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. 2007 Nov 13;104(46):17909-15.
doi: 10.1073/pnas.0708697104. Epub 2007 Oct 30.

Different Mechanisms for Phytoalexin Induction by Pathogen and Wound Signals in Medicago Truncatula

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Free PMC article

Different Mechanisms for Phytoalexin Induction by Pathogen and Wound Signals in Medicago Truncatula

Marina Naoumkina et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Cell suspensions of the model legume Medicago truncatula accumulated the isoflavonoid phytoalexin medicarpin in response to yeast elicitor or methyl jasmonate (MJ), accompanied by decreased levels of isoflavone glycosides in MJ-treated cells. DNA microarray analysis revealed rapid, massive induction of early (iso)flavonoid pathway gene transcripts in response to yeast elicitor, but not MJ, and differential induction by the two elicitors of sets of genes encoding transcription factors, ABC transporters, and beta-glucosidases. In contrast, both elicitors induced genes encoding enzymes for conversion of the isoflavone formononetin to medicarpin. Four MJ-induced beta-glucosidases were expressed as recombinant enzymes in yeast, and three were active with isoflavone glucosides. The most highly induced beta-glucosidase was nuclear localized and preferred flavones to isoflavones. The results indicate that the genetic and biochemical mechanisms underlying accumulation of medicarpin differ depending on the nature of the stimulus and suggest a role for MJ as a signal for rapid hydrolysis of preformed, conjugated intermediates for antimicrobial biosynthesis during wound responses.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Changes in medicarpin pathway transcripts and metabolites in M. truncatula suspension cells. Green/red color-coded heat maps represent relative transcript levels of different gene family members determined with Affymetrix arrays (red, up-regulated; green, down-regulated). Data represent ratios of expression at 2 and 24 h between treatment and control. Yellow bars show metabolite levels in YE-treated cells, and blue bars show MJ-treated cells (fold-change followed by times after elicitation during which increases or decreases occur). Abbreviations for enzyme names are given in SI Fig. 6. G, glucoside; GG, diglucoside; GM, malonyl glucoside; GT, glucosyltransferase; MT, malonyltransferase.
Fig. 2.
Fig. 2.
Summary of global transcript changes in M. truncatula cell cultures exposed to YE or MJ, or in corresponding unelected controls (C). (A) Heat maps showing induction kinetics of 258 YE-induced genes and 293 MJ-induced genes as revealed by oligo array analysis. (B) Heat maps showing induction kinetics of 2,466 YE-induced genes and 3,078 MJ-induced genes as revealed by Affymetrix array analysis. (C and D) Venn diagrams showing the numbers of genes induced by YE or MJ, or genes induced by both treatments, for oligo array (C) and Affymetrix (D) analyses.
Fig. 3.
Fig. 3.
Expression of β-glucosidases in M. truncatula cell cultures. (A) Induction of β-glucosidases by MJ as revealed by oligo array analysis. C, control. (B) Glucosidase transcript levels in YE- and MJ-elicited cultures determined by Affymetrix arrays. Values represent fold changes compared with control cultures. (C and D) HPLC chromatograms showing product formation after incubation of recombinant glucosidases with flavonoid (C) and isoflavonoid (D) substrates. s = substrate (glycoside) and p = product (corresponding aglycone). G1 and G2 were assayed with purified enzyme, G3 was assayed with extract from cell medium, and G4 was assayed with cell lysates (see SI Text). Controls for G3 and G4 were from cells expressing empty vector. The basal rates of hydrolysis were presumably the result of activity of endogenous Escherichia coli hydrolases.
Fig. 4.
Fig. 4.
Subcellular localization of Medicago β-glucosidases. (A–J) Confocal images show localization of free EGFP (A–C), or EGFP fused to G4 (D and E), G1 (F–I), or to G1 lacking 108 aa from the N terminus (J). Transient expression of EGFP constructs was obtained by particle bombardment of tobacco leaves (A, D, and H), M. truncatula leaves (C and F), or MJ-treated M. truncatula cell suspension cultures (G), or by agro-infiltration of tobacco leaves (B, E, I, and J). A, D, and E are projected stacks of Z-series, and B, E, G, and I are a single median of one confocal plane. C and F also show autofluorescence of cholorplasts. H is a superimposed transmitted light image onto the fluorescent image. (K) A ribbon model of the structure of G1, indicating the N-terminal extension and putative nuclear localization signal (in red).
Fig. 5.
Fig. 5.
Model showing the different biochemical and genetic mechanisms for the induction of medicarpin by pathogen and wound signals. In the absence of elicitation, the cells gradually accumulate constitutive isoflavone glucosides and malonyl glucosides in the vacuole (green arrows). After exposure to the pathogen mimic YE, the pterocarpan phytoalexin medicarpin is synthesized de novo from l-phenylalanine and malonyl CoA (primary metabolism) (red arrows). Wounding induces MJ accumulation, which acts as a signal for transcriptional activation of isoflavone-specific β-glucosidases that localize to the vacuole and/or cytosol, where they convert conjugates to free isoflavones. The glycosides or aglycones may exit the vacuole via MJ-induced transporters (ABC transporters are shown, although the few studied to date operate in the opposite direction). This process may be initiated by removal of malonyl groups from the sugar unit of the isoflavone conjugates via a specific malonylesterase (ME). MJ induces the downstream enzymes of medicarpin biosynthesis (purple arrows), resulting in medicarpin production at the expense of preformed intermediates.

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