Karl Angelo M. Rodrillo

Phytoalexins are specialized metabolites that are synthesized by plants in response to pathogens. A paradigm in transcription factor (TF) biology is that conserved TFs have dedicated roles across plant lineages in regulating specific branches of specialized metabolism. However, the Arabidopsis (Arabidopsis thaliana) NAC family TF ANAC042 (a.k.a. JUNGBRUNNEN1 or JUB1) regulates the synthesis of camalexin, a Trp-derived phytoalexin specifically produced by several Brassicaceae species, whereas its homolog in soybean (Glycine max) regulates the synthesis of glyceollins, which are Phe-derived phytoalexins specific to soybean. The question addressed by this research is whether ANAC042 broadly regulates phytoalexin biosynthetic pathways in Arabidopsis. Using a novel matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS) method, we found that the Arabidopsis loss-of-function mutant anac042–1 elicited with bacterial flagellin (Flg22) is deficient in lineage-specific Trp- and conserved Phe-derived phytoalexins—namely camalexin and 4-hydroxyindole-3-carbonyl nitrile (4OH-ICN), and pathogen-inducible monolignols and scopoletin, respectively. Overexpressing ANAC042 in the anac042-1 mutant restored or exceeded wildtype amounts of the metabolites. The expression of phytoalexin biosynthetic genes in mutant and overexpression lines mirrored the accumulation of metabolites. Yeast-one hybrid and promoter-reporter assays in Nicotiana benthamiana found that the ANAC042 protein directly binds and activates the promoters of CYP71B15, CYP71A12, and PAL1 genes for the synthesis of camalexin, 4OH-ICN, and pathogen-inducible monolignol/scopoletin, respectively. Our results demonstrate that ANAC042 regulates conserved and lineage-specific phytoalexin pathways in Arabidopsis. The latter suggests that it is an opportunistic TF that has coopted lineage-specific genes into phytoalexin metabolism, thus providing an exception to the current paradigm.

Phytoalexins are specialized metabolites that are synthesized by plants in response to pathogens. A paradigm in transcription factor (TF) biology is that conserved TFs have dedicated roles across plant lineages in regulating specific branches of specialized metabolism. However, the Arabidopsis (Arabidopsis thaliana) NAC family TF ANAC042 (a.k.a. JUNGBRUNNEN1 or JUB1) regulates the synthesis of camalexin, a Trp-derived phytoalexin specifically produced by several Brassicaceae species, whereas its homolog in soybean (Glycine max) regulates the synthesis of glyceollins, which are Phe-derived phytoalexins specific to soybean. The question addressed by this research is whether ANAC042 broadly regulates phytoalexin biosynthetic pathways in Arabidopsis. Using a novel matrix-assisted laser desorption ionization high resolution mass spectrometry (MALDI-HRMS) method we found that the Arabidopsis loss-of-function mutant anac042-1 elicited with bacterial flagellin (Flg22) is deficient in lineage-specific Trp- and conserved Phe-derived phytoalexins - namely camalexin and 4-hydroxyindole-3-carbonyl nitrile (4OH-ICN), and pathogen-inducible monolignols and scopoletin, respectively. Overexpressing ANAC042 in the anac042-1 mutant restored or exceeded wildtype amounts of the metabolites. The expression of phytoalexin biosynthetic genes in mutant and overexpression lines mirrored the accumulation of metabolites. Yeast-one hybrid and promoter-reporter assays in Nicotiana benthamiana found that the ANAC042 protein directly binds and activates the promoters of CYP71B15, CYP71A12, and PAL1 genes for the synthesis of camalexin, 4OH-ICN, and pathogen-inducible monolignol/scopoletin, respectively. Our results demonstrate that ANAC042 regulates conserved and lineage-specific phytoalexin pathways in Arabidopsis. The latter suggests that it is an opportunistic TF that has coopted lineage-specific genes into phytoalexin metabolism, thus providing an exception to the current paradigm.