14-3-3 proteins participate in light signaling through association with PHYTOCHROME INTERACTING FACTORs

Abstract

14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ) could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1). 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.

Figure 1

(A) Yeast two-hybrid analysis of 14-3-3κ with PIF3 and PIF3ala1+2. The interaction was determined by filter assay. A combination of pEXP32-Krev1 with pEXP22-RalGDS-wt was used as a positive control and pEXP32-Krev1 with pEXP22-RalGDS-m2 as a negative control; (B) Western blot analysis of HIS-14-3-3κ pulled down with maltose-binding protein (MBP, negative control), MBP-PIF3, MBP-PIF3ala1+2 bound to amylose resin beads and 3% of total input blotted with antiHis antibody; (C) Western blot analysis of His-14-3-3κ pulled down with MBP (negative control), MBP-PIFs bound to amylose resin beads and 5% of total input blotted with antiHis antibody; and (D) Western blot analysis of MBP-PIFs after pull-down and 5% of total input blotted with antiMBP antibody. Asterisks indicate the signal for each MBP-bound PIF protein. These experiments were repeated multiple times and representative data are shown.

Figure 1

(A) Yeast two-hybrid analysis of 14-3-3κ with PIF3 and PIF3ala1+2. The interaction was determined by filter assay. A combination of pEXP32-Krev1 with pEXP22-RalGDS-wt was used as a positive control and pEXP32-Krev1 with pEXP22-RalGDS-m2 as a negative control; (B) Western blot analysis of HIS-14-3-3κ pulled down with maltose-binding protein (MBP, negative control), MBP-PIF3, MBP-PIF3ala1+2 bound to amylose resin beads and 3% of total input blotted with antiHis antibody; (C) Western blot analysis of His-14-3-3κ pulled down with MBP (negative control), MBP-PIFs bound to amylose resin beads and 5% of total input blotted with antiHis antibody; and (D) Western blot analysis of MBP-PIFs after pull-down and 5% of total input blotted with antiMBP antibody. Asterisks indicate the signal for each MBP-bound PIF protein. These experiments were repeated multiple times and representative data are shown.

Figure 2

Subcellular localization of GFP, GFP-PIF3, GFP-PIF3ala1 and GFP-PIF3ala1+2. Fluorescence (left) and light field (right) images of onion epidermis cells bombarded with corresponding DNA are shown. The scale bar indicates 100 μm.

Figure 3

Hypocotyl lengths of 14-3-3 mutants grown in continuous red (cR) light. Col-0 (wild-type), κ1 (salk_148929), κ2 (salk_001375), χ1 (salk_150150), χ2 (salk_142285), κχ double-mutant (salk_001375; salk_142285), pif3-3, MYC-PIF3 and phyB were germinated and grown in cR light for five days. Statistically significant differences compared to the wild-type are indicated with asterisks (n > 11): * p < 0.05, ** p < 0.01, *** p < 0.001. These experiments were repeated multiple times, and representative data are shown.

Figure 4

Model for the mode of action of 14-3-3κ in the context of light signaling. I. Upon light illumination, phytochromes (Phy) rapidly convert to an active form and translocate into a nuclear speckle where they bind to negative regulators of light signalling, PHYTOCHROME INTERACTING FACTORs (PIFs). An E3 ligase, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) polyubiquitinates Phy, but not PIFs, to be degraded by the 26S proteasome, serving as a negative feedback mechanism in light signaling; II. Phy-bound PIFs are phosphorylated, E3 ligases, LIGHT-RESPONSE BTBs (LRBs), polyubiquitinate both Phy and PIFs to be degraded by the 26S proteasome, and consequently photomorphogenesis is initiated. 14-3-3κ is predicted to bind with phosphorylated-PIFs and either escort LRBs to the Phy-PIF complex or stabilize the LRB-Phy-PIF complex to promote PIFs degradation as positive regulators of light signalling.