In order to adapt to stress conditions, plants have evolved complex signaling mechanisms implicating various molecular changes to establish appropriate responses. Since protein translation is an energetically demanding process, stress can cause a global drop of protein synthesis [31, 35]. However, some proteins are still synthesized during stress to enable cells to tolerate the stress conditions more effectively [19]. In such context, regulation of the identity of some components of the ribosomal subunits may be key to the plant survival under stress conditions [3, 23, 32, 44]. Furthermore, Arabidopsis ribosomes are extensively heterogenous, each individual RP being encoded by two to seven paralogous genes [47]. With that in mind, it is interesting to speculate that ribosomal protein composition specializes in response to external stimuli to enable the plant adaptation to specific conditions. To address this hypothesis, we immunoprecipitated FLAG-tagged ribosomes followed by protein identification and quantification by LC-MS/MS. This approach allowed us to characterize the abundance of core RP in the Arabidopsis ribosome. Our proteomic characterization shows that the majority of RPs encoded by A. thaliana are present in the riboproteome. 25 ribosomal proteins (16 RPS and 9 RPL) out of 249 RP-encoding genes showed a significant change in their abundance in response to defense activation. Our results agree with those reported by Hummel et al. (2012), who concluded that different ribosomal protein paralogs are incorporated into the ribosomes depending on growth conditions.
We used untargeted proteomic to address riboproteome (encompassing ribosomal proteins and ribosome-associated proteins) modulation in the context of plant immune activation. Following INA treatment and immunoprecipitation, a total of 1882 non-ribosomal proteins were detected (Table S3). Of those, more than a quarter (508) were only observed following INA treatment, indicating an important rearrangement in ribosome-associated protein following immunity stimulation. These included several proteins with a known link to immunity such as VASCULAR ASSOCIATED DEATH-1 [28], HSP90 [21], PR5 [50], IMP-α [38], BIG and CCT2 [33]. As translational activity and regulation are not solely accomplished by ribosomal protein and require a plethora of accessory proteins, these may represent elements required to fine tune translation in response to stresses.
In eukaryotes, the small subunit of the ribosome makes first contact with the mRNA prior to assembly with the large ribosomal subunit to constitute a translation-competent ribosome. As such, the small subunit is involved in the selection of the mRNAs to be translated and the identity of the ribosomal proteins within the small subunit could impact the identity of the recruited mRNAs. In addition to their crucial roles in translation, specific ribosomal proteins of the small subunit (RPS) are known to play vital roles in abiotic stress and plant-pathogen interactions. In the present study, 11 RPS had increased abundance following the INA treatment (RPS15a, RPS10c, RPS11c, RPS24a, RPS5a, RPS27d, RPS8e, RPS17d, RPS19c, RPS9c and RPS2c, Fig. 3). For some of these RPS, variation in their mRNA levels in response to external stimuli has been previously reported in several plant species. Indeed, the transcript levels of RPS15a, the most deregulated RPS in our data, increased significantly in Arabidopsis in response to phytohormones and heat stress [22]. Similarly, in the transcriptome of vanilla infected with Fusarium oxysporum f. sp. vanillae, differential expression of RPSAA, RPS5a, RPS17d and RPS24a was observed [44], these four RPS also being upregulated in our riboproteome. In addition, it has been documented that RPS are induced in response to stress in Oryza sativa. RPS9c, 10 and 19c are among the early responsive genes upregulated under salt stress [26], all three were also increased in our data. The transcript of some RPS genes accumulated at remarkably high levels (≥ 100 fold) under drought stress (RPS9c, RPS17d, RPS19c, RPS27d) or under oxidative stress (RPS9c and RPS15a) [42]; all of these RPS accumulated in our dataset. RPS gene expression was also studied in response to biotic stress in rice. Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, pathogens that respectively cause very serious Bacterial Leaf Blight and Sheath Blight diseases in rice, induced the upregulation of RPS10c (29 fold), RPS9c (18 fold), and RPS5a (14 fold) [42], and in our data these same RPS accumulated in response to INA treatment. These reports point toward a differential expression of RPS genes in response to stress treatments leading to a differential accumulation of RPS in the ribosomal apparatus, which might help subunit remodeling and selective translation to cope up with unfavorable conditions.
Interestingly, disease and stress resistance functions of RPL have been reported in recent years. Silencing of RPL12, RPL19, RPL30 and RPL10 in Nicotiana benthamiana or Arabidopsis thaliana compromised nonhost disease resistance against multiple bacterial pathogens [36, 40]; of those, only RPL10A was deregulated in our experiment. In the present study, 8 RPL showed increased abundance following INA treatment. Figure 4 shows the resulting changes in RPP1C, RPP1A, RPL30c, RPL9b, RPL8a, RPL5b, RPP2b and RPL15b. Several reports focused on stress induced differential expression of these RPL. Induction of RPL30c with 60S acidic RP was reported in vanilla infected by Fusarium oxysporum f. sp. vanilla [44]. Similarly, the transcript levels of RPL30c increased significantly in response to phytohormones in Arabidopsis and in response to oxidative stress in rice [7]. In our data, we observed a 2,7 fold increased accumulation of RPL30c. In rice, under MeJa and SA treatments, RPL8a showed upregulation up to 100 fold, whereas we observed a 2-fold upregulation of RPL8a. Microarray of rice response to Xanthomonas oryzae pv. oryzae revealed that RPL15b was up-regulated more than 10 fold [34], while we observed a 1,6-fold increased accumulation of RPL15b.
The enrichment in common cis-regulatory elements in the putative promoter regions of RPS and RPL genes (TCA-motifs, APE2 domain, MBS, TC-rich repeats, and DOF domain) suggests that variation in accumulation is the result of transcriptional changes. As discussed above, the observed changes in riboproteome composition can mostly be explained by higher mRNA levels leading to higher accumulation of ribosomal protein.
While there is evidence of ribosomes with varying composition, there is little understanding of how the cell regulates ribosome heterogeneity. It can occur in part during ribosome biogenesis, a complex process taking place in the nucleolus and involving association of ribosomal proteins with rRNA to constitute the ribosomal subunits. Previous studies have measured differential ribosomal proteins levels in the nucleus following immunity elicitation [1, 2, 12, 20]. We previously reported that some ribosomal proteins were overrepresented in the nucleus after chitosan elicitor treatment (RPSA, RPS5a, RPS9c, RPS10c, RPS11c, RPS17d, RPS19c, RPS24a, RPS27d and RPL30c) [12]. RPS5a and RPS11c were also found to have a significant change in their abundance in the Arabidopsis nucleus during PTI [1]. In tomato (Solanum lycopersicum), five RPS (RPSAa, RPS5a, RPS10c, RPS17d and RPS19c) were more abundant in the nucleus during infection by the oomycete pathogen Phytophthora capsici [20]. These reports support a probable role of RPs during the plant immune response. Additionally, switching between RPs which are assembled onto the pre-ribosomal subunits in the nucleolus is possible before the new ribosomes are functional [16].
In summary, the findings of this study open new and interesting avenues for research in ribosome composition during biotic and abiotic stress. Our study expands our molecular knowledge of the ribosomal proteins and ribosome-associated proteins and highlights the importance of studying the function of individual RPs paralogs by genetic analysis of ribosomal protein mutants to clarify their roles in response to stresses. Future work will be aimed at unraveling the specific mechanisms by which RPs affects the plant defense.