DET1 | Regulator of the proteasomal degradation of LHY

The DET1-154 peptide is smaller in det1-154 mutant genotypes.(A) Representative western blot image showing the reduced DET1 peptide size in ccr2 det1-154 and det1-154 (59 kDa) compared to WT and ccr2 (62 kDa). Gel electrophoresis of the gel membrane from Figure 7C (electrophoresis for 38 min at 165 volt) was extended for 120 min at 100 volts to resolve the 3 kDa difference in DET1-154 protein size. Under these conditions, the 37 kDa ACTIN peptide and pre-stained ladder were not detected on the membrane. Proteins were extracted from WT, ccr2 and ccr2 det1-154 etiolated seedlings grown on MS media without (control; Ctrl) or with the chemical inhibitor of CCD activity (D15).

Chemical inhibition of CCD activity revealed how a ccr2 generated apocarotenoid signal transcriptionally up-regulates POR and PIF3 in parallel to det1-154 during skotomorphogenesis.(A) Transcript levels of PORA, PIF3 and HY5 in WT, ccr2, ccr2 det1-154 and det1-154 etiolated seedlings growing on MS media (+ /- D15). Statistical analysis denoted as a star was performed by a pair-wise t-test (p<0.05). Error bars represent standard error of means. (B), (C) and (D) Representative western blot images showing POR, DET1, PIF3 and HY5 protein levels, respectively. Proteins were extracted from WT, ccr2 and ccr2 det1-154 etiolated seedlings grown on MS media without (control; Ctrl) or with the chemical inhibitor of CCD activity (D15). The membrane was re-probed using anti-Actin antibody as an internal loading control. Lattice-like symbol below POR western (B), represents formation of a PLB in etiolated cotyledons from that genotype and treatment. (E) Model describing how a cis-carotene derived cleavage product, ACS, regulates POR, HY5, PIF3 and PLB formation during skotomorphogenesis. DET1 maintains skotomorphogenesis by post-transcriptionally maintaining a higher and lower PIF3 and HY5 protein levels, respectively. HY5 promotes and PIF3 represses PhANG expression. det1 mutants trigger photomorphogenesis in that they lack POR mRNA transcripts, protein and a PLB. ccr2 generates ACS that enhances POR mRNA transcript and protein levels that enable PLB formation in det1-154. det1-154 restores PLB formation in ccr2 by blocking a signalling pathway acting independent of POR.The DET1-154 peptide is smaller in det1-154 mutant genotypes.(A) Representative western blot image showing the reduced DET1 peptide size in ccr2 det1-154 and det1-154 (59 kDa) compared to WT and ccr2 (62 kDa). Gel electrophoresis of the gel membrane from Figure 7C (electrophoresis for 38 min at 165 volt) was extended for 120 min at 100 volts to resolve the 3 kDa difference in DET1-154 protein size. Under these conditions, the 37 kDa ACTIN peptide and pre-stained ladder were not detected on the membrane. Proteins were extracted from WT, ccr2 and ccr2 det1-154 etiolated seedlings grown on MS media without (control; Ctrl) or with the chemical inhibitor of CCD activity (D15).