Identification of phenylpropanoids in methyl jasmonate treated Brassica rapa leaves using two-dimensional nuclear magnetic resonance spectroscopy

Abstract

Metabolic analysis showed a clear increase in phenylpropanoid levels in Brassica rapa leaves after treatment with methyl jasmonate. A fraction of phenylpropanoids was prepared by Diaion HP-20 and Sephadex LH-20 column chromatography after MeOH-water extraction. Even with these purification steps, isolation of each phenylpropanoid for structure elucidation is not easy due to the low levels in the plants (ca. 0.004%). A mixture was analyzed without further purification using HPLC-electrospray ionization mass spectrometry and NMR spectroscopy. Based on the NMR results including ¹H NMR, J-resolved, correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) spectra, both ¹H and ¹³C resonances of the compounds were obtained. Using these NMR data, five phenylpropanoids conjugated with malate were identified: 5-hydroxyferuloyl-, caffeoyl-, coumaroyl-, feruloyl-, and sinapoyl malate. Of the compounds, 5-hydroxyferuloyl malate is a new phenylpropanoid. In addition to the five constitutive phenylpropanoids bearing trans-configuration, their cis forms, which are believed to be artifacts formed in the course of extraction steps, were also identified in the fraction.

Introduction

Brassica rapa (turnip) is one of the oldest cultivated vegetables. It is consumed in enormous quantities throughout the world due to its nutritional benefits and its usefulness for the production of edible/industrial oils. It was introduced in the Greek and Roman period in Europe and spread to Asia and northern China, where it became a common vegetable before the medieval period in Europe [1].

Brassica plants produce characteristic metabolites, the glucosinolates in order to protect themselves from herbivore attack and pathogens [2]. These glucosinolates are thought to be one of the reasons why Brassica plants are so widespread and can survive in a variety of environmental conditions and stresses [3], [4]. In addition to the glucosinolates, Brassica plants defend themselves by producing volatile compounds including isoprenes, terpenes, and alkanes [3], [4], [5]. The induction of these volatiles is most likely to reduce herbivory [5]. However, the function of these volatile metabolites in the mechanisms of plant defense is still unclear because they have been also reported to attract certain herbivores in some occasions [6].

Plant defense can be induced with several plant hormones such as analogues of jasmonic acid. These jasmonic acids are widely distributed in plants and affect a variety of processes, including fruit ripening, production of viable pollen, root growth, and tendril coiling. The most important function of these compounds is to play a role in plant response to wounding and abiotic stress, and in defense against insects and pathogens [7]. The function of jasmonic acids in plant defense was deduced by relationships between wounding caused by insect herbivores, the formation of jasmonic analogues, and the induction of genes for proteinase inhibitors that deter insect feeding [8]. Wounding in plants induces production of linolenic acid, the precursor of jasmonic acids, from membrane lipids [8]. Among the derivatives of the jasmonic acids, methyl jasmonate, a volatile methyl ester, has been widely used in model experiments in order to investigate the function of jasmonic acids in the plant defense mechanism [9], [10], [11], [12], [13]. Methyl jasmonate is known to induce the production of metabolites including aliphatic alcohols in tobacco and cotton [9], [10], [11] and terpenoids in lima bean and gerbera [12], [13]. Of the metabolites induced by methyl jasmonate, glucosinolates have been paid great attention to due to their large structural variation and relatively large increase after jasmonate treatment when compared to other metabolites. For example, an indolyl glucosinolate, N-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in Arabidopsis while few changes were detected in aliphatic glucosinolates [14]. Similar to the case of Arabidopsis, indolyl glusinolates such as glucobrassicin, 4-hydroxyglucobrassicin, and 4-methoxyglucobrassicin are significantly induced in B. rapa after wounding [3], [15], [16].

Recently we found in our NMR-metabolomic studies that phenylpropanoids (Fig. 1) together with indolyl glucosinolates in B. rapa leaves accumulated after treatment with methyl jasmonate. l-Phenylalanine ammonia lyase (PAL), the entry point enzyme into the phenylpropanoid pathway, and downstream enzymes such as caffeic acid O-methyltransferase, are known to be strongly induced following infection of plants (e.g. tobacco) with pathogenic viruses or exposure to fungal elicitor [17], [18], [19]. Among the phenylpropanoids, chlorogenic acid (3-O-caffeoyl quinic acid) has been extensively investigated for its role in plant defense. It serves as a phytoanticipin in many plant species [20]. Tobacco plants over-expressing PAL produce high levels of chlorogenic acid and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cerospora nicotianae [21]. Wounding during the preparation of fresh-cut lettuce induced the synthesis and accumulation of chlorogenic acid [22]. Chlorogenic acid was also proposed as one of the allelochemicals increasing the resistance of cotton to larvae [23]. For other phenylpropanoids, feruloyl malate coupled with coniferyl alcohol was hypothesized as an intermediate that can be transesterified to polysaccharides linking the cell wall [24]. Despite a variety of investigations for the role of phenylpropanoids in defense in various plant species, there are few reports about phenylpropanoids in Brassica species.

The aim of this study is to identify the individual phenylpropanoids in B. rapa leaves, which highly accumulated after treatment with methyl jasmonate. Generally, several isolation steps should be carried out to obtain pure compound prior to structure elucidation. However, the low level of those phenylpropanoids (less than 0.004%) in B. rapa leaves makes it difficult to isolate individual compounds for structure elucidation. We decided to study the possibility of doing the structure elucidation directly in the extract using NMR with assistance of HPLC-MS. In this study, the mixture of phenylpropanoids obtained by column chromatography from the total extract was analyzed using correlated spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), heteronuclear single quantum coherence (HSQC), and J-resolved spectroscopy. Based on these NMR data, ¹H and ¹³C NMR resonance spectra of a series of phenylpropanoids are completely assigned, allowing their unequivocal identification.