GIGANTEA recruits deubiquitylases, UBP12 and UBP13, to regulate accumulation of the ZTL photoreceptor complex

To remain synchronous with the environment, plants constantly survey daily light conditions using an array of photoreceptors and adjust their circadian rhythms accordingly. ZEITLUPE (ZTL), a blue light photoreceptor with E3 ubiquitin ligase activity, communicates end-of-day light conditions to the circadian clock. To function properly, ZTL protein must accumulate but not destabilize target clock transcription factors before dusk, while in the dark ZTL mediates degradation of target proteins. It is not clear how ZTL can accumulate to high levels in the light while its targets remain stable. Two deubiquitylating enzymes, UBIQUITIN-SPECIFIC PROTEASE 12 and UBIQUITIN-SPECIFIC PROTEASE 13 (UBP12 and UBP13), which have opposite genetic and biochemical functions to ZTL, were shown to associate with the ZTL protein complex. Here we demonstrate that the ZTL light-dependent interacting partner, GIGANTEA (GI), recruits UBP12 and UBP13 to the ZTL photoreceptor complex. We show that loss of UBP12 and UBP13 reduces ZTL and GI protein levels through a post-transcriptional mechanism. Furthermore, the ZTL target protein TOC1 is unable to accumulate to normal levels in ubp mutants, indicating that UBP12 and UBP13 are necessary to stabilize clock transcription factors during the day. Our results demonstrate that the ZTL photoreceptor complex contains both ubiquitin-conjugating and -deconjugating enzymes, and that these two opposing enzyme types are necessary for the complex to properly regulate the circadian clock. This work also shows that deubiquitylating enzymes are a core design element of circadian clocks that is conserved from plants to animals.

, and our BiFC results show that UBP12 and UBP13 interact with GI in both 75 compartments with strong signal in the nucleus and weaker but detectable signal in the 76 cytoplasm. The interacting complexes of UBP12 and GI formed nuclear foci, similar to the 77 localization of GI alone (Kim et al., 2013). UBP12 and UBP13 contain a MATH-type (meprin 78 and TRAF homology) protein interaction domain and a ubiquitin-specific protease (USP) 79 domain (Fig. S1). The MATH domains of UBP12 and UBP13 were necessary for interaction 80 with GI while the protease domain and the C-terminal portions did not mediate GI-interaction 81 (Fig. 1c). This suggests that the interaction between GI and UBP12 or UBP13 is not dependent 82 on the UBP USP domains binding to poly-ubiquitylated GI protein.

84
We next determined whether GI was necessary to bridge the interaction between UBP12 or 85 UBP13 and ZTL in vivo by performing IP-MS on wild type (Col-0) and gi-2 mutant transgenic 86 lines expressing the decoy ZTL protein (Fig. S2). We collected samples at 9 hours after dawn 87 from plants grown in 12h light/12h dark cycles to capture the time when ZTL and GI are 88 normally interacting. We found that UBP12 and UBP13 were enriched in the Col-0 samples (p-89 value= 3.58E-5 and 0.0113 for UBP12 and UBP13 respectively), but not in the gi-2 mutant (p-90 value= 1 for both) ( Fig.1d and Table S1). These results indicate that GI is required for 91 UBP12/UBP13 to form a complex with ZTL, substantiating our interaction studies in 92 heterologous systems. Notably, LKP2, a known ZTL interacting partner, associated with ZTL in 93 the presence or absence of GI and suggests that the decoy ZTL is able to form biologically  Table S2). LS Periodogram analysis using the Biodare2 platform 121 [biodare2.ed.ac.uk (Zielinski et al., 2014)] showed that the ubp12-1/gi-2 double mutant had a 122 similar phase and amplitude of CCA1 expression to the gi-2 mutant alone and a period more 123 similar to ubp12-1 (Table S3). These results show a non-additive interaction and suggest they 124 function in the same circadian genetic pathway. The ubp13-1/gi-2 double mutant had a similar 125 amplitude to the gi-2 mutant but had a more similar phase and period to the ubp13-1 mutant 126 (Table S3). This again shows a non-additive genetic interaction but also suggests that the roles of 127 UBP12 and UBP13 have slightly diverged with respect to clock function. We also crossed the gi- confirming that the effects of the UBPs and GI are not additive (Table S3). These results indicate 132 that UBP12 and UBP13 work in the same pathway as GI to control clock function.  (Table S3). Interestingly, the period data showed that the ubp12-1/ztl-4 was more 140 similar to ztl-4 than ubp12-1, but the ubp13-1/ztl-4 is more similar to ubp13-1. This data suggests 141 that ZTL is epistatic to UBP12 and UBP13 but that UBP13 has diverged in function from 142 UBP12. It is important to note that the qRT-PCR data is below the suggested resolution for was able to rescue the short period phenotype of ubp12-1 (Fig. 2i-j). As reference, approximately 161 13% of the ubp12-1 plants themselves and 62% of the wild type plants fell into the rescue 162 category. This is likely due to normal variations in population level data of this type. We further 163 confirmed that UBP12-YFP and UBP12 C208S -YFP were both localized to the cytoplasm and 164 nucleus, suggesting that differences in the clock phenotypes are not due to mislocalization of the UBP12 C208S protein (Fig S4). These results indicate that the deubiquitylating functions of UBP12 166 are necessary for its role in regulating the circadian clock. mutants during a 12h light/12h dark time course (Fig. 3a). GI protein levels were approximately 175 50% lower in the ubp12-1 and ubp13-1. mRNA expression of GI-HA was also approximately 176 25% lower than wild type at the peak of GI mRNA expression, ZT8 (Fig. 3b). This suggests that 177 GI protein accumulation is partially dependent on UBP12 and UBP13, but that altered 178 transcription of GI could also have an effect on GI protein.

180
Next, we measured ZTL protein levels in the ubp12-1 and ubp13-1 mutants (Fig. 3c). ZTL 181 protein levels were substantially decreased in the ubp12-1 and ubp13-1 mutants throughout the 182 entire day/night cycle. Overexposure of the immunoblot showed that a small amount of ZTL 183 protein can still accumulate in the ubp mutants (Fig. 3c). The expression of ZTL mRNA was 184 largely unaffected in these lines (Fig. 3d), suggesting that the decrease in ZTL protein levels was 185 caused by a post-transcriptional mechanism. This is similar to the post-transcriptional control of 186 ZTL reported in gi loss-of-function mutants (Kim et al., 2007), and indicates that UBP12 and 187 UBP13 are necessary for robust accumulation of the ZTL protein.  we hypothesize that UBP enzymes are recruited by GI to the ZTL photoreceptor complex to 209 prevent formation of poly-ubiquitin chains, resulting in increased stability of the protein complex 210 (Fig. S5). Interestingly, ZTL protein levels were severely damped in the ubp12 and ubp13 mutants, but counterintuitively the ZTL target, TOC1, also had reduced levels ( Fig. 3c-f). This 212 effect is similar to what was observed in a gi loss-of-function mutant, and suggests that GI and 213 UBP12 and UBP13 can counterbalance the activity of ZTL during the day, allowing TOC1 to 214 accumulate to high levels before being degraded (Kim et al., 2007). Although ZTL levels were 215 decreased in the ubp mutants, there was still a small amount that could potentially decrease 216 TOC1 levels in the light (Fig. 3c long exposure). This is different than what was seen when 217 HSP90 activity was inhibited, resulting in lower ZTL levels but higher TOC1 levels. This 218 suggests that HSP90 is necessary for ZTL protein maturation and to promote its activity (Kim et
To construct the fragments of UBP12 and UBP13 into yeast two-hybrid pGADT7-GW vectors, the desired fragments were first amplified from the full-length UBP12 or UBP13 entry vectors by PCR and cloned into pENTR™/D-TOPO vectors before being sub-cloned into pGADT7-GW with GATEWAY cloning.
For the UBP12 complementation plasmids, the pENTR™/D-TOPO-UBP12-NS vector served as template for site-directed mutagenesis to introduce a Cys to Ser mutation at a.a. 208 position using Q5® Site-Directed Mutagenesis Kit (NEB, E0554). Subsequently, UBP12-NS and UBP12C208S-NS in the pENTR™/D-TOPO entry vectors were sub-cloned into a modified GATEWAY compatible pGreenBarT vector 12 with 1.7k bp upstream of ATG of UBP12 promoter region in the KpnI/XhoI sites. The primers used for cloning were listed in Table S2.
Yeast two-hybrid ZTL, ZTL decoy, GI, TOC1, PRR5 and CHE were fused to the GAL4-BD in pGBKT7-GW vectors, and the full-length or fragments of UBP12 and UBP13 were fused to the GAL4-AD in pGADT7-GW vectors by GATEWAY cloning. The interactions were tested on synthetic dropout medium as described previously 7 .

Bimolecular fluorescence complementation (BiFC) and confocal microscopy
The coding region of GI, UBP12 or UBP13 in the GATEWAY entry vectors were cloned into protoplast GATEWAY destination vectors pUC-DEST-VYCE®GW and pUC-DEST-VYNE(R)GW 12 respectively for transient transfections into protoplasts. pSAT6-mCherry-VirD2NLS was used as a nuclear marker. The protoplasts were isolated from 3-to 4-week-old Arabidopsis (Col-0) grown at 22°C under 8h light/16h dark and transfected following the protocol of tape-Arabidopsis sandwich method 13 . After 14-18 h incubation in low-light conditions, protoplasts were imaged on a Nikon Ti microscope with using a 60X 1.4 NA plan Apo objective lens as described previously 14 . The images were analyzed with FIJI 15 .

Immunoprecipitation and mass spectrometry (IP-MS)
For the ZTL decoys in Col-0 background, homozygous 35S::FLAG-His-ZTL-decoy transgenic lines along with Col-0 and 35S::FLAG-His-GFP controls were used. For the ZTL decoys in the gi-2 background, three independent T2 transgenic lines of 35S::FLAG-His-ZTL-decoy/gi-2 and 35S::FLAG-His-GFP/gi-2 were selected on ½ strength MS plates with 15 µg/ml ammonium glufosinate before being transferred to soil. Twenty-one-day-old soil-grown plants were entrained in 12 h light/12 h dark at 22°C for 7 days prior to harvest. Leaf tissues were collected at 9 h after dawn for subsequent IP-MS. One-step IP-MS and MS spectral analyses were carried out as documented 7 with minor changes. The MS/MS spectral were searched against the SwissProt_2017 tax:Arabidopsis thaliana (thale cress) database (February 2017) using MASCOT MS/MS ion search engine version 2.6.0 16 with the following parameters: up to 2 missed cleavages; variable modifications included Acetyl (K), GlyGly(K), Oxidation (M), Phospho (ST), Phospho (Y); peptide tolerance ± 10 ppm; MS/MS tolerance ± 5 Da; peptide charge 2+ and 3+. The protein lists identified by MASCOT were first filtered out non-specific interactions by removing proteins only present in the controls (Col-0, gi-2, 35S::FLAG-His-GFP/Col-0 and 35S::FLAG-His-GFP/gi-2). The SAINTexpress algorithm 17,18 were further performed to determine the significance of protein-protein interactions.

Bioluminescent assays
The Arabidopsis seedlings bearing pCCA1::LUC in the wild type (Col-0), ubp12 or ubp13 mutants were grown in ½ strength MS medium and entrained in 12h light/12h dark for 7 days prior to being transferred to new ½ strength MS plates and constant light (LL) for circadian freerun experiments. For the various pUBP12::UBP12-YFP complementation T1 lines in the pCCA1::LUC/ubp12-1 background, seedlings were first screened and entrained on the ½ strength MS plates containing 7.5 µg/ml ammonium glufosinate prior to being transferred to ½ strength MS medium and LL. The measurement of luciferase activities and analyses were described as previously 7 .

Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)
RNA extraction, reverse-transcription and constitution of qPCR reactions were followed as described previously 7 , except for minor modifications. Four hundred ng total RNA were used for reverse transcription reactions. For semi-quantification of gene expression, IPP2 (AT3G02780) was used as an internal control. The relative expression represents means of 2 (-ΔCT) from three biological replicates, in which ΔCT = (CT of Gene of Interest -CT of IPP2). The primers were listed in Table S2.

Transient expression in Nicotiana benthamiana and confocal microscopy
UBP12-NS and, UBP12C208S-NS in the pENTR™/D-TOPO vectors were subcloned into inducible GATEWAY destination pABindGFP vectors 20 and transformed into the Agrobacterium tumefaciens strain GV3101 for transient expression in Nicotiana benthamiana. The Agrobacterium culture of pABindGFP-UBP12 or pABindGFP-UBP12C208S and the nuclear marker pABindcherry-AS2 21 were pelleted and resuspended in the infiltration solution (5% (w/v) Sucrose, 450 µM acetosyringone and 0.01% (v/v) Silwet). The bacterial infiltration solution was incubated at 4°C for 2h before infiltrated into 5-week-old Nicotiana benthamiana leaves. After 20h of infiltration, the protein expression was induced by spaying leaves with 20 µM β-estradiol in 0.1% Tween 20. The leaves were imaged after 18h of induction.
The leaf samples were imaged on a Zeiss LSM510 confocal microscope with a Plan-Apochromat 40x/1.3 Oil objective. GFP was excited using 488 nm Argon laser and observed through a 505/530 nm bandpass filter. mCherry was excited using 543 nm HeNe laser and observed through a 585/615 nm bandpass filter. The images were processed with FIJI 15 .

Data Availability
The raw data of mass spectrometry experiments will be deposited to PRIDE (https://www.ebi.ac.uk/pride/archive/).