Root system responses to low temperature acclimation of Arabidopsis reil ribosome biogenesis double mutants. 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. Overall design: 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.

REIL蛋白是拟南芥（Arabidopsis thaliana）成熟叶片在低温驯化过程中晚期核糖体生物发生（late ribosomal biogenesis）以及60S核糖体大亚基（60S large ribosome subunit）积累所必需的。为验证其在根部的功能，我们开展了多维度系统分析，旨在解析低温驯化前以及温度转移后reil突变体的缺陷与代偿响应。水培根组织可实现对真核核糖体复合物的分析，且几乎不受细胞器核糖体（organelle ribosomes）的干扰。水培培养缓解了reil突变体在低温下的生长缺陷，并为拟南芥REIL蛋白的核心功能提供了新的研究视角。

拟南芥会严格调控非翻译型40S与60S亚基（non-translating 40S and 60S subunits）的平衡。reil突变体在转移至10℃环境后，最初会耗尽这两类非翻译亚基，随后才会缓慢补充这些亚基库。reil突变通过提高非翻译型40S与60S亚基的基础水平，并耗竭低温下可能存在的、对KCl敏感的80S亚组分（KCl-sensitive 80S sub-fraction），代偿了60S生物合成缺陷。我们推断，在从头合成（de novo synthesis）满足温度诱导的需求之前，拟南芥通过激活非翻译型核糖体组分，缓冲温度变化带来的翻译需求波动。

reil1 reil2双突变体会积累43S前起始复合物（43S-preinitiation complexes）与前60S成熟复合物（pre-60S-maturation complexes），并影响非翻译型核糖体组分的旁系同源物组成。除少数例外（如RPL3B与RPL24C）外，这些变化并不受转录调控（transcriptional control）。本研究表明，真核核糖体的从头合成对于长期低温驯化是必需的。双突变体分析显示，REIL介导的60S成熟过程会反馈调控NUC2与eIF3C2的转录，并暗示了两种此前未被报道的蛋白在植物晚期核糖体生物发生中的功能。我们提出，拟南芥需要合成特化核糖体才能成功完成低温驯化。

整体实验设计：本转录组数据集描述了拟南芥Col-0野生型、reil1-1 reil2-1及reil1-1 reil2-2双突变体的完整根系在温度转移前（20℃/昼，18℃/夜）、转移至10℃/昼，8℃/夜环境后1天和7天时的总mRNA差异。实验采用水培培养体系，使用pH值调节至5.7的液体MS培养基，添加2%（w/v）蔗糖（Murashige和Skoog，1962）。温度转移时，植株的发育阶段约为1.10（Boyes等人，2001）。我们开展了3次独立的生物学重复实验以进行基于微阵列的转录组分析。每次收获的生物学重复样本均来自单个水培容器中4株植株的完整根系混合而成。