Acquiring competence for shoot development in Arabidopsis: ARR2 directly targets A-type ARR genes that are differentially activated by CIM preincubation.

Shoots can be regenerated from roots in Arabidopsis by treating root explants with cytokinin, however, shoot regeneration requires preincubation on callus induction medium (CIM) prior to induction on cytokinin-rich shoot induction medium (SIM). A cytokinin-inducible marker gene, RESPONSE REGULATOR 15 (ARR15), was identified through a "CIM dropout experiment" with similar requirements for CIM preincubation. The requirements for ARR15 contrasted to ARR5, another cytokinin-inducible ARR gene that does not require CIM preincubation. We show here that despite their differences, both ARR5 and ARR15 are direct targets of the transcriptional B-type response regulator, ARR2. This was demonstrated by identifying genes upregulated following beta estradiol induced nuclear relocation of an ARR2-estradiol receptor fusion protein. The differences in CIM preincubation requirements for ARR5 and ARR15 expression indicate an additional layer of control for these A-type ARR genes during SIM incubation. For ARR15, the CIM requirement is a transcriptional effect, because the expression of ARR15 promoter:GUS reporter gene constructs is also affected by CIM preincubation. A testable model is that transcription of ARR15, but not ARR5, is blocked by a repressor and that the effects of the repressor are relieved by CIM preincubation.