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. 2022 Mar 28;188(4):1966-1978.
doi: 10.1093/plphys/kiac013.

2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana

Monika Chodasiewicz  1   2 , Olga Kerber  2 , Michal Gorka  2 , Juan C Moreno  1   2 , Israel Maruri-Lopez  1 , Romina I Minen  3 , Arun Sampathkumar  2 , Andrew D L Nelson  3 , Aleksandra Skirycz  2   3
Affiliations

2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana

Monika Chodasiewicz et al. Plant Physiol. .

Abstract

The role of the RNA degradation product 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) is poorly understood. Recent studies have identified 2',3'-cAMP in plant material and determined its role in stress signaling. The level of 2',3'-cAMP increases upon wounding, in the dark, and under heat, and 2',3'-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2',3'-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2',3'-cAMP treatment. We demonstrated that 2',3'-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2',3'-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2',3'-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2',3'-cAMP treatment influences the movement of processing bodies, confirming the role of 2',3'-cAMP in the formation and motility of membraneless organelles.

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Figures

Figure 1
Figure 1
Br-2′,3′-cAMP treatment induces stress-responsive changes at the metabolome level. A, Schematic of the experimental design. Arabidopsis wild-type plants were treated with mock, 1-μM Br-2′,3′-cAMP, or 1-µM Br-adenosine (for RNAseq analysis: orange rectangle/dashed lines). A total of three to four biological samples were collected at five time points for proteomics and metabolomics analyses (blue rectangle)—15 min, 30 min, 1 h, 6 h, and 24 h—and at only two time points for transcriptome analysis (orange dashed line/circles)—15 min and 6 h. Samples were extracted and prepared for proteomics, metabolomics, and RNAseq analyses. Data were analyzed with a focus on significant 2′,3′-cAMP-induced changes. B, Change in the levels of Br-2′,3′-cAMP and Br-adenosine in plants treated with 1 µM Br-2′,3′-cAMP. Data are presented as mean of log2 median normalized intensity. Error bars represent standard deviation, n = 4. C, The groups of metabolites which level significantly changed upon 2′,3′-cAMP treatment (two-way ANOVA, P-value FDR corrected  ≤ 0.05). D, Heat map representing the overall significant changes in metabolite levels after Br-2′,3′-cAMP treatment. Data are presented as log2 fold change (two-way ANOVA, P-value FDR corrected ≤ 0.05).
Figure 2
Figure 2
Differential gene expression analysis revealed major transcriptional reprogramming associated with 2′,3′-cAMP and adenosine. A, The number of genes found to be upregulated or downregulated after 30 min and 6 h of treatment. Adeno—corresponds to adenosine. B, Venn diagram of all significantly upregulated genes after 30 min of 2′,3′-cAMP and adenosine treatments. C, Venn diagram of all significantly downregulated genes after 30 min of 2′,3′-cAMP and adenosine treatments. The numbers (B and C) correspond to the DEGs identified as significantly changed compared with the control samples in each experiment. D, Overrepresentation of biological processes in the dataset of upregulated genes in the 2′,3′-cAMP (light orange bars) and adenosine (light blue bars) experiments (30 min time-point). E, Overrepresentation of biological processes in the dataset of downregulated genes in the 2′,3′-cAMP (light orange bars) and adenosine (light blue bars) experiments (30 min time point). In (D and E), overrepresentation is shown as a significant fold enrichment based on the PANTHER overrepresentation test (Mi et al., 2017) using Fisher’s exact test with FDR multiple correction (P ≤ 0.05) and A. thaliana as the reference organism. F, Venn diagram showing an overlap between downregulated genes after adenosine treatment and upregulated genes after 2′,3′-cAMP treatment. G, A network of enriched genes was retrieved by the STRING database (Szklarczyk et al., 2017) but visualized in Cytoskape. Experimental and database evidence and a low confidence cutoff were used to visualize protein–protein interactions. Gene encoding for proteins involved in stress (violet) and response to jasmonic acid (JA) (pink) are highlighted.
Figure 3
Figure 3
2′,3′-cAMP treatment induces stress-related changes in the proteome of A. thaliana. A, The cellular compartment distribution of the identified, significantly upregulated, and downregulated proteins after Br-2′,3′-cAMP treatment. Subcellular localizations for each protein were identified using the SUBA3 database (http://suba3.plantenergy.uwa.edu.au/). B, Enriched biological processes in the dataset of significantly upregulated (dark green) and downregulated (light green) proteins after Br-2′,3′-cAMP treatment. Overrepresentation is shown as a significant fold enrichment based on the PANTHER overrepresentation test (Mi et al., 2017) using Fisher’s exact test with FDR multiple correction (P ≤ 0.05) and A. thaliana as reference organism. C, Changes in the abundance of SG proteins are presented as heat maps. Significant changes were determined using a two-way ANOVA (P-value FDR corrected ≤ 0.05, n = 4 biological replicates). D, Heat map represents significantly accumulated putative aliphatic and indole glucosinolate compounds together with glucosinolate biosynthetic enzymes in response to Br-2′,3′-cAMP treatment. Significant changes were determined using a two-way ANOVA (P-value FDR corrected ≤ 0.05, n = 4 biological replicates).
Figure 4
Figure 4
Br-2′,3′-cAMP treatment induces motility of PBs. A, Maximum projection images from 61 time-points collected from GFP-DCP1 Arabidopsis seedlings under control and after Br-2′,3′-cAMP treatment. Scale bar = 10 µm. B, Particle tracking in the control and Br-2′,3′-cAMP-treated cells. Scale represents the color code for the displacement length for each PB. C, The average displacement length of the PBs in the cell is expressed in micrometer. D, The average speed of PB movement in the control and Br-2′,3′-cAMP-treated seedlings. Speed is expressed in µm/s. For C and D, control n = 1,583 and 2′,3′-cAMP n = 898. Asterisk in C and D indicates significant differences defined by student′s t test, P ≤ 0.05.
Figure 5
Figure 5
2′,3′-cAMP treatment resembles stress responses. Schematic representation of the effects of 2′,3′-cAMP (purple panel) treatment in A. thaliana at the transcriptomics, proteomics, and metabolomics levels. Transcriptomics data (blue panel). Differential gene expression analysis showed the 2′,3′-cAMP upregulation of genes involved in JA homeostasis and stress responses (e.g. wounding, defense to bacteria, and water deprivation), whereas those genes involved in RNA machinery and processing were downregulated during 2′,3′-cAMP treatment. Proteomics data (green panel) revealed that 2′,3′-cAMP triggered stress-related changes in the A. thaliana proteome. Briefly, the accumulation of proteins involved in amino acid biosynthetic pathways and auxin transport increased upon 2′,3′-cAMP treatment, in contrast to translational machinery, which was downregulated. 2′,3′-cAMP treatment also led to the accumulation of key SG proteins and induced PB movement. Metabolites (orange panel) such as dipeptides, amino acids, nucleotides, and RNA-degradation products showed accumulation after 2′,3′-cAMP treatment. Arrows indicate upregulation, and bars depict downregulation. Dotted lines denote the associated process.
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