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

Figures & data

Figure 1. Targeted mutagenesis of 25S rDNA led to mosaic mutation patterns on chromosome 4. (A) Schematical distribution of the rDNA copies on five Arabidopsis chromosomes and their associated VAR polymorphic rRNAs. (B) 3’-ETS polymorphism in the A. thaliana col-0 accession and the oligonucleotide’s binding positions for the detection and identification of mutated VARs in the genome. (C) Location of the targeted bases, consisting of the P-loop and helix-82 regions, in the secondary structure of the 25S LSU ribosomal RNA [9] is indicated in magenta. Numbers 2600 and 2800 in cyan denotes the positions of nucleotides in 25S rRNA (D) Heteroduplex PCR products of three genotypes electrophoresed under 15% native PAGE conditions. (E) Quantification of the mutated and intact rDNA copies of the PCR products analysed in panel C (n = 6 linearly increasing PCR cycles). Data are shown as means ± SD among the total % of rDNA. The P-value indicates the statistical significance with the unpaired student t-test. (F) Cloning and sequencing of the heteroduplex PCR products of panel C resulting in mosaic mutation patterns in the rDNA. (G) Electrophoresis of the PCR products on 2.5% TTE gels from the three genotypes; PCR was carried out using the mut-F and ETS-R oligos, followed by cloning and sequencing of 33 independent clones – their linked VAR subtypes are summarized.

Figure 2. Mutation caused dosage-dependent growth defects linked to a single locus. (A) Seedling growth phenotypes of segregating heterozygous plants. Scale bar: 10 mm (B) root length variations analysed during early growth (n = 18, BG; n = 29, ploop KD ±; n = 16, ploop KD -/- vertically grown seedlings). (C) Rosette morphology of soil-grown mutants. Scale bar: 30 mm (D) rosette diameters of mutants (n = 18 replicates for each genotype). (E) Seed morphology of ploop KD -/- mutants. (F) Genotype-phenotype correlation of 120 randomised seedlings based on the phenotypical segregation as in figure 2A and further genomic PCR analysis of the ploop allelic band (+, present; -, absent) of all seedlings. (G) Venn analysis of the identified proteome based on 4 biological replicates of 10 DAS BG and ploop KD -/- seedlings. (H) Bicluster analysis of the abundance of varying levels of GO terms-based classified proteins between 4 replicates. Data in (B) and (D) are presented as means ± SD.

Table 1. List of proteins distinctly absent in ploop KD -/- seedlings.

At Locus ID	Protein Description	Name^1
AT5G17870	plastid-specific 50S ribosomal protein 6	AtPSRP6
AT2G42530	cold regulated 15b	AtCOR15B
AT5G58110	chaperone binding; ATPase activators	-
AT5G20935	Chloroplast NADH dehydrogenase assembly protein	AtCRR42
AT5G66550	Maf-like protein	-
AT3G10620	nudix hydrolase homolog 26	AtNUDX26
AT1G48610	AT hook motif-containing protein	-
AT4G16500	Cystatin/monellin superfamily protein	ATCYS4
AT5G15530	biotin carboxyl carrier protein 2	At BCCP2
AT3G52730	ubiquinol-cytochrome C reductase UQCRX/QCR9-like family protein	-
AT4G36430	Peroxidase superfamily protein	-
AT5G39210	chlororespiratory reduction 7	AtCRR7
AT3G10860	Cytochrome b-c1 complex, subunit 8 protein	-
AT1G77710	Ubiquitin-like, Ufm1	ATCCP2
AT1G01170	ozone-responsive-stress-like protein	-
AT5G20140	SOUL haem-binding family protein	AtHBP5
AT1G13730	Nuclear transport factor 2 (NTF2) family protein with RNA binding (RRM-RBD-RNP motifs) domain	-
AT2G05310	transmembrane protein	-
AT2G30695	bacterial trigger factor	-
AT3G05020	acyl carrier protein 1	AtACP1
AT4G39860	hematological/neurological-like protein	-
AT2G35390	Phosphoribosyltransferase family protein	-
AT5G26210	alfin-like 4	AtAL4
AT3G58990	isopropylmalate isomerase 1	AtIPM1
AT5G22580	Stress responsive A/B Barrel Domain	-
AT2G47580	spliceosomal protein U1A	AtU1A
AT1G32380	phosphoribosyl pyrophosphate (PRPP) synthase 2	ATPRS2
AT2G16850	plasma membrane intrinsic protein 2;8	AtPIP2, AtPIP3B
AT3G09150	phytochromobilin:ferredoxin oxidoreductase, chloroplast/phytochromobilin synthase (HY2)	ATHY2, ATGUN3
AT4G05520	EPS15 homology domain 2	ATEHD2
AT3G51820	UbiA prenyltransferase family protein	AtG4, AtPDE325
AT5G16810	Protein kinase superfamily protein	-
AT5G63140	purple acid phosphatase 29	ATPAP29
AT1G16410	cytochrome p450 79f1	ATBUS1, ATBUSHY1, ATSPS1, ATCYP79F1
AT4G36390	Methylthiotransferase	-
AT2G35795	Chaperone DnaJ-domain superfamily protein	AtPAM18–1
AT4G18970	GDSL-like Lipase/Acylhydrolase superfamily protein	AtGGL22
AT4G31780	monogalactosyl diacylglycerol synthase 1	AtMGD1, EMB2797
AT1G08530	chitinase-like protein	-
AT3G09250	Nuclear transport factor 2 (NTF2) family protein	-
AT3G25680	SLH domain protein	-
AT4G08360	KOW domain-containing protein	-
AT1G04970	lipid-binding serum glycoprotein family protein	AtLBR-1
AT3G51670	SEC14 cytosolic factor family protein/phosphoglyceride transfer family protein	AtPATL6
AT3G18240	Ribosomal protein S24/S35, mitochondrial	AtRPS24/35
AT3G20680	Domain of unknown function (DUF1995)	-
AT1G50140	P-loop containing nucleoside triphosphate hydrolases superfamily protein	-
AT1G06070	Basic-leucine zipper (bZIP) transcription factor family protein	AtBZIP69
AT4G20010	plastid transcriptionally active 9	ATOSB2, ATPTAC9
AT4G34020	Class I glutamine amidotransferase-like superfamily protein	ATDJ1C
AT3G13560	O-Glycosyl hydrolases family 17 protein	-
AT4G27180	kinesin 2	AtK2, ATKATB, ATKINESIN2
AT5G20360	Octicosapeptide/Phox/Bem1p (PB1) domain-containing protein/tetratricopeptide repeat (TPR)-containing protein	AtPHOX3

¹The At Locus ID refers to the accession numbers, and names given according to the latest information on The Arabidopsis Information Resource (TAIR) in www.arabidopsis.org.

Figure 3. Mutation caused dosage compensation from the usually inactive NOR2 locus leading to other rRNA defects.

(A) (B) Electrophoresis (A) and quantification of PCR products (B) following amplification with the ETS-F and ETS-R oligos (Figure 1B) from genomic DNA (upper panels) and cDNA, reverse transcribed from total RNA (lower panels) of 14-days-old BG and ploop KD mutants. PCR products (VAR1-4) are marked on the right-hand side and the DNA marker (bp) on the left. Data in (B) are presented as means ± SD % of each subtype variant to the total % of rDNA or rRNA. (C) SNV analysis of the 35S pre-rRNA from the BG (cyan) and ploop KD -/- (magenta) seedlings denotes the frequency of each position relative to the reference rDNA [22]. (D) Northern hybridization analysis of the total RNA from the BG and the ploop KD mutants with complementary radioactively-labelled oligonucleotides (p3, p4 and p5 position indicated in panel C) binding to the immature regions of ITS1 and ITS2; the EtBr stained on the left-hand side shown as the loading control. Identified precursors are indicated on the right. The asterisk denotes an abnormal decay product as reported earlier [45]. (E) Quantification of each pre-rRNA precursor relative to the radiolabelled 25S rRNA signal according to the colour scheme in panel E (n = 4 biological replicates). ploop KD ± and -/- are simply indicated by ± and -/-, respectively. Data are shown as means ± SD fold change values after normalizing with radiolabelled 25S rRNA signal and BG the genotype. The P-values denote the statistical significance with the unpaired student t-test.

Figure 4. Mutated copies are not incorporated into the translating polysomes. (A) Absorbance profiles at 254 nm of the floral tissues from BG (black) and ploop KD ± (magenta) seedlings plotted with time of detection from top to bottom following sucrose-density gradient centrifugation. (B) The RNA from the corresponding fractions in panel a for both genotypes was purified, reverse transcribed with the 25S rRNA-specific oligo and the PCR products with the mutation-specific forward oligo and 25S-specific reverse oligo resolved by 12% native PAGE gels. The black arrow denotes the PCR-run through product and the magenta arrow indicates the mutated rRNA product. (C) The proteins from the fractions in panel a of both genotypes were resolved on 10% SDS-PAGE and blotted with the indicated antibodies shown on the right-hand side.

Figure 5. Mutants display elevated autophagic flux. (A) Schematics of the GFP-ATG8e fusion protein localisation under routine growth and autophagic conditions. (B) Localisation of the GFP signals from the GFP-ATG8e transgenic fusion proteins in the BG (top) and ploop KD -/- (bottom) seedlings. Scale bars: 50 µm (C) immunoblotting analysis with the anti-GFP antibodies of the GFP-ATG8e levels in the BG and ploop KD -/- seedlings. Anti-HSC70 blotting served as the loading control. (D) Quantification of the full length GFP-ATG8e and free GFP signals from panel C represented in relation to the total levels after normalisation to the HSC70 loading control (n = 3 biological replicates). Data are presented as means ± SD % of each GFP variant to the total GFP signal intensity after normalizing with the HSC70 loading control signals.

Sweeney BA, Hoksza D, Nawrocki EP, et al. R2DT is a framework for predicting and visualising RNA secondary structure using templates. Nat Commun. 2021;12:1–12. doi: 10.1038/s41467-021-23555-5

 PubMed Web of Science ®Google Scholar

Sims J, Sestini G, Elgert C, et al. Sequencing of the Arabidopsis NOR2 reveals its distinct organization and tissue-specific rRNA ribosomal variants. Nat Commun. 2021;12:1–13. doi: 10.1038/s41467-020-20728-6

 PubMed Web of Science ®Google Scholar

Wang M, Parshin AV, Shcherbik N, et al. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA. RNA. 2015;21:1240–1248. doi: 10.1261/rna.051169.115

 PubMed Web of Science ®Google Scholar

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.