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

ABSTRACT

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.

KEYWORDS:

• rRNA mutation
• plant development
• CRISPR
• dosage compensation
• autophagy

Acknowledgments

We thank Dr. Nicola Patron (The Earlham Institute, UK) for her gift of the CRISPR vectors used in this study. We thank Dr. Yasin Dagdas (Gregor Mendel Institute, Vienna) for his kind gift of the GFP-ATG8e transgenic marker seeds. A.G. is thankful to EU Horizon 2020 research and innovation programme under grant agreement number GA 2020 862-858 (ADAPT). P.C is thankful to Austrian Science Fund (FWF) under grant agreement number I 5234.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The RNA-seq data presented in this study are deposited in GEO repository under accession no. GSE213764 and the proteome data are deposited in PRIDE repository with accession no. PXD035623.

Author contributions

T.S. and E.S. conceived and developed the study; T.S. performed all the experiments with inputs from D.S., T.B. and E.S.; P.C. and A.G. performed the mass spectrometry and proteome data analysis; W.W. contributed the reagents; S.S. analysed the RNA-seq data and performed the SNV analysis; T.S. and E.S. wrote the article; all authors read, edited and approved the manuscript.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2023.2298532

Additional information

Funding

This work was supported through grants from Deutsche Forschungsgemeinschaft’s (DFG) SFB902 to E.S.