SLOMO controls AUX1-mediated gravitropism through a non-proteolytic role for AUX1 ubiquitination

doi:10.21203/rs.3.rs-4616599/v1

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SLOMO controls AUX1-mediated gravitropism through a non-proteolytic role for AUX1 ubiquitination

https://doi.org/10.21203/rs.3.rs-4616599/v1

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Gravity-directed growth ensures that shoots and roots grow upwards and downwards, respectively. To achieve this, the organ’s angle with respect to gravity drives the asymmetric redistribution of the plant hormone auxin and consequently differential elongation creating a curvature of the organ. In addition to efflux transporters, the auxin influx transporter AUXIN RESISTANT 1 (AUX1) is essential for auxin redistribution during root gravitropism. Our results provide a biochemical framework that includes the F-box protein SLOMO affecting, possibly indirectly, AUX1 levels, and SLOMO controlling the ubiquitination of K261, K264, and K266, which affects AUX1 auxin transport properties. This reveals a novel, non-proteolytic role for SLOMO-mediated ubiquitination of AUX1.

Biological sciences/Plant sciences/Plant hormones/Auxin

Biological sciences/Cell biology/Post-translational modifications/Ubiquitylation

Biological sciences/Plant sciences/Tropism

Plant growth and development are affected by environmental signals, which can be variable (e.g. heat or drought) or constant (e.g. gravity). Gravity-directed growth (or gravitropism), ensures that shoots and roots typically grow upwards and downwards, respectively. To achieve this, the organ’s angle with respect to gravity drives the asymmetric redistribution of the plant hormone auxin and consequently differential elongation creating a curvature of the organ ^1–5. This auxin redistribution requires regulation of auxin influx and efflux carriers at multiple levels including transcription, polarized subcellular protein localization, protein modification and protein recycling and degradation, with several of these processes controlled by post-translational modifications ^6–8. In the Arabidopsis root, the auxin influx transporter AUXIN RESISTANT 1 (AUX1) and the efflux transporters, PIN-FORMED 2 (PIN2) and ATP-BINDING CASSETTE B1 (ABCB1), are essential for auxin redistribution during root gravitropism ^9–12. For example, the reduction of PIN2 at the upper side is due to PIN2 ubiquitination and subsequent vacuolar targeting of PIN2 ^13,14. However, little is known about the post-transcriptional regulation of AUX1 in the control of root gravitropism ^9,15.

Ubiquitination of lysine residues involves three main steps, namely ubiquitin activation, conjugation, and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively ^16,17. Ubiquitination marks proteins for degradation via the proteasome, alters their cellular location, affects their activity, and promotes or prevents interactions with other proteins ^18–20. Among the E3 ligases, F-box proteins are part of to the Skp-Cullin-F-box (SCF) complex and, in Arabidopsis, there are around 700 F-box proteins ^17,21. One of these F-box proteins, SLOW MOTION (SLOMO), is involved in organ initiation and hypocotyl growth ^22,23. While it was proposed that SLOMO is required for normal auxin distribution in the plant, the underlying mechanism is largely unknown ²². Here, we used the novel role of SLOMO in gravitropism to unravel its role in ubiquitination-mediated control of AUX1 levels and activity.

To gain deeper insight into the roles and targets of the hardly explored F-box protein SLOW MOTION (SLOMO), we revisited slomo loss-of-function phenotypes. In addition, to other typical auxin-mediated phenotypes (Supplementary Fig. 1) ^22,24, we observed a faster and exaggerated primary root response to gravity (gravitropism) of slomo mutants, compared to Col-0 (Fig. 1a-c and Supplementary Fig. 2). In contrast, 35S-mediated overexpression of mCherry:SLOMO (35S::mCherry:SLOMO) (Supplementary Fig. 3) resulted in a delayed primary root response to gravity compared to Col-0 (Fig. 1d-e). Root gravitropic bending is accompanied by asymmetric auxin distribution and response with the maximum at the lower side of gravity-stimulated roots, which can be monitored with the transcriptional auxin response reporter pDR5::GUS ^15,25–27. To assess auxin distribution and response, vertically grown Col-0 and slomo-3 seedlings expressing pDR5::GUS were subjected to a 90° gravity stimulus and the expression pattern of pDR5::GUS was examined over time. The typical increased GUS activity at the lower side of the root was observed significantly earlier in slomo-3 compared to Col-0 (Fig. 1f). Since the above-mentioned phenotypes are associated with auxin transport and in agreement with a genetic interaction of SLOMO and components of polar auxin transport ^22,28, we analyzed the effect of an auxin efflux inhibitor (NPA) and an auxin influx inhibitor (2-NOA) on primary root length. The response of slomo-3 roots to NPA treatment was similar to Col-0 (Fig. 1g). In contrast, slomo-3 displayed a higher reduction in primary root length upon 2-NOA treatment than Col-0 (Fig. 1g). The auxin transporters AUXIN RESISTANT 1 (AUX1, auxin influx carrier) and PIN2 (auxin efflux carrier) are required for the asymmetric distribution of auxin driving the root gravitropic response ^9,29,30. The higher sensitivity of slomo-3 roots to 2-NOA suggested that auxin influx rather than auxin efflux is altered in slomo-3. This is further supported by an increased sensitivity of slomo-3 to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), the uptake of which is facilitated by AUX1 ^31,32 (Fig. 1h).

We next showed that SLOMO is expressed in the root tip, with strong expression in the root cap and root epidermis, which overlaps with the cells where the AUX1 protein is found ³³ (Supplementary Fig. 4). However, we did not observe any differential expression of SLOMO between the upper and the lower side of the root tip (Supplementary Fig. 4). We subsequently explored if the F-box protein SLOMO directly interacts with AUX1. To stabilize the interaction with putative E3-targets, we used a SLOMO-decoy protein variant that lacks the F-box domain and is still able to bind to substrates, but lacks the ability to be recruited into CULLIN 1 ligase complexes to mediate substrate ubiquitination and degradation ^24,34. Using co-immunoprecipitation (co-IP) following transient co-expression of mCherry:SLOMO-Decoy with AUX1:YFP in Nicotiana benthamiana, we revealed a direct interaction (Fig. 2a). Moreover, the association of SLOMO and AUX1 was further confirmed by a co-IP assay using transgenic aux1-22 plants carrying YFP-tagged AUX1 expressed under the control of its endogenous promoter (pAUX1::AUX1:YFP) and mCherry-tagged SLOMO expressed under the control of the 35S promoter (35S::mCherry:SLOMO) (Fig. 2b).

F-box proteins, such as SLOMO, are responsible for substrate recognition by multi-protein E3 ubiquitin ligase complexes that typically catalyze the ubiquitination of proteins destined for 26S proteasomal degradation ³⁵. Furthermore, ubiquitination can also have non-proteolytic roles, such as membrane trafficking, protein kinase activation, DNA repair, and chromatin dynamics ³⁶. For example, ubiquitin marks plasma membrane cargo for internalization ³⁷, and the closely related ubiquitin-conjugating enzymes UBC35 and UBC36 are the main sources of K63-linked ubiquitin chains that mediate vacuolar degradation of plasma membrane proteins in Arabidopsis ³⁸. Given that SLOMO and AUX1 interact, it is likely that SLOMO ubiquitinates AUX1. To evaluate whether a fraction of AUX1 is post-translationally modified by ubiquitination, immunoprecipitated AUX1:YFP was probed with the general P4D1 anti-ubiquitin antibody that recognizes monoubiquitin and several forms of polyubiquitin chains. We observed a high molecular weight smear (55 kDa – higher), typical of ubiquitinated proteins, in immunoprecipitates from aux1-22 pAUX1::AUX1:YFP plants (Fig. 2c-d). In contrast, we observed a much weaker high molecular weight smear in aux1-22 pAUX1::AUX1:YFP slomo-3 compared to aux1-22 pAUX1::AUX1:YFP (Fig. 2c-d). Taken together, this supports that SLOMO ubiquitinates AUX1.

Since SLOMO ubiquitinates AUX1, we explored if the protein level of AUX1 was affected in slomo mutants. Indeed, we observed higher AUX1 levels in slomo mutant roots compared to Col-0 through Western blot (Fig. 3a-b). Microscopic analyses of pAUX1::AUX1:YFP in slomo-3 aux1-22 compared to pAUX1::AUX1:YFP in aux1-22 supported the higher AUX1 levels in slomo-3 (Fig. 3c-d and Supplementary Fig. 5). In contrast, overexpression of SLOMO resulted in lower AUX1 levels (Fig. 3e-f). We confirmed that these differences in AUX1 levels in slomo mutants and SLOMO overexpression lines are not caused by elevated AUX1 expression (Supplementary Fig. 6). Finally, similar to AUX1 protein accumulation in slomo-3, treatment with concanamycin A (ConA), which inhibits vacuolar degradation in Arabidopsis roots ³⁹ led to accumulation of AUX1 protein in aux1-22 pAUX1::AUX1:YFP (Fig. 3g-h). In contrast, treatment with the proteasome inhibitor MG132 ⁴⁰ did not affect AUX1 protein levels (Fig. 3g-h). These results support that SLOMO regulates AUX1 protein levels, likely through vacuolar targeting.

However, mathematical modelling predicted that globally increasing or decreasing AUX1-mediated auxin flux had limited effect on auxin redistribution upon a gravitropic stimulus (Fig. 3i-j and Supplementary Fig. 7). In contrast, the model predicted that increasing AUX1-mediated auxin flux through increased AUX1 activity only on the lower side of the root tip resulted in a faster establishment of the auxin asymmetry, with higher auxin levels on the lower side (Fig. 3i-j). Simulation of other possible scenarios for AUX1 activity between the upper and the lower root sides either disrupted or did not change the speed in establishing the auxin asymmetry (Supplementary Fig. 7). Therefore, the change in AUX1 levels downstream of SLOMO does not explain the observed increase in auxin asymmetry and gravitropic response and might be an (indirect) side effect. We therefore hypothesized that (some) SLOMO-mediated ubiquitination sites could have other roles.

Therefore, we next attempted to identify in planta ubiquitinated AUX1 residues, but so far, we were not able to identify these (data not shown). Therefore, to pinpoint relevant ubiquitination sites for further functional analyses, we focused on highly conserved lysines as ubiquitination target sites in the AUX1 intracellular hydrophilic regions (Fig. 4a and Supplementary Fig. 8) and especially in the region that was most associated with agravitropic mutants ⁴¹ (Fig. 4a and Supplementary Fig. 8). Next, we evaluated the impact on incorporation of HA:UBQ1 when these lysines (K) were replaced with an arginine (R), by generating AUX1^3K > R (mutating amino acids K261, K264, and K266) and AUX1^5K > R (mutating amino acids K261, K264, K266, K339, and K347) protein variants. This revealed that the AUX1^3K > R variant was less ubiquitinated than AUX1 (Fig. 4b and Supplementary Fig. 9). Since the AUX1^5K > R variant, compared to AUX1^3K > R, did not display an additional decrease in ubiquitination, we concluded that K261, K264, and K266 are the major target sites for ubiquitination and we therefore focused on the AUX1^3K > R variant for subsequent analyses. While SLOMO-mediated AUX1 ubiquitination was largely absent on the AUX1^3K > R variant (Fig. 4c), the AUX1^3K > R variant did not result in highly altered AUX1 levels (Fig. 4d and Supplementary Fig. S10). Furthermore, the AUX1^3K > R variant could only partially rescue the aux1-22 mutant (Fig. 4e). These results suggest that the AUX1^3K > R variant is, at least partially, impaired in its activity.

We therefore speculated that the AUX1 ubiquitination on K261, K264, and K266 is not impacting AUX1 degradation, but could affect AUX1 auxin transport properties. To explore this further, we used the AlphaFold2-predicted AUX1 structure to analyse the impact of the K > R mutations (Supplementary Fig. S11). The root-mean-square deviation (RMSD) from the in silico molecular dynamics (MD) simulation showed that the motility of the AUX1^3K > R variant is reduced (Supplementary Fig. S11). The root-mean-square fluctuation (RMSF) points out that several AUX1 transmembrane helices are restricted in the AUX1^3K > R variant, such as the helices near the mutated region including the 233 to 253 and 267 to 287 residues (Fig. 5a). These residues are crucial for the functionality of membrane channels and transporters ^42–44 that switch between open and closed states by having highly flexible transmembrane helices. This was further confirmed through auxin transport assays using tobacco protoplasts, where auxin influx activity was dramatically impaired in the AUX1^3K > R variant compared with the AUX1 wildtype (Fig. 5b and Supplementary Fig. 12), without affecting the AUX1 protein levels (Supplementary Fig. 12). In addition, while pAUX1::AUX1:YFP was able to partially restore sensitivity to 2,4-D, this was not the case for pAUX1::AUX1^3K > R:YFP (Fig. 5c). Taken together, this suggests that replacing lysines with arginines at K261, K264, and K266, and thus preventing ubiquitination, impairs AUX1 transport activity.

It was previously suggested that PINs control the velocity of auxin transport, while AUX1 controls which tissues have high auxin levels ⁴⁵. Furthermore, a higher level of auxin will lead to a faster regulation of – at least – the transcriptional auxin response ^46,47. Previously, genetic evidence suggested that SLOMO plays an important role in controlling auxin transport ²². Our results provide a biochemical framework that includes SLOMO affecting, possibly indirectly, AUX1 levels, and SLOMO controlling the ubiquitination of K261, K264, and K266, which affects AUX1 auxin transport properties. This reveals a novel, non-proteolytic role of SLOMO-mediated ubiquitination of AUX1. However, to fully incorporate a role for SLOMO in ubiquitin-mediated AUX1 activity on the lower side of the root tip, another regulatory layer is required. This is, for example, a differential incorporation in or activity of the SCF complex that SLOMO is a part of. Alternatively, this involves differential activity of a deubiquitinating enzyme (DUB). With respect to the latter, we previously found that SLOMO interacts with UBP12/13 ²⁴. We therefore checked if the ubp12-2 mutant, in which the levels of both UBP12 and UBP13 transcripts are down-regulated ^48,49 displayed altered gravitropism. However, we did not observe any obvious differences (Supplementary Fig. 13). Taken together, our results point to a model where SLOMO controls AUX1 activity through ubiquitination of K261, K264, and K266, and this is likely counteracted upon a gravity stimulus by the activity of a so far unknown DUB or locally regulated SCF complex activity at the upper side of the root tip.

Plant materials and growth conditions

All Arabidopsis thaliana plants used in this study were in the Col-0 reference accession genetic background and referred to as wild-type. The following Arabidopsis thaliana lines were used: slomo-3, slomo-4, slomo-5, slomo-3 pSLOMO::GPF:SLOMO, slomo-4 pSLOMO::GPF:SLOMO, slomo-5 pSLOMO::GPF:SLOMO ^22,24; Col-0 35S::mCherry:SLOMO, aux1-22 pAUX1::AUX1:YFP 35S::mCherry:SLOMO; pDR5::GUS ⁵⁰; aux1-22 and aux1-22 pAUX1::AUX1:YFP ⁵¹; ubp12-2 ⁴⁹. For the phenotypic assay, Arabidopsis seeds were sown on ½ Murashige and Skoog (MS) growth medium containing 1% sucrose (per litre: 2.15 g of MS salts, 0.1 g of myo-inositol, 0.5 g of MES, 10 g of sucrose and 8 g of plant tissue culture agar; pH 5.7). For the NPA, NOA and 2,4-D treatments to Col-0 and slomo-3, NPA and NOA were dissolved in DMSO. The ½ MS medium was then supplemented with 5µM NPA or 1µM NOA, 50 nM 2,4-D or an equal volume of DMSO as Mock. For the gravitropism treatment, seedlings were vertically grown on ½ MS medium under continuous light conditions for 3 days after germination (DAG), and subsequently, the seedlings were transferred, with their root tips positioned vertically, to a new ½ MS plate and then rotated 90 degrees, and were kept in the dark during the gravitropic experiment (time course of 6 hours gravistimulation). Images were taken with a Canon scanner and measurements were performed using Fiji ImageJ (https://imagej.net/Fiji). For AUX1 protein level and ubiquitination assays with Arabidopsis thaliana plants, seedlings were grown on sugar-free ½ MS plates. For ConA and MG132 treatment, seedlings grew on ½ MS sugar-free medium under continuous light conditions for 5 days after germination, then transferred the seedlings to liquid ½ MS sugar-free medium with DMSO, 50 µM MG132 and 1 µM ConA respectively treated for 5 h in darkness.

2,4-D treatment for aux1-22 complementing lines

Seedlings were grown vertically on MS growth medium (per litre: 4.3g MS salts, 1% sucrose, 1% plant tissue culture agar, pH to 6.0 with KOH) for 3 days after germination before being transferred to a new MS containing 100 nM 2,4-D for additional 3 days. The elongated primary root was measured by using Fiji ImageJ.

GUS staining assay

3 days-after-germination-old pDR5::GUS and pDR5::GUS slomo-3 seedlings after the gravistimulation were fixed for 30 min in ice-cold 90% (v/v) acetone and rinsed with NT buffer (100 mM Tris-HCl (pH = 7.0), 50 mM NaCl), incubated in X-Gluc (500 µg ml^− 1 5-bromo-4-chloro-3-indolyl-β-D-glucuronide) solution (500 µl 100 mM K₃[Fe(CN)₆] + 600 µl X-Gluc + 28.8 ml NT-buffer), and incubated at 37°C in the dark for 3 h. The reaction was stopped by rinsing with NT-buffer, and then seedlings were mounted in 80% lactic acid on a slide and imaged with an Olympus microscope.

Confocal microscopy

Seedlings were mounted in Milli-Q water between the slide and cover glass for imaging using a Zeiss inverted LSM710 confocal laser scanning microscope, equipped with a LD-Plan Neofluar ×40/0.6 Korr M27 or C-Apochromat ×40/1.20 W Korr M27. A 488 nm laser excitation (at 2% power) of a 20 mW argon laser (LASOS, Jena, Germany) and a spectral detection bandwidth of 493–532 or 493–579 nm were used for detecting eGFP. For propidium iodide (PI) staining, whole seedlings were mounted in PI solution. The PI signal was detected using a 561 nm laser excitation (at 2% power), together with a spectral detection bandwidth of 596–645 nm. Fluorescence intensity of AUX1:YFP signal was measure by Fiji ImageJ (https://imagej.net/Fiji).

Nicotiana benthamiana infiltration

For transient co-agroinfiltration of Nicotiana benthamiana leaves, Agrobacteria containing the indicated constructs and P19 were grown in 5 ml LB supplemented with appropriate antibiotics at 28°C for 1–2 days. Then 500 µl was inoculated in 10 ml LB supplemented with 10 mM MES pH 5.6, 10 µM acetosyringone as well as the antibiotics and incubated at 28°C overnight. The pellet was spun down and resuspended with infiltration buffer (10 mM MgCl₂, 10 mM MES [pH 5.6], 100 µM acetosyringone) to a final OD600 of 1.0. Equal volumes of three agrobacteria with selected constructs were mixed well and the mix was infiltrated in 5–6 weeks old tobacco leaves using a syringe. The signal was checked by confocal microscopy after 72 h to make sure the protein expressed well. For the ConA treatment, 1 µM ConA in infiltration buffer was infiltrated into the same Nicotiana benthamiana leaves 16 hours before harvesting.

Co-immunoprecipitation (co-IP) and ubiquitination assay in Nicotiana benthamiana

Total proteins were extracted with buffer containing 150 mM Tris·HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 100 mM EDTA, 1 mM sodium molybdate, 1 mM NaF, 10 mM DTT, 1% NP-40, 1 mM PMSF and EDTA-free protease inhibitor mixture complete (Roche). The protein concentration was measured using the Qubit™ Protein Assay Kit (Qubit™ Protein Assay Kit) and an equal amount of total protein was used for immunoprecipitation. 25 µl of pre-equilibrated GFP-Trap® MA beads (ChromoTek) or HA-Trap®_MA beads (Thermo Fisher) was prewashed 3 times with 700 µl wash buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl and 0.5% NP-40). The protein homogenates were incubated with GFP-Trap® MA beads or HA-Trap®_MA beads and rotated for 2 h at 4°C to maximize protein binding. Subsequently, separating protein homogenates and beads utilizing a magnet, the beads were washed three times with wash buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.5% NP-40). Finally, 1× sample buffer (Bio Rad) was added to elute protein from the beads.

Microsomal fraction preparation

Isolation of microsomal fractions was performed as previously described ⁵² with minor modifications. Seedlings were ground in liquid nitrogen and re-suspended in ice-cold sucrose buffer [100 mM Tris-HCl, pH 7.5, 810 mM sucrose, 5% (vol/vol) glycerol, 10 mM EDTA, pH 8.0, 10 mM EGTA, pH 8.0, 5 mM KCl, and complete protease inhibitor mixture and the PhosSTOP phosphatase inhibitor mixture (both from Roche)]. The homogenate was transferred to a pre-cold 5% (vol/vol) polyvinylpolypyrrolidone pellet, mixed and then kept on ice for 5 min. Samples were then centrifuged for 5 min at 600 g at 4°C and the supernatant was collected. This extraction step was repeated two more times. The clear supernatant was combined with the same volume of distilled water and centrifuged at 4°C for 2 h at 21000 g to pellet microsomes. The pellets were washed with 1 ml of wash buffer (10 mM Tris-HCl, pH 7.5) and centrifuged at 4°C for 30 min at 21000 g and stored at -70°C until further use.

Co-immunoprecipitation in Arabidopsis thaliana

For co-immunoprecipitation (co-IP) and ubiquitination assays, re-suspension of the membrane pellets with extraction buffer [50 mM Tris-HCl, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% SDS and protease inhibitor (Roche), to which 1 mM of PMSF and 10 mM of iodoacetamide were freshly added]. This suspension was centrifuged at 4°C for 30 min at 21,000 × g to obtain a clear supernatant. The solubilized microsomal proteins were first diluted 10x with extraction buffer containing without SDS and then incubated with 25 µL pre-equilibrated GFP-Trap®_MA beads (Chromotek) and rotated for 2 h at 4°C to maximize protein binding. The samples were cleared on a magnetic separation rack and remove supernatant. To remove unspecific binding, the beads were washed in 3 × 1 mL wash buffer [25 mM Tris-HCl, PH = 7.5, 250 Mm NaCl]. Finally, 1× sample buffer (Bio Rad) was added to elute protein from the beads.

Western blot

For SLOMO protein abundance analysis, protein samples were heated at 70°C for 10 min. For AUX1 protein detection, protein samples were heated at 28°C for 45 min, then separated on 4–20% SDS-PAGE stain-free protein gel (Bio-Rad Laboratories, Inc., USA), followed by transferring onto a Trans-Blot® Turbo™ Mini PVDF Transfer Packs (Bio-Rad Laboratories, Inc., USA). For blocking and antibody dilutions, 5% milk powder in TBST solution was used. For protein detection the following antibodies were used: monoclonal α-GFP horseradish peroxidase coupled (1:5,000; Miltenyi Biotech), α-HA (1:5000; abcam), α-AUX1 (1:5000, Agrisera), α-RFP (1:2000, ChromoTek), α-P4D1 (1:2000; Santa Cruz), mouse IgG HRP linked whole antibody (GE Healthcare, Chicago, IL, USA; 1 : 10 000), goat IgG HRP linked whole antibody (1:10000, Dako) and α-Tubulin (1:5000, Sigma). The proteins were detected by ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc., USA). When required, the membranes were stripped using stripping buffer (1:1–1.5 ml 10% SDS: 1.5 ml 100 mM Glycine mix) for 90 s, were washed 3×5 min with T-BST, and then again subjected to the procedure described above.

qPCR analyses

Three biological replicates were performed for each condition. Total RNA was extracted with the RNeasy Mini Kit (Promega) according to the manufacturer’s instructions. DNA digestion was done on columns with RNase-free DNase I (Promega). The iScript cDNA Synthesis Kit (Biorad) was used for cDNA synthesis from 1 µg of RNA. qRT-PCR was performed on a LightCycler 480 (Roche Diagnostics) in 384-well plates with LightCycler 480 SYBR Green I Master (Roche) according to the manufacturer's instructions. Two housekeeping genes, ACTIN and the EF1α were used for normalization of the expression level of the tested genes. All the primers are listed in Supplemental Table 1.

Generation of constructs and transgenic lines

35S::mCherry:SLOMO was described previously ²⁴. AUX1 promoter got from Ranjan Swarup ⁵¹. The DNA sequences of AUX1:YFP(116 aa) and AUX1:YFP(116 aa) ^3K>R refer as AUX1:YFP and AUX1:YFP^3K > R were synthesized by Twist Bioscience, and the synthesized sequences were assembled into the entry vector pEN-L1-AG-L2 and then ligated to the relevant destination vector via a gateway cloning system. Plant vectors were transformed in Agrobacterium tumefaciens C58C1 using a freeze–thaw method ⁵³. 35S::mCherry:SLOMO transformed to Col-0 and aux1-22 pAUX1::AUX1:YFP respectively performed using floral dip method ⁵³.

Mathematical modelling

Simulations were based on the model of auxin dynamics presented previously ⁵⁴, which provides full details of the model assumptions, equations and parameter values used. In brief, the model uses a multicellular root tip geometry extracted from a confocal image of a root tip, and distributions of PIN, AUX1/LAX, and plasmodesmata based on experimental observations. The modelling framework integrates these data into a system of linear ordinary differential equations (ODEs) for the auxin concentration within each cell and cell-wall compartment, which contain terms representing passive diffusion of protonated auxin across cell membranes, carrier-mediated transport of anionic auxin across cell membranes, auxin diffusion within the cell wall, auxin diffusion through plasmodesmata, auxin synthesis and degradation. These ODEs are simulated using python ODE solver, and the matplotlib library is used to present the model solutions. To simulate gravitropic reorientation, we assume that at time t = 0, PIN3 and PIN4 in the columella cells are repositioned to the membrane on the lower side of the cells. Further simulations considered a change in AUX1 activity after reorientation and were produced by prescribing a different value for AUX1 activity on the upper/lower side of the root at time t = 0. Python code that produces the model simulation results is available at

https://gitlab.com/leahband/auxin_gravitropism_SLOMO .

AUX1 lysine sites conservation assay

AUX1 (AT2G38120) protein sequence downloaded from TAIR (https://www.arabidopsis.org/). 560 AUX1 orthologous genes from 96 species was collected form Dicots PLAZA 5.0 by blasting ⁵⁵. AUX1 alignment was performed with CLC Main Workbench 8 (CLC Bio-Qiagen, Aarhus, Denmark).

Molecular dynamics (MD) simulations and analysis

All-atom MD simulations were performed using NAMD 3.0b6 ⁵⁶ with the CHARMM36m force field ⁵⁷. Protein system building was performed using CHARMM-GUI ^57,58. The predicted AUX1 structure was downloaded from the AlphaFold protein structure database ⁵⁹ and trimmed from residue 35 to 471 to remove low confidence protein tails. The wild-type or mutated AUX1 was inserted into a phosphatidylcholine (POPC) bilayer membrane and solvated with TIP3P water molecules ⁶⁰ at both sites of the bilayer lipid membrane in periodic rectangular boxes to a depth of at least 80 Å. The systems were minimized for 1000 steps and then simulated under the NVT (constant number of atoms, volume and temperature) system at 295 K using a Langevin thermostat. Four independent 50 ns simulations of both wild-type and mutated AUX1 systems were performed with structure saving per 100 ps. The resulting simulation was analyzed using the Python package MDAnalysis ⁶¹. The RMSD of C-alpha was calculated from the structure after minimization. The RMSF of C-alpha was calculated over all residues. The structural representations of the proteins were generated using Chimera X ⁶².

Auxin transport measurements

Simultaneous ³H-IAA and ¹⁴C-BA import into Nicotiana benthamiana mesophyll protoplasts was analyzed as described previously ⁶³. Tobacco mesophyll protoplasts were prepared 4 days after Agrobacterium-mediated transfection of 35S::AUX1:YFP or 35S::AUX1:YFP^3K > R, respectively. Relative import into protoplasts is calculated from imported radioactivity into the protoplast as follows: (radioactivity in the supernatant at time t = 15 min.) - (radioactivity in the protoplast at time t = 0) * (100%)/ (radioactivity in the protoplast at t = 0 min).

Statistics and reproducibility

Phenotypic quantification, western blot quantification, and relative gene expression data were analyzed using either One-way or Two-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) in GraphPad Prism 8.0.1. In some cases, data were analyzed using Students’ t-test with two-sample with parametric distribution in GraphPad Prism 8.0.1 as well. For box plots, the lower Tukey-based whisker shows the smallest value that is greater than the lower quartile minus 1.5× interquartile range, the upper Tukey-based whisker shows the greatest value that is smaller than the upper quartile plus 1.5× interquartile range and data points outside this range (outliers) are plotted as individual dots.

Acknowledgements

We thank Ke Xu for assistance with gravitropism and lateral root phenotyping. L.P. and S.Z. were supported by the Chinese Scholarship Council (201806870020, 201506230173) and L.P. also received support from the Ghent University Special Research Fund (01CD0523). M.M.G. was supported by the Swiss National Funds (310030_197563). L.R.B.’s work was supported by the Biotechnology and Biological Sciences Research Council (grant number BB/M019837/1).

Contributions

I.D.S. initiated and managed the project, designed experiments, analyzed and interpreted data, and wrote the manuscript; L.P. designed and performed experiments, analyzed data, and wrote the manuscript; S.Z. designed and performed experiments, and analyzed data; F.R.I., S.Y., P.N., B.v.d.C. performed experiments; L.R.B. performed modelling; K.G., R.S., M.M.G., contributed ideas, interpreted results and critically revised the manuscript. All authors discussed the results and approved the manuscript.

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SLOMO controls AUX1-mediated gravitropism through a non-proteolytic role for AUX1 ubiquitination

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Abstract

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INTRODUCTION

RESULTS AND DISCUSSION

MATERIALS AND METHODS

Plant materials and growth conditions

GUS staining assay

Confocal microscopy

Nicotiana benthamiana infiltration

Co-immunoprecipitation (co-IP) and ubiquitination assay in Nicotiana benthamiana

Microsomal fraction preparation

Co-immunoprecipitation in Arabidopsis thaliana

Western blot

qPCR analyses

Generation of constructs and transgenic lines

Mathematical modelling

AUX1 lysine sites conservation assay

Molecular dynamics (MD) simulations and analysis

Auxin transport measurements

Statistics and reproducibility

Declarations

References

Additional Declarations

Supplementary Files

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