Fig 1.
FIN219 and CRY1 regulate each other in modulating hypocotyl elongation of Arabidopsis seedlings under different light conditions.
(A) FIN219 function in regulating light-mediated inhibition of hypocotyl elongation requires functional CRY1 and CRY2. Hypocotyl growth responses of 4-day-old seedlings under different light conditions. (Left to right) Wild type (Col-0), FIN219-OE, cry1, FIN219-OE/cry1, cry2, FIN219-OE/cry2, cry1cry2 and FIN219-OE/cry1cry2. Blue light: 2.2 μmol•m-2•s-1, far-red light (FR): 3.1 μmol•m-2•s-1, red light: 7.5 μmol•m-2•s-1, white light: 8 μmol•m-2•s-1. Scale bar = 2 mm. (B) Quantification of hypocotyl lengths of seedlings shown in (A). Data are mean ± SD (n ≥ 20). Different lowercase letters represent significant differences by one-way ANOVA at P <0.05. (C) FIN219 overexpression in GUS-CCT1 transgenic plants can rescue the short-hypocotyl phenotype of GUS-CCT1 under all light conditions except red light. Hypocotyl growth responses of 4-day-old seedlings under various light conditions, including the dark. (Left to right) wild type (Col-0), fin219, FIN219-OE, FIN219-OE/GUS-CCT1 #11–21, #13–4, GUS-CCT1 and cry1. Blue light: 2.2 μmol•m-2•s-1, far-red light (FR): 3.1 μmol•m-2•s-1, red light: 7.5 μmol•m-2•s-1, white light: 8 μmol•m-2•s-1. Scale bar = 2 mm. (D) Quantification of hypocotyl lengths of seedlings shown in C. Data are mean ± SD (n ≥ 20). Different lowercase letters represent significant differences by one-way ANOVA at P <0.05.
Fig 2.
FIN219 and CRY1 antagonize each other under blue light.
(A) CRY1 and CRY2 negatively regulate FIN219 protein level under blue light. Western blot analysis of FIN219 protein level in wild-type Col-0, cry1, cry2 mutants and transgenic seedlings grown under blue light. The signal was detected by FIN219 monoclonal antibody. Blue light: 2.2 μmol•m-2•s-1. The number below each blot represents the level of the indicated protein. The level of wild-type Col-0 was arbitrarily set to 1. The asterisk (*) indicates nonspecific bands. (B) FIN219 overexpression in GUS-CCT1 seedlings abolishes GUS-CCT1 fusion protein in blue light. Western blot analysis of protein levels in Col-0, fin219-2, FIN219 overexpression line (FIN219-OE), FIN219-OE/GUS-CCT1, GUS-CCT1 and cry1 seedlings grown in blue light for 3 days. The blots were detected by antibodies against FIN219 and GUS-CCT1 and HY5. Blue light: 2.2 μmol•m-2•s-1. RPN8 was a loading control. The asterisk (*) indicates nonspecific bands. The number below each blot represents the level of the indicated protein. The level of wild-type Col-0 was arbitrarily set to 1. (C) GUS-CCT1 transcripts detected in transgenic seedlings of FIN219-OE/GUS-CCT1 under blue light. Quantitative Real-time PCR (qPCR) analysis of transgenic seedlings shown in B. Total RNAs were extracted from transgenic seedlings shown in the figure and subjected for qPCR analysis. Ubiquitin 10 (UBQ10) was an internal control. (D) GUS-CCT1 fusion proteins were stable in fin219-2/GUS-CCT1 seedlings under blue light. Western blot analysis of GUS-CCT1 level in Col-0, FIN219-OE/GUS-CCT1 (#13–4), fin219-2/GUS-CCT1 and PGR219/GUS-CCT1 seedlings grown in blue light for 4 days. Total proteins extracted from seedlings were probed with GUS antibody. M, protein size markers.
Fig 3.
Degradation of GUS-CCT1 fusion proteins in FIN219-OE/GUS-CCT1 seedlings is mediated by 26S proteasome under blue light.
(A) Western blot analysis of GUS-CCT1 level in FIN219-OE/GUS-CCT1 seedlings transferred from the dark to blue light for different times (B15: 15 min blue light; B60: 60 min blue light) as shown above the figure. “+”: with MG132; “—”: without MG132. (B) Western blot analysis of GUS-CCT1 protein level in FIN219-OE/GUS-CCT1 seedlings transferred from the dark to blue light for different times shown above the figure. fin219-2/GUS-CCT1 seedlings were grown for 4 days under blue light. B1: 1-h blue light; B3: 3-h blue light; B24: 24-h blue light; B24M: 24-h blue light plus MG132; 4DB: 4 days of blue light.
Fig 4.
FIN219 affects GUS-CCT1 stability under the dark and blue light.
(A) Exogenous MeJA slightly reduced GUS-CCT1 level in GUS-CCT1 seedlings under continuous blue light and dark. Western blot analysis of GUS-CCT1 and FIN219 protein levels in GUS-CCT1 seedlings grown in continuous blue light and the dark without or with MeJA for 3 days. (B) Exogenous MeJA greatly decreased GUS-CCT1 level in fin219-2/GUS-CCT1 seedlings under continuous blue light and dark. Western blot analysis of GUS-CCT1 level in fin219-2/GUS-CCT1 seedlings grown in dark and continuous blue light without or with MeJA for 3 days. GUS-CCT1 was detected by antibodies against GUS.
Fig 5.
MeJA greatly enhanced FIN219 and GUS-CCT1 interaction under continuous blue light.
(A) Schematic diagrams of different constructs corresponding to the full-length FIN219 (GST-FIN219), the N terminus and C terminus of FIN219 (GST-FIN219N and GST-FIN219C) and the full-length cryptochrome CRY1 (CBP-CRY1), the N terminus and C terminus of CRY1 (CBP-CNT1 and CBP-CCT1). (B) Pull-down assay of FIN219 interacting with CRY1 via its C terminus. Recombinant proteins GST, GST-FIN219, GST-FIN219N or GST-FIN219C were mixed with CBP-CRY1 and underwent protein pull-down assays (left panel). The asterisk (*) indicates nonspecific bands. The arrowhead represents GST-FIN219C. Right panel: Recombinant proteins CBP-CRY1, CBP-CNT, or CBP-CCT1 were mixed with GST-FIN219 and underwent protein pull-down assays. The mixtures were immunoprecipitated with glutathione sepharose for GST-tag, then probed with antibodies against CBP-tag. Upper and lower arrowheads represent CBP-CRY1 and CBP-CCT1, respectively. (C) BiFC assays showing FIN219 and CRY1 or COP1 interaction in the dark (top panel) and blue light (BL) (bottom panel). The protoplasts isolated from short-day grown Col-0 were transfected with YN-CRY1 or COP1 and YC-FIN219 without (–) or with (+) 50 μM MeJA treatment. Blue light (BL): 2.2 μmol•m-2•s-1. (D) Co-immunoprecipitation assay showing FIN219 and GUS-CCT1 interaction greatly enhanced by MeJA under blue light. GUS-CCT1 transgenic seedlings were grown in the dark and blue light for 4 days with (+) or without (–) MeJA. Total proteins 2 mg extracted from seedlings were immunoprecipitated with FIN219 monoclonal antibodies, then probed with GUS and COP1 polyclonal antibodies. (E) Co-immunoprecipitation assay showing FIN219 and CRY1 interaction greatly enhanced by MeJA under the dark. Wild-type Col-0 and fin219-2 seedlings were grown in the dark and blue light for 4 days with (+) or without (–) MeJA. Total proteins 2 mg extracted from seedlings were immunoprecipitated with FIN219 monoclonal antibodies, and then probed with CRY1 polyclonal antibodies.
Fig 6.
Changes in CRY1 levels affected MeJA-mediated physiological responses to various light conditions.
The cry1 mutant showed a hypersensitive response to MeJA-inhibited hypocotyl elongation under dark (A), low blue light (2 μmol•m-2•s-1) (B) and high blue light (15 μmol•m-2•s-1) as compared to the wild-type Col (C). Seedlings of wild-type Col, fin219-2, cry1, FIN219-OE, GUS-CCT1, FIN219-OE/GUS-CCT1, and fin219-2/GUS-CCT1 were grown on GM plates without or with 50 μM MeJA for 3 days, then hypocotyl elongation with MeJA was analyzed. Different lowercase letters represent significant differences by one-way ANOVA at P <0.05. (D) Ectopic expression of FIN219 reduced anthocyanin content in GUS-CCT1 seedlings under different light conditions, including the dark. Seedlings were grown in various light conditions for 3 days, then anthocyanin content was determined. Different lowercase letters represent significant differences by one-way ANOVA at P <0.05. (E) Ectopic expression of FIN219 reduced GUS-CCT1 seedling resistance to Pseudomonas syringae pv. tomato (Pst.) DC3000 infection. Leaves of 5-week-old Arabidopsis plants grown under short-day conditions were inoculated with Pst. DC3000. Phenotypic response was shown in top panel and quantification of the bacterial number in each sample was in lower panel. Different lowercase letters represent significant differences by one-way ANOVA at P <0.05.
Fig 7.
A model illustrates the effect of FIN219 levels on cry1 functions.
(A) In the dark, FIN219 interacts with the C terminus of CRY1 (CCT1) to likely abolish CRY1 functions. Meanwhile, the COP1–SPA1 complex interacts with HY5, thereby degrading HY5 protein for skotomorphogenic development of Arabidopsis seedlings. (B) In blue light, FIN219 interacts closely with CRY1 as well as COP1, resulting in cry1-mediated photomorphogenesis. (C) In the presence of blue light, induction of FIN219 by MeJA strongly interacts with CCT1, and weakly with COP1–SPA1 complex, leading to a rescued phenotype of GUS-CCT1 seedlings by suppressing hypersensitive short-hypocotyl phenotype.