Biosynthesis and Analysis of 3-Isobutyl-2-methoxypyrazine in Capsicum annuum (Bell Pepper) Plants

Abstract

3-Alkyl-2-methoxypyrazines (MPs) are highly odorous compounds occurring in a wide range of raw vegetables. Due to their high odor activity, they have been of great research interest. The most prominent MPs are 3-isopropyl-2-methoxypyrazine (IPMP), 3-sec-butyl2-methoxypyrazine (sBMP) and 3-isobutyl-2-methoxypyrazine (IBMP). While their occurrence has been widely studied, less is known about their biosynthesis. The identification of Vitis vinifera O-methyltransferases (VvOMTs) catalyzing the O-methylation of 3-alkyl2-hydroxy-pyrazines (HPs) gave an important insight into the last step of MP-biosynthesis. However, precursors and earlier biosynthetic steps are still unknown. Different pathways of MP-biosynthesis have been proposed, but none of them has been confirmed yet.<br /> The main objective of this study was thus to identify precursors of MPs. For this purpose, in vivo feeding experiments were conducted using stable isotope labeled compounds. Feeding experiments were conducted focusing on the IBMP-biosynthesis in bell pepper fruits (Capsicum annuum L.). Volatiles were extracted by headspace solid-phase microextraction (HS-SPME) and analyzed by comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (ToF-MS). Feeding studies revealed an incorporation of L-leucine and α-ketoisocaproic acid (α-KIC) into IBMP. It could be shown that L-serine plays a key role in the biosynthesis of IBMP not only as a C2-building block, but also as nitrogen source. Indeed, the incorporation of L-serine as well as a C1-unit of glycine and glyoxylic acid into IBMP gave evidence for a metabolic interface of the IBMP-biosynthesis and photorespiration.<br /> Besides, the distribution of IBMP within the bell pepper plant was examined. Therefore, a method combining a stable isotope dilution assay (SIDA) with HS-SPME, GC and ToF-MS was developed and validated. IBMP was found in all plant organs. However, unripe bell pepper pericarp turned out to be the main site of IBMP-accumulation. In vivo feeding experiments revealed that IBMP is biosynthesized in all plant parts apart from roots and ripe bell pepper fruits. A long-distance transport of IBMP via the phloem was not detected.