Identification of novel meristem factors involved in shoot regeneration through the analysis of temperature-sensitive mutants of Arabidopsis

Adventitious organogenesis in plant tissue culture involves de novo formation of apical meristems and should therefore provide important information about the fundamentals of meristem gene networks. We identified novel factors required for neoformation of the shoot apical meristem (SAM) through an analysis of shoot regeneration in root initiation defective3 (rid3) and root growth defective3 (rgd3) temperature-sensitive mutants of Arabidopsis. After induction of callus to regenerate shoots, cell division soon ceased and was then reactivated locally in the surface region, resulting in formation of mounds of dense cells in which adventitious-bud SAMs were eventually constructed. The rgd3 mutation inhibited reactivation of cell division and suppressed expression of CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and SHOOT MERISTEMLESS (STM). In contrast, the rid3 mutation caused excess ill-controlled cell division on the callus surface. This was intimately related to enhanced and broadened expression of CUC1. Positional cloning revealed that the RGD3 and RID3 genes encode BTAF1 (a kind of TATA-binding protein-associated factor) and an uncharacterized WD-40 repeat protein, respectively. In the early stages of shoot regeneration, RGD3 was expressed (as was CUC1) in the developing cell mounds, whereas RID3 was expressed outside the cell mounds. When RID3 was over-expressed artificially, the expression levels of CUC1 and STM were significantly reduced. Taken together, these findings show that both negative regulation by RID3 and positive regulation by RGD3 of the CUC-STM pathway participate in proper control of cell division as a prerequisite for SAM neoformation.