New Results

Arabidopsis inositol polyphosphate kinases regulate COP9 signalosome deneddylase functions in phosphate-homeostasis

Yashika Walia, Mritunjay Kasera, Kishor D Ingole, Swaroop Peddiraju, Abhisha Roy, Debabrata Laha, Gabriel Schaaf, Saikat Bhattacharjee
doi: https://doi.org/10.1101/2020.10.02.323584

Abstract

Targeted protein degradation is essential for physiological development and adaptation to stress. Mammalian INOSITOL PENTAKISPHOSPHATE 2-KINASE (IP5K) and INOSITOL HEXAKISPHOSPHATE KINASE 1 (IP6K1) pair generates inositol polyphosphates (InsPs) to modulate association/dissociation equilibrium of Cullin RING Ubiquitin E3 ligases (CRLs) on the COP9 signalosome (CSN) platform. Deneddylase activity of the CSN5 subunit protects cullins from self-ubiquitination ensuring their functional continuity. In plants, similar regulations by InsP-kinases are not known. Here, we show conserved interactions of Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1) and INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), counterparts of the above mammalian InsP-kinase pair, with selective CSN subunits. In ipk1 or itpk1 mutants, deneddylation deficiencies not only cause increased neddylated Cullin1 (CUL1Nedd8) pools more prone to degradation but also impair CSN5 entry/exit shuttles on the CSN holo-complex. Constitutive phosphate-starvation response (PSR), previously known for these mutants are suppressed by pharmacological inhibition of neddylation thus linking CSN-CRL functions to phosphate (Pi)-sensing. Similarly, in wild-type plants exposed to compounds that impair CSN5 deneddylase function affects its dynamics and mimic PSR properties of the mutants. We further show that under Pi-deprivation more CSN5 retentions on the CSN holo-complex and the resulting enhanced CUL1Nedd8 pools is essential for induction of downstream Pi-starvation inducible (PSI) genes. Overall, with our data we present InsP-kinase involvements in maintenance of Pi-homeostasis in plants via CRL-CSN(5) functional synergism.

Significance Neddylation modifications on Culling-RING E3 ligases (CRLs) while essential for their role in proteostasis, also threaten their own stability. Selective inositol polyphosphates (InsPs) aid the constitutive photomorphogenesis 9 signalosome (CSN) functions in protecting, deneddylating, and facilitating CRL recycling. Here we demonstrate that plant mutants deficient in these InsPs have disturbed CSN subunit partitioning, are deficient in deneddylase activity, and hyperaccumulate neddylated cullins that lead to constitutive phosphate-starvation response (PSR). Inhibition of CSN functions/consequences mirror the InsP mutant properties indicating pivotal involvement of CSN in phosphate homeostasis. These raises promising possibilities of targeted intervention on CSN functions for nutritional benefit of plants.

Introduction

The versatile functions of inositol polyphosphates (InsPs) in cellular processes of all eukaryotes are owed to its propensity for differential degree of phosphorylation catalyzed by relatively conserved set of InsP-kinases (Stevenson-Paulik et al., 2005; Monserrate and York, 2010; Shears and Wang, 2019). In plants, selective InsPs have been linked to phytohormone networks, inorganic phosphate (Pi)-homeostasis, mRNA export, (a)biotic stress responses, among others (Stevenson-Paulik et al., 2005; Murphy et al., 2008; Gillaspy, 2013; Lee et al., 2015; Williams et al., 2015; Jia et al., 2019). With all six-hydroxyl group of the myo-inositol ring phosphorylated, InsP6 (myo-inositol-1,2,3,4,5,6-hexakisphosphate or phytic acid), produced by INOSITOL PENTAKISPHSPHATE 2-KINASE 1 (IPK1) is sequentially (pyro)-phosphorylated by INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASES (ITPK1/2) and then by DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASES (VIH1/2) to generate energy-rich phosphoanhydride bond-containing inositol pyrophosphates InsP7 and InsP8, respectively (Desai et al., 2014; Laha et al., 2015; Adepoju et al., 2019; Laha et al., 2019). Though bona fide receptors of plant InsPs remain unidentified, their co-factor functions are being increasingly elaborated (Shears et al., 2012; Williams et al., 2015). While InsP6, or more recently speculated InsP7, promotes auxin-dependent degradation of AUX/IAA repressors, InsP8 targets JASMONATE ZIM-DOMAIN (JAZ) repressors in a jasmonic acid (JA)-dependent manner (Tan et al., 2007; Sheard et al., 2010; Laha et al., 2015; Laha et al., 2020). Interestingly, the cognate substrate receptors TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or CORONATINE INSENSITIVE 1 (COI1) belong to F-box family proteins and are components of typical SCF (ASK1/SKP1-Cullin-F-Box; SCFTIR1 or SCFCOI1)-type E3 ubiquitin ligases (Tan et al., 2007; Sheard et al., 2010). The ipk1 or itpk1 mutants with reduced InsP7 or vih2 plants with reduced InsP8, are auxin- or JA-insensitive, respectively (Laha et al., 2015; Laha et al., 2020).

InsPs have also been recently implicated as sensor of phosphate (Pi)-status in plants (Kuo et al., 2018; Dong et al., 2019; Ried et al., 2019; Zhu et al., 2019). Necessity of phosphates in central dogma processes, protein modifications, and most importantly as indicators of adenylate currencies risk detrimental consequences when cellular Pi levels are low (Szijgyarto et al., 2011; Wundenberg and Mayr, 2012). Under a low Pi-availability from soil, a highly organized adaptive cascade of events collectively termed as PSR is activated and includes increased remobilization of Pi from root to shoot, harnessing of organellar Pi-storage reserves, accompanied by upregulated expression of PSI genes for Pi-transporters (PHTs), phosphatases (PAPs), and several micro-(MIR399) or non-coding RNAs (IPS1) (Puga et al., 2014; Ham et al., 2018). In Arabidopsis, PSR-associated transcriptional changes are majorly orchestrated by the MYB-transcription factors PHOSPHATE STARVATION RESPONSE 1 (PHR1) and its closest homolog PHR1-LIKE (PHL1) (Bustos et al., 2010). These transcription factors bind to partially conserved sequences (P1BS elements) present on PSI gene promoters to activate their transcriptions (Rubio et al., 2001). A phr1 phl1 double mutant is deficient in PSI-gene expressions both basally as well as upon exposure to Pi-deplete conditions (Kuo et al., 2014).

A class of stand-alone SPX (SYG1/Pho81/XPR1)-domain containing proteins SPX1/2 act as negative regulators of PHR1/PHL1 functions in maintaining phosphate balances in a cell. Following the first report that SPX domains can bind specific InsPs it was subsequently demonstrated that under Pi-replete conditions, SPX1 requires InsP8 co-factor to bind PHR1, sequester it away from the nucleus, and suppress its activity (Wild et al., 2016; Jung et al., 2018; Dong et al., 2019; Zhu et al., 2019). When starved for Pi, intracellular InsP8 levels reduce because of the phosphatase activity of VIH2 relieving SPX1-tethered inhibitions on PHR1, thus allowing successful transduction of PSR (Dong et al., 2019). In InsP8-deficient ipk1-1, itpk1-2, or vih1 vih2 and in the spx mutants liberated PHR1 causes constitutive activation of PSI-gene expressions even under Pi-sufficient conditions accumulating high endogenous Pi levels (Kuo et al., 2014; Puga et al., 2014; Dong et al., 2019; Zhu et al., 2019). Decreased InsP8 levels that occur upon Pi-starvation also promotes ubiquitinylation of at least two SPX proteins (Lv et al., 2014; Zhong et al., 2018; Osorio et al., 2019). Recent identification of E3 ubiquitin ligases that target rice SPX4 upon Pi-starvation (Ruan et al., 2019) taken together with specific InsP-requirement in determination of fate of hormonal repressors, InsP7/InsP8 clearly are also biochemical regulators of stimulus-dependent substrate stabilities.

In a typical eukaryotic cell, ∼20% of targeted protein degradations are mediated by Cullin RING ubiquitin ligases (CRLs), the largest member among the E3 ubiquitin ligase family (Soucy et al., 2009; Yi Sun, 2020). A modular CRL contains a Cullin (CUL) scaffold (CUL1-4 in Arabidopsis), an E2-interacting RING-type Rbx/Roc protein, CUL member-specified substrate adaptor and a ubiquitination target-specified substrate receptor. CRLs require covalent modification of a Nedd8 moiety on CULs (a process termed as neddylation) for function (Yi Sun, 2020). Neddylations improve CRL efficiencies in complex formation with the ubiquitin E2 enzyme for subsequent degradation of targeted substrates. In absence of cognate substrates self-ubiquitinylation-vulnerable CRLs are protected on Constitutive photomorphogenesis 9 signalosome (CSN), an evolutionarily conserved eight-subunit (CSN1-8) macromolecular complex (Chamovitz and Segal, 2001; Dubiel et al., 2015). The CSN5 subunit in this holo-complex contains an MPN+/JAMM metalloprotease motif that catalytically removes Nedd8 (deneddylation) from CULs (Cope et al., 2002) causing CRL disassembly and promoting newer associations with substrate receptors and adaptors required for continuum of its functions. Interaction changes between CRL-CSN affect cellular ratio of neddylated CUL: unneddylated CUL (CULNedd8:CUL) (Scherer et al., 2016). Biochemical modes of a CSN operation including its dynamic rearrangements upon CRL binding have been gradually unraveled at structural and functional levels (Enchev et al., 2012; Lingaraju et al., 2014; Dubiel et al., 2015). From these studies, it is evident that optimal CRL activity necessitates regulated neddylation/deneddylation cycles of CULs. Neddylated CRLs are captured on the CSN holo-complex only in deneddylation-deficient csn mutants inferring that deneddylation-dependent disassembly is very rapid (Enchev et al., 2012). The ‘CSN paradox’ (Dubiel, 2009), an apparent contradiction between genetic requirement of a CRL for CSN-protection at the cost of its deneddylation-dependent inactivation is now better understood at a molecular level. Recent breakthroughs show that highly locale-specific coordinated activities of mammalian IP5K with IP6K1, functional homologs of Arabidopsis IPK1 and ITPK1, provide InsP6 to strengthen and InsP7 to loosen CRL-CSN associations (Rao et al., 2014; Scherer et al., 2016; Lin et al., 2020). Through intricate orchestrations, a CRL tether-promoting InsP6 is converted to the liberator InsP7 by an inert IP6K1 that gets activated at CSN only upon a stimulus. This synchronization ensures functional demands of CRLs meet the response requirements proportionately without a compromise on its own stabilities. With reported interactions between Arabidopsis ITPK1 and CSN1 (Qin et al., 2005), direct implication of (any) InsP-kinase on CSN-activities from a plant system has not been forthcoming.

Here we show that Arabidopsis IPK1-ITPK1 pair retains conserved CSN interactions and modulate CUL1 deneddylation efficiencies impacting association/dissociation kinetics of CSN5 on the CSN holo-complex. Unlike noted in animal cells, CRL-CSN associations remain mostly unaffected in ipk1 or itpk1 mutants. Nevertheless, enhanced level of CULNedd8 result in constitutive PSR in these mutants. Further, we reveal that Pi-deprivation in wild-type plants is associated with CSN(5) perturbations and increased CUL1 neddylation that precedes PHR1/PHL1 role in inducing PSI gene expressions. Mutational or pharmacological inhibitions of CSN5 result in constitutive PSR even under sufficient Pi-availability. However, when actually Pi-starved, PSR responses in these plants or in the ipk1 or itpk1 mutants are suppressed indicating that equilibrium shifts of CSN5 deneddylase activity rather than its complete inhibition is essential to execute PSR. Our investigations overall reveal activities of plant IPK1-ITPK1 pair in regulating CSN dynamics, its consequences on CRLs functions and requirement in maintaining Pi-homeostasis or responding to its deficiencies.

Results

Arabidopsis IPK1 and ITPK1 interact with CSN holo-complex subunits

Mammalian IP5K co-precipitates with CSN2/5 whereas IP6K1 interacts with CSN1/2 (Rao et al., 2014; Scherer et al., 2016). Arabidopsis ITPK1 associates with CSN1 (Qin et al., 2005). Interactions of Arabidopsis IPK1 with CSN subunits have not been investigated earlier. We immuno-enriched Myc-IPK1 or ITPK1-GFP from ipk1-1:Myc-IPK1 and itpk1-2:ITPK1-GFP transgenic lines, respectively and probed for the presence of selective CSN subunits, CUL1 or the substrate adaptor ASK1. These transgenic lines restore high endogenous Pi and elevated PSI gene SPX1 expressions to wild-type (Col-0) levels implicating the functionality of the corresponding fusion proteins (Laha et al., 2020) (Supplemental Figure 1). In immunoprecipitation assays, CSN1/2/4/5, CUL1 and ASK1 co-purified with IPK1, whereas ITPK1 enrichments detected CSN1/2 and ASK1 (Figure 1A). Through Bimolecular Fluorescence Complementation (BiFC) assays, IPK1 or ITPK1 association with CSN1 and CSN2 are detected as nucleocytoplasmic complexes similar to localization of the individual proteins (Adepoju et al., 2019) (Figure 1B). A negative control ββ-Glucuronidase (GUS) protein does not interact with IPK1, ITPK1 or CSN1/2 (Supplemental Figure 2). These data suggest that likewise to animals, CSN associations of IPK1 and ITPK1 are conserved in Arabidopsis. Although biochemically coupled by substrate-product relationship, interaction between IPK1 and ITPK1 has not been demonstrated in planta. To this end, BiFC detects IPK1-ITPK1 complexes at nucleocytoplasmic locales (Figure 1B). Further, we performed co-immunoprecipitation assays via transient co-expression of Myc-IPK1 and GFP-ITPK1 in Nicotiana benthamiana leaves. Enrichment of GFP-ITPK1 with anti-GFP affinity beads co-elutes Myc-IPK1 indicating their associations in vivo (Figure 1C). Overall, our results recapitulate IPK1 and ITPK1 interactions with CSN and with each other.

Fig 1.
Fig 1. IPK1 and ITPK1 interact with each other and with selective CSN or CRLs components in planta.

(A) Immuno-enrichments of IPK1 (top panel) or ITPK1 (bottom panel) from corresponding complemented lines (ipk1-1:Myc-IPK1 or itpk1-2:ITPK1-GFP, respectively) was probed with anti-CSN1/2/4/5, anti-CUL1, or anti-ASK1 antibodies. IgG-agarose enrichments were used as negative controls. (B) Bimolecular Fluorescence Complementation (BiFC) assays showing in planta IPK1 or ITPK1 interaction with CSN1/2 and with each other. Indicated binary vectors combinations were co-expressed via Agrobacterium in Nicotiana benthamiana leaves and fluorescence observed under a confocal microscope with YFP filter. Scale bar=50 µm. (C) Co-immunoprecipitation of transiently expressed GFP-ITPK1 with Myc-IPK1 from N. benthamiana leaves. Anti-GFP pulldown was probed with anti-GFP or anti-Myc antibodies.

To determine cellular proportions of IPK1 or ITPK1 that associate with the CSN holo-complex, we subjected Col-0, ipk1-1:Myc-IPK1 and itpk1-2:ITPK1-GFP extracts to size-exclusion chromatography followed by immunoblots for representative CSN subunits, Myc-IPK1 or ITPK1-GFP. The elution patterns of molecular weight standards and selective CSN subunits with demarcation of CSN holo-complex fractions, as noted in earlier reports are shown (Gusmaroli et al., 2004; Dohmann et al., 2005) (Figure 2A). CSN1, exclusively associates with the CSN holo-complex and elutes accordingly in higher molecular weight pools (>600-400 kDa). CSN4 and CSN5 fractionates both as a part of CSN holo-complex as well as in lower molecular weight pools (monomers, 100-50 kDa). Partitioning profiles of CSN5 and CSN4 are comparable between Col-0, ipk1-1:Myc-IPK1 and itpk1-2:ITPK1-GFP (Figure 2B; Supplemental Figure S3A). Remarkably, neither Myc-IPK1 nor ITPK1-GFP proteins are detected in fractions corresponding to the CSN holo-complex implying that their associations are either transient or represent only marginal proportions of these proteins (Figure 2A). Co-elution of Myc-IPK1 and ITPK1-GFP in overlapping fractions is likely indicative of their in vivo associations we noted earlier.

Fig 2.
Fig 2. CSN5 deneddylase deficiencies with increased holo-complex retention in ipk1-1 or itpk1-2 cause enhanced CUL1Nedd8 levels.

(A) IPK1 or ITPK1 does not co-elute with the CSN holo-complex. (B) Enhanced CSN5 engagements on the CSN holo-complex in ipk1-1 and itpk1-2. Extracts from marked plants fractionated by size-exclusion chromatography were immuno-blotted with indicated antibodies. Fraction numbers and elution positions of molecular weight standards (in kDa) are shown. Fractions corresponding to CSN holo-complex and CSN subunit monomeric elutions are marked. (C) Immuno-enrichment of CUL1 detects stoichiometrically similar amounts of CSN1 and CSN2 in ipk1-1 or itpk1-2 to Col-0. Pulldowns with either IgG or anti-CUL1 antibodies were probed with anti-CUL1, anti-CSN1 or anti-CSN2 antibodies. (D) Increased CUL1Nedd8 levels (top panel) and enhanced global polyubiquitin-conjugates (bottom panel) in ipk1-1 or itpk1-2. Total protein extracts were probed with anti-CUL1, anti-actin, or anti-Ubiquitin antibodies as indicated. (E) CSN5-deneddylation deficiencies and lower CUL1 half-life in ipk1-1 or itpk1-2. In-lysate CUL1 deneddylation assay (Lin et al., 2020) at the indicated time points (minutes post-incubation; mpi) probed with anti-CUL1 antibodies. Migration position of molecular weight standards (in kDa) and neddylated (CUL1Nedd8) or unneddylated (CUL1) Cullin1 are shown. Anti-actin or Ponceau S staining demonstrates loadings controls. Migration position of molecular weight standards (in kDa) and neddylated (CUL1Nedd8) or unneddylated (CUL1) cullin1 are shown. Anti-actin or Ponceau S staining demonstrate loadings controls.

CSN5 deneddylase deficiencies cause enhanced CUL1Nedd8 pools in ipk1 or itpk1 plants

Depletion of IP5K or IP6K1 reduces CRL-CSN associations in mammalian systems (Rao et al., 2014; Lin et al., 2020). To examine whether in ipk1-1 or itpk1-2 lower CUL1 pools co-fractionate with the CSN holo-complex, we immuno-enriched CUL1 from Col-0, ipk1-1 or itpk1-2 extracts and probed for the presence of CSN1/2. Although slightly lower CUL1 was immunoprecipitated from ipk1-1 or itpk1-2 than Col-0, nonetheless proportionately similar amounts of CSN1/2 as in Col-0 co-eluted suggesting that CRL-CSN associations are likely not perturbed in these mutants (Figure 2C). Surprisingly, when we compared elution profiles, CSN5 but not CSN4 in ipk1-1 or itpk1-2 extracts showed increased presence in CSN holo-complex fractions with marked decrease in monomeric elutions, relative to Col-0 (Figure 2B). Total CSN5 levels however remained comparable between all plants.

Resident CSN5 performs the deneddylase role in a CSN holo-complex (Cope et al., 2002). With changed partitioning of CSN5 noted in ipk1-1 or itpk1-2 we tested whether deneddylation efficiencies on CULs are impacted. In mammalian system depleting IP5K or IP6K1 causes strong increments in CULNedd8 levels (Rao et al., 2014; Scherer et al., 2016). Remarkably, in ipk1-1 or itpk1-2 lysates, enhanced CUL1Nedd8 levels relative to Col-0 are strikingly prominent (Figure 2D). These increases are specific to ipk1-1 or itpk1-2 and extracts from ipk2ββ-1 with low InsP6 levels as ipk1-1 (Stevenson-Paulik et al., 2005), vih2-4 plants with elevated InsP7 (Laha et al., 2015), or itpk4-1 with low InsP6 and InsP7 levels (Kuo et al., 2018) do not show elevated CUL1Nedd8 pools (Supplemental Figure S3B). In the respective complemented lines of ipk1-1 or itpk1-2, CULNedd8:CUL ratios are restored comparable to Col-0 (Figure 2D).

Global ubiquitinylation is enhanced due to loss of mammalian IP6K1 (Rao et al., 2014). As a possible consequence to increased CULNedd8 levels, we tested relative levels of ubiquitinylation in Col-0, ipk1-1, or itpk1-2 extracts. Clear increase in global ubiquitin-conjugates in appearance of a smear when probed with anti-ubiquitin antibodies is detected in the mutant plants than Col-0 (Figure 2D). In complemented lines, wild-type ubiquitinylation levels are noted. Further, in-lysate deneddylation assay to determine deneddylase defects (Lin et al., 2020) reveal decreased efficiencies of CUL1-deneddylation by endogenous CSN5 simultaneous with increased CUL1 turnover rates in ipk1-1 or itpk1-2 extracts (Figure 2E). Decreased CUL1 stability likely accounts for the reduced immuno-enrichments from the mutants observed earlier. Taken together, our results parallel animal studies demonstrating impaired CSN5 deneddylase activity in ipk1-1 or itpk1-2 that leads to augmented CUL1Nedd8 levels prone to increased turnover due to self-ubiquitinylation.

Pi-starvation alters dynamics of CSN5 and its deneddylase efficiencies

Known consequences in ipk1-1 and itpk1-2 are auxin-insensitivity and constitutive PSR (Kuo et al., 2014; Kuo et al., 2018; Laha et al., 2020; Riemer et al., 2020). Reduced InsP8 in these mutants likely disrupt SPX1-PHR1 interactions that lead to activation of PSI-genes (Ried et al., 2019). MLN4924, a small molecule pharmacological inhibitor of CUL neddylation (Soucy et al., 2009) is functional in Arabidopsis, inhibits degradation of CRL substrates and confers auxin insensitivity (Hakenjos et al., 2011). To test whether increased PSI-gene expressions in ipk1-1 or itpk1-2 are consequences of enhanced CUL1Nedd8 levels via augmenting CRL functions, we treated these mutants with MLN4924. Remarkably, upregulated expression of PSI-genes such as IPS1, SPX1, or PHT1;3 as well as high endogenous Pi levels in ipk1-1 or itpk1-2 were suppressed by MLN4924-treatments (Figure 3A-B; Supplemental Figure 4A). On Col-0 grown under Pi-sufficient conditions, MLN4924 treatments did not cause detectable change in endogenous Pi-levels or IPS1 expression (Figure 3A-B). Most surprisingly, MLN4924 treatments on Col-0 suppressed the induction of PHT1;3 or IPS1 when subjected to Pi-starvation implying that neddylation processes drive PSI-gene expressions (Figure 3C).

Fig 3.
Fig 3. Enhanced CUL1Nedd8 levels associated with PSR is essential for PSI-gene inductions.

MLN4924 suppresses (A) elevated PSI-gene expressions, and (B) higher endogenous Pi-levels in ipk1-1 or itpk1-2. (C) Induction of PHT1;3 or IPS1 expressions in Col-0 upon Pi-starvation is inhibited by MLN4924. (D) Increments in CUL1Nedd8 levels during Pi-deprivation is PHR1/PHL1-independent. Plants grown in Pi-replete medium or transferred to Pi-deplete medium were treated with or without MLN4924. Gene expression changes were relative to MON1 transcripts and reported as fold-change to Col-0 levels. Data is mean ± SD (n=3 for qPCRs; n=6-7 for Pi-estimations). Statistical significance indicated with different alphabets is according to post-hoc Tukey’s test (p<0.05). For PHT1;3 or SPX1 expression data, statistical comparisons were to respective DMSO-treated Col-0. For immunoblots, anti-CUL1 antibodies were used. Ponceau S staining shows loadings controls.

To test whether neddylation efficiencies of cullins are affected upon Pi-deprivation, extracts from Col-0 grown under Pi-replete or Pi-deplete conditions were immunoblotted with anti-CUL1 antibodies. Prominent increase in CUL1Nedd8 levels, sensitive to MLN4924 inhibition, was noted under Pi-starvation (Figure 3D). Interestingly, these enhancements were induced even in the phr1phl1 plants when subjected to Pi-deplete conditions implying that neddylation readjustments are upstream of PHR1/PHL1 role in inducing PSI-gene expressions. Already enhanced CUL1Nedd8 levels in ipk1-1 or itpk1-2 remained unaffected under Pi-deplete exposures (Supplemental Figure 4B). Taken together, our results identify CUL1 hyperneddylation as an early response to Pi-starvation essential for transducing downstream signaling.

As tested earlier for ipk1-1 or itpk1-2, to correlate increased CUL1Nedd8 levels to decreased CSN5 deneddylase activity, we performed in-lysate deneddylation assay on Pi-replete or starved Col-0. Diminished CSN5 deneddylase activity was clearly apparent in Pi-deprived Col-0 extracts (Figure 4A). In the same extracts CUL1 instabilities were not evident perhaps reflecting tight regulations between its increased utilization and turnover rates. When profiled, CSN5 elution patterns under Pi-deplete conditions were peculiarly similar to ipk1-1 or itpk1-2 with more engagements on the CSN holo-complex and lower monomeric pools (Figure 4C). Total CSN5 protein remained comparable between Pi-replete and Pi-deplete extracts. Profiles of CSN1 remained unaltered by the Pi-changes (Supplemental Figure 4C). Resupplied phosphate restored both CSN5 partitioning changes and increased CUL1Nedd8 amounts to Pi-replete pattern suggesting that observed changes were due to Pi-starvation (Figure 4B, C) Curiously, unlike ipk1-1 or itpk1-2, global ubiquitinylation was not enhanced upon Pi-starvation although resupplying phosphate did cause a modest increase (Figure 4D). Together, these results suggest intricate coordination between CSN-CRL activities to maintain specificity in responding to reduced Pi-availability. Also, our data indicates that plants modulate CSN5 deneddylase activity and affect its dynamics during Pi-deprivation to induce PSI-gene expressions, a phenomenon that is constitutive in ipk1-1 or itpk1-2. In Pi-starved ipk1-1:Myc-IPK1 or itpk1-2:ITPK1-GFP plants, marked increase in ITPK1-GFP, but not very prominent for Myc-IPK1 protein, was detected in comparison to non-starved extracts (Supplemental Figure 4D). These at least are not due to increased transcriptions as reported earlier (Riemer et al., 2020) and may instead be in accordance with increased stability the mammalian IP6K1 acquires when associated with an active CRLs (Rao et al., 2014).

Fig 4.
Fig 4. Pi-starvation reduces endogenous CSN5 deneddylase activity and is restored upon phosphate resupply.

(A) Extracts at indicated time points (minutes post-incubation; mpi) from in-lysate CUL1 deneddylation assay on Col-0 from Pi-replete or Pi-deplete media were probed with anti-CUL1 antibodies. (B) Restoration of enhanced CUL1Nedd8 levels occur upon Pi-resupply post-starvation. (C) Increased retention of CSN5 on the CSN holo-complex under Pi-deplete exposure is reversed with Pi-resupply. (D) Global levels of polyubiquitin-conjugates remain unchanged during PSR. For these assays, plants were subjected to Pi-starvation for 3 days and phosphate re-supplementation was done for 12hrs. Comparable loadings are shown by Ponceau S staining. Elution positions of molecular weight standards (in kDa) are indicated.

Functional inhibition of CSN5 phenocopy ipk1 or itpk1 responses

To determine whether PSI-gene expressions are globally affected by CSN functions, we mined the available microarray data of differentially expressed transcripts in csn null mutants (csn3, csn4 and csn5ab) (Dohmann et al., 2008). Strong upregulation in expressions compared to wild-type plants are especially noted in genes responsible for Pi-remobilization (PAP1, PAP10, PAP23, DFR, F3H) and several Pi-transporters (PHT3;2, PHT3;3, OCT1) (Supplemental Figure 5). Expressions of PHR1 or PHL1 remain unaffected in these mutants similar to ipk1-1 or itpk1-2 (Kuo et al., 2014; Kuo et al., 2018). Also, neither IPK1 nor ITPK1 transcriptions are changed in the csn mutants. Thus, CSN holo-complex dysfunctions affect several PSI-gene expressions likely because of its deneddylation deficiencies. To explore CSN5 roles further, we utilized the csn5a-2 plants harboring a T-DNA insertion in the last exon of the CSN5A isoform (At1g22920). Similar to ipk1-1 or itpk1-2, csn5a-2 extracts accumulate higher CUL1Nedd8 levels and display auxin-insensitivity (Gusmaroli et al., 2004; Dohmann et al., 2005; Dohmann et al., 2008; Laha et al., 2020). Under Pi-replete growth, increased basal Pi-levels accompanied by mild up-regulated expressions of PHT1;3 or SPX1 is detected in the csn5a-2 plants (Figure 5A-B). Also, primary root lengths of csn5a-2 plants are slightly, but not statistically shorter than Col-0 in this growth media (Supplemental Figure 6). These features are very similar to ipk1-1 or itpk1-2 (Riemer et al., 2020). Growth under Pi-starvation led to strong inhibition of primary root length in Col-0, while in csn5a-2 (and in ipk1-1 or itpk1-2) plants these were prominently less. With exposure to Pi-deplete conditions, csn5a-2 plants are completely impaired in inducing PHT1;3 or SPX1 (Figure 5B). These results resemble PSI gene induction deficiencies in itpk1-2 when subjected to Pi-starvation (Riemer et al., 2020) suggesting that CSN5 functions are necessary in maintenance of Pi-homeostasis and successful transduction of PSR.

Fig 5.
Fig 5. Inhibition of CSN5 functions activates PSR under Pi-replete but confers non-responsiveness under Pi-deplete growth.

csn5a-2 plants have (A) elevated endogenous Pi levels, (B) increased PHT1;3 or SPX1 transcript levels under Pi-replete growth but suppressed expression upon Pi-starvation. (C) CSN5i-3 inhibits Arabidopsis CUL1 deneddylation, (D) elevates endogenous Pi-levels, and (E) suppresses PHT1;3 induction during phosphate-deprivation. Immunoblots were probed with anti-CUL1 antibodies. Ponceau S staining shows loading controls. Inhibitor treatments were done for 24 hrs and doses used are indicated. Expression levels of MON1 transcripts was used as an internal reference for qPCRs and reported as fold-change relative to DMSO-treated Col-0 under Pi-replete growth. Error bars are mean ± SD (n=3 for qPCRs; n=6-7 for Pi-estimations). Different alphabets denote statistical significance determined by post-hoc Tukey’s test (p<0.05). PHT1;3 and SPX1 data are compared to respective Pi-replete Col-0.

To directly relate CSN5 deneddylase role in Pi-homeostasis, we tested the effects of recently reported CSN5 inhibitors Azaindole#6 and CSN5i-3 on Arabidopsis (Schlierf et al., 2016; Altmann et al., 2017). In animal cell lines, these inhibitors interfere with the CSN5 deneddylase activity, and trap CRLs in their neddylated states. Because they remain untested on any plant systems, we first determined their efficacy on Col-0 seedlings. Parallel MLN4924 treatments were used as neddylation-inhibition controls. Dose-dependent increments in CUL1Nedd8 levels with collateral decrease in unneddylated CUL1 was detected at 24hr post-treatment with CSN5i-3 (Figure 5C). At 48-hrs post-treatment, efficiency of CSN5i-3 inhibitions intensified (Supplemental Figure 7A). At all doses tested, Azaindole#6 remained ineffective in changing CUL1Nedd8:CUL1 ratios. As expected, MLN4924 treatments in a dose-dependent manner reduced CUL1Nedd8 levels. For subsequent experiments, we continued with 10µM dose of CSN5i-3 and the same for MLN4924, as used in our earlier assays. On Pi-replete Col-0, CSN5i-3 caused a modest increase in basal Pi levels and upregulation in expression of PHT1;3, but not significantly for SPX1 or IPS1 (Figure 5D-E; Supplemental Figure 7B-C). Under Pi-deplete exposure, CSN5i-3 suppressed the induction of PHT1;3, SPX1 or IPS1 in Col-0 and mirrored MLN4924 effects (Figure 5D-E; Supplemental ">Figure 7B-C). CSN5i-3-treated Col-0 extracts when fractionated, CSN5 was markedly enriched in the CSN holo-complex pools with negligible detection in monomeric elutions, a pattern resembling ipk1-1, itpk1-2, or Pi-starved profiles (Figure 6A). With these results, we reason strong correlation between CSN5 deneddylation deficiencies to its enhanced retention on the CSN holo-complex, and trigger of PSR as observed in ipk1-1 or itpk1-2 plants. Most interestingly, ipk1-1 or itpk1-2 seedlings grown under sufficient Pi when treated with CSN5i-3 had reduced IPS1 expressions than untreated plants indicating that skewed kinetics of CSN5 activity rather than its complete inhibition were attributed to these mutants (Supplemental Figure 7D). MLN4924-treatments on ipk1-1 or itpk1-2 in Pi-deplete medium offered relief by elevating IPS1 expressions further supporting our hypothesis that inadequacies in these mutants to support PSR is related to dynamics of CSN5-regulated neddylation/deneddylation cycles of CRLs (Supplemental Figure 7E). From these data, it is suggestive that IPK1/ITPK1 functions influence deneddylase efficiencies and shuttling modes of CSN5 thereby affecting CRL requirements in executing PSR.

Fig 6.
Fig 6. CSN5i-3 or TNP treatments on Col-0 mirrors ipk1-1 or itpk1-2 properties.

(A) CSN5 partition shifts to CSN holo-complex pools upon CSN5i-3 or TNP treatment. TNP (B) enhances pools of CUL1Nedd8, (C) increases endogenous Pi-content, (D) upregulates IPS1 or PHT1;3 expressions under Pi-replete, but suppresses them under Pi-deplete growth, and (E) decreases endogenous deneddylation rates with increased CUL1 instability. Immunoblots were probed with anti-CUL1 or anti-CSN5 antibodies as indicated. Ponceau S staining shows loading controls. Expression levels of MON1 transcripts was used as internal reference for qPCRs. Error bars are mean ± SD (n=3 for qPCRs; n=6-7 for Pi-estimations). Statistical significance according post-hoc Tukey’s test (p<0.05) is shown with different alphabets.

IP6K1-like activity, but not InsP6 regulates CSN5 deneddylase functions of CSN

InsP6 is implicated as the physiological glue in potentiating CSN2-CRL associations (Scherer et al., 2016). That a kinase-dead IP5K interacts weakly with CSN2/5 further indicates that InsP6 augmenting these interactions is produced locally. Supplementing InsP6 to IP5K-depleted cell lysates strengthens CRL-CSN2 associations, and increases deneddylation activities of recombinant CSN on purified neddylated cullins (Scherer et al., 2016). Apparently, the externally added metabolite gets access its designated binding pocket at the CRL-CSN2 interface. Even though CRL-CSN associations are not affected in ipk1-1 or itpk1-2, we investigated whether adding InsP6 to lysates recover deneddylation deficiencies in these mutants, or augments it in Col-0. In lysate deneddylation assays showed that supplemented InsP6 did not alter deneddylation deficiencies or CUL1 instability in ipk1-1 or itpk1-2 (Supplemental Figure 8). In similar assays on Col-0 extracts, InsP6 addition did not change deneddylation or CUL1 turnover rates indicating that unlike mammalian systems, CSN functions in plants are not influenced by InsP6.

As a CRL-CSN release factor, InsP7 when supplemented to mammalian cell extracts does not cause complex disassembly meaning that IP6K1 activity is very tightly regulated spatiotemporally (Rao et al., 2014). With observed ITPK1-CSN2 interactions, this instigated us to investigate for a similar mechanism in plants. The cell-permeable compound N2-(m-(trifluoromethyl)benzyl) N6-(p-nitrobenzyl) purine (TNP) inhibits animal IP6K1 functions and reduce InsP7 levels (Padmanabhan et al., 2009). TNP effects so far have remained untested on Arabidopsis InsPs. In extracts of 10µM TNP-treated Col-0, distinct increase in CUL1NEDD8 levels were observed although less potent than a similar dose of CSN5i-3 (Figure 6B). To improve TNP efficacies, we used 25µM doses for subsequent assays. In Col-0 grown in Pi-sufficient media, TNP presence replicated CSN5i-3 results in augmenting basal Pi levels, upregulation of IPS1 or PHT1;3 expressions, and more retention of CSN5 in the CSN holo-complex pools (Figure 6A-C-D). TNP also suppressed endogenous deneddylation rates with increased CUL1 instability (Figure 6E). And likewise, to CSN5i-3, TNP also suppressed PSI-gene inductions in Col-0 under Pi-deplete circumstances (Figure 6D). Curiously, CSN5i-3, TNP or MLN4924 treatments on itpk1-2:ITPK1-GFP resulted in increased ITPK1-GFP levels, similar to observed in Pi-deplete media (Supplemental Figure 9). Although direct inhibitions of TNP on Arabidopsis ITPK1 or other InsP(6)-kinases still remains to be tested, nevertheless our results imply that similar to mammals an IP6K1-like activity affects CSN5 performance on regulating CUL1 deneddylation rates and its consequences on PSR. Overall, with our data we present localized coordinated involvement of InsP-kinase pair in modulating CSN dynamics, defining molecular concept of the CSN paradox, and underlining CSN role in phosphate stress responses of plants.

Discussion

In animal systems, CRL-CSN tether is augmented by InsP6 and released via stimulus-driven InsP7 synthesis. Here, we show that although Arabidopsis IPK1 and ITPK1 retain conserved CSN interactions, InsP6/InsP7 changes in the corresponding mutants do not overall affect CRL1-CSN associations. Thus, either the residual levels of these InsPs suffice at the CRL-CSN junctures or the association/dissociation cycles involve an altogether different InsP(s) not locally altered in the above mutants. Unlike mammalian results, that added InsP6 does not rescue ipk1-1 or even alter CUL1 deneddylation rates/stability in Col-0 supports our speculation. Further, an ITPK4 mutant (itpk4-1) with reduced InsP6/InsP7 as in ipk1-1 or itpk1-2 has normal PSR (Kuo et al., 2018) and matches Col-0 ratio of CUL1Nedd8:CUL1 also lends credence to our hypothesis. One striking distinction in ipk1-1 or itpk1-2 is that these mutants accumulate increased levels of Ins(3,4,5,6)P4 which neither the itpk4-1 nor Col-0 subjected to PSR does (Kuo et al., 2018). Whether this increased abundance of Ins(3,4,5,6)P4 or another unidentified InsP altered in ipk1-1 or itpk1-2 satisfy CRL-CSN interaction requirements remains a possibility to explore further. Nevertheless, analogous to mammals our observations overall parallel that decreased CSN5 deneddylase performance with lower CUL1 deneddylation rates associate with the loss of IPK1 or ITPK1 and implicates their distinct roles on CSN functions.

Mammalian ITPK1 over-expression partitions more monomeric CSN5 free from the CSN holo-complex (Sun et al., 2002). We demonstrate here that absence of Arabidopsis ITPK1 or TNP inhibitions antagonize CSN5 release from the CSN holo-complex. Even with yet to be tested TNP effects on ITPK1 (or another InsP-kinase) functions in plants, nonetheless a mammalian IP6K1-like activity in choreographing CSN5 entry-exit shuttles seems to be conserved in Arabidopsis. Overall structural integrity of an inert CSN holo-complex is remarkably maintained without CSN5, albeit for deneddylation CSN5 incorporation is mandatory (Lingaraju et al., 2014). When CSN5 is integrated into the holo-complex, its interfaces with CSN4-CSN6 inhibit deneddylase activation in the absence of a bound CRL (Enchev et al., 2012; Lingaraju et al., 2014). Upon binding a neddylated CRL, a series of conformational change prompted by CSN4 orients CSN5 active site towards the neddylated cullins. Closely proximal to this active site, conserved lysines in CSN2 alpha helices coordinate bound InsP6 to bridge for more stable associations with CRL. The bound InsP6 in this electropositive environment is uniquely oriented with its 5’-phosphate exposed, amenable for subsequent pyrophosphorylation. Contrastingly, when an InsP7 is modeled into the same active site strong steric clashes especially at CRL-CSN junctures are noted (Lin et al., 2020). These steric intolerances may underlie InsP7-triggered CSN5 deneddylase activation, followed by CRL and perhaps CSN5 release from the CSN holo-complex. When added to cell lysates, since free InsP7 fails to enhance cullin neddylation or disrupt CRL-CSN associations (Rao et al., 2014), its localized synthesis achieved through site-specific availability of InsP6, and its accessibility to CSN5 active site is hence crucial for its proposed role. Thus, similar to the mammalian IP6K1 a plant ITPK1 activity (via InsP7 or an unknown InsP) may possess propensity to reconfigure CSN, initiating deneddylation coupled to CRLs and CSN5 disassembly from the holo-complex. These roles may be supportive of the recent implication that a plant ITPK1 has evolutionarily integrated functional signatures of both mammalian ITPK1 and IP6K1 (Whitfield et al., 2020).

With obvious InsP8 reductions in ipk1-1, itpk1-2 or upon VIH2 phosphatase activity during Pi-starvation (Dong et al., 2019; Zhu et al., 2019), PHR1 liberated from SPX1 causes activation of PSI-gene expressions. Here we demonstrate that a mode of perception of Pi-deprivation in addition to the above also occurs at CSN, involving changes in associated InsP-kinase activities, and is responded by decreased deneddylation rates with pronounced CSN5 retentions on the CSN holo-complex. Since the resulting increase in CUL1Nedd8 levels remain independent of PHR1/PHL1 presence, these observations likely indicate parallel responsive routes of a Pi-starved plant to facilitate more available pools of neddylated CRLs to ubiquitinylate substrates including the recently identified SPX4, released from PHR1-hold (Osorio et al., 2019). These features we show are constitutive in ipk1-1, itpk1-2, and mimicked in wild-type plants with CSN5i-3 or TNP treatment, and suppressed by the neddylation inhibitor MLN4924. Our observation that phosphate re-supplementation post-starvation restores CSN5 partitioning and CULNedd8:CUL1 ratios to Pi-replete patterns in wild-type plants implies that the noted shifts are only transitory adaptations. That, unlike in ipk1-1 or itpk1-2, global polyubiquitin-conjugates remains unchanged in Col-0 under low-Pi availability additionally emphasize on the strictly regulatory PSR mechanisms to controls ubiquitinylation-specificities on intended targets. Not the least, observed failure in itpk1-2 (Riemer et al., 2020) or csn5a-2 to respond to low-Pi by anticipated augmentation of already upregulated PSI-gene expressions, or in CSN5i-3 or TNP treated Col-0 indicates that CSN5 partitioning re-adjustments are only shifts in neddylation/deneddylation equilibrium and not a complete inhibition of the CSN5 deneddylase. Also, that MLN4924 presence causes sub-optimal induction of PSI-gene expressions in Pi-depleted Col-0, suggests continued requirements of CRL neddylation/deneddylation cycles to successfully transduce PSR. Thus, sustained and successful response to Pi-starvation is likely achieved by a shift in equilibrium of CSN5-mediated deneddylation dynamics on CUL1 and not complete inhibition of its activities as described in our model (Figure 7). Similar phenomenon is mirrored by the shuttling requirements of CSN5 in cell proliferation wherein its perturbations cause complete arrest (Tomoda et al., 2002; Yoshida et al., 2010).

Fig 7.
Fig 7. Schematic representation of CRL-CSN dynamics in plants.

CSN holo-complex platform protects CRLs in the absence of bound ubiquitinylation substrate (steric hindrance prevents E2 enzyme binding (Enchev et al., 2012; Mosadeghi et al., 2016) utilizing coordinated activities (bi-directional white arrows) of the IPK1-ITPK1 pair. Equilibrium is maintained between CSN-mediated deneddylation cycles and CUL1Nedd8 requirements in steady-state CRL functions. Balanced assembly/disassembly cycles of CSN5 on the CSN holo-complex are also linked to these regulations. Perception (via unknown mode; question mark) of a stimulus, such as Pi-starvation, reduces CSN5 deneddylase functions, likely via more facilitation of the InsP-kinase functions to CRL-SCF complex roles in targeted ubiquitinylation (Ubn) and degradation of specific substrates (such as SPX4 or Aux/IAAs). IPK1-ITPK1 pair provides necessary InsP co-factors to these activities. Inhibition modes of TNP, CSN5i-3 and MLN4924 are indicated at the respective processes. CSN subunits are presented as numbers; CUL1, Cullin1; RBX, RING H2 protein1; E2, Ubiquitin cojugating enzyme E2; SA, substrate adapter; SR, substrate receptor; N8, Nedd8; IPK1, Inositol pentakisphosphate 2-kinase1; ITPK1, Inositol 1,3,4-trisphosphate 5/6-kinase 1.

Distinct increase in ITPK1 protein but not gene expression under Pi-starvation (Riemer et al., 2020) and as shown here with CSN5i-3, TNP or MLN4924 treatments is conceivably similar to the gain of stability the mammalian IP6K1 acquires upon CRL binding (Rao et al., 2014). Thus, similar to CUL1 vulnerability for self-ubiquitinylation, ITPK1 fates may also be regulated in the absence of CRL substrates. Increased stabilities hence may therefore reflect ITPK1’s priority to accommodate stimulus-appropriate co-factor requirements in downstream responses not limited only for modulating CRL functions. The mammalian phosphate exporter Xenotropic and polytropic retrovirus receptor 1 (XPR1) relies on InsP7 synthesized by IP6K1/2 for function (Wilson et al., 2019). Arabidopsis XPR1 ortholog PHO1 does not complement the pho1 mutant if mutations are introduced in its InsP7-binding pocket (Wild et al., 2016). And as is known during PSR, augmented auxin responses involving SCFTIR1 also require InsP7 (Laha et al., 2020). Whether these new engagements of ITPK1 are adaptation strategies to transiently deprive CSN (and CSN5 release from the holo-complex) and shift balances towards more neddylated CRL availability and/or for providing InsP7 for other cellular processes are encouraging possibility to explore. In conclusion, with our data we highlight remarkable conservation, albeit with slight distinction, in plant InsP-kinase roles in regulating CRL-CSN functions and its implications in responding to Pi-imbalances.

Materials and Methods

Plant material and growth conditions

Details of Arabidopsis thaliana (ecotype Col-0) T-DNA insertion lines ipk1-1, itpk1-2, ipk2ββ-1, itpk4-1, csn5a-2, vih2-4, itpk1-2:ITPK1-GFP, and phr1 phl1 plants have been described earlier (Kuo et al., 2014; Jia et al., 2015; Kuo et al., 2018; Laha et al., 2020). T-DNA and respective gene-specific primers are listed in Supplementary Table 1. Seeds were stratified for 2-days at 4°C in dark and then sterilized using 30% bleach solution. They were then germinated either directly on soil or on ½ strength Murashige and Skoog (MS) Pi-replete (625 µM KH2PO4) agar plates in growth chambers maintained at 22°C with 70% Relative humidity (RH) and 16hrs: 8hrs; light: dark having light intensity 100 µmol µm-2s-1. For fractionation, Pi-starvation assays, and inhibitor treatments, plants grown on media plates as above were transferred 7-days post-germination to liquid ½ MS medium in 12-well sterile culture plates are proceeded accordingly as indicated in respective experiments. For immuno-precipitation assays 4-week-old soil-grown plants were used.

Size-exclusion chromatography of plant extracts

Gel-filtration protocol was according to Huang et al (2013). Briefly, 14-day-old plants ground to a fine powder with liquid nitrogen were homogenized in Gel filtration buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA, 10% Glycerol, 10mM MgCl2, 1mM DTT, 1mM PMSF and 1X Protease inhibitor cocktail). Homogenates were centrifuged for 10 mins at 4°C and supernatant passed through 0.2 µM syringe filters. Total protein extracts were fractionated through Superdex 10/300 GL Column (GE-Healthcare, USA) and eluted in the fractionation buffer at 0.2mL/min flow rate. The eluants were TCA (Trichloroacetic acid) precipitated, washed with acetone and resuspended in 1X Laemmli loading dye and used for indicated immunoblots.

In-lysate deneddylation assay

In-lysate deneddylayion assay was according to Lin et al (2020). Briefly, 14-day-old seedlings were quickly homogenized in deneddylayion buffer (10mM Tris-HCl pH7.5, 150mM NaCl and 0.5% NP-40) in absence of protease inhibitors. The extracts were incubated at 37°C for indicated times, reaction stopped by adding Laemmli loading dye to 1X, and then used for immunoblots.

Phosphate-starvation/re-supplementation assays

After germination of seeds as described earlier, seedlings were transferred to liquid ½ MS medium in 12-well sterile culture plates with Pi-replete conditions (625 µM KH2PO4) and placed in the growth chamber. After 4-days, the plants were either maintained with Pi-replete or transferred to Pi-deplete (10 µM KH2PO4) liquid media and continued for another 3 days (total 14-days post-germination) before being used for Pi-estimation, qRT-PCR, total protein extraction, or fractionation assays. For Pi-resupply, plants in Pi-deplete medium were transferred to Pi-replete medium for 12 hrs before analysis.

Inhibitor treatments

Stock solutions of MLN4924 (Cayman Chemicals, USA), CSN5i-3 (Novartis Inc, Switzerland), or TNP (Sigma-Aldrich, USA) was prepared in DMSO. For treatments, 10µM final concentration of MLN4924 or CSN5i-3 were used. TNP was used at 25µM final unless otherwise indicated. For assays on Col-0, ipk1-1, or itpk1-2 or for fractionations, inhibitors were treated for 24hrs. For effects on PSR, inhibitors were added during the shift to Pi-deplete medium.

Ubiquitination assays

For determining global ubiquitination levels, extracts from indicated plants prepared in Gel filtration buffer in the presence of 10µM proteasome inhibitor MG132 were incubated on ice for 2hrs. Immunoblotting was then performed with anti-ubiquitin antibodies (P4D1; SantaCruz Biotechnology, USA).

Statistical analysis

All Data presented here are representative of at least three biological and technical replicates for each. Post-hoc Tukey’s test (p<0.05) were applied for statistical analysis. Additional details of cloning methodologies for generation of constructs, and transgenic lines, BiFC assays, immuno-enrichments, immunoblotting, Pi-estimations, and qRT-PCRs are described in Supplementary Materials and Methods.

Acknowledgements

We thank Regional Centre for Biotechnology (RCB), Faridabad, for financial support, lab infrastructure, and central instrumental facilities. S.B. expresses gratitude to Department of Biotechnology (DBT), Govt. of India for the award of Ramalingaswami re-entry fellowship and Grant (No. BT/PR23666/AGIII/103/1039/2018) that supported this research. The phr1 phl1 double mutant seeds were from Prof. Tzyy-Jen Chiou, Academia Sinica, Taipei. A special thanks to Dr. Eva Altmann, Novartis Institutes for BioMedical Research, Switzerland for the CSN5i-3 gift. Also to Dr. Divya Chandran (RCB) for help with transcriptomic data analysis of csn mutants. Y.W. thanks DBT for Young Investigator award. M.K. and K.D.I thank UGC and DBT for fellowships, respectively. D.L. acknowledges the Indian Institute of Science for start-up funds. All authors thanks Dr. Nripendra Singh and his team at Advanced Technology Platform Centre (ATPC), Faridabad, India for constant support with sample fractionations.

Footnotes

  • Competing interest statement The authors declare no conflict of interest for this study.

  • Some new results have been added to the manuscript and the text has been accordingly revised.

References

  1. ↵
    Adepoju O, Williams SP, Craige B, Cridland CA, Sharpe AK, Brown AM, Land E, Perera IY, Mena D, Sobrado P, Gillaspy GE (2019) bioRxiv:724914; doi: https://doi.org/10.1101/724914
  2. ↵
    Altmann E, Erbel P, Renatus M, Schaefer M, Schlierf A, Druet A, Kieffer L, Sorge M, Pfister K, Hassiepen U, Jones M, Ruedisser S, Ostermeier D, Martoglio B, Jefferson AB, Quancard J (2017) Azaindoles as Zinc-Binding Small-Molecule Inhibitors of the JAMM Protease CSN5. Angew Chem Int Ed Engl 56: 1294–1297
    OpenUrlCrossRef
  3. Bhattacharjee S, Halane MK, Kim SH, Gassmann W (2011) Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators. Science 334: 1405–1408
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Perez-Perez J, Solano R, Leyva A, Paz-Ares J (2010) A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genet 6: e1001102
    OpenUrlCrossRefPubMed
  5. Chakrabarty R, Banerjee R, Chung SM, Farman M, Citovsky V, Hogenhout SA, Tzfira T, Goodin M (2007) pSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions. Mol Plant Microbe Interact 20: 740–750
    OpenUrlCrossRefPubMedWeb of Science
  6. ↵
    Chamovitz DA, Segal D (2001) JAB1/CSN5 and the COP9 signalosome. A complex situation. EMBO Reports 2: 96–101
    OpenUrlAbstract/FREE Full Text
  7. Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Su CL (2006) Regulation of phosphate homeostasis by MicroRNA in Arabidopsis. Plant Cell 18: 412–421
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Cope GA, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ (2002) Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298: 608–611
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Desai M, Rangarajan P, Donahue JL, Williams SP, Land ES, Mandal MK, Phillippy BQ, Perera IY, Raboy V, Gillaspy GE (2014) Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants. Plant J 80: 642–653
    OpenUrlCrossRefPubMed
  10. ↵
    Dohmann EM, Kuhnle C, Schwechheimer C (2005) Loss of the CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome subunit 5 is sufficient to cause the cop/det/fus mutant phenotype in Arabidopsis. Plant Cell 17: 1967–1978
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Dohmann EM, Levesque MP, Isono E, Schmid M, Schwechheimer C (2008) Auxin responses in mutants of the Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome. Plant Physiol 147: 1369–1379
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Dong J, Ma G, Sui L, Wei M, Satheesh V, Zhang R, Ge S, Li J, Zhang TE, Wittwer C, Jessen HJ, Zhang H, An GY, Chao DY, Liu D, Lei M (2019) Inositol Pyrophosphate InsP8 Acts as an Intracellular Phosphate Signal in Arabidopsis. Mol Plant 12: 1463–1473
    OpenUrl
  13. ↵
    Dubiel D, Rockel B, Naumann M, Dubiel W (2015) Diversity of COP9 signalosome structures and functional consequences. FEBS Lett 589: 2507–2513
    OpenUrlCrossRefPubMed
  14. ↵
    Dubiel W (2009) Resolving the CSN and CAND1 paradoxes. Mol Cell 35: 547–549
    OpenUrlCrossRefPubMed
  15. ↵
    Enchev RI, Scott DC, da Fonseca PC, Schreiber A, Monda JK, Schulman BA, Peter M, Morris EP (2012) Structural basis for a reciprocal regulation between SCF and CSN. Cell Rep 2: 616–627
    OpenUrlCrossRefPubMed
  16. ↵
    Gillaspy GE (2013) The role of phosphoinositides and inositol phosphates in plant cell signaling. Adv Exp Med Biol 991: 141–157
    OpenUrlCrossRefPubMed
  17. ↵
    Gusmaroli G, Feng S, Deng XW (2004) The Arabidopsis CSN5A and CSN5B subunits are present in distinct COP9 signalosome complexes, and mutations in their JAMM domains exhibit differential dominant negative effects on development. Plant Cell 16: 2984–3001
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Hakenjos JP, Richter R, Dohmann EM, Katsiarimpa A, Isono E, Schwechheimer C (2011) MLN4924 is an efficient inhibitor of NEDD8 conjugation in plants. Plant Physiol 156: 527–536
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Ham BK, Chen J, Yan Y, Lucas WJ (2018) Insights into plant phosphate sensing and signaling. Curr Opin Biotechnol 49: 1–9
    OpenUrl
  20. ↵
    Huang H, Quint M, Gray WM (2013) The eta7/csn3-3 auxin response mutant of Arabidopsis defines a novel function for the CSN3 subunit of the COP9 signalosome. PLoS One 8: e66578
    OpenUrlCrossRef
  21. ↵
    Jia Q, Kong D, Li Q, Sun S, Song J, Zhu Y, Liang K, Ke Q, Lin W, Huang J (2019) The Function of Inositol Phosphatases in Plant Tolerance to Abiotic Stress. Int J Mol Sci 20
  22. ↵
    Jia X, Chanda B, Zhao M, Brunner AM, Beers EP (2015) Instability of the Arabidopsis mutant csn5a-2 caused by epigenetic modification of intronic T-DNA. Plant Sci 238: 53–63
    OpenUrl
  23. ↵
    Jung JY, Ried MK, Hothorn M, Poirier Y (2018) Control of plant phosphate homeostasis by inositol pyrophosphates and the SPX domain. Curr Opin Biotechnol 49: 156–162
    OpenUrlCrossRefPubMed
  24. Kim SH, Gao F, Bhattacharjee S, Adiasor JA, Nam JC, Gassmann W (2010) The Arabidopsis resistance-like gene SNC1 is activated by mutations in SRFR1 and contributes to resistance to the bacterial effector AvrRps4. PLoS Pathog 6: e1001172
    OpenUrlCrossRefPubMed
  25. ↵
    Kuo HF, Chang TY, Chiang SF, Wang WD, Charng YY, Chiou TJ (2014) Arabidopsis inositol pentakisphosphate 2-kinase, AtIPK1, is required for growth and modulates phosphate homeostasis at the transcriptional level. Plant J 80: 503–515
    OpenUrlCrossRefPubMed
  26. ↵
    Kuo HF, Hsu YY, Lin WC, Chen KY, Munnik T, Brearley CA, Chiou TJ (2018) Arabidopsis inositol phosphate kinases IPK1 and ITPK1 constitute a metabolic pathway in maintaining phosphate homeostasis. Plant J 95:613–630
    OpenUrl
  27. ↵
    Laha D, Johnen P, Azevedo C, Dynowski M, Weiss M, Capolicchio S, Mao H, Iven T, Steenbergen M, Freyer M, Gaugler P, de Campos MK, Zheng N, Feussner I, Jessen HJ, Van Wees SC, Saiardi A, Schaaf G (2015) VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis. Plant Cell 27: 1082–1097
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Laha D, Parvin N, Hofer A, Giehl RFH, Fernandez-Rebollo N, von Wiren N, Saiardi A, Jessen HJ, Schaaf G (2019) Arabidopsis ITPK1 and ITPK2 Have an Evolutionarily Conserved Phytic Acid Kinase Activity. ACS Chem Biol 14: 2127–2133
    OpenUrlCrossRef
  29. ↵
    Laha NP, Dhir YW, Giehl RFH, Schäfer EM, Gaugler P, Shishavan ZH, Gulabani H, Mao H, Zheng N, von Wirén N, Jessen HJ, Saiardi A, Bhattacharjee S, Laha D, Schaaf G (2020) bioRxiv: 2020.04.23.058487
  30. ↵
    Lee HS, Lee DH, Cho HK, Kim SH, Auh JH, Pai HS (2015) InsP6-sensitive variants of the Gle1 mRNA export factor rescue growth and fertility defects of the ipk1 low-phytic-acid mutation in Arabidopsis. Plant Cell 27: 417–431
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Lin H, Zhang X, Liu L, Fu Q, Zang C, Ding Y, Su Y, Xu Z, He S, Yang X, Wei X, Mao H, Cui Y, Wei Y, Zhou C, Du L, Huang N, Zheng N, Wang T, Rao F (2020) Basis for metabolite-dependent Cullin-RING ligase deneddylation by the COP9 signalosome. Proc Natl Acad Sci U S A 117: 4117–4124
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Lingaraju GM, Bunker RD, Cavadini S, Hess D, Hassiepen U, Renatus M, Fischer ES, Thoma NH (2014) Crystal structure of the human COP9 signalosome. Nature 512: 161–165
    OpenUrlCrossRefPubMedWeb of Science
  33. ↵
    Lv Q, Zhong Y, Wang Y, Wang Z, Zhang L, Shi J, Wu Z, Liu Y, Mao C, Yi K, Wu P (2014) SPX4 Negatively Regulates Phosphate Signaling and Homeostasis through Its Interaction with PHR2 in Rice. Plant Cell 26: 1586–1597
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Monserrate JP, York JD (2010) Inositol phosphate synthesis and the nuclear processes they affect. Curr Opin Cell Biol 22: 365–373
    OpenUrlCrossRefPubMed
  35. ↵
    Mosadeghi R, Reichermeier KM, Winkler M, Schreiber A, Reitsma JM, Zhang Y, Stengel F, Cao J, Kim M, Sweredoski MJ, Hess S, Leitner A, Aebersold R, Peter M, Deshaies RJ, Enchev RI (2016) Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle. Elife 5: e12102
    OpenUrl
  36. ↵
    Murphy AM, Otto B, Brearley CA, Carr JP, Hanke DE (2008) A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens. Plant J 56: 638–652
    OpenUrlCrossRefPubMedWeb of Science
  37. ↵
    Osorio MB, Ng S, Berkowitz O, De Clercq I, Mao C, Shou H, Whelan J, Jost R (2019) SPX4 Acts on PHR1-Dependent and -Independent Regulation of Shoot Phosphorus Status in Arabidopsis. Plant Physiol 181: 332–352
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Padmanabhan U, Dollins DE, Fridy PC, York JD, Downes CP (2009) Characterization of a selective inhibitor of inositol hexakisphosphate kinases: use in defining biological roles and metabolic relationships of inositol pyrophosphates. J Biol Chem 284: 10571–10582
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Puga MI, Mateos I, Charukesi R, Wang Z, Franco-Zorrilla JM, de Lorenzo L, Irigoyen ML, Masiero S, Bustos R, Rodriguez J, Leyva A, Rubio V, Sommer H, Paz-Ares J (2014) SPX1 is a phosphate-dependent inhibitor of Phosphate Starvation Response 1 in Arabidopsis. Proc Natl Acad Sci U S A 111: 14947–14952
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Qin ZX, Chen QJ, Tong Z, Wang XC (2005) The Arabidopsis inositol 1,3,4-trisphosphate 5/6 kinase, AtItpk-1, is involved in plant photomorphogenesis under red light conditions, possibly via interaction with COP9 signalosome. 43: 947–954
    OpenUrl
  41. ↵
    Rao F, Xu J, Khan AB, Gadalla MM, Cha JY, Xu R, Tyagi R, Dang Y, Chakraborty A, Snyder SH (2014) Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4-signalosome complex to regulate DNA repair and cell death. Proc Natl Acad Sci U S A 111: 16005–16010
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Ried MK, Wild R, Zhu J, Broger L, Harmel RK, Hothorn LA, Fiedler D, Hothorn M (2019) Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis. bioRxiv 2019.12.13.875393
  43. ↵
    Riemer E, Laha D, Harmel RK, Gaugler P, Pries V, Frei M, Hajirezaei M-R, Laha NP, Krusenbaum L, Schneider R, Jessen HJ, Saiardi A, Fiedler D, Schaaf G, Giehl RFH (2020) ITPK1 is an InsP6/ADP phosphotransferase that controls systemic phosphate homeostasis in Arabidopsis. bioRxiv 2020.05.18.100297
  44. ↵
    Ruan W, Guo M, Wang X, Guo Z, Xu Z, Xu L, Zhao H, Sun H, Yan C, Yi K (2019) Two RING-Finger Ubiquitin E3 Ligases Regulate the Degradation of SPX4, An Internal Phosphate Sensor, for Phosphate Homeostasis and Signaling in Rice. Mol Plant 12: 1060–1074
    OpenUrl
  45. ↵
    Rubio V, Linhares F, Solano R, Martin AC, Iglesias J, Leyva A, Paz-Ares J (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev 15: 2122–2133
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Scherer PC, Ding Y, Liu Z, Xu J, Mao H, Barrow JC, Wei N, Zheng N, Snyder SH, Rao F (2016) Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function. Proc Natl Acad Sci U S A 113: 3503–3508
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Schlierf A, Altmann E, Quancard J, Jefferson AB, Assenberg R, Renatus M, Jones M, Hassiepen U, Schaefer M, Kiffe M, Weiss A, Wiesmann C, Sedrani R, Eder J, Martoglio B (2016) Targeted inhibition of the COP9 signalosome for treatment of cancer. Nat Commun 7: 13166
    OpenUrlCrossRefPubMed
  48. ↵
    Sheard LB, Tan X, Mao H, Withers J, Ben-Nissan G, Hinds TR, Kobayashi Y, Hsu FF, Sharon M, Browse J, He SY, Rizo J, Howe GA, Zheng N (2010) Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468: 400–405
    OpenUrlCrossRefPubMedWeb of Science
  49. ↵
    Shears SB, Ganapathi SB, Gokhale NA, Schenk TM, Wang H, Weaver JD, Zaremba A, Zhou Y (2012) Defining signal transduction by inositol phosphates. Subcell Biochem 59: 389–412
    OpenUrlCrossRefPubMed
  50. ↵
    Shears SB, Wang H (2019) Inositol phosphate kinases: Expanding the biological significance of the universal core of the protein kinase fold. Adv Biol Regul 71: 118–127
    OpenUrlCrossRefPubMed
  51. ↵
    Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, Brownell JE, Burke KE, Cardin DP, Critchley S, Cullis CA, Doucette A, Garnsey JJ, Gaulin JL, Gershman RE, Lublinsky AR, McDonald A, Mizutani H, Narayanan U, Olhava EJ, Peluso S, Rezaei M, Sintchak MD, Talreja T, Thomas MP, Traore T, Vyskocil S, Weatherhead GS, Yu J, Zhang J, Dick LR, Claiborne CF, Rolfe M, Bolen JB, Langston SP (2009) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458: 732–736
    OpenUrlCrossRefPubMedWeb of Science
  52. ↵
    Stevenson-Paulik J, Bastidas RJ, Chiou ST, Frye RA, York JD (2005) Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases. Proc Natl Acad Sci U S A 102: 12612–12617
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Sun Y, Wilson MP, Majerus PW (2002) Inositol 1,3,4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1. J Biol Chem 277: 45759–45764
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Szijgyarto Z, Garedew A, Azevedo C, Saiardi A (2011) Influence of inositol pyrophosphates on cellular energy dynamics. Science 334: 802–805
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Tan X, Calderon-Villalobos LI, Sharon M, Zheng C, Robinson CV, Estelle M, Zheng N (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446: 640–645
    OpenUrlCrossRefPubMedWeb of Science
  56. ↵
    Tomoda K, Kubota Y, Arata Y, Mori S, Maeda M, Tanaka T, Yoshida M, Yoneda-Kato N, Kato JY (2002) The cytoplasmic shuttling and subsequent degradation of p27Kip1 mediated by Jab1/CSN5 and the COP9 signalosome complex. J Biol Chem 277: 2302–2310
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Whitfield H, White G, Sprigg C, Riley AM, Potter BVL, Hemmings AM, Brearley CA (2020) An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels. Biochem J 477: 2621–2638
    OpenUrlCrossRef
  58. ↵
    Wild R, Gerasimaite R, Jung JY, Truffault V, Pavlovic I, Schmidt A, Saiardi A, Jessen HJ, Poirier Y, Hothorn M, Mayer A (2016) Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science 352: 986–990
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Williams SP, Gillaspy GE, Perera IY (2015) Biosynthesis and possible functions of inositol pyrophosphates in plants. Front Plant Sci 6: 67
    OpenUrlPubMed
  60. ↵
    Wilson MS, Jessen HJ, Saiardi A (2019) The inositol hexakisphosphate kinases IP6K1 and -2 regulate human cellular phosphate homeostasis, including XPR1-mediated phosphate export. J Biol Chem 294: 11597–11608
    OpenUrlAbstract/FREE Full Text
  61. ↵
    Wundenberg T, Mayr GW (2012) Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis. Biol Chem 393: 979–998
    OpenUrlCrossRefPubMed
  62. ↵
    Yi Sun WW, Jianping Jin (2020) CRL Book Sun et al 2020. Advances in Experimental Medicine and Biology Biology and Therapeutics 1217
  63. ↵
    Yoshida A, Yoneda-Kato N, Panattoni M, Pardi R, Kato JY (2010) CSN5/Jab1 controls multiple events in the mammalian cell cycle. FEBS Lett 584: 4545–4552
    OpenUrlCrossRefPubMed
  64. ↵
    Zhong Y, Wang Y, Guo J, Zhu X, Shi J, He Q, Liu Y, Wu Y, Zhang L, Lv Q, Mao C (2018) Rice SPX6 negatively regulates the phosphate starvation response through suppression of the transcription factor PHR2. 219: 135–148
    OpenUrl
  65. ↵
    Zhu J, Lau K, Puschmann R, Harmel RK, Zhang Y, Pries V, Gaugler P, Broger L, Dutta AK, Jessen HJ, Schaaf G, Fernie AR, Hothorn LA, Fiedler D, Hothorn M (2019) Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis. Elife 8: e43582
    OpenUrl
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Arabidopsis inositol polyphosphate kinases regulate COP9 signalosome deneddylase functions in phosphate-homeostasis
Yashika Walia, Mritunjay Kasera, Kishor D Ingole, Swaroop Peddiraju, Abhisha Roy, Debabrata Laha, Gabriel Schaaf, Saikat Bhattacharjee
bioRxiv 2020.10.02.323584; doi: https://doi.org/10.1101/2020.10.02.323584
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