Jasmonic acid/ethylene signaling coordinates hydroxycinnamic acid amides biosynthesis through ORA59 transcription factor

Abstract

Hydroxycinnamic acid amides (HCAAs) are a class of antimicrobial metabolites involved in plant defense against necrotrophic pathogens, including Alternaria brassicicola and Botrytis cinerea. The agmatine coumaryl transferase (AtACT) is the key enzyme that catalyzes the last reaction in the biosynthesis of HCAAs, including p-coumaroylagmatine (CouAgm) and feruloylagmatine in Arabidopsis thaliana. However, the regulatory mechanism of AtACT gene expression is currently unknown. Yeast one-hybrid screening using the AtACT promoter as bait isolated the key positive regulator ORA59 that is involved in jasmonic acid/ethylene (JA/ET)-mediated plant defense responses. AtACT gene expression and HCAAs biosynthesis were synergistically induced by a combination of JA and ET. In the AtACT promoter, two GCC-boxes function equivalently for trans-activation by ORA59 in Arabidopsis protoplasts, and mutation of either GCC-box abolished AtACT mRNA accumulation in transgenic plants. Site-directed mutation analysis demonstrated that the specific Leu residue at position 228 of the ORA59 EDLL motif mainly contributed to its transcriptional activity on AtACT gene expression. Importantly, MEDIATOR25 (MED25) and ORA59 homodimer are also required for ORA59-dependent activation of the AtACT gene. These results suggest that ORA59 and two functionally equivalent GCC-boxes form the regulatory module together with MED25 that enables AtACT gene expression and HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways.

Jasmonate-regulated ERF109-MYB51-MYC3 ternary complexes control indolic glucosinolates biosynthesis

“…To identify more factors binding to the CYP83B1 promoter, a yeast one-hybrid (Y1H) assay was performed using the various CYP83B1 promoter fragments ( Supplemental Fig. S2 ) as bait to screen a JA-treated Arabidopsis cDNA library (Li et al, 2018). Screening of the library with the fragment I (−19 bp to −214 bp) of the CYP83B1 promoter gives positive colonies with yielding clones i.e.…”

“…The promoter fragments of CYP83B1proI (−214 to −1), CYP83B1proII (−430 to −193), CYP83B1proIII (−648 to −410) and CYP83B1proIV (−889 to −628) were PCR-amplified from genomic DNA of A. thalinana digested with NotI and SmaI and fused to a TATA box-His3 gene in plasmid pHIS3NX, respectively. Through homologous recombination, the His3 gene constructs were integrated in the yeast (strain Y187) genome (Zhou et al, 2015; Li et al, 2018). After transformation of the JA-treated Arabidopsis cDNA libraries in the different CYP83B1pro yeast strains, cells were grown on synthetic dextrose minimal medium lacking Leu and His (SD/-LH) supplemented with increasing 3 amino-1,2,4-triazole (3-AT; Sigma) concentrations ranging from 0 to 15 mM, respectively.…”

“…To produce His-tagged proteins, ERF109 and MYB51 were PCR amplified and cloned in pASK-IBA45plus. These constructs were transformed into Escherichia coli strain BL21 (DE3) pLysS for protein extraction according to the protocol as previously described (Zhou et al, 2017; Li et al, 2018). EMSAs were performed using biotin-labeled double-stranded probes (CYP83B1 promoter fragment I and its mutants) and the Light Shift Chemiluminescent EMSA Kit according to the manufacturer’s instructions (Thermo Fisher).…”

“…The plasmid pRT101-ERF109-HA and pRT101-MYB51-HA (5 μg) were transformed into Arabidopsis cell suspension protoplasts. After 18 h incubation at 28°C under dark condition, protoplasts were harvested and washed with PBS buffer (1% formaldehyde, pH 7.4) to crosslink the proteins to the DNA at 4°C (Li et al, 2018). HA antibody (Roche, Mannheim, Germany) was used for immunoprecipitation of chromatin-bound proteins.…”

“…The coding region of CYP83B1 gene and the reference gene UBQ10 promoter were used as a negative control. ChIP assays were performed as previously described (Zhou et al, 2017; Li et al, 2018). Total RNA extraction and reverse transcription (Revert Aid first-strand cDNA synthesis kit; Fermentas) were performed according to the manufacturer’s instructions and qRT-PCR was performed as previously described (Zhou et al, 2017; Li et al, 2018).…”