The Efg1-Bud22 dimer associates with the U14 snoRNP contacting the 5' rRNA domain of an early 90S pre-ribosomal particle.

The DEAD-box helicase Dbp4 plays an essential role during the early assembly of the 40S ribosome, which is only poorly understood to date. By applying the yeast two-hybrid method and biochemical approaches, we discovered that Dbp4 interacts with the Efg1-Bud22 dimer. Both factors associate with early pre-90S particles and smaller complexes, each characterized by a high presence of the U14 snoRNA. A crosslink analysis of Bud22 revealed its contact to the U14 snoRNA and the 5' domain of the nascent 18S rRNA, close to its U14 snoRNA hybridization site. Moreover, depletion of Bud22 or Efg1 specifically affects U14 snoRNA association with pre-ribosomal complexes. Accordingly, we concluded that the role of the Efg1-Bud22 dimer is linked to the U14 snoRNA function on early 90S ribosome intermediates chaperoning the 5' domain of the nascent 18S rRNA. The successful rRNA folding of the 5' domain and the release of Efg1, Bud22, Dpb4, U14 snoRNA and associated snoRNP factors allows the subsequent recruitment of the Kre33-Bfr2-Enp2-Lcp5 module towards the 90S pre-ribosome.

Characterization of MdMYB68, a suberin master regulator in russeted apples.

Introduction: Apple russeting is mainly due to the accumulation of suberin in the cell wall in response to defects and damages in the cuticle layer. Over the last decades, massive efforts have been done to better understand the complex interplay between pathways involved in the suberization process in model plants. However, the regulation mechanisms which orchestrate this complex process are still under investigation. Our previous studies highlighted a number of transcription factor candidates from the Myeloblastosis (MYB) transcription factor family which might regulate suberization in russeted or suberized apple fruit skin. Among these, we identified MdMYB68, which was co-expressed with number of well-known key suberin biosynthesis genes. Method: To validate the MdMYB68 function, we conducted an heterologous transient expression in Nicotiana benthamiana combined with whole gene expression profiling analysis (RNA-Seq), quantification of lipids and cell wall monosaccharides, and microscopy. Results: MdMYB68 overexpression is able to trigger the expression of the whole suberin biosynthesis pathway. The lipid content analysis confirmed that MdMYB68 regulates the deposition of suberin in cell walls. Furthermore, we also investigated the alteration of the non-lipid cell wall components and showed that MdMYB68 triggers a massive modification of hemicelluloses and pectins. These results were finally supported by the microscopy. Discussion: Once again, we demonstrated that the heterologous transient expression in N. benthamiana coupled with RNA-seq is a powerful and efficient tool to investigate the function of suberin related transcription factors. Here, we suggest MdMYB68 as a new regulator of the aliphatic and aromatic suberin deposition in apple fruit, and further describe, for the first time, rearrangements occurring in the carbohydrate cell wall matrix, preparing this suberin deposition.

Cms1 coordinates stepwise local 90S pre-ribosome assembly with timely snR83 release.

Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S. Further investigations detected Cms1 at the 18S rRNA 3' major domain of an early 90S that carried H/ACA snR83, which is known to guide pseudouridylation at two target sites within the same subdomain. Cms1 co-precipitates with many 90S factors, but Rrp12-Enp1 encircling the 3' major domain in the mature 90S is decreased. We suggest that Cms1 associates with the 3' major domain during early 90S biogenesis to restrict premature Rrp12-Enp1 binding but allows snR83 to timely perform its modification role before the next 90S assembly steps coupled with Cms1 release take place.

Spatially Enriched Paralog Rearrangements Argue Functionally Diverse Ribosomes Arise during Cold Acclimation in Arabidopsis.

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.

Arabidopsis REI-LIKE proteins activate ribosome biogenesis during cold acclimation.

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10 °C, reil mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while controlling the balance of non-translating 40S- and 60S-subunits. Reil mutations respond by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis may buffer fluctuating translation by pre-existing non-translating ribosomes before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and alter paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.

Multiplexed Profiling and Data Processing Methods to Identify Temperature-Regulated Primary Metabolites Using Gas Chromatography Coupled to Mass Spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of plant material that was exposed to temperature stress. The method can be combined with stable isotope labeling and tracing experiments and is equally applicable to preparations of plant material and microbial photosynthetic organisms. The described methods are modular and can be multiplexed, that is, the same sample or a paired identical backup sample can be analyzed sequentially by more than one of the described procedures. The modules include rapid sampling and metabolic inactivation protocols for samples in a wide weight range, sample extraction procedures, chemical derivatization steps that are required to make the metabolite fraction amenable to gas chromatographic analysis, routine GC-MS methods, and procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The methods have two applications: (1) The rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification by exact quantification using GC-MS protocols that are extended by internal and external standardization.

Systematic Review of Plant Ribosome Heterogeneity and Specialization.

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.

Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes.

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

Plant Temperature Acclimation and Growth Rely on Cytosolic Ribosome Biogenesis Factor Homologs.

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.

Profiling methods to identify cold-regulated primary metabolites using gas chromatography coupled to mass spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and finally the procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification of cold-regulated metabolites by exact quantification using a GC-MS protocol with extended internal and external standardization.

REIL proteins of Arabidopsis thaliana interact in yeast-2-hybrid assays with homologs of the yeast Rlp24, Rpl24A, Rlp24B, Arx1, and Jjj1 proteins.

The REIL1 and REIL2 proteins of Arabidopsis thaliana are evolutionarily conserved homologs of the cytosolic 60S ribosomal maturation factors Rei1 and its paralog Reh1 of Saccharomyces cerevisiae. We previously demonstrated that the REIL proteins like the yeast homologs are required for the growth of both organisms at suboptimal temperatures. In addition, the cold sensitivity of the yeast Δrei1 mutant was almost fully rescued by heterologous expression of the REIL1 protein. These phenomena and conservation of co-expressed genes linked the function of REIL proteins to the maturation of the eukaryotic ribosome in A. thaliana. Here we demonstrate that REIL proteins interact in yeast-2-hybrid assays with A. thaliana homologs of the yeast proteins, Rlp24, Rpl24A, Rlp24B, Arx1, and Jjj1. These proteins take part in the cytosolic 60S ribosomal maturation process within yeast and physically interact with Rei1. Our study does not provide proof but is consistent with a conserved role of the A. thaliana REIL proteins in ribosomal maturation and demonstrates the potential of future investigations that aim to unravel the protein interactions of REIL proteins in planta.

The REIL1 and REIL2 proteins of Arabidopsis thaliana are required for leaf growth in the cold.

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.