Root system responses to low temperature acclimation of Arabidopsis reil ribosome biogenesis double mutants

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. The transcriptome data set describes the total mRNA differences between complete root systems of Arabidopsis thaliana Col-0 wild type and of the reil1-1 reil2-1 and reil1-1 reil2-2 double mutants at 20°C (day)/ 18°C (night) immediately before temperture shift and at 1 day and 7 days after shift to 10°C (day)/ 8°C (night). Experiments were performed in hydroponic culture with liquid MS-media adjusted to pH 5.7 containing 2% sucrose (w/v) (Murashige and Skoog, 1962). Plants had developmental stage ~1.10 (Boyes et al. 2001) at the time of temperature shift. Three independent biological replicate experiments were harvested for microarray based transcriptome analyses. Each of the three harvested biological replicates was the pool of the complete root systems from four plants of a single hydroponic container.