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, 120 (6), 911-922

Cell Growth and Homeostasis Are Disrupted in Arabidopsis rns2-2 Mutants Missing the Main Vacuolar RNase Activity

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Cell Growth and Homeostasis Are Disrupted in Arabidopsis rns2-2 Mutants Missing the Main Vacuolar RNase Activity

Stephanie C Morriss et al. Ann Bot.

Abstract

Background and aims: Enzymes belonging to the RNase T2 family are essential for normal rRNA turnover in eukaryotes. In Arabidopsis thaliana, this function is performed by RNS2. The null mutant rns2-2 has increased rRNA half-life and constitutive autophagy. The aim of this work was to determine the molecular changes that take place in the rns2-2 mutant that may lead to altered cellular homeostasis, manifested by the observed cellular phenotype.

Methods: To determine the effect of defective rRNA turnover on cellular homeostasis, comparative transcriptome and metabolome analyses of 10-day-old wild-type and rns2-2 seedlings were used to identify molecular processes affected in the mutant. Bioinformatics analyses suggested additional phenotypes that were confirmed through direct plant size measurements and microscopy.

Key results: Few genes were differentially expressed in the rns2-2 mutant, indicating that control of autophagy in this genotype is mainly achieved at the post-transcriptional level. Among differentially expressed genes, transcripts related to carbon flux processes, particularly the pentose phosphate pathway (PPP), were identified. Metabolite analyses confirmed changes in the levels of PPP intermediates. Genes related to cell wall loosening were also differentially expressed in the mutant, and a decrease in monosaccharide components of cell wall hemicellulose were found. As a potential effect of weaker cell walls, rns2-2 plants are larger than wild-type controls, due to larger cells and increased water content. Elevated levels of reactive oxygen species (ROS) were also measured in rns2-2, and the constitutive autophagy phenotype was blocked by preventing ROS production via NADPH oxidase.

Conclusions: Lack of rRNA recycling in rns2-2 cells triggers a change in carbon flux, which is redirected through the PPP to produce ribose-5-phosphate for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential for cellular homeostasis, probably through maintenance of nucleoside levels as part of the salvage pathway.

Keywords: Arabidopsis thaliana; RNS2; autophagy; carbon flux; cell wall; nucleoside homeostasis; rRNA turnover.

Figures

Fig. 1.
Fig. 1.
Verification of microarray results. Expression of genes identified as differentially expressed in the microarray analysis was analysed by quantitative RT-PCR (qPCR). RNA was extracted from WT and rns2-2 seedlings grown identically to the material used for transcriptome analysis. Genes selected for testing were ATEXLA1 (AT3G45970), glycosyl transferase (AT2G41640), GAPCP-1 (AT1G79530) and GAPCP-2 (AT1G16300). Results were normalized using the expression of TIP41-like (AT4G34270) as loading control and then to the average of the WT expression. The analysis was performed using four different RNA samples for each genotype. t-test. P-values are indicated above rns2-2–WT comparisons with a significant difference in expression level.
Fig. 2.
Fig. 2.
Genes differentially expressed in the rns2-2 mutant are regulated by the energy status of the cell. The heatmap shows the regulation of the DEGs identified in the microarray analysis and regulation by carbon starvation, glucose re-feeding or nicotinamide treatments, each of which changes the energy status of the cell, obtained from public databases and the literature. Expression in protoplasts overexpressing KIN10 is also included in the comparisons.
Fig. 3.
Fig. 3.
Differentially expressed genes in the rns2-2 mutant suggest changes in carbon flux and cell wall modifications. Network analysis of DEGs was carried out using Virtual Plant and the Aracyc database. Diamonds represent genes, while circles represent metabolites. Not all DEGs were associated with an enriched category.
Fig. 4.
Fig. 4.
Metabolite analysis indicates changes in levels of sugars in the rns2-2 mutant. (A) Non-targeted metabolite analysis. Polar metabolites were extracted from WT and rns2-2 mutant seedlings grown using the same conditions applied for microarray analysis. Metabolites were separated using gas chromatography and identified by mass spectrometry. Only metabolites with significantly (t-test) different levels are shown. At least four different samples were analysed for each genotype. (B) Targeted metabolite analysis to identify changes in the PPP. Ten-day-old WT and rns2-2 seedlings were grown as in (A). Metabolites were extracted, labelled using 13C, and analysed by RPLC-MS. Significant differences (t-test) are indicated. Results are the average of four independent experiments with at least three replicates per genotype. S7P, sedoheptulose-7-phosphate; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; 6PGA, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; Xu5P, xylulose-5-phosphate, Ri5P, ribose-5-phosphate.
Fig. 5.
Fig. 5.
Cell wall composition is affected in the rns2-2 mutant. Seedlings were grown as described in Fig. 4, and cell wall was extracted. The monosaccharide composition of WT and rns2-2 cell walls after α-amylase treatment was analysed by HPAEC-PAD. Results were obtained as mol % and then normalized to the average of the WT level for each metabolite. Significant differences (t-test) are indicated. At least six independent samples for each genotype were analysed.
Fig. 6.
Fig. 6.
Phenotypic characterization of rns2-2 plants. (A) Root length was measured in 10-day-old WT and rns2-2 seedlings grown on vertical agar plates. t-test P-value is indicated. Three independent plates containing the two genotypes (15 plants for each genotype per plate) were analysed. Both plant size and root length were normalized to the average of the WT values. (B) Roots were stained with propidium iodide and Oregon Green dyes, and imaged using a confocal microscope. After image capture, cell lengths were measured using ImageJ. t-test P-value is indicated. More than 500 cells were measured for each genotype. (C) Water content was determined as the weight difference of basal rosette tissue from WT and rns2-2 seedlings grown as in Fig. 4, before and after 4 d of lyophilization. The dry weight/wet weight ratio was normalized to the WT average. t-test P-value is indicated. Five independent samples for each genotype were analysed.
Fig. 7.
Fig. 7.
ROS signalling controls autophagy in rns2-2 plants. (A) Measurement of ROS. Seven-day-old seedlings were stained with DCFDA to detect ROS. Fluorescence was measured using microscopy. and statistical comparison between WT and mutant was performed using Student’s t-test (P < 0.05). From 44 to 60 total measurements per genotype were taken from six independently grown sets. (B) Effect of DPI treatment on the rns2-2 autophagy phenotype. Roots of WT or rns2-2 seedlings were treated with DPI (NADPH oxidase inhibitor) or DMSO and then stained with MDC and imaged using fluorescence microscopy. The number of autophagosomes per frame was quantified. Statistical analysis was performed using Student’s t-test (P > 0.05) in comparisons against each treatment and genotype
Fig. 8.
Fig. 8.
Model of the effect of the rns2-2 mutation on arabidopsis metabolism. Integration of transcriptome and metabolome data suggests that carbon flux is shuttled through the PPP. Since rns2-2 plants are deficient in rRNA turnover and nucleotide salvage, the increased carbon flux through the PPP pathway may be needed to generate ribose-5-phosphate for de novo synthesis of nucleotides to maintain cellular homeostasis. Changes in metabolism also result in increased NADPH levels. This increase causes accumulation of reactive oxygen species (ROS) that in turn signal the activation of the autophagy machinery. Green boxes indicate enzymes encoded by a gene downregulated in rns2-2, and red boxes upregulated. Green and red arrows indicate metabolites with lower and higher levels in rns2-2, respectively. F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; 6PGL, 6-phosphogluconolactone; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; Xu5P, xylulose-5-phosphate; GAP, glyceraldehyde-3-phosphate; E4P, erythrose-4-phosphate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; RuBP, ribulose-1,5-bisphosphate; 3PG, 3-phosphoglycerate; BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone-phosphate; SBP, sedoheptulose-1,7-bisphosphate; FBP, fructose-1,6-bisphosphate; pgi, glucose phosphate isomerase; g6pdh, glucose-6-phosphate dehydrogenase; pgl, 6-phosphogluconolactonase; rpe, ribulose-5-phosphate epimerase; tal, transaldolase; tkl, transketolase; fbpase, fructose-2,6-bisphosphatase; fba, fructose-bisphosphate aldolase; rpi, ribose-5-phosphate isomerase; sbpase, sedoheptulose-1,7-bisphosphatase; tpi, triosephosphate isomerase; gap, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphoglycerate kinase; rbc, ribulose-1,5-bisphosphate carboxylase/oxygenase; prk, phosphoribulokinase.

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