Light Regulates the RUBylation Levels of Individual Cullin Proteins in Arabidopsis thaliana

Abstract

In plants, the small protein related to ubiquitin (RUB) modifies cullin (CUL) proteins in ubiquitin E3 ligases to allow for efficient transfer of ubiquitin to substrate proteins for degradation by the 26S proteasome. At the molecular level, the conjugation of RUB to individual CUL proteins is transient in nature, which aids in the stability of the cullins and adaptor proteins. Many changes in cellular processes occur within the plant upon exposure to light, including well-documented changes in the stability of individual proteins. However, overall activity of E3 ligases between dark- and light-grown seedlings has not been assessed in plants. In order to understand more about the activity of the protein degradation pathway, overall levels of RUB-modified CULs were measured in Arabidopsis thaliana seedlings growing in different light conditions. We found that light influenced the global levels of RUBylation on CULs, but not uniformly. Blue light had little effect on both Cul1 and Cul3 RUBylation levels. However, red light directed the increase in Cul3 RUBylation levels, but not Cul1. This red-light regulation of Cul3 was at least partially dependent on the activation of the phytochrome B signaling pathway. The results indicate that the RUBylation levels on individual CULs change in response to different light conditions, which enable plants to fine-tune their growth and development to the various light environments.

Keywords: Cullin; NEDD8; Photomorphogenesis; Phytochrome; RUB; Ubiquitin.

Fig. 1

The RUBylation level of CULs increase in light. a, b Immunoblot analysis of total protein extracts from 4-day-old Col-0 or HSN seedlings grown in Dk, W (56 μMol m⁻² s⁻¹), B (10 μMol m⁻² s⁻¹) or R (50 μMol m⁻² s⁻¹). HSN–Dex seedlings were grown in W. Total RUB and RUB-CUL or HSN and HSN-CUL were detected with anti-Nedd8 or anti-HA antibody respectively. PBA1 was used as a loading control. c, d Quantification of the relative change in band intensity in light vs Dk for PBA1 or RUB-CUL displayed as a ratio of light (W, B, or R)/Dk. Asterisks identify significant differences in the fold change between PBA1 and the RUB/HSN-CUL (p ≤ 0.05, Student’s T test). Error bars represent standard deviation (n = 3)

Fig. 2

The RUBylation level of AtCul1 in response to light. a, b Immunoblot analysis with anti-Cul1 antibody of total protein extracts from 4-day-old Col-0 or HSN seedlings grown in Dk, W (56 μMol m⁻² s⁻¹), B (10 μMol m⁻² s⁻¹), or R (50 μMol m⁻² s⁻¹). HSN–Dex seedlings were grown in W. PBA1 was used as a loading control. c, d Quantification of RUB-Cul1 or HSN-Cul1 levels displayed as a ratio of RUB-Cul1 or HSN-Cul1 to unmodified Cul1 levels. Asterisks identify significant differences between D and light samples (p ≤ 0.05, Student’s T test). Error bars represent standard deviation (n = 3)

Fig. 3

The RUBylation level of Cul3 increases in W and R light. Immunoblot analysis of total protein extracts from 4-day-old seedlings with anti-Cul3 antibody. a Col-0, cul3a-1, and cul3b-1 were exposed to R (50 μMol m⁻² s⁻¹). PBA1 was used as a loading control. b, c Col-0 or HSN seedlings grown in Dk, W (56 μMol m⁻² s⁻¹), B (10 μMol m⁻² s⁻¹), or R (50 μMol m⁻² s⁻¹). HSN–Dex seedlings were grown in W. PBA1 was used as a loading control. The short exposure shows the relative levels of unmodified Cul3. d, e Quantification of RUB-Cul3 or HSN-Cul3 levels displayed as a ratio of RUB-Cul3 or HSN-Cul3 to unmodified Cul3 levels. Asterisks identify significant differences between Dk and light samples (p ≤ 0.05, Student’s T test). Error bars represent standard deviation (n = 3)

Fig. 4

The RUBylation level of Cul3 and total CUL increases with increasing R intensity. a, c Immunoblot analysis of total protein extracts from 4-day-old Col-0 or HSN seedlings grown in Dk or R (5, 20, 50, and 100 μMol m⁻² s⁻¹). HSN–Dex seedlings were grown in R (50 μMol m⁻² s⁻¹). Total RUB and RUB-CUL or HSN and HSN-CUL were detected with anti-Nedd8 or anti-HA antibody respectively. Cul3 was detected with anti-Cul3 antibody. PBA1 was used as a loading control. The short exposure shows the relative levels of unmodified Cul3. b, d Quantification of the relative change in band intensity in R vs Dk of RUB-CUL, HSN-CUL, or RUB-Cul3/Cul3 displayed as a ratio of R/Dk. Error bars represent standard deviation (n = 3)

Fig. 5

cul3 mutants do not show significantly lower levels of RUB-CUL in R. a Immunoblot analysis of total protein extracts from 4-day-old Col-0, cul3a-1, and cul3b-1 seedlings grown in Dk or R (50 μMol m⁻² s⁻¹). Total RUB and RUB-CUL were detected with anti-Nedd8 antibody. PBA1 was used as a loading control. b, c Quantification of the relative change in band intensity in cul3a-1 or cul3b-1 vs Col-0 for PBA1 or RUB-Cul displayed as a ratio of cul3/Col-0 in Dk or R. Error bars represents standard deviation (n = 3)

Fig. 6

The RUBylation level of Cul3, but not Cul1 increases in R, but not FR light. a, d Immunoblot analysis of total protein extracts from 4-day-old seedlings grown in, Dk, R (50 μMol m⁻² s⁻¹) and FR (10 μMol m⁻² s⁻¹). HSN–Dex seedlings were grown in R. Total RUB and RUB-CUL or HSN and HSN-CUL were detected with anti-Nedd8 or anti-HA antibody respectively. Cul1 and 3 were detected with anti-Cul1 or -Cul3 antibody respectively. PBA1 was used as a loading control. The short exposure shows the relative levels of unmodified Cul3. b, e Quantification of RUB-Cul or HSN-Cul levels displayed as a ratio of RUB-Cul or HSN-Cul to unmodified Cul levels. Asterisks identify significant differences between D and light samples (p ≤ 0.05, Student’s T test). c, f Quantification of the relative change in band intensity in light (R or FR) vs Dk for PBA1, RUB-CUL, and HSN-CUL displayed as a ratio of light (R or FR)/Dk. Asterisks identify significant differences in the fold change between PBA1 and the RUB/HSN-CUL (p ≤ 0.05, Student’s T test). Error bars represents standard deviation (n = 3)

Fig. 7

The RUBylation levels of Cul3 in R are partially dependent on active phyB. a Immunoblot analysis of total protein extracts from 4-day-old Col-0 and phyB-9 seedlings grown in D and R (50 μMol m⁻² s⁻¹). Total RUB and RUB-CUL were detected with anti-Nedd8 antibody, respectively. Cul3 was detected with anti-Cul3 antibody. PBA1 was used as a loading control. b Quantification of RUB-Cul3 levels displayed as a ratio of RUB-Cul3 to unmodified Cul3 levels. Asterisks identify significant differences between D and light samples (p ≤ 0.05, Student’s T test). c Quantification of the relative change in band intensity in phyb-9 vs Col-0 for PBA1 or RUB-Cul displayed as a ratio of phyB-9/Col-0 in Dk or R. Asterisks identify significant differences in the fold change between PBA1 and the RUB-CUL (p ≤ 0.05, Student’s T test). Error bars represents standard deviation (n = 3)