Abstract
APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato (Solanum lycopersicum) and potato (Solanum tuberosum), nicotine in tobacco (Nicotiana tabacum), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (Catharanthus roseus). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the C. roseus AP2/ERF regulatory circuit, which consists of ORCA3, ORCA4, and ORCA5 ORCA3 and ORCA5 activate ORCA4 by directly binding to a GC-rich motif in the ORCA4 promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates ORCA3 In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco NICOTINE2 (NIC2) ERF cluster. Moreover, overexpression of ORCA5 in tobacco and of NIC2 ERF189 in C. roseus hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.
Highlights
Plants produce a vast array of bioactive specialized metabolites in response to various biotic and abiotic stresses
Phylogenetic analysis of group IX ERFs from tomato, tobacco, potato and C. roseus showed that ORCAs are grouped together with NIC2 and GAME9 ERFs, which are involved in nicotine and steroidal glycoalkaloids (SGA) biosynthesis in tobacco and tomato, respectively (Shoji et al, 2010; Cardenas et al, 2016) (Supplemental Figure S1)
To determine the effects of ET alone or in combination with methyl jasmonate (MeJA) on ORCA gene expression, C. roseus seedlings were treated with MeJA, ACC, or both for 2h, and transcript accumulation were measured by reverse-transcription quantitative PCR (RT-qPCR)
Summary
Plants produce a vast array of bioactive specialized metabolites in response to various biotic and abiotic stresses. The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family transcription factors (TFs) have emerged as key regulators of specialized metabolite biosynthesis, including nicotine in tobacco (Nicotiana tabacum) (Shoji et al, 2010; De Boer et al, 2011), terpenoid indole alkaloids (TIAs) in Madagascar periwinkle (Catharanthus roseus) (van der Fits and Memelink, 2000; Paul et al, 2017) and Ophiorrhiza pumila (Udomsom et al, 2016), artemisinin in Artemisia annua (Yu et al, 2012; Lu et al, 2013), and steroidal glycoalkaloids (SGA) in tomato (Solanum lycopersicum) and potato (S. tuberosum) (Cardenas et al, 2016; Thagun et al., 2016; Nakayasu et al, 2018). ORCA5 overexpression in tobacco hairy roots upregulated nicotine biosynthetic genes and nicotine accumulation, and reciprocal overexpression of NIC2 ERF189 in C. roseus hairy roots induced the TIA biosynthetic genes, suggesting that the ORCAs and NIC2 ERFs are functionally equivalent and are likely interchangeable
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