Multi-Omics Analysis Reveals That SlERF.D6 Synergistically Regulates SGAs and Fruit Development

Abstract

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules that contribute to the pathogen defense in tomato but are toxic and considered to be antinutritional compounds to humans. APETALA2/Ethylene Responsive Factor (AP2/ERF) family transcription factors (TFs) play an indispensable role in various biological processes, such as plant growth and development, fruit ripening, biotic and abiotic stresses responses, and SGA biosynthesis. In this study, we identified 176 AP2/ERF genes that were domesticated or improved SlAP2/ERF in the tomato variome (Solanum lycopersicum) within either domestication or improvement sweeps, respectively. According to the RNA-sequencing data, 93 of the ERF genes with high transcriptional level (Transcripts Per Million, TPM > 1) belong to six clusters. Weighted gene co-expression network analysis (WGCNA) and metabolite-based genome-wide association study (mGWAS) analyses revealed that the expression level of the Solyc04g071770 (SlERF.D6) gene in the cluster six gradually increased as the fruit matured. Transient transformation verified that the overexpression of SlERF.D6 significantly promoted fruit ripening and regulated the expression of multiple genes in the SGA synthesis pathway, thereby affecting the SGA content of the fruit. Virus-induced gene silencing (VIGS) showed that the silencing of SlERF.D6 delayed fruit ripening and influenced the content of SGAs. Our data provide new insights into AP2/ERF TFs in tomato, offer a candidate TF for fruit development and steroidal glycoalkaloids, and provide new resources for tomato breeding and improvement.

SlERF.H6 mediates the orchestration of ethylene and gibberellin signaling that suppresses bitter‐SGA biosynthesis in tomato

“…GAME genes responsible for SGA biosynthesis and regulation were also found near two major signals on chromosomes 1 and 7, including GAME1, GAME2, GAME6, GAME11, GAME17, GAME18, and GAME9. Phylogenetic analysis showed that SlERF.H6 belongs to the DREB (dehydration-responsive element binding) subfamily of ERFs, which is different from the reported ERF subfamily that regulates the biosynthesis of distinct alkaloid classes (Shoji & Yuan, 2021;Guo et al, 2022;Fig. 1b).…”

“…Phylogenetic analysis showed that SlERF.H6 belongs to the DREB (dehydration‐responsive element binding) subfamily of ERFs, which is different from the reported ERF subfamily that regulates the biosynthesis of distinct alkaloid classes (Shoji & Yuan, 2021; Guo et al ., 2022; Fig. 1b).…”

“…Previous studies had identified SlGAME9/JRE4, SlMYC2, and SlbHLH114 as positive regulators of the SGA biosynthesis pathway (Cardenas et al ., 2016; Li et al ., 2020), whereas the negative regulation was relatively less well‐studied. A recent study identified that SlERF.D6 negatively regulates the biosynthesis of tomatidine via the direct repression of GAME12 (Guo et al ., 2022). Our study revealed that SlERF.H6 represses bitter‐SGA accumulation by inhibiting the expression of multiple GAME genes, including GAME6 , GAME11 , GAME25 , and GAME1 .…”

“…The GAMEs and SlGA2ox12 promoters were amplified and inserted into the upstream of the luciferase (LUC) sequence to yield the reporter vectors. The cDNA of SlERF.H6 was amplified and inserted into the pEAQ vector as the effector (Guo et al ., 2022). The mixture of A. tumefaciens strain GV3101 harboring effectors and reporters was infiltrated into 5‐wk‐old N. benthamiana leaves.…”

The SlDOG1 Affect Biosynthesis of Steroidal Glycoalkaloids by Regulating GAME Expression in Tomato

“…Furthermore, SlGAME9 co-binds with the SlMYC2 transcription factor to also regulate SGA biosynthesis genes [ 13 ]. In addition, SlERF.D6 can not only promote fruit ripening as AP2/ERF family transcription factors but also regulate the expression of genes in several SGAs synthesis pathways [ 37 ]. In this study, we identified a SlDOG1-like protein using GWAS and mGWAS.…”