ADAP is a possible negative regulator of glucosinolate biosynthesis in Arabidopsis thaliana based on clustering and gene expression analyses

Glucosinolates (GSLs) are plant secondary metabolites consisting of sulfur and nitrogen, commonly found in Brassicaceae crops, such as Arabidopsis thaliana. These compounds are known for their roles in plant defense mechanisms against pests and pathogens. 'Guilt-by-association' (GBA) approach predicts genes encoding proteins with similar function tend to share gene expression pattern generated from high throughput sequencing data. Recent studies have successfully identified GSL genes using GBA approach, followed by targeted verification of gene expression and metabolite data. Therefore, a GSL co-expression network was constructed using known GSL genes obtained from our in-house database, SuCComBase. DPClusO was used to identify subnetworks of the GSL co-expression network followed by Fisher's exact test leading to the discovery of a potential gene that encodes the ARIA-interacting double AP2-domain protein (ADAP) transcription factor (TF). Further functional analysis was performed using an effective gene silencing system known as CRES-T. By applying CRES-T, ADAP TF gene was fused to a plant-specific EAR-motif repressor domain (SRDX), which suppresses the expression of ADAP target genes. In this study, ADAP was proposed as a negative regulator in aliphatic GSL biosynthesis due to the over-expression of downstream aliphatic GSL genes (UGT74C1 and IPMI1) in ADAP-SRDX line. The significant over-expression of ADAP gene in the ADAP-SRDX line also suggests the behavior of the TF that negatively affects the expression of UGT74C1 and IPMI1 via a feedback mechanism in A. thaliana.