The Arabidopsis RETARDED ROOT GROWTH gene encodes a mitochondria-localized protein that is required for cell division in the root meristem - PubMed (original) (raw)

The Arabidopsis RETARDED ROOT GROWTH gene encodes a mitochondria-localized protein that is required for cell division in the root meristem

Xiaojing Zhou et al. Plant Physiol. 2011 Dec.

Abstract

To develop a growing root, cell division in the root meristem has to be properly regulated in order to generate or propagate new cells. How cell division is regulated in the root meristem remains largely unknown. Here, we report the identification and characterization of the Arabidopsis (Arabidopsis thaliana) RETARDED ROOT GROWTH (RRG) gene that plays a role in the regulation of root meristem cell division. In the root, RRG is predominantly expressed in the root meristem. Disruption of RRG function reduced numbers of dividing cells, the rate of cell production, and endoreduplication, and thus affected meristem size and root growth. Quantitative reverse transcription-polymerase chain reaction and marker-assisted analyses revealed that expression levels of several cell cycle genes were decreased in the mutant roots, indicating a defect in cell cycle progression. Mutations in RRG, however, did not affect the expression of key root-patterning genes and an auxin-responsive marker, suggesting that RRG is not essential for root patterning and auxin signaling. RRG is a mitochondria-localized protein conserved in plants and shares a DUF155 domain with proteins related to cell division in yeast, and rrg mutants displayed extensive vacuolization in mitochondria. We propose that Arabidopsis RRG is a conserved mitochondrial protein required for cell division in the root meristem.

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Figures

Figure 1.

Isolation of rrg mutants and map-based cloning of the RRG gene. A, Seven-day-old seedlings of the wild type (WT), rrg-1, and rrg-2 grown vertically on Murashige and Skoog agar medium. B, Physical mapping of the RRG locus. Vertical lines on the top of the image indicate the positions of markers (from left to right: nga80, CER451198, CER461580, CER473407, CER473866, CER481032, CER480837, CER469950, CER450582, and nga111). The numbers below the vertical lines correspond to the recombinants that have a recombination break point between the marker and the RRG locus. BACS, Bacterial artificial chromosomes. C, Structure of the RRG gene and schematic representation of the point mutation in rrg-1 and the T-DNA insertion in rrg-2 in the RRG gene. The black boxes represent the exons, and lines indicate introns. D, Complementation of the rrg-1 mutant by the RRG gene. rrg-1/RRG gDNA indicates an rrg-1 mutant transformed with a vector containing RRG genomic DNA. E, RT-PCR analysis of RRG expression in wild-type and rrg mutant plants.

Figure 2.

Protein sequence analysis and subcellular localization of RRG. A, Deduced amino acid sequence of the RRG gene product and prediction of the protein structure. Mitochondrial signal sequence is indicated by the dashed underline, the DUF155 domain is depicted by the solid underline, and the transmembrane segment is boxed. B to D, Subcellular localization of RRG-GFP in roots of Arabidopsis. B, RRG-GFP. C, MitoTracker Orange staining. D, Colocalization (yellow) of RRG-GFP and MitoTracker Orange. Bars = 5 μm. E to G, Subcellular localization of RRG-GFP in epidermal cells of tobacco leaves. RRG-GFP labels granular structures that colocalize with the mitochondrial marker mt-rk CD3-991. Bars = 30 μm. Arrows point to examples of particles labeled by RRG-GFP, MitoTracker Orange, and mt-rk CD3-991.

Figure 3.

Histochemical analysis of GUS expression in transgenic Arabidopsis plants carrying the RRG::GUS fusion construct. A, GUS expression in 7-d-old RRG::GUS seedlings. The arrow points to the root tip with GUS staining. Bar = 1 mm. B, GUS expression in the tip region of the primary root. C, Cortex; En, endodermis; Ep, epidermis; S, stele. Bar = 100 μm. C, GUS expression in the tip region of the lateral root. Bar = 100 μm. D, GUS expression in the flower. Bar = 0.5 mm.

Figure 4.

Characterization of root phenotypes of rrg mutants. A to C, Confocal optical sections through the root meristem of the wild type (WT; A), rrg-1 (B), and rrg-2 (C). White arrows indicate the transition zone between the meristem and the elongation-differentiation zone. Bars = 50 μm. D to G, Root cellular parameter analyses. Meristem size, mature cortex cell length, cortex cell number in the root meristem, and meristem cortex cell length were obtained as described in “Materials and Methods.” Values shown represent means of at least 30 seedlings. Error bars indicate

. ** P < 0.01 by Student’s t test. H and I, Growth kinematics and cell production analysis of rrg mutant roots. H, Root meristem cell number of wild-type plants and rrg mutants measured over time. I, Rate of cortex cell production in primary roots. At least 15 primary roots from each genotype were assayed for their growth and cell production at each time point indicated. Data shown are average values ±

. J and K, Transmission electron micrographs of mitochondria in meristematic cells of wild-type (J) and rrg-2 (K) roots. Arrows indicate mitochondria. Mitochondria in wild-type cells are structurally normal, while mitochondria in rrg-2 cells display internal vacuolization. Bars = 1 μm.

Figure 5.

Analyses of cell division parameters and expression of cell cycle-related genes in rrg mutants. A, Cell cycle durations in wild-type (WT) and rrg mutant plants, which were estimated according to Tapia-López et al. (2008). B, Expression levels of cell cycle-related genes in the wild type and rrg-2. Data were collected from the qRT-PCR analysis and are shown as averages from triplicate repeats and three biological replicates. Error bars indicate

. * P < 0.05 by Student’s t test. C, CYCB1;1-GFP expression in 7-d-old wild-type and rrg-2 roots. Bars = 50 μm. D, Statistical evaluation of the number of dividing cells per root meristem and the relative GFP-positive cells per unit of root meristem area (the wild-type value of 8.9 × 10−4 ± 0.7 × 10−4 μm2 was set as 100%). Error bars represent

. E and F, Ploidy distributions in 9-d-old seedling roots of wild-type and rrg-2 plants. Average data of three independent measurements are shown in F. Error bars represent ±

Figure 6.

Expression of root patterning and auxin-responsive markers in rrg-2. Expression is shown for SHR::SHR-GFP (A and B), SCR::H2B-YFP (C and D), WOX5::ERGFP (E and F), and auxin-responsive marker DR5rev::GFP (G and H) in 7-d-old seedling roots of the wild type (WT; A, C, E, and G) and rrg-2 (B, D, F, and H).

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The Arabidopsis RETARDED ROOT GROWTH gene encodes a mitochondria-localized protein that is required for cell division in the root meristem - PubMed (original) (raw)