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Functions of Arabidopsis REI1-LIKE cytosolic ribosome biogenesis factor homologs

ABSTRACT: Functions of Arabidopsis REI1-LIKE (REIL) proteins, two homologs of a yeast ribosome biogenesis protein (RBP) that takes part in the late cytoplasmic steps of 60S ribosomal subunit maturation, were characterized by systems analyses of the 10°C cold acclimating reil1-1 reil2-1 double mutant compared to Col-0 wildtype. The mutant lacked both REIL proteins, was strongly growth inhibited in the cold and complemented by constitutive expression of N-terminal FLUORESCENT PROTEIN (FP)-REIL1 and FP-REIL2 fusion proteins under control of the UBIQUITIN 10 promoter. Wildtype acclimation to 10°C causes relative accumulation of cytosolic ribosome subunits and rRNA. Expression of cytosolic ribosomal genes, known cytosolic RBPs, translation initiation- and elongation-factors was activated. Conserved function of Arabidopsis REIL proteins was indicated by delay of these processes in reil1-1 reil2-1, cytosolic localization of FP-REIL proteins and native REIL protein interactions with 60S containing ribosome fractions. Non-acclimated reil1-1 reil2-1 triggered plant specific metabolic and transcriptomic cold acclimation responses that included activation of the DREB/CBF-regulon with a preference for the cold acclimation factors, CBF1/DREB1B, CBF2/DREB1C, and CBF3/DREB1A. Cold-acclimating reil1-1 reil2-1 maintained cellular integrity and acquired freezing tolerance but did not activate FLOWERING LOCUS T expression in mature leaves. This block was independent of FLOWERING LOCUS C and AGAMOUS-LIKE 19 mediated vernalization. We conclude that Arabidopsis REIL proteins enhance accumulation of cytosolic ribosome subunits after cold shift and either directly or indirectly feedback on temperature perception by suppression of premature cold acclimation at optimized temperature and by triggering growth and the vegetative to generative phase transition in the cold. Transcriptomic profiling demonstrated a hidden acclimation phenotype of the morphologically inconspicuous reil1-1reil2-1 mutant under optimized temperature conditions. Premature triggering of cold acclimation, a severe growth defect at 10°C and compensation responses indicate that REIL function may extend beyond cytosolic ribosome biogenesis towards translation initiation.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE101111 | GEO | 2018/02/01

REPOSITORIES: GEO

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Project description:The REIL proteins are required for late ribosomal biogenesis and accumulation of the 60S large ribosome subunit in mature leaves of Arabidopsis thaliana upon acclimation to low temperature. To validate these functions in roots, we conducted a multi-level system analysis targeted at understanding defects and compensations responses of reil mutants before acclimation to low temperature and following temperature shift. Hydroponic root tissue enabled analysis of eukaryotic ribosome complexes with negligible interference of organelle ribosomes. Hydroponic cultivation attenuated the growth defect of reil mutants at low temperature and provided new insights into the primary functions of Arabidopsis REIL proteins. Arabidopsis tightly controls the balance of non-translating 40S and 60S subunits. Reil mutants initially deplete both non-translating subunits upon shift to 10°C and subsequently replenish these pools slowly. Reil mutations compensate the 60S biosynthesis defect by increased baseline levels of non-translating 40S and 60S subunits and depletion of a likely non-translating, KCl-sensitive 80S sub-fraction in the cold. We infer that Arabidopsis buffers fluctuating translation demands following temperature cues by activating non-translating ribosome fractions before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation complexes and pre-60S-maturation complexes and affect the paralog composition of non-translating ribosome fractions. With few exceptions, e.g. RPL3B and RPL24C, these changes were not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation. Double mutant analysis indicates feedback control of REIL-mediated 60S maturation on NUC2 and eIF3C2 transcription and implies functions of two so far non-described proteins in late plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for successful cold acclimation.

Project description:Purpose: We aimed at i) obtaining insight into how a thermophile organism reacts to cold stress, and ii) evaluating the impact of HGT candidates on the acclimation process to temperature decrease. Methods: The experimental design followed a temperature shift timeline: after two weeks of cultivation at 42°C, constant illumination (90 μE) and constant shaking (160 rpm) in photoautotrophic conditions the first sampling took place (Hot_T48_1) and the cultures of G.sulphuraria were swiftly moved to 28°C ( = cold temperature). "Cold" samples were taken after 3h (Cold_T3_2), 12h (Cold_T12_3) and 48h (Cold_T48_4). After cold treatment at 28°C for 48 hours, the G. sulphuraria was then switched to 46°C for 48 hours (="Hot"). Again, samples were taken after 3h (Hot_T3_5), 12h (Hot_T12_6) and 48h (Hot_T48_7). Altogether, a 48h temperature timeshift at 28°C and successive recovery at 46°C were targeted for sampling. Results: Galdieria sulphuraria is a unicellular red alga that lives in hot, acidic, toxic metal-rich, volcanic environments, where few other organisms survive. Its genome harbours up to 5% of genes most likely acquired through horizontal gene transfer. These genes probably contributed to G. sulphuraria’s adaptation to its extreme habitats, resulting in today’s polyextremophilic traits. Here, we applied RNA-sequencing to obtain insights into the acclimation of a thermophilic organism towards temperatures below its growth optimum and to study how horizontally acquired genes contribute to cold acclimation. A decrease in growth temperature from 42 °C/46 °C to 28 °C resulted in an upregulation of ribosome biosynthesis, while excreted proteins, probably components of the cell wall, were downregulated. Photosynthesis was suppressed at cold temperatures, and transcript abundances indicated that C-metabolism switched from gluconeogenesis to glycogen degradation. Folate cycle and S-adenosylmethionine cycle (one-carbon metabolism) were transcriptionally upregulated, probably to drive the biosynthesis of betaine. All these cold-induced changes in gene expression were reversible upon temperature increase. Numerous genes acquired by horizontal gene transfer displayed pronounced temperature-dependent expression changes, corroborating the view that these genes contributed to adaptive evolution in G. sulphuraria.

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Functions of Arabidopsis REI1-LIKE cytosolic ribosome biogenesis factor homologs

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