CLCuMuB βC1 hinders the degradation of YFP-GAI in vivo.

(A) CLCuMuB βC1 attenued degradation of YFP-GAI in vivo. YFP-GAI expression construct was coinfiltrated with constructs expressing HA-nLUC or HA-βC1 into seven to eight-week-old N. benthamiana plant leaves. Around 48 hpi, agroinfiltrated leaves were sprayed with 100 μM GA₃ or mock solution (ethonal) and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scales represents 200 μm. DMSO and MG132 (50 μM) were applied into plant leaves 12 h before observation. Protein level was analyzed via SDS-PAGE and western blot analysis with the anti-GFP antibody, which also recognizes YFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control. (B) Real-time RT-PCR detected the mRNA level of YFP-GAI. Total RNA was extracted from each N. benthamiana leaves and then subjected to quantitative RT-PCR (means±SEM, n = 3) to quantify YFP-GAI mRNA level. eIF4a was used as the internal reference. (C) CLCuMuB βC1 didn’t affect stability of GFP in vivo. Detection of GFP (as an internal control) in N. benthamiana leaves coinfiltrated with the construct expressing GFP together with constructs expressing HA-nLUC or HA-βC1 and treated with 100 μM GA₃ or mock (ethanol) solution and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scale represents 200 μm. Protein level was analyzed via SDS-PAGE and immunoblot analysis with anti-GFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control.