Poster: Hormones

Abs # 578: A pOp/LhGR glucocorticoid-inducible system allows tight control of expression of a cytokinin biosynthesis gene, ipt, in tobacco

Presenter:	Brzobohaty, Bretislav , brzoboha@ibp.cz
Authors	Brzobohaty, Bretislav  (A) (B)  Samalova, Marketa  (A) (C)  Moore, Ian  (C)   Craft, Judith  (C)   Genkov, Todor  (B)   Malbeck, Jiri  (D)
Affiliations:	(A): Academy of Sciences of the Czech Republic, Institute of Biophysics (B): Masaryk University, Department of Functional Genomics and Proteomics (C): University of Oxford, Department of Plant Sciences (D): Academy of Sciences of the Czech Republic, Institute of Experimental Botany

The potential of a novel glucocorticoid-inducible system, pOp/LhGR, to achieve tightly regulatable expression of a cytokinin biosynthetic gene, ipt, was investigated in tobacco. In LhGR, the transcription activation capacity of a chimeric transcription activation factor LhG4 is brought under chemical control through fusion to the hormone-binding domain (HBD) of the rat glucocorticoid receptor. Near complete tightness of the system was proven by analysis of pOp-ipt::LhGR plants. Three LhGR forms were tested carrying the HBD at the N-terminus of LhG4 (LhGR-N), in between the DNA binding and transcription activation domains (LhGR-I), and at the C-terminus of LhG4 (LhGR-C). When pOp-ipt lines were re-transformed with CaMV35S-LhGR-N and CaMV35S-LhGR-I, no phenotype alterations characteristic of increased levels of cytokinins were observed in the absence of dexamethasone (DEX) while severe phenotype alterations were induced by DEX application. Partial phenotype alterations were observed even in the absence of DEX in pOp-ipt plants retransformed with CaMV35S-LhGR-C indicating partial leakiness of LhGR-C fusion. LC-MS analysis confirmed a dramatic increase in endogenous cytokinins upon DEX application, with cytokinin-N7-glucosides being the prevailing metabolites. Under greenhouse conditions, no phenotype abnormalities were observed in pOp-ipt::CaMV35S-LhGR-I lines, while DEX watering resulted in a pleiotropic cytokinin overproduction syndrome. Dramatic reduction of apical dominance could be separated from inhibition of internode elongation when DEX was applied only to leaf axils. Thus, pOp/LhGR system has proven potential to achieve both spatial and temporal control of ipt expression. Supported by grants IAA5004001, MSM143100008, LN00A081, Z5004920, Royal Society and BBSRC.