Cytokinin Response Factor 9 Represses Cytokinin Responses in Flower Development.

A multi-step phosphorelay system is the main conduit of cytokinin signal transduction. However, several groups of additional factors that also play a role in this signaling pathway have been found-among them the Cytokinin Response Factors (CRFs). In a genetic screen, CRF9 was identified as a regulator of the transcriptional cytokinin response. It is mainly expressed in flowers. Mutational analysis indicates that CRF9 plays a role in the transition from vegetative to reproductive growth and silique development. The CRF9 protein is localized in the nucleus and functions as a transcriptional repressor of Arabidopsis Response Regulator 6 (ARR6)-a primary response gene for cytokinin signaling. The experimental data suggest that CRF9 functions as a repressor of cytokinin during reproductive development.

Novel in vivo screening design for the rapid and cost-effective identification of transcriptional regulators.

Genetic screens are a common tool to identify new modulators in a defined context, e.g. hormonal response or environmental stress. However, most screens are either in vitro or laborious and time-and-space inefficient. Here we present a novel in planta screening approach that shortens the time from the actual screening process to the identification of a new modulator and simultaneously reduces space requirements and costs. The basic features of this screening approach are the creation of luciferase reporter plants which enable a non-invasive readout in a streamlined multiplate reader process, the transformation of those plants with an inducible, Gateway™-compatible expression vector, and a screening setup, in which whole plants at the seedling stage are screened in 96-multiwell plates in the first transformed generation without the use of an expensive charge-coupled device (CCD) camera system. The screening itself and the verification of candidates can be done in as little as 2-3 weeks. The screen enables the analysis of reporter gene activity upon different treatments. Primary positive plants can immediately be selected and grown further. In this study a fast, simple, cost- and space-efficient in planta screening system to detect novel mediators of a given transcriptional response was developed and successfully tested using the cytokinin signal transduction as a test case.