Specific alterations in riboproteomes composition of isonicotinic acid treated arabidopsis seedlings

Abstract

Plants have developed strategies to deal with the great variety of challenges they are exposed to. Among them, common targets are the regulation of transcription and translation to finely modulate protein levels during both biotic and abiotic stresses. Increasing evidence suggests that ribosomes are highly adaptable modular supramolecular structures which remodel to adapt to stresses. Each Arabidopsis thaliana ribosome consists of approximately 81 distinct ribosomal proteins (RPs), each of which is encoded by two to seven genes. To investigate the identity of ribosomal proteins of the small subunit (RPS) and of the large subunit (RPL) as well as ribosomes-associated proteins, we analysed by LC/MS/MS immunopurified ribosomes from A. thaliana leaves treated with isonicotinic acid (INA), an inducer of plant innate immunity. We quantified a total of 2084 proteins. 165 ribosome-associated proteins showed increased abundance while 52 were less abundant. Of the 52 identified RPS (from a possibility of 104 encoding genes), 15 were deregulated. Similarly, from the 148 possible RPL, 80 were detected and 9 were deregulated. Our results revealed potential candidates involved in innate immunity that could be interesting targets for functional genomic studies.

Keywords: Arabidopsis thaliana; Plant immunity; Proteomics; Ribosomal protein large subunit (RPL); Ribosomal protein small subunit (RPS); Translation regulation.

Fig. 1

Ribosome enrichment following immunity activation (A) qRT-PCR analysis of INA-induced PR1 and PR2 expression. At1G13320 was used as the reference gene. The error bars represent ± SD (n = 3). The asterisks represent a significant difference between treated and mock-treated samples. *P-values < 0.01, Student’s t-test. (B) Western blot analysis of ribosome enriched protein extracts of untreated and INA-treated leaf tissues of the FLAG-RPL18 transgenic A. thaliana line. Top panel: PR1 accumulation in INA samples, middle panel: FLAG-RPL18, bottom panels: detection of the small subunit protein RPS6

Fig. 2

The riboproteome is deregulated by INA treatment. (A) PCA of all the mass spectra matched peaks obtained from immunopurified ribosomal preparations of INA treated and control leaves. Ellipses encircle biological replicates. (B) Volcano plot of the deregulated proteins. Significant deregulation was set as an absolute log₂ value of fold change ≥ 1.5 and a q-values < 0.05. (C) GO functional analysis of the deregulated proteins, retrieved using the PANTHER Classification System

Fig. 3

Small subunit ribosomal protein levels change in response to INA treatment (A) Volcano plots of deregulated RPS: downregulated in blue and upregulated in red. Significant deregulation was set as an absolute log₂ value of fold change ≥ 1.5 and a q-values < 0.05 (B, C) Box plots showing the differences and replicate distribution of replicates for each statistically significant downregulated (B) and upregulated (C) proteins. The asterisks represent a significant difference between treated and mock-treated samples. *P-values < 0.05, **P-values < 0.01, limma t-test. (C) Heat map showing the ten most upregulated and downregulated RPS paralogs detected in this study

Fig. 4

RP remodeling potential of Arabidopsis 80S ribosomes upon INA treatment. The visualization outlines mapped changed protein abundances in response to INA treatment compared to control conditions. Proteomic data were statistically evaluated across individual paralogs within RP families as reported in Supplementary Table S4 and mapped to the wheat 80S monosome used as reference (Armache et al. 2010). Homology of wheat and Arabidopsis RP families was confirmed by protein BLAST matching (Martinez-Seidel et al. 2020a, b). Red and blue represent RP families with increased (red) or decreased (blue) protein abundances of at least one of the RP paralogs. The paralog identities and specific protein changes are reported in Figs. 3 and 4

Fig. 5

Large subunit ribosomal protein levels change in response to INA treatments Volcano plots of deregulated RPS: downregulated in blue and upregulated in red. Significant deregulation was set as an absolute log₂ value of fold change ≥ 1.5 and a q-values < 0.05 (B, C) Box plots showing the differences and replicate distribution of replicates for each statistically significant downregulated (B) and upregulated (C) proteins. The asterisks represent a significant difference between treated and mock-treated samples. *P-values < 0.05, **P-values < 0.01, limma t-test. (C) Heat map showing the ten most upregulated and downregulated RPS paralogs detected in this study

Fig. 6

Venn diagram showing of the regulatory elements enriched in the 5’ upstream region of the INA-deregulated ribosomal proteins