressed with the MAX1 gene responsible for CLA formation for biochemical characterization. The recombinant At4g36470 protein expressed in Escherichia coli exhibited exclusive activity against naturally occurring (11R)-CLA among the substrates, including CLA enantiomers and a variety of acidic plant hormones. The apparent Km value for (11R)-CLA was 1.46 µM, which was relatively smaller than that of the other Arabidopsis SABATH MTs responsible for the carboxymethylation of acidic plant hormones. The strict substrate specificity and high affinity of At4g36470 suggested it is an (11R)-CLA MT. We also confirmed the function of the identified gene by reconstructing MeCLA biosynthesis using transient expression in Nicotiana benthamiana. Phylogenetic analysis demonstrated that At4g36470 and its orthologs in non-canonical SL-producing plants cluster together in an exclusive clade, suggesting that the SABATH MTs of this clade may be involved in the carboxymethylation of CLA and the biosynthesis of non-canonical SLs.

Identification of α-Tomatine 23-Hydroxylase Involved in the Detoxification of a Bitter Glycoalkaloid.

Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.

CYP722C from Gossypium arboreum catalyzes the conversion of carlactonoic acid to 5-deoxystrigol.

MAIN CONCLUSION: CYP722C from cotton, a homolog of the enzyme involved in orobanchol synthesis in cowpea and tomato, catalyzes the conversion of carlactonoic acid to 5-deoxystrigol. Strigolactones (SLs) are important phytohormones with roles in the regulation of plant growth and development. These compounds also function as signaling molecules in the rhizosphere by interacting with beneficial arbuscular mycorrhizal fungi and harmful root parasitic plants. Canonical SLs, such as 5-deoxystrigol (5DS), consist of a tricyclic lactone ring (ABC-ring) connected to a methylbutenolide (D-ring). Although it is known that 5DS biosynthesis begins with carlactonoic acid (CLA) derived from ß-carotene, the enzyme that catalyzes the conversion of CLA remains elusive. Recently, we identified cytochrome P450 (CYP) CYP722C as the enzyme that catalyzes direct conversion of CLA to orobanchol in cowpea and tomato (Wakabayashi et al., Sci Adv 5:eaax9067, 2019). Orobanchol has a different C-ring configuration from that of 5DS. The present study aimed to characterize the homologous gene, designated GaCYP722C, from cotton (Gossypium arboreum) to examine whether this gene is involved in 5DS biosynthesis. Expression of GaCYP722C was upregulated under phosphate starvation, which is an SL-producing condition. Recombinant GaCYP722C was expressed in a baculovirus-insect cell expression system and was found to catalyze the conversion of CLA to 5DS but not to 4-deoxyorobanchol. These results strongly suggest that GaCYP722C from cotton is a 5DS synthase and that CYP722C is the crucial CYP subfamily involved in the generation of canonical SLs, irrespective of the different C-ring configurations.

Concise synthesis of heliolactone, a non-canonical strigolactone isolated from sunflower.

Heliolactone is one of the earliest identified non-canonical strigolactones. Its concise synthesis was achieved by employing Knoevenagel-type condensation and semi-reduction of a malonate intermediate as the key steps. This synthesis was performed in a non-stereoselective manner, and thus a racemic and diastereomeric mixture of heliolactone was obtained. The developed synthetic route is fairly concise and straightforward.

Identification of a 3ß-Hydroxysteroid Dehydrogenase/ 3-Ketosteroid Reductase Involved in α-Tomatine Biosynthesis in Tomato.

α-Tomatine and dehydrotomatine are major steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves and flowers of tomato (Solanum lycopersicum), and function as defensive compounds against bacteria, fungi, insects and animals. The aglycone of dehydrotomatine is dehydrotomatidine (5,6-dehydrogenated tomatidine, having the Δ5,6 double bond; the dehydro-type). The aglycone of α-tomatine is tomatidine (having a single bond between C5 and C6; the dihydro-type), which is believed to be derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 reduction and C3 reduction; however, these conversion processes remain uncharacterized. In the present study, we demonstrate that a short-chain alcohol dehydrogenase/reductase designated Sl3ßHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3ßHSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of α-tomatine are accumulated. Biochemical analysis of the recombinant Sl3ßHSD1 protein revealed that Sl3ßHSD1 catalyzes the C3 oxidation of dehydrotomatidine to form tomatid-4-en-3-one and also catalyzes the NADH-dependent C3 reduction of a 3-ketosteroid (tomatid-3-one) to form tomatidine. Furthermore, during co-incubation of Sl3ßHSD1 with SlS5αR1 (steroid 5α-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3ßHSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in α-tomatine content. Furthermore, the constitutive expression of Sl3ßHSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3ßHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in α-tomatine biosynthesis.

A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in Steroidal Glycoalkaloid Biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16ß-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

Evidence for species-dependent biosynthetic pathways for converting carlactone to strigolactones in plants.

Strigolactones (SLs), comprising compounds with diverse but related chemical structures, are determinant signals in elicitation of germination in root parasitic Orobanchaceae and in mycorrhization in plants. Further, SLs are a novel class of plant hormones that regulate root and shoot architecture. Dissecting common and divergent biosynthetic pathways of SLs may provide avenues for modulating their production in planta. Biosynthesis of SLs in various SL-producing plant species was inhibited by fluridone, a phytoene desaturase inhibitor. The plausible biosynthetic precursors of SLs were exogenously applied to plants, and their conversion to canonical and non-canonical SLs was analysed using liquid chromatography-tandem mass spectrometry. The conversion of carlactone (CL) to carlactonoic acid (CLA) was a common reaction in all investigated plants. Sorghum converted CLA to 5-deoxystrigol (5-DS) and sorgomol, and 5-DS to sorgomol. One sorgomol-producing cotton cultivar had the same SL profile as sorghum in the feeding experiments. Another cotton cultivar converted CLA to 5-DS, strigol, and strigyl acetate. Further, 5-DS was converted to strigol and strigyl acetate. Moonseed converted CLA to strigol, but not to 5-DS. The plant did not convert 5-DS to strigol, suggesting that 5-DS is not a precursor of strigol in moonseed. Similarly, 4-deoxyorobanchol was not a precursor of orobanchol in cowpea. Further, sunflower converted CLA to methyl carlactonoate and heliolactone. These results indicated that the biosynthetic pathways of hydroxy SLs do not necessarily involve their respective deoxy SL precursors.

Stereospecific reduction of the butenolide in strigolactones in plants.

Reductive metabolism of strigolactones (SLs) in several plants was investigated. Analysis of aquaculture filtrates of cowpea and sorghum each fed with four stereoisomers of GR24, the most widely used synthetic SL, revealed stereospecific reduction of the double bond at C-3' and C-4' in the butenolide D-ring with preference for an unnatural 2'S configuration. The cowpea metabolite converted from 2'-epi-GR24 and the sorghum metabolite converted from ent-GR24 had the methyl group at C-4' in the trans configuration with the substituent at C-2', different from the cis configuration of the synthetic H2-GR24 reduced with Pd/C catalyst. The plants also reduced the double bond in the D-ring of 5-deoxystrigol isomers with a similar preference. The metabolites and synthetic H2-GR24 stereoisomers were much less active than were the GR24 stereoisomers in inducing seed germination of the root parasitic weeds Striga hermonthica, Orobanche crenata, and O. minor. These results provide additional evidence of the importance of the D-ring for bioactivity of SLs.

Identification of (Z)-3:(E)-2-Hexenal Isomerases Essential to the Production of the Leaf Aldehyde in Plants.

The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.

Essential role of the PSI-LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions.

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m-2 s-1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI-LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.

Novel steroidal saponins from Dioscorea esculenta (Togedokoro).

Fifteen steroidal saponins 1-15, which include 4 furostanol glycosides 1-3 and 15, and 11 spirostanol glycosides 4-14, were isolated from the tubers and leaves of lesser yam (Dioscorea esculenta, Togedokoro). Their structures were identified by nuclear magnetic resonance and liquid chromatography mass spectroscopy. Four steroidal saponins 9, 11, 14, and 15 were found to be novel compounds.

Planteose as a storage carbohydrate required for early stage of germination of Orobanche minor and its metabolism as a possible target for selective control.

Root parasitic weeds in Orobanchaceae cause serious damage to worldwide agriculture. Germination of the parasites requires host-derived germination stimulants, such as strigolactones, as indicators of host roots within reach of the parasite's radicles. This unique germination process was focused on to identify metabolic pathways required for germination, and to design a selective control strategy. A metabolomic analysis of germinating seeds of clover broomrape, Orobanche minor, was conducted to identify its distinctive metabolites. Consequently, a galactosyl-sucrose trisaccharide, planteose (α-d-galactopyranosyl-(1→6)-ß-d-fructofuranosyl-(2→1)-α-d-glucopyranoside), was identified as a metabolite that decreased promptly after reception of the germination stimulant. To investigate the importance of planteose metabolism, the effects of several glycosidase inhibitors were examined, and nojirimycin bisulfite (NJ) was found to alter the sugar metabolism and to selectively inhibit the germination of O. minor. Planteose consumption was similar in NJ-treated seeds and non-treated germinating seeds; however, NJ-treated seeds showed lower consumption of sucrose, a possible intermediate of planteose metabolism, resulting in significantly less glucose and fructose. This inhibitory effect was recovered by adding glucose. These results suggest that planteose is a storage carbohydrate required for early stage of germination of O. minor, and NJ inhibits germination by blocking the supply of essential glucose from planteose and sucrose. Additionally, NJ selectively inhibited radicle elongation of germinated seeds of Orobanchaceae plants (Striga hermonthica and Phtheirospermum japonicum). Thus, NJ will be a promising tool to develop specific herbicides to the parasites, especially broomrapes, and to improve our understanding of the molecular mechanisms of this unique germination.