Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) is a leucine-rich repeat receptor-like kinase (LRR-RLK) involved in the acquisition of embryogenic competence and in male sporogenesis. To determine the composition of the SERK1 signaling complex in vivo, we generated plants expressing the SERK1 protein fused to cyan fluorescent protein under SERK1 promoter control. The membrane receptor complex was immunoprecipitated from seedlings, and the coimmunoprecipitating proteins were identified using liquid chromatography/matrix-assisted laser desorption ionization-time of flight/mass spectrometry of the trypsin-released peptides. This approach identified two other LRR-RLKs, the BRASSINOSTEROID-INSENSITIVE1 (BRI1) receptor and its coreceptor, the SERK3 or BRI1-ASSOCIATED KINASE1 protein. In addition, KINASE-ASSOCIATED PROTEIN PHOSPHATASE, CDC48A, and 14-3-3n were found. Finally, the MADS box transcription factor AGAMOUS-LIKE15 and an uncharacterized zinc finger protein, a member of the CONSTANS family, were identified as part of the SERK1 complex. Using blue native gel electrophoresis, we show that SERK1 and SERK3 are part of BRI1-containing multiple protein complexes with relative masses between 300 and 500 kD. The SERK1 mutant allele serk1-1 enhances the phenotype of the weak BRI1 allele bri1-119. Collectively, these results suggest that apart from SERK3, SERK1 is also involved in the brassinolide signaling pathway.

The regioselectivity of phase II conjugation of flavonoids is expected to be of importance for their biological activity. In the present study, the regioselectivity of phase II biotransformation of the model flavonoids luteolin and quercetin by UDP-glucuronosyltransferases was investigated. Identification of the metabolites formed in microsomal incubations with luteolin or quercetin was done using HPLC, LC-MS, and (1)H NMR. The results obtained demonstrate the major sites for glucuronidation to be the 7-, 3-, 3'-, or 4'-hydroxyl moiety. Using these unequivocal identifications, the regioselectivity of the glucuronidation of luteolin and quercetin by microsomal samples from different origin, i.e., rat and human intestine and liver, as well as by various individual human UDP-glucuronosyltransferase isoenzymes was characterized. The results obtained reveal that regioselectivity is dependent on the model flavonoid of interest, glucuronidation of luteolin and quercetin not following the same pattern, depending on the isoenzyme of UDP-glucuronosyltransferases (UGT) involved. Human UGT1A1, UGT1A8, and UGT1A9 were shown to be especially active in conjugation of both flavonoids, whereas UGT1A4 and UGT1A10 and the isoenzymes from the UGTB family, UGT2B7 and UGT2B15, were less efficient. Due to the different regioselectivity and activity displayed by the various UDP-glucuronosyltransferases, regioselectivity and rate of flavonoid conjugation varies with species and organ. Qualitative comparison of the regioselectivities of glucuronidation obtained with human intestine and liver microsomes to those obtained with human UGT isoenzymes indicates that, in human liver, especially UGT1A9 and, in intestine, UGT1A1 and UGT1A8 are involved in glucuronidation of quercetin and luteolin. Taking into account the fact that the anti-oxidant action as well as the pro-oxidant toxicity of these catechol-type flavonoids is especially related to their 3',4'-dihydroxyl moiety, it is of interest to note that the human intestine UGT's appear to be especially effective in conjugating this 3',4' catechol unit. This would imply that upon glucuronidation along the transport across the intestinal border, the flavonoids loose a significant part of these biological activities.

Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.

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