ANAC042 Regulates the Biosynthesis of Conserved- and Lineage-Specific Phytoalexins in Arabidopsis

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 (<i>Arabidopsis thaliana</i>) NAC family TF ANAC042 (a.k.a. JUNGBRUNNEN1 or JUB1) regulates the synthesis of camalexin, a Trp-derived phytoalexin specifically produced by several <i>Brassicaceae</i> species, whereas its homolog in soybean (<i>Glycine max</i>) 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 <i>anac042-1</i> 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 <i>ANAC042</i> in the <i>anac042-1</i> 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 <i>Nicotiana benthamiana</i> found that the ANAC042 protein directly binds and activates the promoters of <i>CYP71B15</i>, <i>CYP71A12</i>, and <i>PAL1</i> 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.