Metabolomics reveals comprehensive reprogramming involving two independent metabolic responses of Arabidopsis to UV-B light.

Because of ever-increasing environmental deterioration it is likely that the influx of UV-B radiation (280-320 nm) will increase as a result of the depletion of stratospheric ozone. Given this fact it is essential that we better understand both the rapid and the adaptive responses of plants to UV-B stress. Here, we compare the metabolic responses of wild-type Arabidopsis with that of mutants impaired in flavonoid (transparent testa 4, tt4; transparent testa 5, tt5) or sinapoyl-malate (sinapoylglucose accumulator 1, sng1) biosynthesis, exposed to a short 24-h or a longer 96-h exposure to this photo-oxidative stress. In control experiments we subjected the genotypes to long-day conditions as well as to 24- and 96-h treatments of continuous light. Following these treatments we evaluated the dynamic response of metabolites including flavonoids, sinapoyl-malate precursors and ascorbate, which are well known to play a role in cellular protection from UV-B stress, as well as a broader range of primary metabolites, in an attempt to more fully comprehend the metabolic shift following the cellular perception of this stress. Our data reveals that short-term responses occur only at the level of primary metabolites, suggesting that these effectively prime the cell to facilitate the later production of UV-B-absorbing secondary metabolites. The combined results of these studies together with transcript profiles using samples irradiated by 24-h UV-B light are discussed in the context of current models concerning the metabolic response of plants to the stress imposed by excessive UV-B irradiation.

Biosynthetic gene-based secondary metabolite screening: a new diterpene, methyl phomopsenonate, from the fungus Phomopsis amygdali.

The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2. Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate. The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal genomic data.

MS/MS spectral tag-based annotation of non-targeted profile of plant secondary metabolites.

The MS/MS spectral tag (MS2T) library-based peak annotation procedure was developed for informative non-targeted metabolic profiling analysis using LC-MS. An MS2T library of Arabidopsis metabolites was created from a set of MS/MS spectra acquired using the automatic data acquisition function of the mass spectrometer. By using this library, we obtained structural information for the detected peaks in the metabolic profile data without performing additional MS/MS analysis; this was achieved by searching for the corresponding MS2T accession in the library. In the case of metabolic profile data for Arabidopsis tissues containing more than 1000 peaks, approximately 50% of the peaks were tagged by MS2Ts, and 90 peaks were identified or tentatively annotated with metabolite information by searching the metabolite databases and manually interpreting the MS2Ts. A comparison of metabolic profiles among the Arabidopsis tissues revealed that many unknown metabolites accumulated in a tissue-specific manner, some of which were deduced to be unusual Arabidopsis metabolites based on the MS2T data. Candidate genes responsible for these biosyntheses could be predicted by projecting the results to the transcriptome data. The method was also used for metabolic phenotyping of a subset of Ds transposon-inserted lines of Arabidopsis, resulting in clarification of the functions of reported genes involved in glycosylation of flavonoids. Thus, non-targeted metabolic profiling analysis using MS2T annotation methods could prove to be useful for investigating novel functions of secondary metabolites in plants.

Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene-metabolite correlations in Arabidopsis.

To complete the metabolic map for an entire class of compounds, it is essential to identify gene-metabolite correlations of a metabolic pathway. We used liquid chromatography-mass spectrometry (LC-MS) to identify the flavonoids produced by Arabidopsis thaliana wild-type and flavonoid biosynthetic mutant lines. The structures of 15 newly identified and eight known flavonols were deduced by LC-MS profiling of these mutants. Candidate genes presumably involved in the flavonoid pathway were delimited by transcriptome coexpression network analysis using public databases, leading to the detailed analysis of two flavonoid pathway genes, UGT78D3 (At5g17030) and RHM1 (At1g78570). The levels of flavonol 3-O-arabinosides were reduced in ugt78d3 knockdown mutants, suggesting that UGT78D3 is a flavonol arabinosyltransferase. Recombinant UGT78D3 protein could convert quercetin to quercetin 3-O-arabinoside. The strict substrate specificity of UGT78D3 for flavonol aglycones and UDP-arabinose indicate that UGT78D3 is a flavonol arabinosyltransferase. A comparison of flavonol profile in RHM knockout mutants indicated that RHM1 plays a major role in supplying UDP-rhamnose for flavonol modification. The rate of flavonol 3-O-glycosylation is more affected than those of 7-O-glycosylation by the supply of UDP-rhamnose. The precise identification of flavonoids in conjunction with transcriptomics thus led to the identification of a gene function and a more complete understanding of a plant metabolic network.

Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali.

Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16 alpha-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16 alpha-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16 alpha-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11 alpha,16 alpha,18-triol, which is a possible metabolite of phyllocladan-16 alpha-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.

Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi.

Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H(+)-ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a C(20) precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal C(5) isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (+)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the C(5) isoprene units by an unusual multifunctional enzyme, P. amygdali fusicoccadiene synthase (PaFS), which shows both prenyltransferase and terpene cyclase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the C(5) precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca-2,10 (14)-diene in Escherichia coli cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi ent-kaurene synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene.

Identification of a flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome coexpression analysis and reverse genetics.

Glycosylation plays a major role in the remarkable chemical diversity of flavonoids in plants including Arabidopsis thaliana. The wide diversity encoded by the large family-1 glycosyltransferase (UGT) gene family makes it difficult to determine the biochemical function of each gene solely from its primary sequence. Here we used transcriptome coexpression analysis combined with a reverse genetics approach to identify a gene that is prominent in determining the flavonoid composition of Arabidopsis. Using transcriptome coexpression analysis accessible on the ATTED-II public data base, the expression pattern of a UGT gene, UGT89C1, was found to be highly correlated with known flavonoid biosynthetic genes. No C-7 rhamnosylated flavonols were detected in either of two T-DNA ugt89c1 mutants. This specific metabolite deficiency in the mutants was complemented by stable transformation with the genomic fragment containing intact UGT89C1. Glutathione S-transferasefused recombinant UGT89C1 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucoside-7-O-rhamnoside and recognized 3-O-glycosylated flavonols and UDP-rhamnose as substrates, but not flavonol aglycones, 3-O-glycosylated anthocyanins or other UDP-sugars. These results show that UGT89C1 is a flavonol 7-O-rhamnosyltransferase. The abundance of UGT89C1 transcripts in floral buds was consistent with the flavonoid accumulation of C-7 rhamnosylated flavonols in Arabidopsis organs. Our present study demonstrates that the integration of transcriptome coexpression analysis with a reverse genetic approach is a versatile tool for understanding a multigene family of a metabolic pathway in Arabidopsis.