Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis

Arabidopsis contains hundreds of ribosomal DNA copies organized within the nucleolar organizing regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1–4, based on the polymorphisms of 3’ external transcribed sequences. The variants are known to be differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~ 25 nt from predominantly NOR4 ribosomal DNA copies, obtaining mosaic mutational events on ~ 5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies by elevated expression of VAR1 in mutants and further associated single-nucleotide variants, thus, resulting in altered rRNA sub-population. Furthermore, the mutants exhibited rRNA maturation defects specifically in the minor pathway typified by 32S pre-rRNA accumulation. Density-gradient fractionation and subsequent RT-PCR of rRNA analyses revealed that mutated copies were not incorporated into the translating ribosomes. The mutants in addition displayed an elevated autophagic flux as shown by the autophagic marker GFP-ATG8e, likely related to ribophagy.

拟南芥（Arabidopsis）的2号和4号染色体的核仁组织区（nucleolar organizing regions, NORs）内携带有数百个核糖体DNA（ribosomal DNA, rDNA）拷贝；基于3'外转录序列（3’ external transcribed sequences）的多态性，rDNA可分为VAR1至VAR4四种主要变异型，且已知这些变异型在植物发育过程中呈差异表达。我们通过CRISPR-Cas9介导的序列切除，从以NOR4为主的rDNA拷贝中切除了约25 nt的序列片段，获得了在约5%的总rDNA拷贝上存在嵌合突变的突变体株系；被切除的区域包含25S核糖体RNA（25S rRNA）的P环（P-loop）与螺旋82（Helix-82）区段。该突变引发了等位基因依赖性、剂量敏感性缺陷，具体表现为侧根生长受抑制、植株体型减小以及叶片呈尖形，这些表型此前均在核糖体功能缺陷的植株中被观测到。NOR4位点的突变通过提高突变体中VAR1的表达水平以及相关的单核苷酸变异，触发了来自NOR2拷贝的剂量补偿效应，最终导致rRNA亚群组成发生改变。此外，该突变体呈现出rRNA成熟缺陷，该缺陷特异性地发生在以32S前核糖体RNA（32S pre-rRNA）积累为特征的次要成熟途径中。密度梯度分级分离结合后续的rRNA逆转录聚合酶链式反应（RT-PCR）分析结果显示，发生突变的rDNA拷贝并未整合进入正在进行翻译的核糖体中。此外，该突变体还表现出自噬流增强，这一现象可通过自噬标记物GFP-ATG8e得到验证，该现象可能与核糖体自噬（ribophagy）相关。