COP1-mediated degradation of BBX22/LZF1 optimizes seedling development in Arabidopsis

Abstract

Light regulates multiple aspects of growth and development in plants. Transcriptomic changes govern the expression of signaling molecules with the perception of light. Also, the 26S proteasome regulates the accumulation of positive and negative regulators for optimal growth of Arabidopsis (Arabidopsis thaliana) in the dark, light, or light/dark cycles. BBX22, whose induction is both light regulated and HY5 dependent, is a positive regulator of deetiolation in Arabidopsis. We found that during skotomorphogenesis, the expression of BBX22 needs to be tightly regulated at both transcriptional and posttranslational levels. During photomorphogenesis, the expression of BBX22 transiently accumulates to execute its roles as a positive regulator. BBX22 protein accumulates to a higher level under short-day conditions and functions to inhibit hypocotyl elongation. The proteasome-dependent degradation of BBX22 protein is tightly controlled even in plants overexpressing BBX22. An analysis of BBX22 degradation kinetics shows that the protein has a short half-life under both dark and light conditions. COP1 mediates the degradation of BBX22 in the dark. Although dispensable in the dark, HY5 contributes to the degradation of BBX22 in the light. The constitutive photomorphogenic development of the cop1 mutant is enhanced in cop1BBX22ox plants, which show a short hypocotyl, high anthocyanin accumulation, and expression of light-responsive genes. Exaggerated light responsiveness is also observed in cop1BBX22ox seedlings grown under short-day conditions. Therefore, the proper accumulation of BBX22 is crucial for plants to maintain optimal growth when grown in the dark as well as to respond to seasonal changes in daylength.

Figure 1.

Both BBX22 protein and transcript are transiently accumulated. A, A BBX22-specific antibody was generated and used to determine the steady-state protein level by immunoblot analysis. Proteins were isolated from 4-d-old etiolated (dark; D) and 12-h light-treated (L) ecotype Columbia (Col) or bbx22 seedlings. Endogenous α-tubulin was a loading control (TUB). B and C, BBX22 protein (B) and mRNA (C) are transiently accumulated during photomorphogenesis. Immunoblotting was used to detect the accumulation of BBX22 in etiolated seedlings and seedlings illuminated with light for 3 to 24 h. Real-time quantitative RT-PCR was used to monitor the expression of endogenous BBX22 in C. The expression of UBQ10 in each sample was used as an internal control. The BBX22 expression in etiolated seedlings was set to 1. The expression of BBX22 is presented as the amount of increase at each time point relative to that in etiolated seedlings and represented as “ratio.” The means and sd were calculated from three replicates and plotted.

Figure 2.

Transient accumulation of BBX22 is regulated at the protein level. The stability of BBX22 was determined in 4-d-old bbx22 expressing 35S:BBX22 (bbx22BBX22ox) under the conditions indicated. A, Northern blot showing the overexpression of BBX22 in etiolated (dark; D) bbx22BBX22ox seedlings or etiolated seedlings illuminated with light for 6, 9, or 24 h. The ethidium bromide-stained image was used to show equal loading of the total RNA samples. B, Immunoblot showing the transient accumulation of BBX22 in plant samples used in A. Endogenous α-tubulin was a loading control (TUB).

Figure 3.

BBX22 is a short-lived protein degraded by the 26S proteasome. The degradation kinetics of BBX22 protein were determined as a percentage of full-length BBX22 remaining relative to BBX22 at 8 h of light treatment in the presence of cycloheximide. Half-life (t_1/2) was calculated by regression analysis. The half-life of BBX22 is 60 min in the light (white circles, solid lines) and 20 min in the dark (black circles, solid lines). The degradation of BBX22 was blocked by treatment with MG132 under both light (white circles, dashed lines) and dark (black circles, dashed lines) conditions. n = 3.

Figure 4.

BBX22 protein functions in the inhibition of hypocotyl elongation under SD conditions. A, The bbx22 mutant is hyposensitive to light only under SD conditions. Hypocotyl length was measured for 4-d-old seedlings grown under SD or LD conditions. * P < 0.01, Student’s t test; n = 38 to 76. B, BBX22 protein accumulates to a higher level in SD-grown Col plants (SD; white triangles, solid lines) compared with that in plants under LD conditions (white circles, dashed lines). BBX22 protein levels at different times of the day were determined by immunoblot analysis. Results are presented as values relative to that at dawn for 4-d-old seedlings grown under LD conditions. White bars indicate light periods and black bars indicate dark periods. n = 4. AUC, Areas under the SD or LD protein curves. C, bbx22 shows an increased growth rate during the dark period of SD conditions, with the most noticeable difference at day 4 after germination. Time 0 indicates dawn of day 4 after germination. Shaded and black areas indicate night (darkness), and white areas indicate day (lights on). Shaded areas around each growth trace (blue and red) show se. n = 29.

Figure 5.

COP1 is required for selective degradation of BBX22 in the dark. A and B, The expression of both BBX22 transcript (A) and BBX22 protein (B) is higher in dark-grown cop1-4 plants than in wild-type (Col) plants. C, Northern-blot analysis was used to confirm the comparable BBX22-GFP transgene expression in these lines. The ethidium bromide-stained image was used to show equal loading of the total RNA samples. D, BBX22-GFP protein accumulates to high levels in cop1 mutants. Endogenous α-tubulin was a loading control (TUB).

Figure 6.

BBX22 degradation depends in part on HY5 in the light but is independent of HY5 and HYH in the dark. A, BBX22 decreases in hy5 due to a lack of HY5-dependent transcriptional activation of BBX22. B, Northern-blot analysis was used to confirm the comparable expression of BBX22 transgene in Ler, hy5, and hy5hyh plants expressing 35S:BBX22 and grown in the dark (D) for 4 d or in 4-old etiolated seedlings illuminated with light for 24 h (L₂₄). The ethidium bromide-stained image was used to show the amount of total RNA samples loaded in each lane. C, Immunoblotting was used to detect BBX22 protein in the plant samples used in B. The detection of endogenous α-tubulin was performed as a loading control (TUB).

Figure 7.

cop1BBX22-GFPox has exaggerated light responsiveness. Exaggerated cop phenotypes (top panels), the extra inhibition of hypocotyl growth and excess anthocyanin accumulating both in cotyledons and hypocotyls (marked by arrowheads), were seen when BBX22-GFP was overexpressed under both dark (A) and SD (B) conditions. Hypocotyl length (bottom panels) was measured in 4-d-old seedlings grown under dark (A) and SD (B) conditions. ^*,⁺ Significantly different from Col and the corresponding cop1 allele, respectively (P < 0.01, Student’s t test; n = 20–32). Bars = 1 mm.

Figure 8.

A model illustrating the biological impact of BBX22 degradation on seedling development. Protein abundance of the positive regulator BBX22 is tightly controlled by COP1 to regulate the development of Arabidopsis grown in the dark and under SD conditions. Overaccumulated BBX22 in cop1 gives rise to an exaggerated light responsiveness by altering the expression of genes responsive to light and hormone signals.