Plants dedicate a high amount of energy and resources to the production of ribosomes. Historically, these multi-protein ribosome complexes have been considered static protein synthesis machines that are not subject to extensive regulation but only read mRNA and produce polypeptides accordingly. New and increasing evidence across various model organisms demonstrated the heterogeneous nature of ribosomes. This heterogeneity can constitute specialized ribosomes that regulate mRNA translation and control protein synthesis. A prominent example of ribosome heterogeneity is seen in the model plant, Arabidopsis thaliana, which, due to genome duplications, has multiple paralogs of each ribosomal protein (RP) gene. We support the notion of plant evolution directing high RP paralog divergence toward functional heterogeneity, underpinned in part by a vast resource of ribosome mutants that suggest specialization extends beyond the pleiotropic effects of single structural RPs or RP paralogs. Thus, Arabidopsis is a highly suitable model to study this phenomenon. Arabidopsis enables reverse genetics approaches that could provide evidence of ribosome specialization. In this review, we critically assess evidence of plant ribosome specialization and highlight steps along ribosome biogenesis in which heterogeneity may arise, filling the knowledge gaps in plant science by providing advanced insights from the human or yeast fields. We propose a data analysis pipeline that infers the heterogeneity of ribosome complexes and deviations from canonical structural compositions linked to stress events. This analysis pipeline can be extrapolated and enhanced by combination with other high-throughput methodologies, such as proteomics. Technologies, such as kinetic mass spectrometry and ribosome profiling, will be necessary to resolve the temporal and spatial aspects of translational regulation while the functional features of ribosomal subpopulations will become clear with the combination of reverse genetics and systems biology approaches.

Arabidopsis (Arabidopsis thaliana) REI1-LIKE (REIL) proteins, REIL1 and REIL2, are homologs of a yeast ribosome biogenesis factor that participates in late cytoplasmic 60S ribosomal subunit maturation. Here, we report that the inhibited growth of the reil1-1 reil2-1 mutant at 10°C can be rescued by the expression of amino-terminal FLUORESCENT PROTEIN (FP)-REIL fusions driven by the UBIQUITIN10 promoter, allowing the analysis of REIL function in planta. Arabidopsis REIL1 appears to be functionally conserved, based on the cytosolic localization of FP-REIL1 and the interaction of native REIL1 with the 60S subunit in wild-type plants. In contrast to its yeast homologs, REIL1 also was present in translating ribosome fractions. Systems analysis revealed that wild-type Arabidopsis remodels the cytosolic translation machinery when grown at 10°C by accumulating cytosolic ribosome subunits and inducing the expression of cytosolic ribosomal RNA, ribosomal genes, ribosome biogenesis factors, and translation initiation or elongation factors. In the reil1-1 reil2-1 mutant, all processes associated with inhibited growth were delayed, although the plants maintained cellular integrity or acquired freezing tolerance. REIL proteins also were implicated in plant-specific processes: nonacclimated reil1-1 reil2-1 exhibited cold-acclimation responses, including activation of the DREB/CBF regulon. In addition, acclimated reil1-1 reil2-1 plants failed to activate FLOWERING LOCUS T expression in mature leaves. Therefore, in the wild type, REIL function may contribute to temperature perception by suppressing premature cold responses during growth at nonstressful temperatures. In conclusion, we suggest that Arabidopsis REIL proteins influence cold-induced plant ribosome remodeling and enhance the accumulation of cytosolic ribosome subunits after cold shift either by de novo synthesis or by recycling them from the translating ribosome fraction.

The evolutionarily conserved proteins REI1-LIKE (REIL1) and REIL2 have four conserved zinc finger domains and are Arabidopsis thaliana homologs of the cytosolic 60S ribosomal maturation factor Rei1p (for Required for isotropic bud growth1 protein) from yeast (Saccharomyces cerevisiae) and its paralog Reh1p (for REI1 homologue1 protein). The yeast and A. thaliana paralogs result from independent gene duplications. The A. thaliana REIL paralogs are required specifically in the cold (10°C) but not for growth at optimal temperature (20°C). A reil1-1 reil2-1 double mutant is arrested at 10°C prior to the emergence of the first rosette leaf. Two allelic reil2 mutants, reil2-1 and reil2-2, form small spoon-shaped leaves at 10°C. This phenomenon reverts after emergence of the inflorescence in the cold or upon shift to 20°C. Except for a slightly delayed germination, a reil1-1 mutant shows no further growth phenotype under the currently investigated conditions. A comparative analysis demonstrates conserved coexpression of orthologous genes from yeast and A. thaliana that are coregulated with yeast rei1 or with A. thaliana REIL2, respectively. The conserved correlations point to a role of A. thaliana REIL proteins in the maturation of the eukaryotic ribosomal 60S subunit. We support this conclusion by heterologous complementation of the cold-induced growth defect of the yeast Δrei1 deletion.

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.