In order to elucidate the biochemical mechanism of BZS1 function, we performed a SILIAIP-MS analysis of the BZS1 protein complex. We transformed Arabidopsis with a construct that over expresses a BZS1 protein fused with the yellow fluorescence protein at the C-terminus driven by the constitutive 35S promoter . A transgenic line that showed mild dwarf and dark-green-leaf phenotypes, resembling the bzs1-D mutant , was selected for the analysis. Pair-wised comparison was designed to seperately compare BZS1-YFP and 35S::YFP transgenic plants with non-transgenic wild type, to determine proteins associated with BZS1-YFP and YFP alone, respectively. To obtain complete 15nitrogen labeling of young seedlings, we first grew BZS1-YFP, YFP and wild-type plants hydroponically in medium containing 15N, and obtained stable isotope-labeled seeds . These 15N-labeled seeds and regular 14N seeds were grown again on corresponding 15N or 14N medium to obtain 5-day-old seedlings for further analysis . For each pair of isotope-labeled sample and control, equal amount of tissues was mixed, and the protein extract was used for immuno precipitation using the GFPtrap beads. The immuno precipitated proteins were separated in SDS-PAGE, gel bands were in-gel digested, and the tryptic peptides were analyzed by mass spectrometry . Mass spectrometry analyses of the two BZS1-YFP immuno precipitation experiments identified 514 and 383 proteins, respectively, with 279 proteins identified in both repeats . A smaller number of proteins were identified in the YFP experiments . Quantitation of isotope ratios showed median ratios of 1.16 and 1.23 for the two BZS1-YFP experiments,planting gutter and 1.0 and 0.92 for the two YFP control experiments. The protein ratios of the YFP control datasets had standard deviation of 0.23 and 0.57 .
Using 2× median as cutoff, 16 proteins were enriched in BZS1-YFP compared to wild-type control in the two repeat experiments. The YFP and wild type comparison identified 2 proteins that were enriched over 2× median, presumably due to association with YFP or false discovery, suggesting a false discovery rate <0.8% . The 15 proteins enriched by BZS1-YFP were not enriched by YFP alone, and thus were considered BZS1-associated proteins . Among the BZS1-associated proteins are COP1 and HY5, two key regulators of the light signaling pathways, as well as BZS1/BBX20’s homologs STH2/BBX21 and STO/BBX24 . To verify the interaction between BZS1 and COP1 in vivo, we performed immuno precipitation of BZS1-YFP from the BZS1-YFP transgenic Arabidopsis seedlings using anti-GFP antibody, and probed the immunoblot with anti-COP1 antibody. The results showed that COP1 co-immuno precipitates with BZS1-YFP , confirming that BZS1 interacts with COP1 in plants. Consistent with BZS1’s interaction with the COP1 E3 ubiquitin ligase, the immuno precipitated BZS1-YFP can be detected by anti-ubiquitin antibody, and the level of ubiquitination was increased by treatment with proteasome inhibitor MG132 . We further confirmed the direct interaction of BZS1 and HY5 by yeast two-hybrid assays . Further, when transiently co-expressed in Nicotiana benthamiana, the BZS1-myc protein was co-immuno precipitated by the HY5-YFP protein , confirming their interaction in plant cells. Similarly, the STH2-myc protein was co-immuno precipitated by BZS1-YFP . These results confirmed the SILIA-IP-MS results that BZS1 interacts with COP1, HY5, and STH2/BBX21. To determine the functional relationship between BZS1 and HY5, we first compared previously published transcriptomic data from BZS1-overexpression plants with chromatin immuno precipitation-microarray data of HY5 direct target genes . The result showed that 56.3% of BZS1-activated genes are HY5 targets while only 13% of BZS1-repressed genes are HY5 targets . Such significant overlap betweenBZS1-activated and HY5-bound genes suggests that BZS1 interacts with HY5 to activate gene expression.
Fusing a transcription repressor domain, such as the SRDX domain, to a transcription activator has been shown to have a dominant negative effect . Over expression of the BZS1-SRDX fusion sequence driven by 35S promotor in Arabidopsis caused a long-hypocotyl phenotype and reduced anthocyanin accumulation , which were similar to the phenotypes of loss-of-function mutant hy5-215 but opposite to the phenotypes caused by BZS1 over expression, further supporting that BZS1 functions as a transcription activator together with HY5. The BZS1-SRDX plants grown in the dark did not show any obvious phenotype , consistent with HY5 and BZS1 being degraded in the dark. To further investigate whether BZS1 function requires HY5, we crossed BZS1-YFP with hy5-215. The BZS1-YFP/hy5-215 plants showed similar phenotypes of long hypocotyls and low anthocyanin accumulation as hy5-215 , demonstrating that BZS1 activity requires HY5. Interestingly, the BZS1-YFP protein accumulates at a higher level in the hy5-215 mutant than in wild-type background , suggesting that HY5 negatively regulates BZS1 accumulation while required for BZS1 function. On the other hand, the RNA levels of HY5 and HYH are higher in BZS1-YFP line but lower in BZS1-SRDX seedlings as compared with those in wild type . Immunoblot analysis also confirmed that the HY5 protein level was increased in the BZS1-YFP line and reduced in the BZS1-SRDX line . These results indicated that BZS1 and HY5 proteins not only interact directly, but also influence each other’s protein abundance. A previous study showed that HY5 is required for SL inhibition of hypocotyl elongation. The HY5 protein level is increased by SL treatment and the hypocotyl elongation of hy5 is partially insensitive to SL . Since BZS1’s function is dependent on HY5 in the light, we examined if BZS1 is also involved in SL signaling. As reported previously , treatment with 1 μM GR24, an analog of SL, dramatically inhibited the hypocotyl elongation of wild-type seedlings but had no effect on the SL insensitive mutant max2-3 . We found that the hypocotyl elongation of BZS1-SRDX seedlings was partially insensitive to GR24, similar to the hy5-215 mutant.
The GR24 treatment decreased the hypocotyl length of wild-type seedlings by about 72% compared to the untreated control, but only by about 17% for hy5-215 and 30% for the BZS1-SRDX seedlings . GR24 also increased the chlorophyll content in wild-type plants by about 24%, but had no significant effect in max2-3, hy5-215 and BZS1- SRDX seedlings . Additionally, GR24 induced HY5 accumulation in wild-type background but not in the BZS1-SRDX seedlings . These results indicated that, like HY5, BZS1 also plays an important role in SL regulation of hypocotyl elongation and chlorophyll accumulation. We then tested if SL regulates the expression of BZS1/BBX20 and its homologs. Real-time reverse transcription PCR analysis showed that GR24 increased the expression level of BZS1/BBX20 mRNA in wild type, but not in the max2-3 mutant . Interestingly, expression levels of other members of BBX IV family, including STH2/ BBX21, were not dramatically affected by GR24. Immunoblot analysis confirmed that GR24 treatment increased the levels of the BZS1-myc protein expressed from the BZS1 native promoter and the BZS1-YFP protein expressed from the constitutive 35S promoter, suggesting that SL regulates BZS1 at both transcriptional and post transcriptional levels . These results indicated that BZS1 plays a positive role in SL signaling downstream of MAX2 at the early stage of seedling development. Seedling development is crucial for establishment of life for a plant, and is thus highly responsive to a wide range of environmental and hormonal signals. The signaling pathways that transduce these signals are highly integrated at the molecular level to ensure coherent cellular responses and optimal growth according to environmental condition and endogenous physiology . This study uncovers additional mechanisms for such signal integration. Our quantitative proteomic analysis of the BZS1 complex reveals BZS1’s interaction with HY5,gutter berries as well as provides direct evidence for in planta BZS1-COP1 interaction. Genetic analyses using over expression and dominant negative loss-of-function transgenic plants demonstrate that BZS1 interacts with HY5 to activate gene expression and promote photomorphogenesis. Further, we find that BZS1 also mediates SL regulation of HY5 level and hypocotyl elongation. Together with previous finding of BZS1 function downstream of the BR pathway , our study establishes BZS1 as a key integrator of light, BR, and SL signals for regulating seedling morphogenesis. IP-MS is a powerful method for identification of interacting proteins, which has been widely used in dissecting signal transduction pathways . With increased sensitivity of modern mass spectrometers, IP-MS tends to identify not only specific interacting proteins but also large numbers of non-specific proteins. Under our experimental conditions, over 300 proteins were identified in each IP-MS analysis. Distinguishing specific from non-specific interactors is challenging without quantitative measurement. SILIA-IP-MS provides an ideal quantitative method for this purpose, as the sample and negative control can be mixed at an early step of the immuno precipitation experiment to avoid technical variations. Indeed, among the large numbers of proteins identified by mass spectrometry, only 29 showed enrichment by the BZS1-YFP fusion protein, and thus were considered BZS1-associated proteins. The interactions of BZS1 with HY5, COP1, and its homolog STH2/BBX21 were confirmed by yeast two-hybrid or coimmuno precipitation assays. Consistent with COP1-mediated ubiquitination of BZS1, our BZS1-interactome data includes ubiquitin and one proteasome activating protein PA200 .
In theory, the ratio between sample and negative control should be infinite for proteins that specifically interact with the bait protein in SILIA-IP-MS. However, due to background signals in the control samples, either from non-specific binding of proteins in immuno precipitation or interfering signals in MS1, the ratios actually distribute within a wide range. For example, Hubner et al. observed that pull-down with Aly-GFP leads to only moderate enrichment because Aly itself binds to control beads as well. In our study, only 2 of the 254 proteins identified in the YFP sample were enriched over 2× median, suggesting that even 2-fold cutoff yields low false discovery rate when two reverse-labeled replicates are used. Our genetic analyses support that BZS1 interacts with HY5 to activate gene expression and promote photomorphogenesis. First, comparison of genome-wide data shows that BZS1 tends to activate, rather than repress, HY5 direct target genes . Second, dominant inactivation of BZS1 causes similar phenotypes as the hy5-215 mutant , supporting that BZS1 and HY5 act in the same or overlapping pathway. Third, the phenotypes of BZS1-YFP plants are suppressed by hy5-215 , confirming that BZS1 functions in a HY5-dependent manner. These results together provide strong evidence for a model that BZS1 interacts with HY5 to activate HY5-bound target genes. BBX proteins contain one or two B-box zinc finger motifs in their N-terminal regions, and are organized into five subfamilies . The fourth subfamily includes eight B-box proteins containing two tandem B-boxes without CCT domain . Our study together with previous studies show that five members of the BBX subfamily IV interact with COP1 and HY5 . Thus, interaction with HY5 seems to be a common mechanism for these B-box proteins to regulate gene expression. Interestingly, BZS1/BBX20, STH2/BBX21 and LZF1/STH3/BBX22 are positive regulators in photomorphogenesis, while BBX19, STO/BBX24 and STH/BBX25 are negative regulators . Our finding of STH2/BBX21 and STO/BBX24 as interactors of BZS1/BBX20 suggests that these factors form hetero-dimers. The dominant negative effect of the BZS1-SRDX fusion indicates that BZS1/BBX20 normally functions as a transcription activator, which is consistent with previous finding that STH2/BBX21 functions as a transcription activator . It has been reported that STO/BBX24 and STH/BBX25 interact with HY5 and most likely inhibit HY5 function by forming inactive heterodimers . Our identification of STO/BBX24 as a BZS1-associated protein suggests another possibility that STO/BBX24 may form a non-functional heterodimer with BZS1/BBX20 and hence inhibit BZS1/BBX20 activity. In addition to direct interaction between BZS1 and HY5 proteins in regulating target gene expression, BZS1 and HY5 also regulate each other’s expression level. BZS1 positively regulates the RNA and protein levels of HY5 . Recent studies have shown that HY5 binds to its own promoter to regulate its own level , thus BZS1 may regulate HY5 transcription through interaction with HY5 protein. In contrast, the BZS1 protein level is increased in hy5-215, suggesting a negative regulation by HY5 at the protein level. HY5 may promote BZS1 degradation by interacting with COP1. Similarly, a previous study showed that the degradation of BBX22 is also promoted by both COP1 and HY5 , whereas BBX22 transcription is directly activated by HY5 and repressed by BBX24 . Such positive and negative regulation between interacting partners potentially contributes to the signaling dynamics during dark-to-light transition and fluctuating light intensities.Our study uncovers a major role for BZS1 in SL response.