Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis    

Abstract

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventyfive compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapatederived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.The emergence of metabolomics has for good reasons attracted enormous attention in recent years (Fiehn, 2002;Sumner et al., 2003;Bino et al., 2004). Comprehensive detection of small molecules in a biological system has huge potential as a tool for functional genomics and systems biology. Also, metabolome analysis is expected to contribute significantly to economically important endeavors such as the discovery of bioactive compounds or improving food quality (Dixon et al., 2006;Ryan and Robards, 2006). Currently, however, there is no single analytical approach in sight that would cover the chemical diversity of metabolomes (Dunn and Ellis, 2005), and, perhaps even more problematic, most of the metabolites in any higher eukaryote are as yet unknown (Fernie et al., 2004). Plants in particular synthesize myriad compounds. Plasticity and diversity of metabolism are hallmarks of plant biology, yet we are far from understanding the complex networks of plant metabolism (Last et al., 2007). An essential first step toward a better comprehension of regulation and dynamics is the large-scale discovery of metabolites in Arabidopsis (Arabidopsis thaliana) and other model plants or crops and the cataloging of all of the metabolites that are synthesized in a system under investigation (Last et al., 2007). A large fraction of the thousands of unknowns in any given plant species are secondary metabolites. Fifteen percent to 20% of the genes in Arabidopsis, for instance, are pre...

Auxin and Ethylene Induce Flavonol Accumulation through Distinct Transcriptional Networks

“…DPBA fluoresces with distinct spectral properties in complex with kaempferol and quercetin (Neu, 1956;Sheahan and Rechnitz, 1992;Peer et al, 2001), with Q-DPBA having a longer wavelength fluorescence emission than K-DPBA. In Arabidopsis seedlings, DPBA fluorescence is largely due to binding to glycosylated flavonols, as aglycone flavonols are of low abundance or nondetectable in these tissues, as determined by HPLC analyses (Burbulis et al, 1996;Bö ttcher et al, 2008;Yonekura-Sakakibara et al, 2008). We optimized laser scanning confocal microscope (LSCM) settings to spectrally separate the fluorescence of K-DPBA and Q-DPBA (which reflect the fluorescence of the glycosylated forms of kaempferol and quercetin, respectively) by capturing a window of the emission spectrum for each compound where they do not overlap.…”

“…In addition to the synthesis of the core aglycone flavonols outlined in this figure, flavonols can be extensively glycosylated to regulate both their structure and function (Winkel-Shirley, 2001). In young Arabidopsis seedlings, aglycone flavonols are of low abundance or nondetectable, as determined by HPLC analyses (Burbulis et al, 1996;Bö ttcher et al, 2008;Yonekura-Sakakibara et al, 2008).…”

The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network ofArabidopsis thaliana

“…Product ions were detected using the following parameter settings: collision RF 150/400 Vpp (timing 50/50); transfer time, 70 ms; prepulse storage, 5 ms; pulser frequency, 10 kHz; spectra rate, 1.5 Hz. For CID of in-source fragment ions (pseudo-MS 3 ), in-source CID energy was increased from 0 to 100 V. API QSTAR Pulsar: Acquisition of MS 2 and pseudo-MS 3 spectra was performed as described by Bö ttcher et al (2008). Hyphenation of UPLC with the QSTAR was achieved via a TurboIonSpray ion source (nebulizer gas [nitrogen], 2.0 liters min 21 ; dry gas [nitrogen], 6.5 liters min 21 , 4008C; curtain gas [nitrogen], 2.2 liters min 21 , ion spray voltage +5000 V).…”

An approach to peak detection in GC-MS chromatograms and application of KNApSAcK database in prediction of candidate metabolites

“…Using this function, a list of candidate metabolites is feasibly constructed in any species including model plants, because metabolites reported to closely related species as well as a target species are simultaneously retrieved by molecular weight in ionization mode (m/z). Several researchers utilized this function to examine metabolites such as in tomato fruit (Iijima et al 2008;Mintz-Oron et al 2008), Arabidopsis thaliana (Oikawa et al 2006;Nakamura et al 2007;Yonekura-Sakakibara et al 2007;Ohta et al 2007;Böttcher et al 2008), strawberry (Hanhineva 2008) and bacterium Escherichia coli (Takahashi et al 2008 …”