An intact heterotrimeric G-Protein complex is required for the N-acylethanolamine-induced, transcriptionally-mediated chloroplast response in developing Arabidopsis seedlings

Purpose: Using RNA-seq and differential expression analysis, we examined the NAE 18:3-induced and AGB1-dependent and -independent transcriptional responses involved in transducing early signals into downstream physiological changes involved in Arabidopsis seedling growth. Methods: Seedling mRNA profiles of 4-day-old Arabidopsis seedlings treated with DMSO or 40 µM NAE 18:3 for 1 h or 3 h were generated by deep sequencing, in triplicate, using the Illumina Next-Seq 500 system. The sequence reads that passed quality filters were analyzed using STAR followed by DESeq2. qRT–PCR validation was performed using SYBR Green assays. Results: We show that the activity of N-linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex; specifically, genetic ablation of the Gß dimer or loss of the full set of atypical G? subunits strongly attenuates an NAE 18:3 de-greening of cotyledons in Arabidopsis seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll synthesis gene expression. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. Conclusions: These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems. Overall design: Seedling mRNA profiles of 4-day-old Arabidopsis Col-0 and agb1 seedlings treated with DMSO or 40 µM NAE 18:3 for 1 h or 3 h were generated by deep sequencing, in triplicate, using the Illumina Next-Seq 500 system.