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.
Highlights
In the course of shoot regeneration from calli, callus-derived cells are newly assembled into shoot apical meristems (SAMs), which eventually develop into adventitious buds
In the model plant Arabidopsis thaliana (Arabidopsis), vigorous shoot regeneration can be induced from either root or hypocotyl segments by a two-step tissue culture procedure consisting of pre-culture on callus-inducing medium (CIM), which is rich in both auxin and cytokinin, and the subsequent culture on shoot-inducing medium (SIM), which contains both auxin and cytokinin but in a higher cytokinin/auxin ratio (Valvekens et al, 1988; Akama et al, 1992)
As far as we investigated, contained only one RID3 ortholog, and WD40 proteins whose similarities to the RID3 ortholog were as low as that of RID3 to the closest Arabidopsis homolog (Supplementary Table S2)
Summary
In the course of shoot regeneration from calli, callus-derived cells are newly assembled into shoot apical meristems (SAMs), which eventually develop into adventitious buds. This SAM construction can be regarded as a de novo cell organization process free from the regulation by pre-existing structures in the plant body, and exemplifies the spontaneous nature of plant organogenesis. Transfer onto SIM changes cell proliferation pattern in the calli so that spots of active proliferation emerge and give rise to cell mounds on the callus surface Those cell mounds further form SAMs. Those cell mounds further form SAMs These morphological changes are associated with expression of genes that regulate
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