FHY1 Mediates phyA Nuclear Import Figure 7. Cytoplasmically localized FHY1 CT induces a dominant negative phenotype. (A) Morphology of seedlings expressing FHY1 CT. Wild-type (Ler), fhy1-1, and phyA-201 seedlings as well as transgenic lines expressing different FHY1 167–202 ( = FHY1 CT) constructs in wild-type background were grown for 5 days in the dark or in FR (15 mmol m22 s21). #2590, #2619, #2643; Ler P35S:YFP-FHY1 167–202 (Ler YFP-FHY1 CT) #4520, #4527; Ler P35S:NLS-YFP-FHY1 167–202 (Ler NLS-YFP-FHY1 CT, i.e. artificial FHY1) #4578, #4597; Ler P35S:NES-YFP-FHY1 167–202 (Ler NES-YFP- FHY1 CT) (B) Cytoplasmically localized FHY1 CT inhibits phyA nuclear accumulation. Ler P35S:NES-YFP-FHY1 167–202 was crossed into phyA-201 PPHYA:PHYA-CFP. F1 seedlings were grown for 3 days in the dark, irradiated for 6 h with FR (15 mmol m22 s21) and used for microscopic analysis. The scale bars represent 10 mm. doi:10.1371/journal.pgen.1000143.g007 response to light and how this import is possible under light conditions where the pool of Pfr is extremely small [13]. Such conditions are typically encountered for phyA-controlled light responses, such as the VLFR and the FR-HIR [1]. Two alternative scenarios for FHY1 function have been proposed, in which nuclear transport of phyA would not depend on FHY1-like proteins and may even be light-independent (i.e. both Pr and Pfr are transported) [26]. In these models (hereafter referred to as the ‘‘FHY1 nuclear anchor’’ and ‘‘protection’’ models) phyA could either be trapped in the nucleus or protected from degradation by binding to FHY1. As the phyA/FHY1 interaction is light dependent, these models would explain the light regulated nuclear accumulation of phyA as well. Yet, these hypotheses are inconsistent with our data for several reasons. In etiolated seedlings phyA protein levels are much higher than FHY1 (data not shown). This renders both the ‘‘FHY1 nuclear anchor’’ and the ‘‘protection’’ models difficult to envisage unless one FHY1 molecule would bind to multiple phyA proteins. In the ‘‘import’’ model one FHY1 molecule would transport one phyA dimer per cycle resulting in nuclear accumulation of large numbers PLoS Genetics | www.plosgenetics.org 8 August 2008 | Volume 4 | Issue 8 | e1000143