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

Abstract

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.

Keywords: Arabidopsis thaliana; basal immunity; phytoalexin; pleiotropy; transcription factor.

Figure 1

Phytoalexin metabolite profiles and lignin staining of ANAC042 mutant and gene overexpression lines. (A) ANAC042 expression levels and (B,C) phytoalexin profiles of seedling extracts quantified by matrix-assisted laser desorption ionization high-resolution mass spectrometry (MAL-DI-HRMS) relative to daidzein (internal standard) and qRT-PCR relative to UBIQUITIN2, respectively. Ten-day-old seedlings were elicited with Flg22, and metabolites were measured at 12 h post-elicitation. The significance test was performed by single factor ANOVA, Tukey post hoc test, which is indicated by different letters (p < 0.01). Error bars represent SE (n ≥ 3). For a list of statistical values, see Supplementary Table S1. (D) Lignin staining with phloroglucinol-HCl under 16× magnification. Seven-day-old seedlings were elicited with flg22 and stained 48 h post-elicitation.

Figure 2

Phytoalexin gene expression profiles of ANAC042 mutant and gene overexpression lines. (A) Primary metabolism gene EMB114; (B) Phe pathway phytoalexin genes; (C) Trp pathway phytoalexin genes; and (D) phytoalexin TFs. Gene expression of flg22-treated seedlings was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) relative to ACTIN2. The significance test was performed by single factor ANOVA, Tukey post hoc test, which is indicated by different letters (p < 0.01). Error bars represent SE (n ≥ 3). For a list of statistical values, see Supplementary Table S2. EMB1144, chorismate synthase; PAL1, phenylalanine ammonia–lyase 1; CAD5, cinnamyl alcohol dehydrogenase 5; COMT, caffeic acid O–methyltransferase; F5H, ferulate–5–hydroxylase; F6′H, feruloyl–CoA 6′–hydroxylase; CYP79B2, cytochrome P450 79B2; CYP71A12, cytochrome P450 71A12; CYP71A13, cytochrome P450 71A13; CYP71B15, cytochrome P450 71B15; FOX1, 2-hydroxy-2-(1H-indol-3-yl)acetonitrile oxidase; CYP82C2, cytochrome P450 82C2.

Figure 3

Characterization of the subcellular localization and protein-DNA interactions (PDIs) of ANAC042 protein. (A) Fluorescence microscopy of p35S::GFP::ANAC042-18-12. Seedlings from anac042-1 were used as negative control. DAPI (6 µg/mL) images indicate nuclear staining. Bars in red represent 5 µm. (B) Y1H analysis of strain YM4271 transformed with ANAC042-Gal4AD, WRKY33-Gal4AD, or MYB15-Gal4AD, and pCYP71B15::HIS3, pCYP71A12::HIS3, or pPAL1::HIS3. SD-Leu plates were used as control for TF transformation; SD-Leu-His as control for TF and promoter transformation; and SD-Leu-His + 10 mM 3-aminotriazole (3AT) as indicators for positive PDIs. pDEST-GADT7 was used as the empty ‘Vector’ control. (C) Luciferase transactivation assay of transiently transformed N. benthamiana leaves with p35S::ANAC042, p35S::WRKY33, or p35S::MYB15, and pCYP71B15::LUC, pCYP71A12::LUC, or pPAL1::LUC. Luciferase activity measurements were performed on 48 h post-infiltrated leave lysates. p62GW was used as the empty ‘Vector’ control. The significance test was performed by single factor ANOVA, Tukey post hoc test, which is indicated by different letters (p < 0.01). Error bars represent SE (n ≥ 3). For a list of statistical values, see Supplementary Table S3. (D) Schematic diagram demonstrating promoter fragments of CYP71B15, CYP71A12, and PAL1 used for yeast one-hybrid and luciferase transactivation assays. N-box elements with either 5′-GCCGT-3′ or 5′-ACGGC-3′ sequences (green boxes).

Figure 4

A schematic diagram of Phe- and Trp-derived phytoalexin biosynthetic genes in Arabidopsis and the transcription factors that regulate their expression. Black arrows () indicate the direction of genes involved in phytoalexin biosynthesis starting at PAL1 (Phe-derived phytoalexins) and CYP79B2 (Trp-derived phytoalexins); Purple arrows () indicate direct regulation of the gene; Green dotted arrows () indicated regulation of the gene by qRT-PCR, but that it remains unknown whether the corresponding genes are regulated directly or indirectly. Gene names: GSTF11, GLUTATHIONE S–TRANSFERASE F11; GGP1, Γ–GLUTAMYL PEPTIDASE 1; C4H, CINNAMIC ACID 4–HYDROXYLASE; 4CL, 4–COUMARATE–COENZYME A LIGASE; CCR, CINNAMOYL–COA REDUCTASE; SGT, SCOPOLETIN–GLUCOSYLTRANSFERASE.