Elsevier

Phytochemistry

Volume 59, Issue 6, March 2002, Pages 611-625
Phytochemistry

Chemical defenses of crucifers: elicitation and metabolism of phytoalexins and indole-3-acetonitrile in brown mustard and turnip

https://doi.org/10.1016/S0031-9422(02)00026-2Get rights and content

Abstract

The metabolism of the cruciferous phytoalexins brassinin and cyclobrassinin, and the related compounds indole-3-carboxaldehyde, glucobrassicin, and indole-3-acetaldoxime was investigated in various plant tissues of Brassica juncea and B. rapa. Metabolic studies with brassinin showed that stems of B. juncea metabolized radiolabeled brassinin to indole-3-acetic acid, via indole-3-carboxaldehyde, a detoxification pathway similar to that followed by the “blackleg” fungus (Phoma lingam/Leptosphaeria maculans). In addition, it was established that tetradeuterated brassinin was incorporated into the phytoalexin brassilexin in B. juncea and B. rapa. On the other hand, the tetradeuterated indole glucosinolate glucobrassicin was not incorporated into brassinin, although the chemical structures of brassinins and indole glucosinolates suggest an interconnected biogenesis. Importantly, tetradeuterated indole-3-acetaldoxime was an efficient precursor of phytoalexins brassinin, brassilexin, and spirobrassinin. Elicitation experiments in tissues of Brassica juncea and B. rapa showed that indole-3-acetonitrile was an inducible metabolite produced in leaves and stems of B. juncea but not in B. rapa. Indole-3-acetonitrile displayed antifungal activity similar to that of brassilexin, was metabolized by the blackleg fungus at slower rates than brassinin, cyclobrassinin, or brassilexin, and appeared to be involved in defense responses of B. juncea.

The metabolism of brassinin, indole-3-acetaldoxime, glucobrassicin, and related compounds in Brassica rapa and B. juncea was investigated.

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Introduction

Pathogen attack affects plant development and may have a strong negative impact on the quality and production of crops. Plants fight pathogens with an enormous and complex arsenal of defense mechanisms. A significant aspect of these defense mechanisms involves biosynthesis of secondary metabolites, which may be either constitutive such as phytoanticipins (VanEtten et al., 1994) or biosynthesized de novo, i.e. phytoalexins (Smith, 1996, Brooks and Watson, 1985). The important role of phytoalexins and phytoanticipins in plant defense responses is becoming better understood with the development of new transgenic and mutant organisms. Perhaps one of the most convincing arguments in favor of phytoalexins is the genetic engineering of the phytoalexin stilbene in tobacco plants which became more resistant to fungal infection than the wild-type plants (Hain et al., 1993). On the other hand, a phytoalexin-deficient mutant of Arabidopsis thaliana showed significantly higher susceptibility to the fungus Alternaria brassicicola than the wild-type parental plants (Thomma et al., 1999). Consequently, a large number of biological and chemical studies are directed to secondary metabolism of plants of economic importance. For example, metabolites from the plant family Cruciferae, which comprises a large number of economically important oilseed crops and vegetables, are being widely investigated (Bennett and Wallsgrove, 1994). Besides their economic importance, crucifers are also interesting plant model-systems, containing the first and only example to date of a completely sequenced plant genome (The Arabidopsis Genome Initiative, 2000).

Chemical characterization of secondary metabolites from crucifers has unraveled a remarkable array of phytoalexins (e.g. Scheme 1, Scheme 2, Scheme 5) (Pedras et al., 2000), as well as a major group of secondary metabolites known as glucosinolates (e.g. 5), (Fahey et al., 2001) both of which contain sulfur and nitrogen. In fact, the unique structure of the phytoalexin brassinin (Scheme 1, Scheme 2, Scheme 5) resembles brassicin (5, R=H), the aglycone portion of indole glucosinolate 5 (i.e. glucobrassicin). This structural connection becomes more transparent considering that methoxy derivatives of brassinin (e.g. Scheme 1, Scheme 2, Scheme 5, R=OMe, R1=H and Scheme 1, Scheme 2, Scheme 5, R=H and R1=OMe) and glucobrassicin (e.g. 5, R=OMe, R1=H, R2=β-d-glucosyl and 5, R=H, R1=OMe and R2=β-d-glucosyl) are naturally occurring within the same plant species and that their concentration levels increase simultaneously in plants subjected to stress (Monde et al., 1991b). Furthermore, unambiguous biosynthetic studies have demonstrated that (S)-tryptophan is the precursor of both brassinin (Scheme 1, Scheme 2, Scheme 5) and glucobrassicin (5). Most importantly, the C-2 of [2-13C]-tryptophan was incorporated into the thiocarbonyl carbon of brassinin (Scheme 1, Scheme 2, Scheme 5), demonstrating that potential intermediates must contain a 2-carbon unit at position C-3 of the indole ring (Monde et al., 1994). In this connection a number of suggestions and attempts to establish a biogenetic relationship between indole glucosinolates such as glucobrassicin (5) and cruciferous phytoalexins have been reported (Hanley et al., 1990). Furthermore, a number of studies have demonstrated that (S)-tryptophan is converted to 5 via indole-3-acetaldoxime (6) (Halkier and Du, 1997). Paradoxically, because glucosinolates are considered an undesirable group of metabolites in brassicas, a large number of oilseed breeding programs are directed at obtaining plants containing low amounts of glucosinolates. If, however, indole glucosinolates such as glucobrassicin (5) are precursors of cruciferous phytoalexins (brassinin is a precursor of 24), from a plant defense perspective lowering glucosinolate contents in cruciferous crops may pose a substantial ecological risk. Nonetheless, despite a number of biosynthetic studies in crucifers, it is not ascertained whether or not this biogenetic relationship exists (Pedras et al., 2000). Thus we became interested in establishing the possible biogenetic relationship between indole glucosinolate 5 and brassinin (Pedras et al., 2001).

Moreover, the resistance of brown mustard (Brassica juncea) to the devastating blackleg disease [fungal pathogen Leptosphaeria maculans (Desm.) Ces. et Not. asexual stage Phoma lingam (Tode ex Fr.) Desm] was reported to correlate with the accumulation of the phytoalexin brassilexin (3) (Rouxel et al., 1991). However, since P. lingam can rapidly metabolize and detoxify brassilexin (3) (Pedras and Okanga, 1999), it is suspected that additional antifungal metabolites are produced in brown mustard. We have now discovered that indole-3-acetonitrile (7) is an inducible metabolite likely involved in defense responses of brown mustard (B. juncea). Furthermore, we have also established the biosynthetic relationships among several secondary metabolites of Brassica species, namely indole-3-acetaldoxime (6) and phytoalexins Scheme 1, Scheme 2, Scheme 5, as well as the metabolic degradation of [2-14C]-brassinin in plant tissues. Here we report for the first time the syntheses of tetradeuterated aldoxime (6a), tetradeuterated glucobrassicin (5a), and radiolabeled brassinin (Scheme 1, Scheme 2, Scheme 5), as well as results of the metabolic studies with these compounds and propose a new role for indole-3-acetonitrile (7).

Section snippets

Phytoalexin elicitation in different plant tissues: cell suspension cultures, stems, leaves and roots

Previous experiments with leaves of Brassica carinata elicited with P. lingam demonstrated that both [4,5,6,7-D4]-brassinin (1a) and [4,5,6,7-D4]-cyclobrassinin (2a) were incorporated into brassilexin (3a) (Pedras et al., 1998). These results, however, indicated that experiments involving leaf uptake of solutions containing these phytoalexins were difficult to carry out due to the toxicity of the substrates to leaf petioles. Subsequently, we searched for a tissue to conduct biosynthetic studies

Conclusions

Recent examples of phytoalexin detoxification by crucifer fungal pathogens suggest that enzymes involved in such processes may result from an intergeneric-genetic exchange between brassicas and their pathogens (Pedras et al., 2000). We have now demonstrated that brown mustard converts the phytoalexin brassinin (Scheme 1, Scheme 2, Scheme 5) into nontoxic products via a pathway similar to that occurring in virulent P. lingam. Therefore, we suggest that this pathogen may have acquired a more

General

All chemicals were purchased from Sigma-Aldrich Canada Ltd., Oakville, ON unless otherwise stated. All solvents were analytical grade with the exception that HPLC grade solvents were used for HPLC analysis. [2-14C]-Indole (sp. activity 50 mCi/mmol, 1.1×1011 dpm/mmol) was purchased from Moravek Biochemicals, Inc., Brea, CA. Phytoalexins were identified by comparison of retention times and UV-spectra (photodiode array detector) with authentic samples.

Analytical techniques were carried out as

Acknowledgements

We would like to thank G. Séguin-Swartz (AAFC) for providing isolates of P. lingam, and A. brassicae and J. Shyluk (NRC-PBI) for generously providing cell suspension cultures of B. juncea. Support for the authors’ work was obtained from: the Natural Sciences and Engineering Research Council of Canada (Individual Research grant and NRC-NSERC Research Partnership grant to M.S.C.P.) and the University of Saskatchewan. We would like to acknowledge the technical assistance of Ken Thoms, Department

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