Novel Acidic Sesquiterpenoids Constitute a Dominant Class of Pathogen-Induced Phytoalexins in Maize    

Abstract

Nonvolatile terpenoid phytoalexins occur throughout the plant kingdom, but until recently were not known constituents of chemical defense in maize (Zea mays). We describe a novel family of ubiquitous maize sesquiterpenoid phytoalexins, termed zealexins, which were discovered through characterization of Fusarium graminearum-induced responses. Zealexins accumulate to levels greater than 800 μg g−1 fresh weight in F. graminearum-infected tissue. Their production is also elicited by a wide variety of fungi, Ostrinia nubilalis herbivory, and the synergistic action of jasmonic acid and ethylene. Zealexins exhibit antifungal activity against numerous phytopathogenic fungi at physiologically relevant concentrations. Structural elucidation of four members of this complex family revealed that all are acidic sesquiterpenoids containing a hydrocarbon skeleton that resembles β-macrocarpene. Induced zealexin accumulation is preceded by increased expression of the genes encoding TERPENE SYNTHASE6 (TPS6) and TPS11, which catalyze β-macrocarpene production. Furthermore, zealexin accumulation displays direct positive relationships with the transcript levels of both genes. Microarray analysis of F. graminearum-infected tissue revealed that Tps6/Tps11 were among the most highly up-regulated genes, as was An2, an ent-copalyl diphosphate synthase associated with production of kauralexins. Transcript profiling suggests that zealexins cooccur with a number of antimicrobial proteins, including chitinases and pathogenesis-related proteins. In addition to zealexins, kauralexins and the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-glucose) were produced in fungal-infected tissue. HDMBOA-glucose accumulation occurred in both wild-type and benzoxazine-deficient1 (bx1) mutant lines, indicating that Bx1 gene activity is not required for HDMBOA biosynthesis. Together these results indicate an important cooperative role of terpenoid phytoalexins in maize biochemical defense.

Maize death acids, 9-lipoxygenase–derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators

“…2N). These results indicate that 10-OPEA can function as a broadly active phytoalexin that shares similar antibiotic potencies to maize zealexins and kauralexins (35,36). Because phytoalexins can also harm plant cells (39), we tested the cytotoxic properties of 10-OPEA in comparison with four different maize phytoalexins (zealexin A1/ A3, kauralexin A3/B3).…”

“…S5-S7) concentrations similar to fungal induced levels of terpenoid phytoalexins (35,36). Given that oxylipins can function as phytoalexins, we investigated the potential for 10-OPEA to act as a direct defense (23, 37).…”

“…To validate the microarray results and to compare 10-OPEA activity to other meaningful metabolites, we treated plants with either SA, 12-OPDA, JA, 10-OPEA, DA 0 -4:0, or SLB and compared the expression levels of pathogen-and insect-related genes at 3 h using qRT-PCR. Defense marker genes included ZmOPR2, the maize OPR most strongly up-regulated in response to SLB (40); pathogenesis-related 4b (ZmPR4b) and EF-hand Ca 2+ -binding protein (ZmCCD1), genes involved in the promotion of cell death and pathogen-elicited defense responses (41-43); GST 2 (ZmGST2) and hydrolase (ZmHYD), previously shown to be induced by both insect-and pathogen-related treatments (35,44); a stress-inducible oxidoreductase (ZmOXR); and maize cystatin 9 (ZmCC9), wound inducible protein 1 (ZmWIP1), and serine protease inhibitor (ZmSerPIN), that encode PIs inducible by biotic attack (27,45,46). The defense genes ZmPR4b, ZmOPR2, and ZmCCD1 were strongly induced by 10-OPEA and DA 0 -4:0 treatments with expression levels either statistically equivalent to or higher than those of 12-OPDA, JA, and SA ( Fig.…”

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…To test this hypothesis, we conducted E. coli coexpression assays of ZmCYP71Z16 with ZmCPR2 and ZmTPS11, which form the zealexin precursor b-macrocarpene (Köllner et al, 2008b;Huffaker et al, 2011). This enzyme combination resulted in the formation of zealexin A1 (Supplemental Fig.…”

“…1), are part of a complex repertoire of defense-related specialized metabolites, such as phenylpropanoids, oxylipins, benzoxazinoids, and volatile terpenoids (Schnee et al, 2006;Köllner et al, 2008a;Santiago and Malvar, 2010;Ahmad et al, 2011;Huffaker et al, 2011;Schmelz et al, 2011;Christensen et al, 2015;Richter et al, 2016;Wouters et al, 2016). Inducible zealexin and kauralexin production contributes to defense, both aboveground and belowground, against pathogenic fungi, including Fusarium, Aspergillus, and Colletotrichum spp.…”