Minisymposium 18: Seed Biology

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Abs # M1802: A KELCH REPEAT CONTAINING F-BOX protein targets PHYTOCHROME INTERACTING FACTOR1 (PIF1) and PIF3 for proteasome-mediated destruction.

Presenter:	Downie, Bruce       Contact Presenter
Authors	Majee, Manoj  (A)   Downie, Bruce  (A)
Affiliations:	(A): University of Kentucky

Phytochromes control light-mediated phenomenon by interaction with transcription factors eg. PHYTOCHROME INTERACTING FACTOR 1 (PIF1) and PIF3, thus influencing transcription. These bHLH transcription factors are known to be subject to polyubiquitination upon illumination, leading to their destruction by the 26S proteasome, but how they are recognized for ubiquitination in response to light is still unknown. We identified a KELCH REPEAT CONTAINING F-BOX PROTEIN (KFB) in a screen of activation tagged mutant lines for seeds capable of completing germination at 10⁰C ahead of wild type. This F-BOX protein, one of hundreds encoded in the Arabidopsis genome and important components of E3-SCF ligase complexes, targets both PIF1 and PIF3 for ubiquitination. Subsequent destruction promotes seed germination (PIF1) and positively influences phytochrome-mediated inhibition of hypocotyl elongation under both red (PIF3) and far-red (PIF1) light. The causality between KFB gene up-regulation and rapid seed germination was confirmed in KFB over-expressing lines (kfb-D) while an insertional mutant (kfb) completed germination poorly in the dark. The KFB interacts with Arabidopsis SKP1-like proteins in yeast two hybrid (Y2H) experiments, verifying the existence of the predicted F-BOX. Y2H analysis also identified the target proteins to which the kelch-repeat binds, PIF1 and PIF3. As expected from Y2H results, kfb-D seeds are capable of completing germination in darkness or in cycles of far-red/darkness, similar to pif1 mutant seeds. Similar to pif1-D seeds, kfb mutant seeds complete germination poorly in darkness. kfb-D has a shorter hypocotyl under far-red and red light relative to controls, similar to pif1 and pif3 mutants, respectively. The inability of pif1-D mutant seeds to complete germination in darkness was rescued in pif1-D, kfb-D, double mutant seeds while, in contrast to kfb mutant seeds, the kfb, pif1 double mutant seeds complete germination in darkness. Also consistent with the hypothesis that the KFB is targeting PIF1 for proteasome-mediated destruction, the kfb-D mutants are hyper-sensitive to bioactive GAs and, like the pif1 mutant, transcript amounts for GA3 β HYDROXLASES (GA4, GA4H) are greater, while mRNA for a GA metabolizing enzyme (GA2 OX) are less, than in seeds of wild type or vector controls. These results support our contention that the KFB protein targets PIF1 and PIF3 for ubiquitin-mediated protein degradation.