Inhibition of TOR Represses Nutrient Consumption, Which Improves Greening after Extended Periods of Etiolation. Inhibition of TOR Represses Nutrient Consumption, Which Improves Greening after Extended Periods of Etiolation

Upon illumination, etiolated seedlings experience a transition from heterotrophic to photoautotrophic growth. During this process, the tetrapyrrole biosynthesis pathway provides chlorophyll for photosynthesis. This pathway has to be tightly controlled to prevent the accumulation of photoreactive metabolites and to provide stoichiometric amounts of chlorophyll for its incorporation into photosynthetic protein complexes. Therefore, plants have evolved regulatory mechanisms to synchronize the biosynthesis of chlorophyll and chlorophyll-binding proteins. Two phytochrome-interacting factors (PIF1 and PIF3) and the DELLA proteins, which are controlled by the gibberellin pathway, are key regulators of this process. Here, we show that impairment of TARGET OF RAPAMYCIN (TOR) activity in Arabidopsis (Arabidopsis thaliana), either by mutation of the TOR complex component RAPTOR1B or by treatment with TOR inhibitors, leads to a significantly reduced accumulation of the photoreactive chlorophyll precursor protochlorophyllide in darkness but an increased greening rate of etiolated seedlings after exposure to light. Detailed profiling of metabolic, transcriptomic, and physiological parameters revealed that the TOR-repressed lines not only grow slower, they grow in a nutrient-saving mode, which allows them to resist longer periods of low nutrient availability. Our results also indicated that RAPTOR1B acts upstream of the gibberellin-DELLA pathway and its mutation complements the repressed greening phenotype of pif1 and pif3 after etiolation. Overall design: Wild type or rb10 plants were grown on half MS media without sucrose under dark for 5 days. The etiolated seedling were exposed to white light for 1 hour or not. Microarray analysis was performed using Affymetrix Gene 1.0 ST Array (Thermo Fisher Scientific) with three biological replicates of WT and rb10 samples. Total RNA was isolated from pools of seedlings and labeling was performed using 1 μg RNA as previously described. Affymetrix Gene 1.0 ST Array hybridizations were performed by Atlas Biolabs.

当受到光照时，黄化幼苗会从异养生长（heterotrophic growth）转向光合自养生长（photoautotrophic growth）。在此过程中，四吡咯生物合成途径（tetrapyrrole biosynthesis pathway）为光合作用提供叶绿素（chlorophyll）。该途径必须受到严格调控，以防止光活性代谢物积累，并为光合蛋白复合物的组装提供化学计量比的叶绿素。因此，植物演化出了调控机制，以同步叶绿素与叶绿素结合蛋白（chlorophyll-binding proteins）的生物合成。光敏色素互作因子（phytochrome-interacting factors, PIF1和PIF3）以及受赤霉素途径（gibberellin pathway）调控的DELLA蛋白（DELLA proteins）是该过程的关键调控因子。

本研究发现，在拟南芥（Arabidopsis thaliana）中，雷帕霉素靶蛋白（TARGET OF RAPAMYCIN, TOR）的活性受损——无论是通过突变TOR复合物组分RAPTOR1B，还是通过TOR抑制剂处理——均会导致黑暗环境下光活性叶绿素前体原叶绿素酸酯（protochlorophyllide）的积累量显著降低，但黄化幼苗经光照后的转绿速率显著提升。对代谢组、转录组及生理参数的详细分析显示，TOR抑制株系不仅生长速率更慢，还会以养分节约模式生长，使其能够耐受更长时间的低养分胁迫。此外，本研究结果还表明，RAPTOR1B作用于赤霉素-DELLA途径的上游，其突变可互补黄化后pif1与pif3突变体受抑制的转绿表型。

整体实验设计：将野生型或rb10植株接种于不含蔗糖（sucrose）的半浓度MS培养基（half MS media），黑暗培养5天以获得黄化幼苗。将部分黄化幼苗置于白光（white light）下照射1小时，其余保持黑暗处理。采用Affymetrix Gene 1.0 ST Array（Thermo Fisher Scientific）进行微阵列分析（Microarray analysis），野生型与rb10样本均设置3次生物学重复（biological replicates）。从混合幼苗样本中提取总RNA（Total RNA），并以1 μg总RNA为起始材料，按照此前报道的方法完成标记。Affymetrix Gene 1.0 ST Array的杂交实验由Atlas Biolabs完成。