A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis

Regulated protein degradation contributes to plant development by mediating signaling events in many hormone, light, and developmental pathways. Ubiquitin ligases recognize and ubiquitinate target proteins for subsequent degradation by the 26S proteasome. The multisubunit SCF is the best-studied class of ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). However, the extent of SCF participation in signaling networks is unclear. SCFs are composed of four subunits: CULLIN 1 (CUL1), ASK, RBX1, and an F-box protein. Null mutations in CUL1 are embryo lethal, limiting insight into the role of CUL1 and SCFs in later stages of development. Here, we describe a viable and fertile weak allele of CUL1, called cul1-6. cul1-6 plants have defects in seedling and adult morphology. In addition to reduced auxin sensitivity, cul1-6 seedlings are hyposensitive to ethylene, red, and blue light conditions. An analysis of protein interactions with the cul1-6 gene product suggests that both RUB (related to ubiquitin) modification and interaction with the SCF regulatory protein CAND1 (cullin associated and neddylation dissociated) are disrupted. These findings suggest that the morphological defects observed in cul1-6 plants are caused by defective SCF complex formation. Characterization of weak cul1 mutants provides insight into the role of SCFs throughout plant growth and development.


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Molecular cloning 354 The pMiniT-gAFB1(AT4G03190)-mCitrine constructs were previously described 1 . The domain swap constructs 355 were assembled using the NEBuilder HiFi DNA assembly kit (NEB). Briefly, TIR1(AT3G62980) domains or 356 the entire TIR1 5′ UTR plus coding sequence were amplified with primers with 5′ extensions matching AFB1, 357 then inserted into corresponding PCR-amplified pMiniT-gAFB1 backbone fragments. These chimeric genes 358 were then moved to the pMP535 binary vector as MluI-AscI fragments. For generating gAFB1-NES-mCitrine 359 and gTIR1-NES-mCitrine lines, a double-stranded oligo with the NES fragment was inserted into NheI-digested 360 pMiniT-gAFB1nhe and pMiniT-gTIR1nhe constructs, in which the stop codons were mutated to create NheI 361 restriction enzyme sites 1 . An mCitrine fragment was amplified, digested with XbaI, and then inserted into the 362 NheI site after the NES fragment. For pMiniT-gAFB1-NLS-mCitrine, a fragment containing the SV40 NLS 363 followed by mCitrine was amplified, cut with XbaI, and ligated into the NheI-digested pMiniT-gAFB1nhe 364 plasmid. These pMiniT-gAFB1/gTIR1-NES/NLS-mCitrine were subcloned into the pMP535 binary vector as 365 described previously. 366 The first 53 and 57 amino acids of AFB1 and TIR1, respectively, ending in CYAVS were exchanged to generate 367 the constructs fbATTT and fbTAAA, respectively. The ccvTIR1 and ccvfb-TAAA lines were generated by the 368 substitution mutations F79 > G in TIR1 6 ; ccvAFB1 and ccvfb-ATTT lines were generated by a substitution 369 mutation F75 > G in AFB1. The ccvTIR1 E12K mutation was created as described before 7 . The plasmid 370 constructs of ccvTIR1, ccvAFB1, ccvfb-ATTT, ccvfb-TAAA, fbATTT, fbTAAA, and ccvTIR1 E12K were first 371 cloned into the pUPD2 vector. Then, the genes were cloned into pDGB1alpha1 by combining the TIR1 372 promoter, a C-terminal mScarlet-I 8 or mVenus tag, and a 35s terminator into the pDGB1alpha1 vector. These 373 transcriptional units were then combined with a Basta resistance cassette into the pDGB3omega1 binary vector. 374 Cloning steps were performed using the GoldenBraid method 9 (https://gbcloning.upv.es/). 375 The binary constructs were transformed into Agrobacterium tumefaciens strain pGV3101 by electroporation, 376 and then transformed into Col-0, afb1-3, tir1afb2, or tir1afb345 plants by floral dipping 10  Calcium transients were visualized using the R-GECO1 reporter 11 and imaged using a microfluidic setup 382 combined with a vertical spinning disk microscope 12 . Five-day-old Arabidopsis seedlings were transferred to a 383 sealable single-layer PDMS silicone chip. The PDMS silicone chip containing the seedlings was then placed on 384 a vertical spinning disk microscope for 20 min to recover. The seedlings were imaged with a 20x/0.8 objective 385 every 15 seconds for the first 5 minutes with control medium and then switched to treatment medium for 10 386 minutes. Constant media flow and switching between control and treatment medium was performed using OBI1 387 Elveflow software. Images were acquired with VisiView (Visitron Systems, v.4.4.0.14). Control and treatment 388 . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted January 4, 2023. ; https://doi.org/10.1101/2023.01.04.522696 doi: bioRxiv preprint medium were prepared with 1% (w/v) sucrose containing either 0 or 150 nM IAA. For ccvAFB1 and ccvTIR1 R-389 GECO1 lines, we used 500 nM cvxIAA (#M3141, BDL) prepared from a 10 mM stock in DMSO. 390 R-GECO1 time series images were analyzed in Fiji 13 . Images were stabilized by selecting a ROI in-root 391 transition zone and using imageJ plugin registration followed by correcting 3D drift 14 . We then quantified the R-392 GECO1 intensity in a region of interest (ROI) in several epidermal cells in the root transition zone. Signal 393 intensity at each timepoint (F) was normalized using the average value of 8 timepoints (2 min) before treatment 394 (F 0 ), obtaining the F/F 0 . In the case of tir1afb2 mutant, the R-GECO1 expression was generally lower, therefore 395 we subtracted the background noise level before calculating the F/F0. 396 397 Quantification of gravitropic response 398 The gravitropic experiment was done as described 15 : a thin layer of growth medium (½ MS, 1% (w/v) sucrose in 399 1% plant agar, Duchefa) containing 5-day-old seedlings was placed into a 3D-printed microscopy chamber. The 400 chamber was placed vertically on the vertical stage microscope for 45 minutes to recover. Then the chamber 401 was rotated by 90 degrees. After the two minutes needed to set the imaging, the roots were imaged every minute 402 for 43 minutes. The root tip angles were measured using ACORBA v1.2.

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Root growth assay 405 For the rapid root growth inhibition assay, we used two methods. The first method was previously described 1,16 , 406 and was used for Figure 1a, 1b, 3b, 3f, 4c, 4e, S1b, S1e, S1h. Briefly, 10-15 five-day-old seedlings were 407 transferred to culture chambers containing the growth medium supplemented with EtOH (mock) or 10 nM IAA 408 (stock solution 10 µM in EtOH), and immediately imaged every 25 seconds for 20 minutes or every 72 seconds 409 for 1 hr. The 50 images taken for 20 min or 1 hr were combined and processed by MatLab software using a 410 customized Matlab script called Rootwalker 16 . Rootwalker MATLAB generated growth curves of individual 411 roots (root length increment/time interval) for each treatment (mock and IAA) and exported to jpeg and excel 412 files. In the second method, used in Figure 1c, h, five-day-old seedlings were transferred to growth medium 413 containing either 0 or 10 nM IAA. The seedlings with the media were then immediately transferred to a 3D-414 printed microscopy chamber (24 × 60 mm) and placed on the vertical microscope. 4-5 min were required to set 415 the stage positions on the microscope, and then roots were imaged for 10 minutes after each 5 min using a 416 5x/0.16 objective. Root growth (length increment) measured after stabilizing the background drift of the root tip 417 using the Fiji plugin correct 3D drift, as described 12 . 418 For measuring long-term root growth (3 hr and 6 hr), vertically grown five-day-old seedlings were imaged at 419 1,200 dpi using a flatbed scanner at time 0, 3hr and 6hr after transferring onto treatment medium (mock and 10 420 nM IAA). Root tips of individual seedlings were marked at each time point, and root growth increment was 421 measured using the segmented line tool in FIJI.

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The root growth response to auxin was calculated as the length increment in treatment divided by the length 423 increment in control conditions. The response value 1 indicates that the treatment did not affect root growth.

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For measuring primary root length after long-term auxin treatment, five day-old seedlings were transferred to 425 media containing the ethanol (mock) or 100 nM IAA and incubated for 3 days. The response to IAA was 426 expressed as IAA-treated primary root length divided by the mean value of mock-treated primary root length.

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To acquire subcellular localization of ccvAFB1and ccvTIR1 E12K and ccv-fbATTT five-days old seedlings 456 roots were stained with 2.5 µg/ml propidium iodide for 2 min and mounted on a glass slide with ½ MS, 1% 457 sucrose medium followed by imaging with Zeiss LSM 880.

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Lateral root and primary root measurement 460 Vertically grown eight-day-old seedlings were used for lateral root measurements as previously described 20 . 461 Briefly, seedlings were soaked in pre-heated 0.24 N HCl in 20% methanol, and further incubated at 60 ℃ for 10 462 minutes. overlapping and specialized functions. Elife 9, 1-28 (2020). 491 . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted January 4, 2023. ; https://doi.org/10.1101/2023.01.04.522696 doi: bioRxiv preprint