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. 2014 Apr 28;5:159.
doi: 10.3389/fpls.2014.00159. eCollection 2014.

Involvement of rRNA Biosynthesis in the Regulation of CUC1 Gene Expression and Pre-Meristematic Cell Mound Formation During Shoot Regeneration

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Free PMC article

Involvement of rRNA Biosynthesis in the Regulation of CUC1 Gene Expression and Pre-Meristematic Cell Mound Formation During Shoot Regeneration

Naoki Shinohara et al. Front Plant Sci. .
Free PMC article

Abstract

At an early stage of shoot regeneration from calli of Arabidopsis, pre-meristematic cell mounds develop in association with localized strong expression of CUP-SHAPED COTYLEDON (CUC) genes. Previous characterization of root initiation-defective 3 (rid3), an Arabidopsis mutant originally isolated as being temperature-sensitive for adventitious root formation, with respect to shoot regeneration implicated RID3 in the negative regulation of CUC1 expression and the restriction of cell division in pre-meristematic cell mounds. Positional cloning has identified RID3 as a WD40 repeat protein gene whose molecular function was not investigated before. Here we performed in silico analysis of RID3 and found that RID3 is orthologous to IPI3, which mediates pre-rRNA processing in Saccharomyces cerevisiae. In the rid3 mutant, rRNA precursors accumulated to a very high level in a temperature-dependent manner. This result indicates that RID3 is required for pre-rRNA processing as is IPI3. We compared rid3 with rid2, a temperature-sensitive mutant that is mutated in a putative RNA methyltransferase gene and is impaired in pre-rRNA processing, for seedling morphology, shoot regeneration, and CUC1 expression. The rid2 and rid3 seedlings shared various developmental alterations, such as a pointed-leaf phenotype, which is often observed in ribosome-related mutants. In tissue culture for the induction of shoot regeneration, both rid2 and rid3 mutations perturbed cell-mound formation and elevated CUC1 expression. Together, our findings suggest that rRNA biosynthesis may be involved in the regulation of CUC1 gene expression and pre-meristematic cell-mound formation during shoot regeneration.

Keywords: CUC1; RID2; RID3; WD40 repeat protein; rRNA biosynthesis; shoot regeneration.

Figures

Figure 1
Figure 1
Identification of RID3 orthologs. (A) Graphical representation of a reciprocal best-hit cluster. Dots and curve lines represent protein sequences and reciprocal best-hit relationships, respectively. Each row contains two similar sequences from one species. (B) A maximum-likelihood tree generated by protein sequences. Numbers on branches indicate bootstrap values when more than 70 in 100 repetitions. For each taxon, an organism name, a protein name alias (shaded if its mutant has been reported), and an E-value to RID3 are shown (for details of each sequence, see Supplementary Table S2). (C) Clustal W alignment of amino acid sequences of Arabidopsis RID3, Drosophila WDR18, Dictyostelium WDR18, and budding yeast (Saccharomyces) IPI3. Identical and similar residues are shaded with black and gray, respectively. Blue lines represent WD-40 repeat domains of RID3.
Figure 2
Figure 2
Effect of rid3 mutation on rRNA precursor accumulation. (A) Pre-rRNA processing pathway in Arabidopsis based on Zakrzewska-Placzek et al. (2010). (B) RNA gel blot analysis of rRNA precursors. Seedlings of rid3 and wild type (WT) were cultured at 19, 22, 25, or 28°C for 16 days after sowing. Seedlings of rid2 and rgd3 were also cultured under the same condition as references. Total RNA samples were prepared from these seedlings and 3 μg/lane of RNA was electrophoresed. RNA gel blot analysis was performed with a probe specific to ITS1. rRNA bands visualized by staining with GelRed (Biotium) are shown as an equal loading control.
Figure 3
Figure 3
Morphological phenotypes of seedlings cultured at various temperatures. (A) Cotyledons and first several true leaves of 12-day-old seedlings of rid2, rid3, rgd3, and wild type (WT) grown at 19, 22, 25, or 28°C. Bar = 5 mm. (B) Overall appearances of 16-day-old plants. Bar = 2 cm. (C) Leaf phenotypes 16-day-old plants. Bars = 1 cm.
Figure 4
Figure 4
Shoot regeneration-related phenotypes of hypocotyl explants cultured at various temperatures. (A) Time course of shoot regeneration from hypocotyl explants of rid2, rid3, rgd3, and wild type (WT). Explants were cultured on SIM at 19, 22, 25, or 28°C after 6 days of pre-culture on CIM at 19°C. n = 24. (B) Morphological changes of explants during culture on SIM. Photographs in each row indicate the same explant at different times. Insets show magnified images of the squared regions. Red arrowheads indicate irregularly large mounds of cells. Bar = 5 mm.
Figure 5
Figure 5
Real-time PCR analysis of expression of CUC1 and STM in hypocotyl explants cultured on SIM. Hypocotyl explants of rid2, rid3, rgd3, and wild type (WT) were cultured on SIM at 19, 22, 25, or 28°C after 6 days of pre-culture on CIM at 19°C. Total RNA was prepared from explants at 0, 5, and 8 day after transfer onto SIM and used for real-time PCR analysis of CUC1 and STM expressions. Means and standard deviations of normalized relative quantities (relative to the mean of the 0-day samples of wild type) of biological triplicates are shown.
Figure 6
Figure 6
CUC1p::CUC1:GUS expression in hypocotyl explants of rid2 and wild-type cultured on SIM. Hypocotyl explants of rid2 and wild-type (WT) carrying CUC1p::CUC1:GUS were cultured on SIM at 19°C or 25°C after 6 days of pre-culture on CIM at 19°C and subjected to histochemical detection of GUS activity. Bar = 50 μm.

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