Rapid reversion from monomer to dimer regenerates the UV-B photoreceptor UVR8 in intact Arabidopsis plants

Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor that specifically mediates photomorphogenic responses to UV-B in plants. UV-B photoreception induces the conversion of the UVR8 dimer into a monomer that interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) protein to regulate gene expression. However, it is not known how the dimeric photoreceptor is regenerated in plants. Here we show, by using inhibitors of protein synthesis and degradation via the proteasome, that the UVR8 dimer is not regenerated by rapid de novo synthesis following destruction of the monomer. Rather, regeneration occurs by reversion from the monomer to the dimer. However, regeneration of dimeric UVR8 in darkness following UV-B exposure occurs much more rapidly in vivo than in vitro with illuminated plant extracts or purified UVR8, indicating that rapid regeneration requires intact cells. Rapid dimer regeneration in vivo requires protein synthesis, the presence of a C-terminal 27-amino acid region of UVR8, and the presence of COP1, which is known to interact with the C-terminal region. However, none of these factors can account fully for the difference in regeneration kinetics in vivo and in vitro, indicating that additional proteins or processes are involved in UVR8 dimer regeneration in vivo.

Photoreception leads to both the rapid nuclear accumulation of UVR8 (Kaiserli and Jenkins, 2007) and interaction with the CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) protein (Favory et al., 2009;Rizzini et al., 2011;Cloix et al., 2012). COP1 is a positive regulator of UVR8 mediated gene expression (Oravecz et al., 2006), in contrast to its function as a negative regulator of photomorphogenesis in dark-grown seedlings (Osterlund et al., 2000;Yi and Deng, 2005). Whereas COP1 acts as an E3 ubiquitin ligase to degrade positive regulators such as HY5 in dark-grown seedlings, there is no evidence that it functions as an E3 ubiquitin ligase in UV-B photomorphogenic responses. The REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1) and RUP2 proteins negatively regulate UVR8 mediated photomorphogenic responses and interact with UVR8 (Gruber et al., 2010).
The RUP proteins are expressed in response to UV-B and both UVR8 and COP1 are required for their UV-B induced expression (Gruber et al., 2010).
An important issue is how the functional UVR8 photoreceptor is regenerated following photoreception. Since UV-B photoreception converts the UVR8 dimer into the monomer, how is the dimeric photoreceptor restored? In principle there are two possible mechanisms. First, the monomer could be degraded after it functions and protein synthesis could replace the dimer in the cell. This would entail quite rapid and continual turnover of the UVR8 protein. Second, the monomer could revert to the dimer to reconstitute the functional photoreceptor without any requirement for synthesis and degradation. Experiments with the homogeneous purified protein show that the monomer can revert to the dimer and that the reformed dimer is functional in UV-B photoreception (Christie et al., 2012;Wu et al., 2012). However, the kinetics of reversion in vitro are slow; although some dimer is reformed within a few hours, complete reversion takes 24 to 48 hours. Hence, our aim in this study was to investigate the kinetics and mechanism of regeneration of the UVR8 photoreceptor in plants.

Regeneration of the UVR8 dimer is rapid in intact plants
To examine whether UVR8 is in the dimeric or monomeric form we used a SDS-PAGE assay developed by Rizzini et al (2011). In this assay, protein samples for electrophoresis are prepared using sample buffer containing SDS, but they are not boiled prior to loading on a SDS-PAGE gel.
Interactions that maintain the dimer are sufficiently strong to resist denaturation by SDS under these conditions. Hence, the UVR8 dimer and monomer are clearly resolved on the gels and are visualized by incubating an immunoblot of the gel with an anti-UVR8 antibody.
We used this assay to monitor the relative amounts of UVR8 dimer and monomer following UV-B treatment of intact wild-type Arabidopsis plants. As shown in Figure 1A, essentially all the UVR8 protein is present as a dimer before UV-B exposure and it is then converted to the monomer in response to 3 hours UV-B treatment. When plants are subsequently transferred to darkness, a decrease in the amount of monomer and a concomitant increase in the dimer is seen within 15 minutes. After 1 hour in darkness virtually all the UVR8 protein is present as the dimer. The total amount of UVR8 does not appear to change significantly over the time course at least up to 2 hours following the end of illumination.
In contrast, reappearance of the dimer in darkness following UV-B treatment of purified UVR8 protein shows much slower kinetics ( Figure 1B).
Although reversion to the dimer is detectable 3 hours after transfer to darkness, most of the protein is still in the monomeric form 6 hours after the end of UV-B illumination, and approximately 30 hours are required to see near complete dimer regeneration.
In an attempt to quantify the kinetics of dimer regeneration, the percentage of UVR8 in the monomeric form was determined by measuring band intensities in Western blots from several independent experiments. As shown in Figure 1C, in vivo the monomer declines exponentially in darkness following UV-B exposure whereas in vitro the rate is much slower. The mean time required for 50% loss of the monomer was calculated from the graphical data with 95% confidence limits (Table I). In vivo, 50% monomer is lost within approximately 18 minutes whereas in vitro it takes about 15 hours.
In the Introduction we raised two possible mechanisms of dimer regeneration following monomerisation: (i) reversion of monomer to dimer and (ii) dimerisation of newly synthesized monomer to replace degraded monomer. From the above data we conclude that if the UVR8 dimer is regenerated by reversion, the process occurs much more quickly in intact plants than in vitro. However, if there is monomer degradation and resynthesis, both must occur rapidly in vivo and must be coordinated to maintain a constant amount of UVR8.

Rapid regeneration of the UVR8 dimer requires intact cells
To further explore the mechanism of regeneration we monitored the amounts of UVR8 dimer and monomer following UV-B treatment of plant extracts. UV-B exposure of Arabidopsis extracts in vitro initiates monomerisation of UVR8 ( Figure 1D). However, reappearance of the dimer is much slower in extracts than in intact plants and the kinetics are very similar to those seen for reversion of the purified protein (compare Figures 1B and   1D), with most of the UVR8 protein being in the monomeric form 12 hours after the end of UV-B illumination. Quantification of the loss of monomer (Supplemental Figure S1A) further demonstrates the similarity in kinetics for the purified protein and plant cell extract in vitro (Table I). We conclude that rapid regeneration of the UVR8 dimer requires intact cells, implying the involvement of physiological processes.

Protein synthesis is required for rapid regeneration of the dimer
To examine whether protein synthesis is important in dimer regeneration we treated plants with cycloheximide (CHX). Plants were transferred to liquid medium containing CHX one hour before the start of UV-B exposure to ensure that the chemical entered the cells. To test the effectiveness of the CHX treatment, we monitored the UV-B induction of CHS expression, which is inhibited by CHX (Christie and Jenkins, 1996). Control plants were treated in exactly the same way except that CHX was omitted. As shown in Figure 2A, CHX treatment did not impair UV-B induced conversion of the UVR8 dimer to the monomer. Nevertheless, CHX was active in the tissue because CHS expression, detected by a specific antibody, was prevented ( Figure 1B).
We reasoned that if protein synthesis is required to replenish the UVR8 dimer following hypothetical rapid degradation of the monomer, then the amount of UVR8 should decrease substantially following CHX treatment.
However, as shown in Figure 2A, CHX treatment did not affect the total amount of UVR8 up to at least 3 hours following the end of UV-B exposure, by which time UVR8 is in the dimeric form. This result indicates that synthesis of the UVR8 protein is not required for dimer regeneration following UV-B exposure. Nevertheless, Figure 2A shows that the rate of dimer reappearance is slower in the plants treated with CHX; whereas very little monomer remains in control plants 30 minutes after transfer to darkness following UV-B treatment, in the CHX treated plants a substantial amount of monomer remains after 1 hour and is still detectable after 2 hours of darkness.
Quantification of the loss of monomer (Supplemental Figure S1B) reveals the slower kinetics following CHX treatment (Table I). We conclude that protein synthesis is required to maximize the rate of reversion from monomer to dimer.

No evidence of targeted proteolysis of UVR8 via the proteasome
To complement the experiments with CHX, we examined the effect of MG132, which inhibits protein degradation by the proteasome, on the amount of UVR8 following UV-B exposure. Since several previous studies employed quite long preincubations with MG132 to see inhibition of the proteasome (e.g. Yang et al., 2005;Jang et al., 2005;Dong et al., 2006), plants were transferred to liquid medium containing MG132 eleven hours before the start of UV-B illumination. MG132 did not impair UV-B induced conversion of the UVR8 dimer to the monomer ( Figure 3A). Furthermore, there was no effect on regeneration of the dimer following transfer to darkness ( Figure 3A) and no quantitative difference in the kinetics for monomer loss in MG132 treated and control plants (Supplemental Figure S1C; entered the tissue an immunoblot was incubated with an antibody to ubiquitin. Inhibition of proteasomal degradation should lead to the accumulation of polyubiquitylated proteins in the cell and it is evident that increased amounts of such proteins are present in the plants treated with the inhibitor compared to the control ( Figure 3B). The key observation in this experiment is that the total amount of UVR8 did not change over the time course of illumination and dimer regeneration in darkness ( Figures 3A and 3C). We therefore conclude that UVR8 is not subject to proteasomal degradation following UV-B exposure.

The C-terminus of UVR8 is required for rapid in vivo regeneration of the photoreceptor
To further study regeneration of the UVR8 dimer we examined the importance of the C-terminus of the protein. A 27 amino acid region in the Cterminus from residues 397 to 423 (termed C27) is both necessary and sufficient for interaction with the WD40 region of the COP1 protein and can also interact with other WD40 proteins (Cloix et al., 2012). As shown in Figure   4A, the uvr8-2 mutant, which lacks the C-terminal 40 amino acids including the C27 region (Brown et al., 2005;Cloix et al., 2012) shows normal UV-B induction of monomerisation, but slower regeneration of the dimer in subsequent darkness compared to the wild-type; the monomer is still detectable 4 hours after the end of illumination. A similar observation is seen with a UVR8 mutant protein lacking specifically the C27 region ( Figure 4B). This experiment was undertaken with a transgenic uvr8-1 line expressing UVR8 lacking the C27 region fused to GFP at the N-terminus (uvr8-1/GFP-ΔC27UVR8; Cloix et al., 2012); control plants expressed wild-type UVR8 fused to GFP (uvr8-1/GFP-UVR8; Kaiserli and Jenkins, 2007). Both lines show UV-B induced monomerisation, but regeneration of the dimer was much slower in the uvr8-1/GFP-ΔC27UVR8 plants compared to the uvr8-1/GFP-UVR8 control ( Figure 4B). Quantification (Supplemental Figure S1D; Table I) shows the slower kinetics of the loss of monomer in the uvr8-1/GFP-ΔC27UVR8 plants. We conclude that the C27 region is required to maximize the rate of UVR8 dimer regeneration in vivo.
To test whether the C-terminus affected the rate of dimer regeneration in vitro, purified UVR8 was subjected to a mild trypsin treatment, which removes 40 amino acids from the C-terminus (Christie et al., 2012). This Cterminally truncated protein undergoes normal UV-B induced dimer to monomer conversion, as reported previously (Christie et al., 2012). However, in contrast to the in vivo situation, the rate of dimer regeneration in darkness following UV-B exposure was not slower for C-terminally truncated UVR8 compared to the wild-type protein; the kinetics of regeneration were indistinguishable for the two proteins (compare Figure 4C with Figure 1B).
The same conclusion can be drawn from measurements of monomer loss (Supplemental Figure S1E; Table I). This finding indicates that the absence of the C27 region only slows regeneration in intact cells, suggesting that interaction of one or more proteins with the C27 region may be required to maximize the rate of regeneration.

COP1 is required for rapid dimer regeneration in vivo
Since the C27 region interacts with COP1, we reasoned that this interaction might be important for dimer regeneration. The kinetics of regeneration was therefore examined in the cop1-4 mutant. As shown in Figure 4D, cop1-4 plants show normal UV-B induced UVR8 monomerisation, but the rate of dimer regeneration in subsequent darkness is slower than in wild-type plants; whereas the monomer is no longer detectable in wild-type plants after an hour of darkness, monomer is present in cop1-4 plants after at least 2 hours of darkness (compare Figure 4D with Figures 4A and 1A).
Quantification of monomer loss reveals the slower kinetics for cop1-4 plants compared to wild-type (Supplemental Figure S1F; Table I). Thus COP1 is required to maximize the rate of dimer regeneration in vivo.
the time course of monomerisation and regeneration. The question we addressed here is how the dimer is regenerated. We considered the possibility that the monomer is degraded following its formation and that the dimer is replaced following synthesis of new monomer. If this were the case, the processes would have to be rapid and closely coordinated to maintain a constant amount of UVR8. The experiments presented in Figures 2 and 3 do not provide any evidence of such rapid turnover.
The preincubation with CHX was evidently effective because it prevented the accumulation of CHS in response to UV-B treatment. We previously reported that CHX inhibits CHS transcript accumulation in response to UV-B without generally affecting gene expression (Christie and Jenkins, 1996). Hence the CHX treatment would be expected to inhibit synthesis of UVR8 following UV-B treatment if it was required to replace the dimer. However, CHX treatment did not prevent the regeneration of the dimer and there was no detectable change in the total amount of UVR8 over the duration of the experiment. The fact that the dimer reappeared completely in the presence of CHX indicates that UVR8 protein synthesis is not required for dimer regeneration.
The presence of MG132 in cells causes the general accumulation of polyubiquitylated proteins, which can be seen in the present experiment using an anti-ubiquitin antibody. Although MG132 evidently entered the tissue, there was no effect either on the regeneration of the dimer or on the total amount of UVR8. Clearly, if the monomer was rapidly degraded via the proteasome following UV-B exposure MG132 would have inhibited its disappearance. We therefore conclude that the monomer is not subject to significant proteasomal degradation. Although it is not possible to rule out monomer degradation by other types of proteolysis, the fact that the total amount of UVR8 remained constant throughout all the experiments presented in this study indicates that the protein is not subject to rapid turnover. This conclusion is consistent with previous studies showing that UVR8 is essentially constitutively expressed; the protein is present in all plant tissues analysed to date (Rizzini et al., 2011) and its abundance is not affected by different light qualities (Kaiserli and Jenkins, 2007).
We conclude from the above experiments that the UVR8 dimer is regenerated by reversion of the monomer to the dimer. Since the photoreceptor appears to be relatively stable, we anticipate that in vivo UVR8 will cycle between the dimeric and monomeric forms according to the prevailing ambient level of UV-B. Thus a UVR8 dimer/monomer photoequilibrium may be established analogous to the balance between the inactive Pr and active Pfr forms of phytochromes in daylight. The stability of the UVR8 photoreceptor is similar to that of the light-stable phytochromes and cry1 but contrasts with that of the phyA and cry2 photoreceptors, both of which are subject to proteasomal degradation (Seo et al., 2004;Yu et al., 2007).

Protein synthesis is required to maximize the rate of dimer regeneration
Regeneration of the UVR8 dimer following UV-B exposure occurs much more slowly with the homogeneous purified protein than it does in vivo.
The purified protein is very stable in vitro, and it monomerises in response to UV-B even after 48 hours of incubation in darkness at room temperature (Christie et al., 2012). In addition, dimer regeneration occurs with the same slow kinetics following exposure of plant extracts to UV-B. It therefore appears that a normal cellular environment is required to maximize the rate of regeneration, presumably because cellular compartmentation or particular physiological processes are needed.
The CHX experiment indicates that protein synthesis following UV-B exposure is required to facilitate rapid reversion from monomer to dimer. CHX treatment did not affect the total amount of UVR8 or prevent dimer regeneration, but it did slow the kinetics of monomer disappearance and dimer accumulation. A likely scenario is that one or more proteins synthesized in response to UV-B facilitate reversion of the monomer. Nonetheless, dimer formation still occurred in the presence of CHX, so protein synthesis was evidently not essential for regeneration. Moreover, the rate of dimer formation in vivo in the presence of CHX was still a lot faster than that in vitro, so clearly protein synthesis is not the only factor required for rapid dimer regeneration.
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The C terminus of UVR8 is required for rapid regeneration in vivo.
Deletion of the C-terminal 40 amino acids of UVR8, in the uvr8-2 allele, and specifically the C27 region, in the GFP-ΔC27UVR8 fusion, reduced the rate of dimer regeneration in vivo. To address the possibility that removal of the C-terminal region impaired reversion to the dimer because of an adverse effect on UVR8 structure, we examined regeneration of trypsin-treated purified UVR8, which is equivalent to the uvr8-2 allele because it lacks the Cterminal 40 amino acids. Since there was no difference in the kinetics of dimer regeneration for the wild-type and trypsin-treated UVR8 proteins in vitro we conclude that truncation of the C-terminus does not impair regeneration for structural reasons. That the absence of the C-terminal region only affects regeneration in vivo suggests that this part of UVR8 may interact with proteins that facilitate reversion to the dimer. This could include proteins synthesized in response to UV-B, as discussed above. The presence of pre-existing proteins is not sufficient to maximize the rate of reversion, at least in vitro, because there is no difference in reversion kinetics between purified UVR8 and UV-B treated whole cell extracts. Nevertheless, the absence of COP1, which is present prior to UV-B exposure, does diminish the rate of dimer regeneration, indicating that it is involved, most likely with other proteins, in facilitating reversion of the monomer.
We reported recently that COP1 interacts with the C27 region of UVR8 to initiate signal transduction (Cloix et al., 2012). It is possible that this interaction is required to promote the recruitment of other proteins that facilitate reversion to the dimer. In principle the C-terminus could interact with a range of proteins; it is known to interact with the WD40 domain proteins Moreover, COP1 is required for UV-B induction of many UVR8 regulated genes, so the absence of COP1 in the cop1-4 mutant may impair synthesis of one or more components needed for rapid regeneration. We therefore do not know whether the role of COP1 in dimer regeneration is direct, via its ability to interact with the C-terminus of UVR8, or indirect via its requirement to www.plantphysiol.org on August 29, 2017 -Published by Downloaded from Copyright © 2012 American Society of Plant Biologists. All rights reserved. synthesise one or more other proteins that can interact with the C-terminus. It should be noted, however, that regeneration of the dimer in cop1-4 plants and in UVR8 with C-terminal deletions in vivo is still faster than in vitro, so additional factors are likely to maximize the rate of dimer regeneration.

CONCLUSION
In summary, this study demonstrates that the dimeric UVR8 photoreceptor is regenerated rapidly in vivo and that this is accomplished by reversion of the monomer to the dimer rather than by a mechanism involving rapid turnover of the protein. This process is crucial, as it will enable the photoreceptor to respond rapidly and sensitively to changes in ambient UV-B levels in sunlight to regulate photomorphogenic responses. The data presented suggest that the process of reversion from monomer to dimer is complex and is facilitated by several factors: the presence of intact cells, protein synthesis in response to UV-B and interaction of the C-terminal region of UVR8 with proteins, including COP1. Further research is therefore required to identify the components and processes involved.

Plant Materials and Treatments
Seeds of wild-type Arabidopsis thaliana ecotype Landsberg erecta For direct UV-B illumination of extracts ( Figure 1C), exposure to 1.5 µmol m -2 s -1 narrowband UV-B for 1 h was carried out on ice (control extracts were not illuminated) and the extracts were then transferred to darkness at room temperature.
Preparation of purified UVR8 protein as well as trypsin treatment (TPCK treated, Sigma) were performed as described in Christie et al. (2012).
Purified proteins were exposed to 1.5 µmol m -2 s -1 narrowband UV-B on ice for 1 h and transferred to darkness at room temperature for the indicated times. Protein samples were prepared for electrophoresis without boiling (unless indicated otherwise) and loaded on a 7.5 % SDS-PAGE gel. Gels were stained with Coomassie Blue.
For the analysis of CHS and polyubiquitylated proteins, protein samples were boiled prior to electrophoresis on a 7.5 % SDS-PAGE gel.
Immunoblots were stained with Ponceau S to reveal the Rubisco large subunit (rbcL), which was used as a loading control. The data shown are representative of at least three independent experiments.
Quantification of UVR8 monomer loss in darkness following UV-B exposure was undertaken for representative Western blots from three independent experiments. The immunodetected UVR8 bands were quantified using Image J software. Data were corrected for background and normalized against the value of the monomer after UV-B illumination, taken as 100%.
Points were plotted and fitted using Curve Fitting Toolbox in MATLAB (Version 7.12.0). The best fit was chosen and a 95% confidence level of the fit is shown. To facilitate comparison between treatments and genotypes, the time taken for loss of 50% of the monomer was calculated.

Supplemental Data
The following materials are available in the online version of this article. Figure S1. Kinetics of the loss of UVR8 monomer in darkness following UV-B exposure under the experimental conditions used in this study.

Accession numbers
The Arabidopsis Genome Initiative locus identifier for UVR8 is At5g63860 and for COP1 is At2g32950.
of UV-B treated whole cell extract from WT Ler plants probed with anti-UVR8 antibody. The extract was exposed to 1.5 μmol m -2 s -1 UV-B for 1 h (UV-B +) and then transferred to darkness at room temperature for the indicated times.
Samples were analyzed without boiling on a 7.5% SDS-PAGE gel prior to immunoblotting. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control.   plants probed with anti-UVR8 antibody. Plants were exposed to 2.5 μmol m -2 s -1 UV-B for 3 h (UV-B +) and then transferred to darkness for the indicated times before extracts were prepared. Extract samples were prepared for electrophoresis without boiling and resolved on a 7.5% SDS-PAGE gel prior to immunoblotting. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. B, Immunoblots of whole cell extracts from uvr8-1/GFP-UVR8 and uvr8-1/GFP-ΔC27UVR8 plants probed with anti-GFP antibody. Plants were exposed to UV-B, transferred to darkness and samples analyzed by electrophoresis as in (A). The asterisk (*) indicates a non-specific band recognized by the anti-GFP antibody. C, Coomassie stained SDS-PAGE of purified UVR8 protein digested with trypsin, exposed to 1.5 μmol m -2 s -1 UV-B for 1 h (UV-B +) and then transferred to darkness for the indicated times.
Samples were analyzed without boiling on a 7.5% SDS-PAGE gel. A non-UV-B treated boiled sample is shown as a control. D, Immunoblot of whole cell extract from cop1-4 plants probed with anti-UVR8 antibody. Plants were exposed to UV-B, transferred to darkness and samples analyzed by electrophoresis as in (A). Figure S1. Kinetics of the loss of UVR8 monomer in darkness following UV-B exposure under the experimental conditions used in this study.

Supplemental
UVR8 protein bands were quantified in three representative Western blots for each type of experiment using Image J software. The value for the monomer at each time point was normalized against that after UV-B illumination, taken as 100%. Values of %monomer with time were plotted and fitted using Curve Fitting Toolbox in MATLAB (Version 7.12.0). The line of best fit is shown with the 95% confidence level of the fit. In each panel the inset provides a key to the lines. A, purified UVR8 in vitro compared to UVR8 whole cell extract in Table I. Quantification and statistical analysis of the lifetime of the UVR8 monomer in different experimental conditions UVR8 protein bands were quantified in three representative Western blots for each type of experiment using Image J software. The value for the monomer at each time point was normalized against that after UV-B illumination, taken as 100%. Values of %monomer with time were plotted (see Figure 1C and Supplemental Figure S1) and fitted using Curve Fitting Toolbox in MATLAB (Version 7.12.0). The best fit was chosen and the time point at which the monomer reached 50% is shown +/-the 95% confidence values at that point. The R 2 value indicates how well the line fits the data points, where 1.0 would represent a perfect fit.  1. Regeneration of the UVR8 dimer after UV-B exposure is much more rapid in vivo than in vitro. A, Immunoblot of whole cell extracts from wild-type Ler plants probed with anti-UVR8 antibody. Plants were exposed to 2.5 μ mol m -2 s -1 UV-B for 3 h (UV-B +) and then transferred to darkness for the indicated time periods before extracts were prepared. Extract samples were prepared for electrophoresis without boiling and resolved on a 7.5% SDS-PAGE gel prior to immunoblotting. The UVR8 dimer and monomer are indicated. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. B, Coomassie stained SDS-PAGE gel of purified UVR8 protein exposed to 1.5 μ mol m -2 s -1 UV-B for 1 h (UV-B +) and then transferred to darkness at room temperature for the indicated times. Samples were analyzed without boiling on a 7.5% SDS-PAGE gel. A non-UV-B treated boiled sample is shown as a control. C, Quantification of the loss of monomer in darkness following UV-B exposure for wild-type in vivo (solid line) and purified UVR8 in vitro (solid line with points). Dotted and dashed lines show 95% confidence limits of the best fit curves from 3 replicates. D, Immunoblot of UV-B treated whole cell extract from WT Ler plants probed with anti-UVR8 antibody. The extract was exposed to 1.5 μ mol m -2 s -1 UV-B for 1 h (UV-B +) and then transferred to darkness at room temperature for the indicated times. Samples were analyzed without boiling on a 7.5% SDS-PAGE gel prior to immunoblotting. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control.

Figure 2.
Protein synthesis is required to maximize the rate of dimer regeneration. A, Immunoblots of whole cell extracts from WT Ler plants probed with anti-UVR8 antibody. Plants were placed in medium containing 0.1% DMSO with or without cycloheximide 1 h before exposure to 2.5 μ mol m -2 s -1 UV-B for 3 h (UV-B +), and then transferred to darkness for the indicated times before extracts were prepared. Samples were prepared for electrophoresis without boiling and resolved on a 7.5% SDS-PAGE gel prior to immunoblotting. B, Immunoblots from the experiment shown in (A) probed with anti-CHS antibody. The asterisk (*) indicates a non-specific band recognized by the antibody. In (A) and (B) Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control.  μ mol m -2 s -1 UV-B for 3 h (UV-B +), and then transferred to darkness for the indicated times before extracts were prepared. Samples were prepared for electrophoresis without boiling and resolved on a 7.5% SDS-PAGE gel prior to immunoblotting. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. B, Immunoblots from the experiment shown in (A) probed with anti-ubiquitin antibody. C, Immunoblots prepared as in (A) probed with anti-UVR8 antibody, but with samples of whole cell extract boiled prior to electrophoresis on a 7.5% SDS-PAGE gel.  . The C-terminus of UVR8 is required for rapid regeneration of the dimer in vivo. A, Immunoblots of whole cell extracts from WT Ler and uvr8-2 plants probed with anti-UVR8 antibody. Plants were exposed to 2.5 μ mol m -2 s -1 UV-B for 3 h (UV-B +) and then transferred to darkness for the indicated times before extracts were prepared. Extract samples were prepared for electrophoresis without boiling and resolved on a 7.5% SDS-PAGE gel prior to immunoblotting.
Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. B, Immunoblots of whole cell extracts from uvr8-1/GFP-UVR8 and uvr8-1/GFP-ΔC27UVR8 plants probed with anti-GFP antibody. Plants were exposed to UV-B, transferred to darkness and samples analyzed by electrophoresis as in (A). The asterisk (*) indicates a nonspecific band recognized by the anti-GFP antibody. C, Coomassie stained SDS-PAGE of purified UVR8 protein digested with trypsin, exposed to 1.5 μ mol m -2 s -1 UV-B for 1 h (UV-B +) and then transferred to darkness for the indicated times. Samples were analyzed without boiling on a 7.5% SDS-PAGE gel. A non-UV-B treated boiled sample is shown as a control. D, Immunoblot of whole cell extract from cop1-4 plants probed with anti-UVR8 antibody. Plants were exposed to UV-B, transferred to darkness and samples analyzed by electrophoresis as in (A).