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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.

Involvement of rRNA biosynthesis in the regulation of CUC1 gene expression and pre-meristematic cell mound formation during shoot regeneration