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

ABSTRACT: 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 Gβ 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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE140294 | GEO | 2020/10/02

REPOSITORIES: GEO

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Project description:Purpose: Using RNA-seq and differential expression analysis, we examined the NAE-type and organ-specific genetic pathways involved in transducing ear;y signals into downstream physiological changes involved in Arabidopsis seedling growth. Methods: Intact seedlings and dissected cotyledon and root mRNA profiles of 3-day-old Arabidopsis seedlings treated with DMSO, 40 µM NAE 18:2 or 80 µM NAE 18:3 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: Using RNA-seq and differential expression analysis, we identified early (1 h) transcriptional changes induced by the exogenous treatment of NAE 18:2 and NAE 18:3 in cotyledons, roots and intact seedlings. These two treatments led to a significant enrichment in ABA-response and chitin-response genes in organs where the treatments led to changes in development. In Arabidopsis seedlings, NAE 18:2 treatment led to the repression of genes involved in cell wall biogenesis and organization in roots and seedlings. In addition, cotyledons, roots, and seedlings treated with NAE 18:3 also showed a decrease in transcripts that encode proteins involved in growth processes. NAE 18:3 also led to changes in the abundance of transcripts involved in the modulation of chlorophyll biosynthesis and catabolism in cotyledons. Overall, NAE 18:2 and NAE 18:3 treatment led to lipid-type and organ-specific gene expression changes that include overlapping and non-overlapping gene sets. These data will provide future, rich opportunities to examine the genetic pathways involved in transducing early signals into downstream physiological changes in seedling growth. Conclusions: A detailed transcriptional analyses provided insight into the early organ-specific molecular responses to NAE 18:2 and NAE 18:3. Using this experimental and computational approach, we gained an unbiased view of the NAE 18:2- and NAE 18:3-modulated transcriptome changes that are activated and lead to downstream changes in seedling development. NAE 18:2 and NAE 18:3 induced the expression of several genes also induced by ABA or chitin treatment. The overlap with ABA-response genes corroborates previously work; however, this is the first time chitin-response genes have been identified as part of the NAE-modulation of seedling growth. Overall, the bioinformatic analyses presented here supports the hypothesis that NAE 18:2 and NAE 18:3 elicit organ-specific and signal- specific molecular changes that precede developmental changes in Arabidopsis seedlings. Further, these data provide novel insights into the genetic programs that are modulated by NAE 18:2 and NAE 18:3 at the organ-specific level in Arabidopsis seedlings, and will facilitate future studies of the corresponding signaling networks.

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

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