Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF

SUMMARYCytokinins are involved in many aspects of plant growth and development, and physiological evidence alsoindicates that they have a role in floral transition. In order to integrate these phytohormones into the currentknowledge of genetically defined molecular pathways to flowering, we performed exogenous treatmentsof adult wild type and mutant Arabidopsis plants, and analysed the expression of candidate genes. We used ahydroponic system that enables synchronous growth and flowering of Arabidopsis, and allows the preciseapplication of chemicals to the roots for defined periods of time. We show that the application of N 6 -benzylaminopurine (BAP) promotes flowering of plants grown in non-inductive short days. The response tocytokinin treatment does not require FLOWERING LOCUS T (FT), but activates its paralogue TWIN SISTER OFFT (TSF), as well as FD, which encodes a partner protein of TSF, and the downstream gene SUPPRESSOR OFOVEREXPRESSIONOFCONSTANS 1(SOC1).TreatmentofselectedmutantsconfirmedthatTSFandSOC1arenecessaryforthefloweringresponsetoBAP,whereastheactivationcascademightpartiallyactindependentlyof FD. These experiments provide a mechanistic basis for the role of cytokinins in flowering, and demonstratethat the redundant genes FT and TSF are differently regulated by distinct floral-inducing signals.Keywords: cytokinins, flowering, Arabidopsis, florigen, hydroponics.INTRODUCTIONCytokinins are important phytohormones that were firstidentified as factors promoting cell proliferation and shootformation in vitro. These effects are now at least partlyexplained by the observations that cytokinins activate cell-cycle genes and interact with genetic regulators of stem cellnumber within the shoot apical meristem (SAM) (Riou-Khamlichi et al., 1999; Rupp et al., 1999; Leibfried et al.,2005; Yanai et al., 2005; Gordon et al., 2009). Cytokinins areinvolved in many other aspects of plant growth and devel-opment, including vascular cambium activity, chloroplastdevelopment, response to nutrients and senescence, as wellas shoot and root branching. Despite the elucidation ofseveral pathways that regulate the transition from vegeta-tive growth to flowering in Arabidopsis (Amasino, 2010;Fornara et al., 2010), the role of cytokinins remains unclear.Repeated applications of cytokinin activated the floweringof relatively old vegetative plants, but not of younger ones(Michniewicz and Kamienska, 1965; Besnard-Wibaut, 1981;Dennis et al., 1996). In vitro, positive (Chandler and Dean,1994), null (Brandstatter and Kieber, 1998) or negative(Riefler et al., 2006) effects were reported, suggesting thatprecise environmental conditions might have an effect onthe response (Kinet et al., 1993).The understanding of the regulation of cytokinin synthe-sis, catabolism and signalling has advanced recentlythrough the identification of genes encoding metabolicenzymes, receptors and response regulators (reviewed inSakakibara, 2006; Hirose et al., 2008; Werner and Schmu¨l-ling, 2009; Kudo et al., 2010; Perilli et al., 2010). However,redundancy is an obstacle to using genetics to examine thebiologicalroleofcytokininsinflowering:metabolicenzymesand signalling components are encoded by multigenefamilies, so that single mutants are similar to wild-type(WT) plants, whereas multiple mutants are impaired ingrowth, generating complex pleiotropic phenotypes. Forexample, mutants deficient in all three cytokinin receptors