Ryosuke Sasaki

Metabolic Pathways Involved in Cold Acclimation Identified by Integrated Analysis of Metabolites and Transcripts Regulated by DREB1A and DREB2A

DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold- and dehydration-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, the expression of many genes encoding starch-degrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically; consequently, many kinds of monosaccharides, disaccharides, trisaccharides, and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.

Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode of plant odor reception and defense

Significance Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. To date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. We found that tomato plants absorbed the airborne green leaf alcohol (Z)-3-hexenol emitted by neighboring conspecific plants under attack by herbivores and subsequently converted the alcohol to a glycoside. The glycoside suppressed growth and survival rates of cutworms. The accumulation of glycoside in the receiver plants explained the defense acquired via “smelling” their neighbors. This study showed that the processing of a volatile compound is a mechanism of volatile reception in tomato plants. Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. However, to date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. In this study, we found that undamaged tomato plants exposed to volatiles emitted by conspecifics infested with common cutworms (exposed plants) became more defensive against the larvae than those exposed to volatiles from uninfested conspecifics (control plants) in a constant airflow system under laboratory conditions. Comprehensive metabolite analyses showed that only the amount of (Z)-3-hexenylvicianoside (HexVic) was higher in exposed than control plants. This compound negatively affected the performance of common cutworms when added to an artificial diet. The aglycon of HexVic, (Z)-3-hexenol, was obtained from neighboring infested plants via the air. The amount of jasmonates (JAs) was not higher in exposed plants, and HexVic biosynthesis was independent of JA signaling. The use of (Z)-3-hexenol from neighboring damaged conspecifics for HexVic biosynthesis in exposed plants was also observed in an experimental field, indicating that (Z)-3-hexenol intake occurred even under fluctuating environmental conditions. Specific use of airborne (Z)-3-hexenol to form HexVic in undamaged tomato plants reveals a previously unidentified mechanism of plant defense.

Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis

Leaf oil bodies are the source of a novel phytoalexin produced in response to fungal infection and senescence. Oil bodies are intracellular structures present in the seed and leaf cells of many land plants. Seed oil bodies are known to function as storage compartments for lipids. However, the physiological function of leaf oil bodies is unknown. Here, we show that leaf oil bodies function as subcellular factories for the production of a stable phytoalexin in response to fungal infection and senescence. Proteomic analysis of oil bodies prepared from Arabidopsis (Arabidopsis thaliana) leaves identified caleosin (CLO3) and α-dioxygenase (α-DOX1). Both CLO3 and α-DOX1 were localized on the surface of oil bodies. Infection with the pathogenic fungus Colletotrichum higginsianum promoted the formation of CLO3- and α-DOX1-positive oil bodies in perilesional areas surrounding the site of infection. α-DOX1 catalyzes the reaction from α-linolenic acid (a major fatty acid component of oil bodies) to an unstable compound, 2-hydroperoxy-octadecatrienoic acid (2-HPOT). Intriguingly, a combination of α-DOX1 and CLO3 produced a stable compound, 2-hydroxy-octadecatrienoic acid (2-HOT), from α-linolenic acid. This suggests that the colocalization of α-DOX1 and CLO3 on oil bodies might prevent the degradation of unstable 2-HPOT by efficiently converting 2-HPOT into the stable compound 2-HOT. We found that 2-HOT had antifungal activity against members of the genus Colletotrichum and that infection with C. higginsianum induced 2-HOT production. These results defined 2-HOT as an Arabidopsis phytoalexin. This study provides, to our knowledge, the first evidence that leaf oil bodies produce a phytoalexin under a pathological condition, which suggests a new mechanism of plant defense.

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana.

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi-enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non-exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter-mediated pathway and the vesicle trafficking-mediated pathway. No molecules involved in the vesicle trafficking-mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan-colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis-sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome-like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9-mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.

Suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila.

Camptothecin is a monoterpenoid indole alkaloid that exhibits anti-tumor activity. In Ophiorrhiza pumila, production of camptothecin and its related alkaloids was high in the hairy roots, but not in the cell suspension culture derived from hairy roots. To identify the intermediates in camptothecin biosynthesis, expression of genes encoding tryptophan decarboxylase (TDC) and secologanin synthase (SLS), the two enzymes catalyzing the early steps in camptothecin biosynthesis, were suppressed in the hairy roots of O. pumila by RNA interference (RNAi), and metabolite changes were investigated. In most TDC- and SLS-suppressed lines, accumulation of camptothecin and related alkaloids, strictosidine, strictosamide, pumiloside, and deoxypumiloside was reduced. The accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS. Some hairy root-specific chromatographic peaks detected by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) exhibited positive or negative correlation with TDC expression, suggesting their possible involvement in camptothecin biosynthesis.

Steroidal glycoalkaloid profiling and structures of glycoalkaloids in wild tomato fruit

Steroidal glycoalkaloids (SGAs) constitute one of the main groups of secondary metabolites in tomato fruit. However, the detailed composition of SGAs other than α-tomatine, dehydrotomatine and esculeoside A, remains unclear. Comparative SGA profiling was performed in eight tomato accessions, including wild tomato species by HPLC-Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FTICR/MS). On the basis of molecular formulae obtained from accurate m/z and fragmentation patterns by multistage MS/ MS (MS(n)), 123 glycoalkaloids in total were screened. Detailed MS(n) analysis showed that the observed structural diversity was derived from various chemical modifications, such as glycosylation, acetylation, hydroxylation and isomerization. Total SGA content in each tomato accession was in the range of 121-1986 nmol/gfr.wt. Furthermore, the compositional variety of SGA structures was distinctive in some tomato accessions. While most tomato accessions were basically categorized as α-tomatine-rich or esculeoside A-rich group, other specific SGAs also accumulated at high levels in wild tomato. Here, five such SGAs were isolated and their structures were determined by NMR spectroscopic analysis, indicating three of them were presumably synthesized during α-tomatine metabolism.

Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis

Production of plant lysine-derived alkaloids originates with the convergent evolution of lysine decarboxylase. Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer’s disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata. We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.

Molecular Components of Arabidopsis Intact Vacuoles Clarified with Metabolomic and Proteomic Analyses

We analyzed the metabolites and proteins contained in pure intact vacuoles isolated from Arabidopsis suspension-cultured cells using capillary electrophoresis-mass spectrometry (CE-MS), Fourier transform-ion cyclotron resonance (FT-ICR)-MS and liquid chromatography (LC)-MS. We identified 21 amino acids and five organic acids as major primary metabolites in the vacuoles with CE-MS. Further, we identified small amounts of 27 substances including well-known vacuolar molecules, but also some unexpected substances (e.g. organic phosphate compounds). Non-target analysis of the vacuolar sample with FT-ICR-MS suggested that there are 1,106 m/z peaks that could predict the 5,090 molecular formulae, and we have annotated 34 compounds in these peaks using the KNapSAck database. By conducting proteomic analysis of vacuolar sap, we found 186 proteins in the same vacuole samples. Since the vacuole is known as a major degradative compartment, many of these were hydrolases, but we also found various oxidoreductases and transferases. The relationships between the proteins and metabolites in the vacuole are discussed.

Metabolic variation in the pulps of two durian cultivars: Unraveling the metabolites that contribute to the flavor

Durian (Durio zibethinus M.) is a major economic fruit crop in Thailand. In this study, two popular cultivars, namely Chanee and Mon Thong, were collected from three orchards located in eastern Thailand. The pulp metabolome, including 157 annotated metabolites, was explored using capillary electrophoresis-time of flight/mass spectrometry (CE-TOF/MS). Cultivars and harvest years had more impact on metabolite profile separation than cultivation areas. We identified cultivar-dependent metabolite markers related to durian fruit quality traits, such as nutritional value (pyridoxamine), odor (cysteine, leucine), and ripening process (aminocyclopropane carboxylic acid). Interestingly, durian fruit were found to contain high amounts of γ-glutamylcysteine (810.3 ± 257.5 mg/100 g dry weight) and glutathione (158.1 ± 80.4 mg/100 g dry weight), which act as antioxidants and taste enhancers. This metabolite information could be related to consumer preferences and exploited for durian fruit quality improvement.

WIND1 induces dynamic metabolomic reprogramming during regeneration in Brassica napus

Plants often display a high competence for regeneration under stress conditions. Signals produced in response to various types of stress serve as critical triggers for de novo organogenesis, but the identity of these signaling molecules underlying cellular reprogramming are largely unknown. We previously identified an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION1 (WIND1), as a key regulator involved in wound-induced cellular reprogramming in Arabidopsis. In this study, we found that activation of Arabidopsis WIND1 (AtWIND1) in hypocotyl explants of Brassica napus (B. napus) enhances callus formation and subsequent organ regeneration. Gene expression analyses revealed that AtWIND1 enhances expression of B. napus homologs of ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN (ESR1/DRN), which is a direct target of WIND1 in Arabidopsis. Further, time-course hormonal analyses showed that an altered balance of endogenous auxin/cytokinin exists in AtWIND1-activated B. napus explants. Our mass spectrometry analyses, in addition, uncovered dynamic metabolomic reprogramming in AtWIND1-activated explants, including accumulation of several compounds, e.g. proline, gamma aminobutyric acid (GABA), and putrescine, that have historically been utilized as additives to enhance plant cell reprogramming in tissue culture. Our findings thus provide new insights into how WIND1 functions to promote cell reprogramming.