https://doi.org/10.1038/s41467-026-69810-5
Article in Press
The intracellular inositol (pyro)phosphate receptor AtSPX1 reciprocally binds to P1BS DNA
Received: 28 January 2025
Accepted: 10 February 2026	Hayley L. Whitfield, Megan Gilmartin, Andrew M. Riley, Megan L. Shipton, Barry V. L. Potter, Andrew M. Hemmings & Charles A. Brearley	
Cite this article as: Whitfield, H.L., Gilmartin, M., Riley, Α.Μ. et aL The intracellular inositol (pyro)phosphate receptor AtSPX1 reciprocally binds to PIBS DNA. Nat Commun (2026). https://doi.Org/10.1038/ 541467-026-69810-5	We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.
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The intracellular inositol (pyro)phosphate receptor AtSPXl reciprocally binds to P1BS DNA Hayley L. Whitfield1*, Megan Gilmartin1, Andrew M. Riley2, Megan L. Shipton2, Barry V.L. Potter2, Andrew M. Hemmings13 and Charles A. Brearley1*
1School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
2 Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford ΟΧΙ 3QT, UK
3School of Chemistry, Pharmacy and Pharmacology, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
*Email: h.whitfield@uea.ac.uk
*Email: c.brearley@uea.ac.uk
ABSTRACT
The response of plants to phosphate starvation engages PHR (CC-MYB-PHOSPHATE STARVATION RESPONSE) transcription factors that bind to P1BS (GNATATNC) promoter elements of phosphate-starvation induced (PSI) genes. The encoded proteins include singledomain SPX (SYG1/Pho81/XPR1) proteins. SPX proteins bind PHR proteins. Current models of SPX1: PHR interaction define only a high-phosphate role for SPX1, as an inositol (pyro)phosphate-dependent negative regulator of PHR. Here, by combination of chemical synthesis, orthogonal binding assays and molecular modelling we report that full-length SPX1 binds P1BS promoter elements and inositol (pyro)phosphates with similar affinity. Inositol (pyro)phosphates and DNA are reciprocally competing ligands of SPX1. Structural models of SPX1: inositol (pyrophosphate) and of SPX1: P1BS interaction are provided beside a working hypothesis of SPX1: PHR1 interaction. The results reveal the low-phosphate function of SPX1. These findings proffer a fundamentally different perspective of SPX involvement in the phosphate starvation response (PSR).
INTRODUCTION
Phosphate is an essential growth-limiting plant macronutrient. Estimated at μΜ level in many soils, it accumulates to mM level in plant tissues. Phytate, myo-inositol 1,2,3,4,5,6- hexakisphosphate (InsPe), is the major form of organic phosphate in the biosphere. Both phosphate and phytate form complexes with soil minerals. This limits phosphate bioavailability to plants in most unfertilised soils. When plants sense low levels of phosphate in the rhizosphere, a PSR is initiated. This involves reprogramming of gene expression to maintain core cellular phosphate homeostasis including upregulation of phosphate uptake, mobilization of internal storage forms and modulation of organismal transport processes 1.
A major subset of PSR genes is regulated by phosphate starvation response transcription factors (PHR) which bind to P1BS cis-acting promoter elements to activate expression of PSR genes 2. A second player in the regulation of this process, SPX proteins are a diverse group of proteins critical to regulation of PSR 3. Crystallography of stand-alone SPX domain proteins in eukaryotes revealed high conservation of residues forming a positively charged surface capable of binding InsP6 and diphosphoinositol phosphates (PP-InsPs)4. This affords a mechanism by which SPX proteins sense metabolites whose levels respond to changing phosphate status of tissues.
SPX domain protein 1 (SPX1), one of four Class I (stand-alone) SPX proteins in Arabidopsis, is upregulated in Pi deplete conditions 5. SPX1 has been studied extensively in Arabidopsis and rice, with phosphate 6, InsP6 7,8 or PP-InsP ligand 4,8,9,10 - dependent function emphasised. Collectively, these studies suggest that SPX1 and PHR interact only in the presence of InsP6 or PP-InsP ligands, which leads to the suggestion that SPX1 is an indirect phosphate sensor. Mechanistic explanation is further provided in rice by crystallography whereby on binding of InsP6 to SPX1, the 'mobile' al helix becomes stabilised 4. This offers steric hindrance to the PHR dimer formation that is needed for PHR interaction with P1BS promoter elements 10. In this model, the result is cessation of PSR 8. Several of these studies report difficulties obtaining full length protein 7'8, and consequently use C terminal truncated protein in some experiments.
Here, we generate full length highly purified Arabidopsis thaliana SPX1 (AtSPXl). Using a suite of tools including a chemically synthesised 2-O-linked InsPs affinity matrix, we explore the binding of InsP, PP-InsP, and DNA ligands to AtSPXl, determining that AtSPXl binds DNA only when purified to remove a bound contaminant. AtSPXl-bound DNA is displaced by inositol (pyro)phosphates and conversely, AtSPXl-bound inositol (pyro)phosphates are displaced by DNA. These findings redefine SPX1-PHR1 involvement in PSR, whereby SPX1 is binding PHR1 to disrupt binding to the P1BS promoter in the presence of inositol phosphates, but interacts with DNA itself in the absence of inositol phosphates, offering insight into a new low-phosphate function for SPX1.
RESULTS
Full-length SPX1 shows little discrimination in binding affinity for InsP6, PP-InsPs (InsP7) or [PP]2-lnsP4 (InsPs) species
AtSPXl has most commonly been studied as a truncated protein and/or as a fusion protein. We sought to investigate the binding affinities of different inositol (pyro)phosphates to full-length native AtSPXl. The chemical structures of the inositol (pyro)phosphates are shown in Figure 1. Given the posited variation in binding pose(s) of different inositol (pyro)phosphate ligands of 
plant SPX 4,8,9,10, we initially compared the binding of two inositol phosphate fluorescence polarization probes bearing reporter substitution on the 2- and 5-positions. A newly described synthetic route for the novel molecule myo-inositol 5-[3-(5- fluoresceinylcarboxy)aminopropylphosphate] 1,3,4,6-tetrakisphosphate (5-FAM-InsP5) is shown in Supplementary Figure 1. Comparison of 5-FAM-lnsPs and 2-O-[2-(5- fluoresceinylcarboxy)aminoethyl]-myo-inositol 1,3,4,5,6-pentakisphosphate (2-FAM-InsP5) as probes of AtSPXl is shown (Supplementary Figure 2a, chemical structures Supplementary Figure 2b). 2-FAM-InsPs has proved useful for characterization of diverse proteins 1112 and inositol (pyro)phosphate synthesizing enzymes 13,14,15 and was chosen as the most appropriate probe for AtSPXl, yielding a Kd of 175 nM (Supplementary Figure 2c, d). This allowed use of AtSPXl at 300 nM and 2-FAM-InsPs at 2 nM in competition (displacement) assays with a range of inositol (pyro)phosphates that are potential physiological ligands of AtSPXl (Figure la, b).
Of the inositol phosphates tested, all of which have been identified in plants 16,17, the enantiomeric pair 1D-myo-inositol 1,4,5,6-tetrakisphosphate, Ins(1,4,5,6)P4, and lo-myo- inositol 3,4,5,6-tetrakisphosphate, lns(3,4,5,6)P4, showed weak displacement of 2-FAM-InsPs at concentrations up to 1 μ.Μ (Figure la). By contrast, the symmetrical (meso) compound myoinositol 1,3,4,5,6-pentakisphosphate, lns(l,3,4,5,6)Ps, a precursor of InsP6 in plants 17,18, gave an ICso of 485 nM. With the addition of a further phosphate to the displacing ligand, InsP6 (meso) was a stronger displacing ligand (ICso 82 nM) with an identical ICso value to 1D-5-diphospho- myo-inositol 1,3,4,6-tetrakisphosphate (5-PP-InsP4, meso). The enantiomeric PP-InsPs and [PP]2- InsP4 molecules tested, like InsP6, are substrates of Arabidopsis thaliana inositol tris/tetrakisphosphate kinase 1 (ITPK1) and/or Arabidopsis thaliana diphosphoinositol pentakisphosphate kinase 1/2 (VIH1/2) 13,14,15,19,20 and had similar ICso values (49-75 nM), showing no statistical significance between ligands, nor any difference in affinity between the respective enantiomers, viz. 1D-1-diphospho-myo-inositol 2,3,4,5,6-pentakisphosphate (1- PP- InsPs) and lD-3-diphospho-myo-inositol 1,2,4,5,6-pentakisphosphate (3-PP-InsPs) or 1D-1,5-bis- diphospho-myo-inositol 2,3,4,6-tetrakisphosphate (1,5-[PP]2-InsP4) and lD-3,5-bis-diphospho- myo-inositol 1,2,4,6-tetrakisphosphate (3,5-[PP]2-InsP4) (Figure lb). In this respect, the similarity of ligand affinity is much like that shown by the SPX2 domain of Vacuolar transporter chaperone complex subunit 2 (Vtc2) 21. In summary, full-length AtSPXl shows little enantiomeric preference and cannot, on its own, distinguish PP-InsPs from InsP6, the much more abundant form of InsP in a cellular environment.
It is important to note that the available structure of OsSPXl is that of a truncated form fused at its C-terminus with bacteriophage Τ4 lysozyme (SPX11-198-T4-lysozyme), hereafter SPX1 PDB: 7Ε40, whereas the gel filtration and isothermal titration calorimetry (ITC) experiments accompanying the structure were performed with full length protein (SPX11-259) 8. Therefore, in the case of both SPX1 PDB: 7Ε40 and truncated SPX2 (mH2Al.l181-366-tagged SPX21-202/A47- 59/lnsP6/PHR2225-362), hereafter SPX2 PDB: 7D3Y 22, there are c. 60-80 amino acids of the SPX1/2 protein(s) unaccounted for in the published works. We were also unable to crystallize full length AtSPXl. Modelling software (both AlphaFold 23 and RoseTTAFold 24) places this additional amino acid sequence prominently across the SPX1: PHR2 contact surface residues of the Zhou et al. 8 model (Supplementary Figure 3). It seems likely, therefore, that this unstudied C terminal region could influence SPX: PHR interaction and must be a consideration when interpreting previously published OsSPX crystal structures or modelling described herein.
We performed induced fit docking (IFD) because neither the regiochemical composition of plant PP-InsPs and [PP]2-lnsP4, nor the crystallographic pose of PP-InsPs to plant SPX proteins, is known. We chose to use the truncated structure (SPX1 PDB: 7Ε40) as a receptor, since conservation between AtSPXl and OsSPXl are high in the InsP binding region and a structure offers a more accurate starting point than a model of AtSPXl, such as that from AlphaFold. Predictions of the position of the deleted C terminal region did not appear to interfere with the InsP positive binding patch, thus modelling with the more accurate truncated form was considered appropriate. Induced fit modelling revealed a similar pattern of ligand binding preference to that of the polarisation assays, with 1,5-[PP]2-InsP4, 3,5-[PP]2-InsP4 and 5- diphospho-myo-inositol 1,2,3,4,6-pentakisphosphate (5-PP-lnsPs) showing the lowest IFD score (and thus by implication highest binding affinity) and with InsP6 somewhat (ca. 14 kcal mol1) higher (Supplementary Table 1). The highest scoring poses from IFD were subsequently subjected to molecular dynamics simulations and ligand binding free energies (AG) were estimated by the MM/PBSA (Molecular Mechanics/Poisson-Boltzmann Surface Area) approach (Supplementary Methods, with results in Supplementary Table 1). The AG values obtained (Supplementary Table 1) showed a positive but weak correlation with ICso (Supplementary Figure 4a). In addition, computational scanning alanine mutagenesis allowed a prediction of the roles of individual SPX1 binding site residues in ligand recognition (Supplementary Figure 4b, Supplementary Table 2): the values represent estimates of the change in AG (AAG) on substitution of individual residues with an alanine residue. Finally, representative binding poses were extracted from the resulting trajectories (Supplementary Figure 5). These were taken from the most highly populated clusters of similar structures of each ligand and binding site residues observed during the ten 5 ns molecular dynamics trajectories used to calculate AG (Supplementary Figure 6-13). The representative poses of ligands were similar but with the 'planes' formed by the carbon atoms of the inositol rings 25 displaced relative to each other and, in some cases, rotated. This appears to arise from differential interactions of phosphate and pyrophosphate groups with the protonated N-terminus of the protein and charged sidechains of the numerous lysine residues in this region. The pose predicted for InsP6 differs somewhat from that observed in the X-ray crystal structure (PDB: 7Ε40) and is likely due either to lattice contacts involving InsP6 in the crystal structure of its complex with OsSPXl (PDB: 7Ε40) and/or to the involvement of residues such as serine 0 (SO) present in the OsSPXl crystal structure as a cloning artefact.
Docking of 1,5-[PP]2-InsP4 results in the sidechains of residues Κ2, Κ26, Κ29, Κ33, Κ147, Κ151 and Κ154 forming electrostatic contacts with the ligand, whereas for InsP6 only residues Κ2, Κ5, Κ29, Κ147 and Κ151 do so. The net result is a total of seventeen polar interactions formed by this diphosphoinositol phosphate and only ten by InsP6. In addition, only twelve polar contacts are made by 5-PP-lnsP4 with Κ2, Κ5, Κ29, Κ147, Κ150 and K151, suggesting increased affinity with overall negative charge represents a possible basis for the variation in IC50 values measured for these ligands. Scanning alanine mutagenesis suggests a strong influence of residues Κ2, Υ25, Κ29, and Κ151 in determining the affinity of ligand binding (Supplementary Table 2). Additionally, for 1,5-[PP]2-InsP4, the tail of the al helix (including the N-terminal residue Ml) rotates towards the ligand allowing formation of polar contacts with the presumably protonated SPX1 N-terminus. A similar situation arises with other ligands, such as 3,5-[PP]2-InsP4 (Supplementary Figure 5b, c). Free Energy Landscape analysis was performed for each of the ligands bound to OsSPXl and for the uncomplexed protein (Supplementary Figure 14). These plots suggest that InsP6 and diphosphoinositol phosphate binding to SPX1 can alter the low frequency molecular motions of the protein. This is unsurprising and other authors have noted that ligand binding can remodel the conformational space of a protein, shifting energy minima and stabilizing specific states 26. In summary, the conformations of several SPX1 residue sidechains are dependent on the identity of the bound ligand, whereby each ligand appears to cause differential modulation of the electropositive nature of the binding surface (Supplementary Figure 5). Pipercevic et al 21 have shown that, while InsPe has the most pronounced effect on the global thermal stability of SPX2 of Vtc2, PP-InsP ligands allow motions of the a7 helix that is constrained by InsPe.
AtSPXl binds the P1BS element
Others have noted difficulties obtaining soluble full-length AtSPXl or OsSPX2 proteins 7,8,22, with authors using HA-, GST- or MBP-tagged and/or truncated proteins. Consequently, biophysical analysis of full-length SPX (in isolation) is limited, and that of truncations is qualitative rather than quantitative in nature 22. Given the roles posited for the N-terminal al helix in modulating PHR binding 8,22, we sought to generate stable, tag-free protein using a cleavable N-terminal Histidine (His) tag. In optimization of solubility, it was found that AtSPXl was more stable in high salt buffers. We noted that initial AtSPXl protein preparations had a high Α260: 280, suggesting the presence of a contaminant. Two preparations of AtSPXl were purified side-byside using Ni NTA and Heparin columns (Prep A) or with a high salt wash step prior to these purification steps (Prep B) generating protein with Α260:280 of 1.24 and 0.76, respectively. These two purified stocks of AtSPXl were subjected to fluorescence polarization with 2-FAM-InsPs and although both could bind, the stock with higher Α260:280 showed lower affinity for the probe (Supplementary Figure 15a). Given the possibility that the contaminant was DNA, a 5'-12mer FAM-DNA probe, hereafter FAM-DNA, was used to determine whether AtSPXl binds DNA. All DNA probe sequences used throughout are described in Supplementary Table 3. Indeed, although AtSPXl Prep A did not bind the FAM-DNA probe, Prep Β did (Supplementary Figure 15b). Separately, we used Human Src Homology 2 Domain-Containing Inositol 5-Phosphatase 2 (HsSHIP2) as a control protein. This well-characterized 5-phosphatase 27 has, like SPX1, a substantial positive patch on the surface 12 Despite HsSHIP2 binding 2-FAM-InsPs with IC50 (= Kd) of 121 nM 12, it did not bind the FAM-DNA probe (Supplementary Figure 15c). AtSPXl interaction with DNA was confirmed by native acrylamide gel electrophoresis (Supplementary 
Figure 15d) where interaction of AtSPXl prep Β with FAM-DNA probe was visualized, but minimal interaction was observed between AtSPXl prep A and the DNA probe. The InsP kinase Arabidopsis thaliana inositol tris/tetrakisphosphate kinase 4 (AtlTPK4) 14 was also included in this binding assay as an InsP binding control. It showed no interaction with the DNA probe at equal concentration to AtSPXl. A comparison of 2-FAM-InsPs and S'-FAM-PIBS binding to AtSPXl was performed under identical conditions, yielding Kd of 175 nM (Supplementary Figure 2d) and 630 nM (Supplementary Figure 15e), respectively. Given that SPX1 is a nuclear localized protein, the relatively similar (within 3-4-fold) binding affinities of FAM-InsPs and FAM-DNA, suggest that DNA is accessible to this protein. Indeed, weaker binding affinities underpin current mechanistic explanations of PP-InsP action 4,5,8.
AtSPXl was tested for affinity towards different FAM-DNA probes of P1BS: single copy (P1BS), randomized sequence of a single copy (PIBSr), P1BS repeat (4xPlBS) and P1BS randomized sequence of the P1BS repeat (4xPlBSr) using anisotropy (Figure 2a). One way ANOVA of loglCso values showed that AtSPXl had significantly (P<0.001) weaker affinity towards the PIBSr than PIBS probe. The same was true (Ρ<0.05) of 4xPlBSr and 4xPlBS. The relatively small difference in binding affinities for single repeat DNA probes of the same length and AT/GC content, but with different base-order, suggests that AtSPX: DNA interaction has some sequence-specific contributions. The salt (NaCI) dependence of binding of FAM-P1BS, more pronounced than for 2-FAM-InsPs (Supplementary Figure 15f), suggests that electrostatic contacts are formed between SPX1 and DNA. Comparison of single probe (PIBS) to repeat probe (4xPlBS) logICso revealed significantly weaker binding (P<0.0001) of AtSPXl toward the repeating P1BS probe suggesting that PIBS repeat length also plays a role in AtSPXl binding site specificity.
Data were converted to fraction of probe bound and subjected to a one site total binding fit to give comparative Kd values (Figure 2b). All probes showed binding to AtSPXl, with the strongest affinity for FAM-P1BS (Ka 174 nM), with the randomized sequence showing slightly weaker binding (Ka 244 nM). The 4xPlBS probe had lower affinity for AtSPXl (Kd 474 nM for 4xPlBS and Kd 690 nM for 4xPlBSr).
The interaction between AtSPXl and DNA was further analyzed by EMSA, where a mixed ssDNA and dsDNA pool of a single concentration was incubated with an increasing concentration of AtSPXl. AtSPXl bound both ssDNA and dsDNA (Figure 2c), whereas AtlTPK4 did not.
The deep learning guided modelling software RoseTTAFoldNA 24 generated models of SPX: DNA binding, using 4xPlBS sequence with OsSPXl1198 (Figure 2d), OsSPXl full-length, AtSPXl1198, or AtSPXl full-length (Supplementary Figure 16). All models proposed showed binding of the DNA across the positively charged surface, in which the inositol phosphate binding site is found, close to the al helix. Modelling with the full-length proteins showed the same binding surface as the truncated protein, albeit with the DNA rotated at a different angle (Supplementary Figure 16), suggesting this additional C term region would not alter this binding position. Model outputs from AlphaFold 3 23 predicted the same binding site.
AtSPXl immobilized on inositol phosphate resin is displaced by DNA and InsP6
The discovery of DNA binding function makes the interplay between AtSPXl binding of DNA and inositol (pyro)phosphates a key question when considering the function of SPX1. We employed pull down assays to investigate this further. For this, an InsPs affinity matrix with the inositol ring coupled via the axial 2-oxygen atom (Supplementary Methods and Supplementary Figure 17) was incubated with AtSPXl. The resin was washed with buffer or with increasing concentration of DNA or InsP6 in buffer. Samples analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) showed a progressive displacement of AtSPXl from the matrix as either InsP6 or DNA were increased but not with the addition of buffer alone (Figure 3a). On the final addition of buffer, some protein was displaced, likely due to the prolonged assay period.
AtSPXl-bound inositol phosphate is displaced by DNA
In an orthogonal assay, we observed the displacement of 2-FAM-InsPs from AtSPXl by addition of DNA. An ICso of 2.57 μΜ (Figure 3b) was estimated. The approximate DNA concentration at which displacement occurs in the matrix pull-down assay (Figure 3a, middle) and anisotropy assay are similar. To exclude the possibility that Pi contamination in the DNA could result in an effect on the AtSPXl protein binding, rather than the DNA itself, suppressed ion conductivity high-pressure liquid chromatography (HPLC) was used to determine Pi content of the DNA. The potential interference by Pi was excluded (Supplementary Table 4), with concentration falling substantially below that employed in studies of SPX1: PHR1 interactions 4,6 and Pi concentration in plants 28. Both AlphaFold 3 and RoseTTAFoldNA modelling of SPX1: DNA (the latter shown in Figure 3c) predicted that the binding site of DNA overlaps with that of InsPs, validated here by displacement of InsP from SPX1 with an increase in DNA (Figure 3a, b).
PIBS-immobilized AtSPXl is displaced by inositol phosphates or free PIBS / GC-rich DNA probe
With the knowledge that AtSPXl-bound InsPs are displaced by DNA, the reverse was investigated. Biotin-tagged P1BS dsDNA, bound to Streptavidin Sepharose resin, was incubated with AtSPXl, washed, then boiled in SDS buffer which showed AtSPXl had bound to the P1BS DNA target but not to control resin (Supplementary Figure 18a). Initial tests with a single concentration of InsP6 or dsDNA showed displacement of AtSPXl from the P1BS dsDNA-bound resin (Supplementary Figure 18a, b). To investigate binding affinity, the P1BS DNA-bound matrix was incubated with AtSPXl, washed and increasing concentration of DNA or InsP6 or buffer were applied (Figure 4a). Displacement of AtSPXl from the P1BS matrix occurs with the addition of either InsP6 (Figure 4a, top) or DNA (Figure 4a, middle) above the background control (buffer addition, Figure 4a, bottom) samples.
DNA-bound AtSPXl is displaced by inositol phosphates and inositol pyrophosphates
Displacement of FAM-P1BS (Supplementary Table 3) from AtSPXl by InsPs (Figure 4b) and PP- InsPs (Figure 4b, c) reveals ICso values approximately double those of 2-FAM-InsPs displacement (Figure 1) with a similar pattern of PP-InsPs and [PP]2-InsP4 molecules displacing the probe more efficiently than lnsP4 or InsPs molecules. Here, because Kt= ICso/ (1+ [ligand]/^) 29, ICso approximates very closely to K1. By testing a full, with the exception of 4/6-PP-InsPs, set of potential physiological ligands of full-length SPX1, we observed that in contrast to displacement of 2-FAM-InsPs (Figure 1), 5-PP-lnsP4 was significantly (P<0.0001) less effective in displacement of FAM-DNA than InsP6, PP-InsPs or [PP]2-InsP4. Modelling suggested significant repositioning of Y25, Κ26 and Κ29 compared to their position when InsP6 is bound. These general trends were repeated when FAM-P1BS was used as the displaced probe. Clearly, individual pyrophosphate substituents make little specific contribution to the strength of ligand binding over and above that of InsPs·
DISCUSSION
In Arabidopsis, phosphate starvation reduces the levels of inositol phosphates and diphosphoinositol phosphates 20, and the roles of both in biotic and abiotic stress responses have been reviewed 30. While the role of Pi as 'molecular signal' of PSR 6 has been replaced by InsPs or PP-InsPs 4,8,10,22, not without some debate 28, it should not be forgotten that phosphate starvation reduces multiple organic phosphate species. The especial role of PP-InsPs as agents of PSR, confusingly, arises from phosphate-resupply experiments in which PP-InsPs rise in a more pronounced manner than InsPs, notwithstanding that they all rise - as do other organic phosphates. It seems unlikely that recovery from phosphate deprivation is simply a reversal of cellular response to phosphate starvation or that the only active ligand of SPX is the least abundant of inositol phosphate or inositol pyrophosphates.
Against this debate, it is worth examining the properties of SPX proteins and of PHR1. Crystallography has shown how the isolated coil-coil (CC) domain of PHR1 forms a dimer 10. PHR1 interaction with SPX proteins has been demonstrated in pull-down, gel-filtration, SmallAngle X-ray Scattering (SAXS), ITC and grating-coupled interferometry experiments 6,8,9,22 There is, however, no a priori reason why PHR1 should be the only cognate partner of SPX1. Indeed He et al. 31 have recently described interaction of OsSPXl/2 with OsBZRl, while SPX4 partners include both PHR1 and Production of anthocyanin pigments 1 (ΡΑΡ1) 32. There is, additionally, mixed evidence as to the nature of organic or inorganic phosphate ligands that facilitate interaction of AtSPXl protein with PHR1.
The surface cluster of positive residues that constitute an inositol (pyro)phosphate, first identified in the SPX-domain of the Vtc complex of the filamentous fungus Chaetomium 
thermophilum 4, occupy a much larger surface than needed to bind inositol (pyro)phosphate; with what else might they interact? Restricting ourselves to inositol (pyro)phosphate binding to plant SPX1, recent crystallographic evidence indicates that InsP6 (added into crystallizing solution) binds in a pose that engages a basic surface between helices al, a2, and a4 as well as residues of the mobile helix al 8 that are posited 4,22 to be important in conferring PP-InsP- specific function on SPX1. It is worth mentioning that PP-InsPs have not been crystallized with plant SPX proteins.
Close inspection of the available crystallographic data for SPX1 PDB: 7Ε40 and SPX2 PDB: 7D3Y shows, however, for the former, that contacts are made by InsP6 with a serine residue in the linker in the fusion protein that was crystallized. The requirement of use of a lysozyme fusion reflects the much-vaunted difficulty of the handling of SPX1. The authors 8 reported two other InsP6 ligands in the asymmetric unit, one that participates in crystal packing, and another bound in a different pose to a second SPX1 molecule. For this SPX1 molecule, density for the mobile helix al and adjunct serine is not modelled. This suggests, perhaps, that the mobile helix al is not critical for InsP6 binding, a result confirmed by ITC on deletion of helix al (Figure 4a of Reference 8). Indeed, deletion of the al helix of otherwise full-length SPX1 retains the dimeric form of SPX1-PHR2, using two molecules of each protein (analyzed by SEC Supplementary Figure 8 of Reference 8). This occurs even in the presence of InsP6, which is a pre-requisite (in the authors' model) for repression of PHR2-mediated PSR. Put another way, if for full-length SPX1 the al helix is a restraint to the 'preconditioning' SPX1 dimerization that separates SPX1 dimer from PHR2 allowing PHR2 dimer to activate gene expression, then in the absence of InsP6 the factor maintaining SPX1 in dimeric form could be DNA.
Current models of SPX: PHR interaction are reliant on modelling superposition of the CC domains of separate PHR1 molecules. The separate crystallographic models of SPX: PHR interaction which do not include AtSPXl residues 199-259 or OsSPX2 residues 203-280 either do not take account of the encoded lysozyme of the SPX1 fusion protein 8 or involve swapping of the orientation of the monomers for SPX2. For the latter, helices al and α2 of the N-terminal domain of one monomer interact with helices α4 and α5 of the C-terminal domain of the other monomer, linked by two antiparallel helices α3 from both monomers 22. For the former model, superposition of the CC domain of AtPHRl onto truncated SPX1 places the CC: CC interface (necessary for PHR dimerization) in a position incompatible with the AlphaFold prediction for the missing residues (aa 199-259) of SPX1, while for the latter model, residues 203-280 are not considered. Recently, interrogation of the Vtc complex of yeast, including thermal stability experiments, has revealed dynamic changes in a previously undescribed a7 helix in the C terminal region of the SPX domain of Vtc2 sub-unit 21. The corresponding region in OsSPXl is not included in the truncated crystal structure (PDB: 7Ε40), highlighting the importance of further study of the full-length plant SPX proteins.
The literature offers limited data sets on the relative affinities (as measured by a singular approach) of inositol (pyro)phosphate ligands for manipulable SPX1, while for OsSPX2 no quantitative parameters were reported 22. We have, therefore, examined in some detail the protein purification requirements demanded by full-length AtSPXl. Here, we reiterate that most studies of plant proteins are of truncated proteins and/or fusions and that the characterization of ligand binding to SPX1 in the absence of other proteins is limited, giving Kd 5-6 μΜ for InsP6 by ITC8. In Pi-deplete scenarios in which SPX1 protein is most strongly expressed, PP-InsPs remain a small fraction of total inositol (pyro)phosphates 20. Without partition of InsPs from PP-InsPs, PP-InsPs will be outcompeted by InsP6. Vacuolar compartmentation of InsP6 33, mediated by MRP5 (ABCC5) 34, provides a mechanism by which competition could be obviated. Alternatively, close association of ΙΤΡΚ1 function with the activity of SPX1 targets (PHR1) might in mutants 20, be indicative of localized InsP6 and/or PP- InsP production in the vicinity of SPX.
Employing a wider set of inositol (pyro)phosphate ligands, bearing between 4 and 8 phosphates, we find that SPX1 offers no discrimination between 1,5-[PP]2-InsP4, 3,5-[PP]2-InsP4, 1-PP-InsPs or 3-PP-InsPs, but binds these ligands with slightly lower Kd (Κ) than it does InsPs. Indeed, binding affinity is a function of total charge. Protein with lower A260: A280 ratio binds the inositol phosphate probe 2-FAM-InsPs more tightly than protein with the higher A260: A280 ratio. This led us to speculate that SPX1 binds nucleotide. Such a premise is formally discounted 8 and, for SPX2, is not considered in domain-swap models 22.
Formal testing of the premise that SPX1 binds DNA has been demonstrated by multiple orthogonal approaches: EMSA, non-denaturing PAGE of protein: DNA complexes, fluorescence polarization of FAM-labelled P1BS DNA binding to SPX1, displacement of bound inositol phosphate analog (2-FAM-InsPs) by DNA, affinity purification of SPX1 on resin-immobilized DNA (and displacement by InsP6) and affinity purification of SPX1 on resin-immobilized InsPs (and displacement by DNA).
The results proffer a fundamentally different perspective of SPX-involvement in PSR (Figure 5). One in which inositol (pyro)phosphates and DNA are reciprocally competing ligands for binding to SPX. In other words, SPXl-binding to DNA (the less mobile partner) is modulated by (highly mobile) inositol (pyro)phosphates - InsP6 or PP-InsPs. In one possible iteration of this model, depletion of inositol (pyro)phosphates on phosphate starvation increases SPX1 binding to DNA. The extent to which SPX1 binds to inositol (pyro)phosphate or DNA is a function of the binding constants of competing processes and the prevailing concentrations of different inositol (pyro)phosphate ligands. PHR1 binds to the P1BS element of PSI genes and these include SPX1, but SPX1: DNA interaction is of similar affinity to PHR1: DNA.
The model (Figure 5) solves the conundrum identified by Collins et al. 35 that SPXl/2 gene transcription is induced by Pi-starvation 6, yet SPX1: PHR1 interaction occurs in Pi-replete conditions (high levels of inositol (pyro)phosphates), i.e., conditions of recovery from PSR. By conferring a specific low Pi function for SPX1, DNA-binding, SPX1 gains molecular function consistent with SPX-dependent phenotypes. The model, moreover, is consistent with evidence that SPX1 is nuclear localised (like SPX2) 5,6,36
SPX1 and SPX2 show redundancy 6,37,38. Spxl/spx2 double mutants are severely growth retarded and possess very low levels of Pi in shoots 38. In contrast, the spx4 mutant, another stand-alone SPX protein, accumulates Pi in shoots 37,39. It seems likely that the precedent of DNA-binding applies to SPX4. In mixture with PAP1, SPX4 was shown by electrophoretic mobility shift assay (EMSA) to bind to the Myb-Recognizing Element (MRE1) of dihydroflavonol- 4-reductase (DFR) promoters. Binding was also competed by InsP639. Clearly, inositol (pyro)phosphates have broad roles beyond mediation of SPX1-PHR interaction in Pi-replete scenarios 6,38. Through SPX4, they control expression of PHRl-dependent PSR genes and control PHRl-independent responses 35'37. Here, although SPX4 is not as well characterized (as SPX1) as an inositol (pyro)phosphate receptor, it is positively responsive to phosphate resupply to Pi- limited plants, it shows dynamic turnover, it acts to retain PHR1 in the cytosol and modulates the shoot PSR 37,39.
Nonetheless, returning to SPX1, the inclusion of missing amino acid residues in our structural model of SPX1: inositol (pyro)phosphate interaction accommodates both InsP6 and PP-InsPs as ligands of full-length protein. The model challenges the relevance of CC: CC interaction models of dimerization of (truncated) PHR28or SPX2 dimerization (SPX2 PDB: 7D3Y) 22 to full-length SPX proteins.
The model explains how InsP6 and DNA are competing cognate partners of SPX1, sharing the same binding surface on SPX1. The model accommodates differential expression of SPX1 and PHR1 (PHR1 does not respond to phosphate deprivation 36) and, through differential binding poses of InsP6 and PP-InsP ligands to SPX1, allows for differential interaction of SPX1: PHR1 complexes with potential unidentified partners (as suggested 4) by modulation of the positive binding surface. While current models do 8 or do not 22 invoke a role of InsP6 as 'molecular glue', both appraise InsP6 as a moderator of PHR: SPX interaction (both describe crystallographic coordination of InsPe). PHR: SPX interaction occurs in the absence of added InsP6 6,9, albeit this interaction is strengthened by added InsP68. For truncated PHR2 230-426, PHR2: SPX1 interaction analyzed by gel-filtration was increased after prolonged incubation of protein with c. 20-fold molar excess (1 mM) of InsP6 (Figure la of Reference 8), against Ka of 5.5 μΜ for SPX1: InsP6 measured by ITC (Figure 2b of Reference 8). In this study, a L348A/ L358A/I362A form of PHR2230-426 (neither full-length nor native protein were reported) bound P1BS DNA with Kd = 0.26 μΜ and bound SPX2 with Kd ~0.17 μΜ. Interaction with DNA was largely unchanged (Ka = 0.38 μΜ) by SPX1, but was abolished by additional inclusion of InsP6 at unspecified concentration (Figure le, of Reference 8). The authors posited that PHR2 monomerization induced by InsP6 is a critical step in SPX1- and/or InsP6- induced inactivation of the transcription factor. A similar dependency of SPX: PHR interaction on InsP6 (1 mM) was reported for full
length OsSPX2, Co-expressed with truncated OsPHR2 before pull-down. Without knowledge of protein concentration, and hence mol ratio to InsP6, it is difficult to make comment on enhancement of affinity of OsSPX2 for OsPHR2 mediated by InsP6.
While a broader understanding of whether SPX1, or any other SPX protein, has specific 'target' DNA sequences awaits clarification, comparison of Kd InsP6 and Kd DNA with DNA-binding constants for other proteins is informative. A Kd InsP6 of 4.6 μΜ was reported for the dualdomain protein SPXVTP140. We obtained IC50 InsP6 (for displacement of 2-FAM-InsPs from AtSPXl) of 82 nM. For competitive binding, the limits of Κ\ (effectively Kd) are 0.5-lx ICso 29· In the experiments described here, ICso is approximately equal to Ki, because protein was held at 2 χ Kd with ligand 2-orders of magnitude lower. For SPX1, we obtained Kd FAM-P1BS of 235 nM. This value is not far removed from that (88 nM) obtained for binding of P1BS by PHR141. A value of 16 nM was reported for binding of the ORE1-NAC transcription factor to its cognate DNA binding site 42. Qi et al. 7 estimated Kd for binding of MBP-tagged truncated PHR1 (AtdPHR1208-362) to lxPIBS and 2xPlBS at 707 and 14 nM, respectively. Set against the widely accepted perspective that inositol (pyro)phosphates are cognate ligands of SPX proteins, it is inescapable that the DNA and InsP/PP-InsP binding constants (of SPX1) are similar and of magnitude comparable to the DNA: protein interactions (e.g., of PIBS: PHR1) that support existing mechanistic models of PSR.
The demonstration of DNA- and PIBS-binding by SPX1 raises questions of topology of interaction of SPX1 and PHR1 with cognate DNA partner. While increase in number of P1BS repeats increased affinity of PHR1 to DNA, by reduction of Koff 7, for AtSPXl reduced affinity is seen on 4xPlBS promoters (Figure 2). The binding of SPX1 to a single P1BS sequence motif may sterically preclude binding of another SPX molecule to a closely proximal P1BS sequence. Thus, differential expression of PHR1 targets may depend on PIBS repetition. More excitingly perhaps, SPX1 might modulate PHR1/2 function by control of DNA topology during transcription of PSR genes. We draw support from the role of a distal, surface (non-catalytic) DNA binding site of DNA topoisomerase I, comprising a quartet of lysine and additional serine and arginine residues which is implicated in supercoiling. This provides precedent for protein binding at juxtaposed DNA segments 43, which could for SPX1 (as dimer) represent separated PIBS motifs. Equally, SPX1 through interaction with PHR1 might be considered a 'proximity sensor' for PHR interaction with DNA.
In Figure 6 we summarise how exchange of inositol phosphates between subcellular compartments adds organellar dimension to nuclear, vacuolar and cytosolic processes regulated by inositol (pyro)phosphates, as impacted by the new-found DNA-binding function of the SPX1 transcriptional repressor. To date, the only verified inositol phosphate transporter is MRP5 34. Disruption of mrp5 may isolate [PP]2-InsP4 and PP-InsPs species from competition by InsP6, but this needs further elaboration. Both mrp5 and wild-type (Col-0) show common elevations of [PP]2-InsP4 in shoots on phosphate resupply following starvation, to levels that are a consistent and small fraction of InsP6 20. Moreover, the two genotypes exhibit similar levels of Pi in shoots, irrespective of Pi supply 20. MRP5 aside, the dynamic Pi-status-dependent turnover of SPX4 37 makes it likely that SPX4-dependent retention of PHR1 in the cytosol of shoots is itself an inositol (pyro)phosphate function in Pi-replete scenarios.
Finally, others 30,35 have reviewed the engagement of SPX proteins in the interplay of phosphate starvation and growth factor signalling, including COP signalosome function, in plants. Our findings offer perspectives on inositol (pyro)phosphate involvement beyond the role of inositol (pyro)phosphates as 'molecular glue'. Indeed, taking jasmonate as example, for cells with low levels of jasmonate and its bioactive conjugate JA-lle, Jasmonate-ZIM-Domain (JAZ) proteins repress the transcription factors that regulate JA-responsive genes. JA-lle promotes binding of JAZ proteins to the jasmonate receptor CORONATINE INSENSITIVE! (COII) which is an F-box protein that, as part of the the SCFCO11 E3 ubiquitin ligase complex, targets proteasomal degradation of JAZ repressors 44. The inositol (pyro)phosphate dependence of the interaction of JAZ proteins with COII (reviewed 30) critically places jasmonate signalling responsive to phosphate status 35. The finding of competition between inositol (pyro)phosphates and DNA (promoter elements) likely impacts the network of signalling interactions of biotic and abiotic stress pathways. These hierarchies will include transcriptional phenomena and the protein: protein interactions that underlie them. It is unlikely that direct competition between DNA and inositol (pyro)phosphates is restricted only to PSR. Our methods, particularly fluorescence polarization and affinity matrices, offer opportunity here.
METHODS
Protein expression and purification
Arabidopsis thaliana SPX1 (At5g20150) was cloned into the pOPINF vector using primers 5'- AAGTTCTGTTTCAGGGCCCGATGAAGTTTGGTAAGAGTCTCA-3' and 5'- ATGGTCTAGAAAGCTTTATTTGGCTTCTTGCTCCAA-3. The In Fusion HD enzyme kit (Clontech) was used to recombine Hindlll and Kpnl digested pOPINF plasmid with the PCR product. This was transformed into Escherichia coli Stellar cells (Clontech) and colonies confirmed by PCR amplification. The construct was transformed into ArcticExpress™ RIL cells, with expression cultures grown at 13 °C, 0.3 mM IPTG for 24 hours. Sequence provided in source data.
Lysate recovered from French press of the cell pellet in 50 mM NaH2PO4 pH 6.5, 300 mM NaCI, 20 mM imidazole, 0.5% Triton X-100, was applied to a 5 mL nickel nitriloacetic acid (Ni-NTA) HiLoad column (Qiagen) equilibrated in 50 mM NaH2PO4 pH 6.5, 300 mM NaCI, 20 mM imidazole. The column was washed with 50 mM NaH2PO4 pH 6.5, 1M NaCI, 20 mM imidazole before applying a gradient of Ni-NTA buffer A (50 mM NaH2PO4 pH 6.5, 300 mM NaCI, 20 mM imidazole) to Ni-NTA buffer Β (50 mM NaH2PO4 pH 7.5, 300 mM NaCI, 500 mM imidazole). Subsequently, the 6x His tag was cleaved using HRC 3C protease (ThermoFisher Scientific Pierce) at 4 °C overnight in buffer A. Pooled fractions were diluted with 20 mM Tris-HCI pH 7.5 
and applied to a 1mL Heparin HiTrap column (Qiagen) equilibrated with heparin A buffer (20 mM Tris-HCI pH 6.5, 50 mM NaCI), washed with heparin A buffer, and eluted with a gradient of heparin A to heparin Β buffer (20 mM Tris-HCI pH 6.5, 1 M NaCI). Protein was buffer exchanged into 10 mM sodium phosphate, pH 6.5, 600 mM NaCI, 6 mM DTT, 10 % glycerol for storage at - 80 °C. Protein concentration was quantified by absorbance at 280 nm using the NanoDrop One (Thermofisher) and calculated using the extinction coefficient generated by Protparam (Expasy). The level of nucleotide contamination was observed using the nanodrop one (Thermofisher) A260/A280 ratio. Protein was purified on four separate occasions, the initial purification was split into two, either without (prep A) or with (prep Β) the high salt wash step after protein loading. The three subsequent purifications were performed as described above. Expression and purification of AtlTPK4 (At2g43980) and HsSHIP2 (Uniprot: 015357) for use as control proteins were performed as described previously 14,45
Fluorescence anisotropy and probe synthesis
Assays were performed not less than three times as described 46 in 20 mM 4-(2-hydroxyethyl)- 1-piperazineethane sulfonic acid (HEPES) pH 6.5, 1 mM MgCl2, 100 mM NaCI. Probes were used at 2 ηΜ in 4 replicates of 20 pL volume in Corning nonbinding 384-well plates (product no. 3575). Fluorescence polarisation was recorded on a BMG ClarioSTAR plate reader: excitation 485 nm, 12 nm; dichroic 505 nm; emission 505 nm, 16 nm; and 200 flashes. Individual run settings in source data. For displacement assays, protein was used at = 2 x Kd, 300 ηΜ with 2- FAM-InsPs or 500 ηΜ with 5‘-FAM-DNA. All assays were performed at room temperature. ICso was determined using a 4-parameter fit of anisotropy data and statistical significance using a one-way ANOVA on logICso values, using GraphPad software.
2-FAM-InsPs was synthesised from 2-O-(2-aminoethyl)-lns(l,3,4,5,6)Ps as previously reported 47. 5-FAM-lnsPs was synthesised in a similar way from myo-inositol 5-(3-aminopropylphosphate) 1,3,4,6-tetrakisphosphate (Supplementary Fig. 1). Briefly, 5-FAM-lnsPs consists of lns(l,3,4,5,6)Ps coupled to 5-carboxyfluorescein via a linker attached to a terminal oxygen atom of the 5-phosphate group of the InsPs. This isomer of InsPs is a meso-compound, with a plane of symmetry through C-2 and C-5 atoms and the synthesis of conjugates is therefore simplified by functionalising the InsPs at either 0-2 for 2-FAM-InsPs or 0-5 for 5-FAM-lnsPs; see Supplementary Methods for full details.
Protein modelling
Induced Fit docking (IFD), followed by Molecular Dynamics (MD) simulations, were performed on each ligand and MMPBSA calculations were used to estimate binding free energy. Computational alanine scanning mutagenesis was also performed. Methods for the process can be found in Supplementary Methods.
RoseTTAFold2NA 24 was used to predict the structures of protein: nucleic acid complexes. Default parameters were adopted to predict structures based on the full-length amino acid sequences of Arabidopsis (Uniprot: Q8LBH4) and rice (Uniprot: Q69XJ0) SPX1 or the truncated SPX1-198 sequences of both, with the 54 bp double stranded nucleic acid sequence of P1BS repeat (4xPlBS) (Supplementary Table 3).
Electrophoretic Mobility Shift Assay
Binding assays were performed in 20 μί reactions with 2.5 ηΜ FAM-oligomer and SPX1 (0-10 μΜ) or AtlTPK4 (10 μΜ) as a negative control, in 20 mM HEPES pH 6.5, 1 mM MgCl2, 50 mM NaCI (or lOOmM where stated), 0.1 mg/mL BSA, lmM ethylenediamine tetra-acetic acid (EDTA). Samples were incubated on ice for 1h before adding native loading dye (50 mM Tris- HCI pH 6.8, 0.01% (w/v) bromophenol blue, 10% (v/v) glycerol). An 8% native polyacrylamide gel (8% acrylamide/bisacrylamide, 375 mM Tris pH 8.8, 0.1% APS, 0.05% TEMED) with 5% native stacking gel (5% acrylamide/bisacrylamide, 125 mM Tris pH 6.8, 0.1% APS, 0.05% TEMED) was pre-run at 50 V for 2 h on ice. Samples were loaded and run at 150 V for a further 2-2.5 h on ice. Gels were imaged using a Typhoon™ FLA 9500 system (GE Healthcare) using the preset parameters for FAM detection. Experiment performed three times with varying dsDNA probe sequence and SPX1 concentrations, binding consistent using clean AtSPXl stocks.
Pull-down assays and synthesis of 2-linked InsPs affinity matrix
The 2-linked InsPs affinity matrix was produced by reaction of 2-O-(2-aminoethyl)- lns(l,3,4,5,6)Ps 47 with Affi-Gel 10 (Biorad, UK) using the non-aqueous coupling methodology previously described for 2-O-(2-aminoethyl)-Ins(1,4,5)P348. Trials showed that this method typically gave > 2 umole of immobilised InsPs per mL of settled gel as determined by total phosphate assay, while much lower coupling efficiencies were obtained when aqueous buffers were used. Full details are given in Supplementary Methods.
Three aliquots of 2-linked InsPs beads were washed three times with assay buffer (20 mM HEPES pH 6.5, 50 mM NaCI), 7 μΜ AtSPXl added to each and incubated on ice with gentle agitation for l h. An increasing concentration of InsP6 or dsDNA was added (or the same volume of buffer as a control) and incubated on ice for 15 min after each addition, with a sample of the same volume as the addition taken for analysis before the next addition was made. Samples were analysed by SDS-PAGE using 12% acrylamide gels at 150 V for lh and stained using InstantBlue® Coomassie stain (Abcam). The experiment was performed twice with varying concentrations of InsP6 or dsDNA.
Pull-down assays using PIBS bound matrix
PIBS-bound Sepharose beads were generated using high-capacity Streptavidin Sepharose™ High Performance beads (Cytiva 17-5113-01, obtained from Merck, UK). Beads were washed three times with binding buffer (20 mM Tris pH 7.5, 150 mM NaCI, 1 mM EDTA) and resuspended with an additional 200 μί binding buffer containing 20 μΜ biotin tagged P1BS dsDNA (MWG Eurofins). Beads were incubated for lh at room temperature with gentle agitation. A separate sample of beads was incubated with buffer only, to be used as a control. After 3 washes with assay buffer (20 mM HEPES pH 6.5, 50 mM NaCI), 7 μΜ AtSPXl was added and incubated on ice with gentle agitation for 1 h. Initially, protein-bound DNA-tagged (DNA- conjugated) beads and control beads were analysed by boiling a small sample of beads in SDS loading buffer, with subsequent addition to a final concentration of 100 μΜ InsP6 or an addition of the same volume of water as a control and incubation on ice for 30 min. Both supernatant and bead samples were analysed by SDS-PAGE. Alternatively, DNA-conjugated or control beads were split into aliquots and either an increasing concentration of InsP6 or dsDNA was added (or the same volume of buffer as a control) and incubated on ice for 15 min after each addition, with a sample of the same volume as the addition taken for analysis before the next addition was made. Samples were analysed by SDS-PAGE using 12% acrylamide gels at 150 V for 1 h and stained using InstantBlue* Coomassie stain (Abcam). Experiment performed twice at varying concentrations of InsPe or dsDNA.
Ion exchange HPLC
HPLC quantification of orthophosphate was performed by chromatography on a 2 x 250 mm Dionex™ lonPac™ AS18 column on a Dionex™ ICS-6000 Ion Conductivity System. The column was eluted at a flow rate of 0.5 mL min-1 with a gradient of potassium hydroxide delivered according to the following schedule: time (min), concentration (mM); 0, 0; 12, 12; 20, 34; 25, 34; 26, 0; 36, 0. Orthophosphate was detected at retention time 18 min by suppressed conductivity measurement.
DATA AVAILABILITY
The Molecular dynamics data generated in this study have been deposited in the MDRepo database under accession codes MDR00004441, MDR00004443, MDR00004444, MDR00004445, MDR00004446, MDR00004447, MDR00004448. The free energy calculation data are available at the University of East Anglia Digital Respository. Source Data are provided with this paper.
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41.		Jiang M, et al. Structural basis for the Target DNA recognition and binding by the MYB domain of phosphate starvation response 1. FEBS J 286, 2809-2821 (2019). DOI: 10.1111/febs.14846
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ACKNOWLEDGEMENTS
We thank Hui-Fen Kuo and Tzyy-Jen Chiou, Academia Sinica, Tawian, for the gift of plasmid encoding SPX. Induced fit docking calculations and SPX1: DNA complex structure predictions were carried out on the High-Performance Computing Cluster supported by the Research and Specialist Computing Support service at the University of East Anglia. The molecular dynamics simulations and MMPBSA calculations needed to complete second round review corrections were performed by A.M.H. using the high performance computing facility at the International Center for Food and Health, Shanghai Ocean University.
This research was funded in whole, or in part, by Research Council (Grant number NE/W000350/1) to C.A.B. and the Wellcome Trust [Grant number 101010] to B.V.L.P. as a Wellcome Trust Senior Investigator. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
AUTHOR CONTRIBUTIONS
C.A.B.: conceptualization, funding acquisition, project administration, formal analysis, supervision, investigation, methodology, writing—original draft, writing—review and editing. Α.Μ.Η.: investigation, methodology, writing—review and editing. M.G.: investigation. B.V.L.P.: funding acquisition, supervision, methodology, writing—review and editing. A.M.R.: investigation, supervision, methodology, writing—review and editing. M.L.S.: investigation, methodology, writing—review and editing. H.L.W.: formal analysis, investigation, methodology, writing—original draft, writing—review and editing.
COMPETING INTERESTS
The authors declare no competing interests.
FIGURE LEGENDS
Figure 1. Comparison of inositol phosphates and diphosphoinositol phosphates as ligands of SPX1. Fluorescence anisotropy displacement assays of (full-length) AtSPXl-bound 2-FAM-InsPs with (a) InsPs and 5-PP-lnsP4; (b) PP-InsPs. Ligand structures shown). N= 4 replicates, repeated three times with similar results. One-way ANOVA of log ICso revealed that the differences in ICso were not significant at p<0.05; for InsP4 anisotropy the data could not be fitted to a 4-parameter logistic and for InsPs did not reach saturation within this range of ligand concentrations; (c) Side-by-side comparison of representative bound conformations of OsSPXl1-198 with InsPe (green) and 1,5-[PP]2-InsP4 (lilac). Note that hydrogen atoms, water molecules and counter ions have been removed for clarity.
Figure 2. AtSPXl is a DNA binding protein, (a) AtSPXl binds a range of fluorescently-tagged dsDNA indicated by increasing anisotropy as AtSPXl concentration increases. Ν- 4 replicates, repeated three times with similar results. One-way ANOVA of logICso revealed significant difference between all probe pairwise combinations to at least p<0.001 except between 4xPlBS and 4xPlBSr (ρ<0.05) and between PIBSr and 4xPlBS (ns) (b) Data from a transformed into fraction of probe bound subjected to a one site - total binding model, (c) EMSA analysis of S'-FAM labelled ssDNA and dsDNA mix showing 12mer DNA with increasing concentration of full-length AtSPXl. No shift is seen for the control protein AtlTPK4 at the highest concentration tested (red). Experiment repeated twice with similar results, (d) OsSPXl1198 RoseTTAFoldNA model of binding to 4xPlBS (left) and rotated by 90°in the y axis (right), surface coloured according to electrostatic charge.
Figure 3. AtSPXl-bound to an inositol phosphate matrix is displaced by DNA. (a) Full-length AtSPXl is displaced from a resin-immobilized InsPs (linked via the 2-oxygen atom) by successive aliquots of increasing concentration of InsP6 (top) or P1BS DNA (middle), but not by buffer alone (bottom). Repeated twice with similar results, (b) Displacement of 2-FAM-InsPs from full-length AtSPXl by increasing DNA concentration. Probe (2 ηΜ) was incubated with 300 ηΜ SPX1 in the presence of varying concentration of PIBS DNA. N= 4 replicates, repeated three times with similar results, (c) Proposed RoseTTAFoldNA 24 model of OsSPXl1198 bound with 4x P1BS DNA showing the overlap in binding position of InsPe (yellow spheres) and DNA. Protein surface is coloured according to electrostatic charge.
Figure 4. DNA-bound AtSPXl is displaced by inositol phosphates (a) Full-length AtSPXl is displaced from a P1BS DNA matrix by successive aliquots of increasing concentration of InsP6 up to 100 μΜ (top) or P1BS DNA up to 40 μΜ (middle), but not by buffer alone (bottom). Repeated twice with similar results, (b) Displacement of FAM-P1BS from full-length AtSPXl by increasing inositol polyphosphate or inositol pyrophosphate concentration. Assay performed in triplicate and repeated twice with the same result, (c) Displacement of FAM-P1BS from full-length AtSPXl by increasing inositol pyrophosphate concentration. Probe (2 ηΜ) was incubated with 500 ηΜ SPX1 in the presence of varying concentration of InsP or PP-InsP ligand. One-way ANOVA of logICso revealed significant difference between binding of 5-PP-InsP4 and all other ligands to p<0.0001. Other pairwise comparisons did not show significant difference. For InsP4 and InsPs, fitting to the 4-parameter logistic was imprecise, reflected in the wide confidence interval for InsPs and the lack of fit for InsP4 species N= 4 replicates, repeated twice with similar results.
Figure 5. A model of inositol phosphate involvement in PSR that accommodates competition with DNA, and which explains the 'missing' low phosphate role for SPX1. PSR, the induction of a multitude of Phosphate Starvation Induced (PSI) transcripts, occurs in conditions of low cytosolic phosphate, Picyt, conditions in which expression of the SPX1 gene, encoding a nuclear localized protein, are maximal. Inositol phosphates and inositol pyrophosphates are reduced as Picyt falls. SPX1 binds DNA, this may include PIBS elements of promoters of PSI genes, including SPX1, forming a scaffold for interaction with PHR1, a MYB-CC transcription factor. The extent to which PHR1: SPX1 protein interaction occurs in the absence or at low levels of inositol (pyro)phosphates is not well-defined, but PHR1, in association or not with SPX1, activates PIBS-driven PSI gene expression. Resupply of phosphate increases Picyt with ensuing increase in inositol phosphates and inositol pyrophosphates. InsP6 displaces SPX1 from DNA, including PIBS promoter elements, the mixed equilibria of PHR1: SPX1, PHR1: P1BS and SPX1: DNA interactions are shifted towards SPX1: InsP6 and SPX1: InsPs: PHR1. Displacement of SPX1 from DNA, including P1BS elements, and PHR1 from PIBS ceases the transcription of PSI genes. Model images of SPX1 and PHR1 were generated from (PDB: 7Ε40) 8. We draw support for our model from observations that deletion of the al helix facilitates an SPX dimer PHR1 dimer interaction that is not disrupted by InsPe8 Perhaps, in the absence of InsPe, SPX1 dimer PHR1 dimer interaction, that of full-length SPX1, is stabilized by binding of SPX1 to DNA.
Figure 6. A simplified schematic of InsP metabolism and transport between organelles (nucleus, vacuole) and cytosol in a plant cell. Highlighting IPKs which have been shown to localize in the nucleus from the lipid independent pathway of InsP biosynthesis (green) and InsP binding proteins (orange) with protein partners (yellow). InsPs are shown as pink hexagons. Straight arrows represent exchange of InsPs between subcellular compartments, curly arrows represent phosphorylation of InsPs. Created in BioRender. Brearley, C. (2025) https://BioRender.com/lz942ml.
Editorial summary:
SPX1 is an inositol (pyro)phosphate-dependent negative regulator of DNA binding by the PHR transcription factor. Here the authors show that SPX1 can also bind the promoter element targeted by PHR suggesting a role for SPX1 during phosphate deficiency.
Peer review information: Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

P - AXo pp = AXo.Xo	
0	00	

1,5-[PP]2-lnsP4	3,5-[PP]2-lnsP4

	0	1	2
log [InsP] (nM)
95% Cl (nM)	3
R2	0
Ligand	1
log [Ins
IC50 (nM)	2
P] (nM)
95% Cl (nM)	3
R2	
	Ligand	IC50 (nM)							
— ·—	lnsP6	81.8	77.5 to 86.4	0.9920	——	1-PP-lnsP5	48.8	45.7 to 52.2	0.9856	
•	5-PP-lnsP4	81.7	75.7 to 88.2	0.9754	——	3-PP-lnsP5	59.7	55.0 to 64.9	0.9695	
— ·—	lns(1,3,4,5,6)P5	484.7	293 to 803	0.9776		5-PP-lnsP5	49.8	47.6 to 52.1	0.9920	
•	Ins(1,4,5,6)P4	n/a	n/a	n/a	——	1,5-[PP]2-lnsP4	56.2	52.9 to 59.7	0.9789	
•	lns(3,4,5,6)P4	n/a	n/a	n/a	——	3,5-[PP]2.InsP4	75.1	71.8 to 78.6	0.9824	

HO 7Η
PO IvMi OP POV^
lns(1,4,5,6)P4
po
POivM, OP PO ^^
5 OP
lnsP6

b

2.5-1

-5

+5

a 250-1
II 0	1	II 2	3
log [SPX1] (nM)	I
4	1	1	1	1	1
0	1000 2000 3000 4000
[SPX1] (nM)	5000	
	ICso (ηΜ) 95% Cl (nM)	R2	Kd (nM) 95% Cl (nM)	R2	
-·- 5’FAM-PIBS	174	165 to 184	0.9971	-•- 5’FAM-PIBS	230	189 to 272	0.9927	
-• 5’FAM-PIBSr	244	208 to 286	0.9760	-•- 5’FAM-PIBSr	393	233 to 554	0.9686	
5’FAM-4xP1BS	295	261 to 334	0.9796	5'FAM-4xP1BS	474	291 to 658	0.9726	
•5’FAM-4xP1BSr	377	348 to 410	0.9917	• 5'FAM-4xP1BSr	690	460 to 921	0.9834	

σι-1
MW	X	- 3 z - 3 a - z
(kDa)	%	§ 8	8 - §

I	I	I	I	I	I 	r
0	1 log [InsP
ICso (nM)	2	3
1(nM)	0
Ligand	1 log [InsP]
ICso (nM)	2	3
I(nM)	
	Ligand		95% Cl (nM)	R2			95% Cl (nM)	R2	
•	lnsP6	138.7	93 to 206	0.9442	•	1-PP-lnsP5	72.5	57 to 92	0.9425	
•	5-PP-InsP4	743.1	464 to 1190	0.9526	—e—	3-PP-lnsP5	138.9	94 to 206	0.9369	
——	Ins(1,3,4,5,6)P5	209.2	84 to 522	0.8588		5-PP-lnsP5	118.3	108 to 129	0.9855	
•	lns(1,4,5,6)P4	n/a	n/a	n/a	•	1,5-[PP]2-lnsP4	101.4	88 to 115	0.9823	
•	lns(3,4,5,6)P4	n/a	n/a	n/a		3,5-[PP]2-lnsP4	135.4	121 to 152	0.9871	

Pj and lnsP6 deplete
P|and lnsP6 replete