Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants

Ranjana Yadav; Guizhen Liu; Priyanshi Rana; Naga Jyothi Pullagurla; Danye Qiu; Henning J Jessen; Debabrata Laha

doi:10.1371/journal.pgen.1011838

. 2025 Sep 11;21(9):e1011838. doi: 10.1371/journal.pgen.1011838

Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP₇ in land plants

Ranjana Yadav ^1,^#, Guizhen Liu ^2,^#, Priyanshi Rana ¹, Naga Jyothi Pullagurla ¹, Danye Qiu ², Henning J Jessen ², Debabrata Laha ^1,^*

Editor: Bao-Cai Tan³

PMCID: PMC12445474 PMID: 40934223

Abstract

Inositol pyrophosphates (PP-InsPs) are soluble cellular messengers that integrate environmental cues to induce adaptive responses in eukaryotes. In plants, the biological functions of various PP-InsP species are poorly understood, largely due to the absence of canonical enzymes found in other eukaryotes. The recent identification of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP₇ in the eudicot Arabidopsis thaliana, further highlights the intricate PP-InsP signalling network employed by plants. Yet, the abundance of 4/6-InsP₇ in land plants, the enzyme(s) responsible for its synthesis, and the physiological functions of this species are all currently unknown. In this study, we show that 4/6-InsP₇ is ubiquitous in the studied land plants. Our findings demonstrate that the Arabidopsis inositol polyphosphate multikinase (IPMK) homologs, AtIPK2α and AtIPK2β phosphorylates InsP₆ to generate 4/6-InsP₇ as the predominant PP-InsP species in vitro. Consistent with this, AtIPK2α and AtIPK2β act redundantly to control 4/6-InsP₇ production in planta. Notably, activity of these IPK2 proteins is critical for heat stress acclimation in Arabidopsis. Our parallel investigations using the liverwort Marchantia polymorpha suggest that the PP-InsP synthase activity of IPK2 and role of IPK2 in regulating the heat stress response are conserved in land plants. Furthermore, we show that the transcription activity of heat shock factor (HSF) is regulated by IPK2 proteins, providing a mechanistic framework of IPK2-controlled heat stress tolerance in land plants. Collectively, our study indicates that IPK2-type kinases have played a critical role in transducing environmental cues for biological processes during land plant evolution.

Author summary

Inositol pyrophosphates (PP-InsPs) are eukaryote-specific cellular messengers that control a plethora of critical physiological processes, ranging from cellular metabolism to cellular energetics, and nutrient sensing. The identification of a new inositol pyrophosphate species, 4/6-InsP₇, suggests the presence of a more diverse PP-InsP signalling network in plants. To date, the molecular basis of 4/6-InsP₇ production and its physiological function in plants remained completely elusive. We report the identification of a non-archetypal function of inositol polyphosphate multikinase (IPMK/IPK2) that catalyzes the synthesis of 4/6-InsP₇, and the kinase activity is critical for controlling heat stress acclimation. Furthermore, we show that the role of IPK2 in generating 4/6-InsP₇ and regulating heat stress response is conserved in the studied land plants.

Introduction

Inositol phosphates (InsPs) are phosphate esters of myo-inositol (Ins) that are synthesized by the combinatorial phosphorylation of the hydroxyl group (-OH) of the myo-inositol ring. The fully phosphorylated inositol ring, InsP₆, also known as phytic acid, is one of the most abundant InsP species that controls diverse cellular processes and serves as a substrate for the cellular messengers, inositol pyrophosphates (PP-InsPs). Diphosphate-containing PP-InsPs are characterized by the presence of “high energy” phosphoanhydride bonds, with InsP₇ and InsP₈ being the most characterized species [1–8]. In yeast and metazoans, PP-InsPs serve as the critical cellular messengers controlling a large array of physiological processes including phosphate homeostasis [7,9–11], cellular energetics [12] and metabolism [13–15]. To date, the metabolic pathways leading to the production of PP-InsPs are well established in yeast, amoeba and metazoans [7,16–19]. In Saccharomyces cerevisiae, phospholipase C-dependent pathway generates Ins(1,4,5)P₃ [20]. The yeast IPMK homolog, Ipk2 phosphorylates Ins(1,4,5)P₃ sequentially at the 6-OH and 3-OH positions to generate Ins(1,3,4,5,6)P₅ that serves as a precursor for InsP₆ [21]. In agreement with this, the yeast ipk2 knockout strains lack detectable levels of InsP₆ [21,22]. Subsequently, the yeast Kcs1/ mammalian IP6K -type proteins phosphorylate the C5 position of InsP₆ and 1-InsP₇ to generate 5-InsP₇, and 1,5-InsP₈, respectively [16,23]. Furthermore, mammalian PPIP5K/yeast Vip1 kinases phosphorylate InsP₆ and 5-InsP₇ to generate 1-InsP₇ and 1,5-InsP₈, respectively [17,18,24,25].

Notably, the metabolic pathway of PP-InsP production is partially conserved in plants [8,26–29]. For instance, plants lack the canonical Kcs1/IP6K-type proteins responsible for 5-InsP₇ production found in yeast and metazoans. By contrast, Arabidopsis ITPK1 and ITPK2 that are not sequence related to yeast Kcs1 or mammalian IP6K enzymes, phosphorylate InsP₆ to generate 5-InsP₇ in vitro [29–32] and in planta [33,34]. Notably, Vip1 isoforms could be identified in all available plant genomes [35–37]. Arabidopsis Vip1 isoforms, VIH1 and VIH2 that possess 1-kinase activity, produce 1-InsP₇ and 1,5-insP₈ in vitro and contribute to InsP₈ synthesis in planta [33,35,36,38]. Thanks to these recent advances in understanding PP-InsP metabolism that we can now investigate the physiological functions of various PP-InsP species in plants. For instance, reduction in InsP₈ levels through perturbance of VIH2 function in Arabidopsis leads to the compromised immune response against insect herbivores and necrotrophic fungi [35,39]. Furthermore, VIH-derived PP-InsPs play crucial roles in regulating phosphate starvation responses [38,40–42]. Chlamydomonas Vip1 controls nutrient sensing [37]. Similarly, ITPK1 function is also implicated in phosphate homeostasis [33,43,44] and hormonal responses [34,45] in plants. Enzymes responsible for PP-InsP production are also linked to plant immunity against pathogenic bacteria in Arabidopsis [46]. Collectively, these studies reinforce that PP-InsPs are critical cellular messengers regulating various aspect of plant physiology, immunity and development.

Recent studies have reported the presence of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP₇ in Arabidopsis and M. polymorpha tissue extracts [33,34,44,47,48]. In the social amoeba Dictyostelium discoideum, 4/6-InsP₇ represents the most abundant InsP₇ species and is synthesized by the amoeba Ip6k [19]. Since, plants lack the canonical IP6K, it is unclear how 4/6-InsP₇ is produced in plant cells. Furthermore, whether 4/6-InsP₇ is specific to a certain plant lineage or is ubiquitous in land plants are yet to be explored. Consequently, physiological functions of this newly identified PP-InsP species have not been characterized.

In this study, we demonstrate that 4/6-InsP₇ is the predominant form of InsP₇ present in the studied land plant tissues. To identify the protein(s) responsible for 4/6-InsP₇ production, we performed a structural-based homology screening, where we identified Arabidopsis IPK2, a member of inositol polyphosphate multikinase (IPMK) family [21,22,49–54], as the primary hit. Using in vitro biochemical assays, we show that indeed both Arabidopsis AtIPK2α and AtIPK2β proteins phosphorylate InsP₆ to generate 4/6-InsP₇. Consistent with this finding, analyses of IPK2-deficient plants revealed that Arabidopsis IPK2 isoforms control the synthesis of 4/6-InsP₇ in planta. Furthermore, our analyses revealed that the activity of AtIPK2 proteins is critical for plant adaptation to heat stress. Our parallel investigations using the bryophyte species M. polymorpha, the first plant to conquer the land 500 million years ago [55], allow us to conclude that the functions of IPK2 in controlling cellular levels of 4/6-InsP₇ and facilitating heat stress acclimation are conserved in land plants.

Results

4/6-InsP₇ is ubiquitous in the studied land plants

To investigate whether 4/6-InsP₇ is ubiquitous across land plants, we analyzed the inositol phosphate profile of different plant species representing diverse clades of the embryophytes. To this end, inositol phosphates from the 14-day-old gametophytic thallus of Tak-1 (male plant), Tak-2 (female plant) of M. polymorpha (Liverworts; Bryophyta), mature sporophylls of Nephrolepis sp. and Dryopteris sp. (Ferns; Pteridophyta) and 14-day-old seedlings of wild-type Arabidopsis (Eudicot; Angiosperms) were extracted and analyzed using capillary electrophoresis coupled with mass spectrometry method (CE-MS) [56–58]. Our analyses establish the presence of 4/6-InsP₇ in all the plant tissue extracts used in the study (Fig 1A)_. Furthermore, the quantification of PP-InsP species (in pmol/mg) revealed that the level of 4/6-InsP₇ was significantly higher than that of other InsP₇ isomers, i.e., 5-InsP₇ and 1/3-InsP₇ detected in the plant tissue extracts (Fig 1A). Altogether, these findings revealed that 4/6-InsP₇ is the predominant InsP₇ isomer detected in the specific tissue extracts of the embryophytes used in our study. Our CE-MS measurements further identified various InsP_3-4-5 species that are conserved in land plants (S1 Fig).

Arabidopsis IPK2 proteins phosphorylate InsP₆ to generate 4/6-InsP₇ as the major PP-InsP species in vitro

We aimed to identify the protein(s) responsible for 4/6-InsP₇ production in plants (Fig 1B). Given that green plants lack canonical IP6K-type proteins as found, e.g., in mammals [16,59], we speculated that the plant genome might encode a novel InsP kinase that generates 4/6-InsP₇ from InsP₆. To test this hypothesis, we performed a homology search by Phyre² software [60] using human inositol hexakisphosphate kinase 3 (HsIP6K3) that converts InsP₆ to 5-InsP₇ [16,23], as a query sequence to identify proteins having similar structural fold albeit poor sequence homology. Notably, the Arabidopsis inositol polyphosphate kinase alpha (AtIPK2α) was found as one of the primary hits (PDB ID: 4FRF) (S2 Table and S2A Fig) belonging to the inositol polyphosphate multikinase (IPMK) family [51,53]. Our analyses with structural models of HsIP6K3 and AtIPK2α revealed overall structural similarity between these two proteins, albeit poor sequence homology (S2A Fig). The Arabidopsis genome encodes two IPMK homologs, AtIPK2α and AtIPK2β [51,53,61]. We did not find AtIPK2β as a hit by the Phyre² search likely due to the lack of published crystal structure deposited in protein data bank portal. Similar to the yeast IPK2, both Arabidopsis IPK2 proteins phosphorylate Ins(1,4,5)P₃ and Ins(1,4,5,6)P₄ to produce Ins(1,3,4,5,6)P₅ in vitro [50,51,53]. Interestingly, Arabidopsis mutant seedlings defective in AtIPK2β function display a rather similar InsP profile compared to wild-type plants [50], suggesting that AtIPK2β might have different substrate specificities in vivo and that AtIPK2α and AtIPK2β may act redundantly to control InsP homeostasis. In contrast, previous studies had reported that AtIPK2β deficiency has a distinct effect on seed InsP metabolism than seedlings [36,50,62,63], suggesting that AtIPK2β executes specialized catalytic activity in different plant tissues. Given the catalytic flexibility of IPK2 members in recognizing different substrates such as Ins(1,4,5)P₃ and Ins(1,4,5,6)P₄ [21,22,51,53], we wondered whether Arabidopsis IPK2 proteins have evolved to possess PP-InsP synthase activity. The recent identification of an IP6K member that belongs to the IPMK family, responsible for the synthesis of 4/6-InsP₇ in the social amoeba Dictyostelium discoideum [19], further encouraged us to explore the potential of AtIPK2 proteins to function as a 4/6-InsP₇ synthase in plants.

To determine whether Arabidopsis IPK2 homologs recognize InsP₆ as a substrate, we tested the binding affinities of these proteins for InsP₆ under in vitro conditions. We purified tag-free Arabidopsis IPK2 proteins by subjecting recombinant AtIPK2 proteins having an N-terminal translational fusion with His₈-MBP-TEV to TEV protease followed by affinity-based purification (S2 B and S2C Fig). Quantitative isothermal titration calorimetry (ITC)-binding assays were performed to determine dissociation constants (K_D) for Ins(1,4,5)P₃ (canonical substrate) and InsP₆ to AtIPK2. We performed control ITC assays including injection of InsP ligands into buffer and injection of buffer into enzymes (Figs 1C, S2 D and S2 E). Our ITC assays determined the dissociation constants (K_D) for Ins(1,4,5)P₃ and InsP₆ to AtIPK2α to be ~ 7 µM and ~2 µM, respectively (Fig 1D). Furthermore, our analyses revealed that Ins(1,4,5)P₃ and InsP₆ bound to AtIPK2β in vitro with the K_D of ~6 µM and ~0.5 µM, respectively (Fig 1E), suggesting that InsP₆ could be a substrate for AtIPK2 proteins.

To explore the potential of AtIPK2α and AtIPK2β in phosphorylating InsP₆ to generate InsP₇ species, we incubated InsP₆ and ATP with the purified recombinant AtIPK2 proteins S2F and S2G Fig). Incubation of recombinant AtIPK2α with InsP₆, resulted a clear band that migrates slower than InsP₆ as resolved by highly concentrated polyacrylamide gel electrophoresis (PAGE) [64] (Figs 1F and S2 H). Similarly, when InsP₆ was incubated with AtIPK2β, bands migrating slower than InsP₆ could be detected by PAGE (Fig 1G). In agreement with the previously published report [51], we found that the K_m ATP for Ins(1,4,5)P₃ of both AtIPK2 proteins to be in micromolar range, 62–150 µM (S2I Fig; left panel). Subsequent kinetic parameter analysis revealed that the K_m ATP for InsP₆ of both AtIPK2α and AtIPK2β to be 1.8 – 2.3 mM (S2I Fig; right panel), indicating that Ins(1,4,5)P₃ is a preferred in vitro substrate of AtIPK2. Intriguingly, these high K_m ATP for InsP₆ values resemble the K_m for ATP of mammalian IP6K1 [65,66] and Arabidopsis ITPK1 [29,33]. Additionally, the velocity of InsP₆ kinase reaction was surprisingly slow (S2I Fig), similar to what was previously observed for AtITPK1 proteins [29,33].

Next, the molecular identity of the reaction product was elucidated by CE-MS analyses, wherein the AtIPK2α and AtIPK2β-derived reaction products were spiked with corresponding heavy stable isotope labelled standards (SIL) [¹³C₆]1-InsP₇, [¹³C₆]5-InsP₇ [66] and [¹⁸O₂]4-InsP₇ [67]_. Our CE-MS analyses revealed that AtIPK2α and AtIPK2β-derived reaction products consist of different InsP₇ isomers, with 4/6-InsP₇ being the predominant species (Figs 1H, 1I, S2 J and S2 K). The precise enantiomeric identity of 4/6-InsP₇, i.e., 4-InsP₇ or 6-InsP₇ or a mixture of both species cannot be resolved yet using this conventional CE-MS method [68]. In addition to 4/6-InsP₇, 1/3-InsP₇ was also detected in the AtIPK2α-catalyzed reaction products (S2J Fig). Similarly, the CE-MS analyses of AtIPK2β-derived reaction products identified 4/6-InsP₇ as the major PP-InsP product (Figs 1I and S2 K). Other PP-InsP isomers, including, 5-InsP₇, 1/3-InsP₇ and 4/6,5-InsP₈ could also be detected in the reaction products (Figs 1I and S2 K). Time-course kinetic analyses further revealed that 4/6-InsP₇ is the predominant species produced by AtIPK2β in vitro (S2L Fig). Taken together, these data reflect that AtIPK2 proteins phosphorylate InsP₆ to generate different PP-InsP species in vitro, of which 4/6-InsP₇ is the predominant one.

Furthermore, to elucidate the role of the conserved PXXXDXKXG motif [51] of AtIPK2 proteins in its catalytic activity, translational fusion proteins of the catalytic dead mutants of AtIPK2α and AtIPK2β, i.e., AtIPK2α^D98A and AtIPK2α^K100A and AtIPK2β^D100A and AtIPK2β^K102A were generated (S2 F and S2G Fig). Incubation of these catalytic dead mutants with InsP₆ and ATP did not result in more polar species than InsP₆ as determined by PAGE and CE-MS (Figs 1H, S2 H, S2 J and S2 K), highlighting the importance of the key residues D98 and K100 for AtIPK2α and residues D100 and K102 for AtIPK2β in phosphorylating InsP₆. Collectively, our analyses identified a previously unreported non-canonical function of Arabidopsis IPK2 proteins as PP-InsP synthase, catalyzing the phosphorylation of InsP₆ to generate 4/6-InsP₇ in vitro.

AtIPK2α and AtIPK2β act redundantly to control the synthesis of 4/6-InsP₇ in planta

Given the ability of Arabidopsis IPK2 isoforms to recognize various InsP substrates and to generate diverse InsP and PP-InsP species in vitro, we decided to examine carefully the InsP and PP-InsP profiles of AtIPK2-deficient plants using CE-MS to clarify the specific contribution of AtIPK2 proteins in regulating InsP and PP-InsP metabolism in planta. To this end, we studied the InsP profile of Col-0 (wild-type), atipk2β-1 and atipk2α-1 single knockout lines using CE-MS. The analysis revealed that atipk2β-1 knockout plants exhibit a similar InsP profile when compared with the profile of Col-0 seedling extracts (Fig 2A). This is in agreement with the previously reported InsP profile of the same atipk2β-1 T-DNA knockout line analyzed by SAX-HPLC [50]. Similar to the atipk2β-1 mutant plants, the atipk2α-1 single knockout seedlings also did not exhibit notable differences in the InsP profile compared to Col-0 plants, with the exception of significant accumulation of an InsP₅ isomer (Fig 2A). Additionally, we did not detect significant differences in any of the PP-InsP species between wild-type and the atipk2 single knockout plants (Fig 2A). These results indicate that AtIPK2α and AtIPK2β act redundantly to control cellular InsP and PP-InsP metabolism. To corroborate further, we aimed to characterize Arabidopsis lines defective in both AtIPK2α and AtIPK2β. Notably, the atipk2βatipk2α double knockout plants are embryonic lethal [61], presenting a challenge for functional analysis of AtIPK2 homologs. To mitigate this limitation, we took an approach to generate Arabidopsis lines with compromised expression of both AtIPK2α and AtIPK2β. We aimed to generate knockdown lines with reduced transcript levels of AtIPK2α in the atipk2β-1 knockout plants. Following this strategy, we established stable lines for dexamethasone-inducible RNAi gene silencing of AtIPK2α in the atipk2β-1 knockout plants by introduction of the complete coding DNA sequence (~900 bp) of AtIPK2α into the Hellsgate12 hairpin cassette under the dexamethasone-inducible bidirectional pOp6 promoter of the pOpOFF2(Hyg) vector [69,70] (S3A Fig). Selected independent knockdown (kd) lines were confirmed by genotyping (S3B Fig) and were tested for RNAi induction upon treatment with dexamethasone by means of a β-glucuronidase (GUS) reporter gene under control of the bidirectional pOp6 promoter (S3A and S3C Fig). Furthermore, the independent atipk2βatipk2α^kd knockdown lines showed compromised stability of AtIPK2α transcripts (S3D Fig). The atipk2βatipk2α^kd knockdown lines did not exhibit any obvious growth defects compared to the wild-type plants (Fig 2B and 2C). To illustrate the contribution of AtIPK2α and AtIPK2β in inositol phosphate homeostasis, we purified global InsP species from the extracts of respective genotypes using TiO₂ beads [71], and subjected the extracts to CE-MS analysis. The atipk2βatipk2α^kd seedlings exhibited an approximate two-fold increase in InsP₄ species of unknown isomeric identity and displayed no significant differences in InsP₃ and InsP₆ levels when compared with the wild-type plants (Fig 2D). This result was further supported by our PAGE analysis wherein all the genotypes had equal levels of InsP₆ and the atipk2βatipk2α^kd seedlings showed accumulation of unknown InsP₄ and InsP₅ isomers (Fig 2E). These findings are in agreement with the previously published report that AtIPK2 proteins can phosphorylate different lower InsP species [51,53] and further suggest that Arabidopsis IPK2 activity is not required for maintaining the global pool of InsP₆ in seedlings (Fig 2D and 2E). We would like to point out here that we were not able to detect any InsP₇ isomer in the Arabidopsis seedling extracts using PAGE (Fig 2E). Our CE-MS analyses revealed that the cellular levels of 1/3-InsP₇ were not affected in the atipk2βatipk2α^kd seedlings (Fig 2D), suggesting that AtIPK2 proteins do not contribute to the production of 1/3-InsP₇ species in planta. Although 5-InsP₇ was accumulated in the atipk2βatipk2α^kd line #1, the knockdown line #2 did not show significant changes in this PP-InsP isomer, indicating that the changes in 5-InsP₇ levels of the atipk2βatipk2α^kd line #1 may not be directly associated with AtIPK2 activity. Notably, the level of 4/6-InsP₇ was significantly compromised in the independent knockdown lines compared to Col-0 plants (Fig 2D), suggesting that AtIPK2α and AtIPK2β cooperate together to regulate 4/6-InsP₇ production in planta.

Fig 2 — CE-MS analyses of inositol phosphate extracts from shoot parts of 14-day-old Col-0, *atipk2α-1* and *atipk2β-1* seedlings. Values are ± SEM (n = 4, biological replicates). Statistical significance is determined in one-way ANOVA followed by Dunnett’s test (**P < 0.05). FW denotes fresh weight. B. Representative image of 14-day-old seedling of Col-0, *atipk2*β-1, atipk2α-1 and independent *atipk2βatipk2α*^kd lines grown on solidified half-strength MS media supplemented with 25 µM dexamethasone. Images were taken using Digital Single-Lens Reflex (DSLR) camera (Canon EOS 700D). C. Representative images of 4-week-old Col-0, *atipk2*β-1, atipk2α-1 and *atipk2βatipk2α*^kd plants grown on perlite soil. Images were taken using a DSLR camera. D. AtIPK2-deficient plants exhibit deregulated levels of InsP₄ species and are compromised in 4/6-InsP₇ production. CE-MS analyses of inositol polyphosphate extracts of 2-week-old Col-0 and *atipk2βatipk2α*^kd seedlings. The InsP₅, InsP₆ and InsP₇ species were assigned by mass spectrometry and identical migration time compared with their relative standards. One InsP₄ and two InsP₃ isomers were detected. Data are means ± SEM (n = 3, biological replicates). Asterisk denotes significance determined in one-way ANOVA followed by Dunnett’s test (**P < 0.01, *** P < 0.001, ****P < 0.0001). E. AtIPK2-deficiency does not affect global pool of InsP₆ production in *Arabidopsis* seedlings. PAGE analysis of inositol phosphates extracted from shoots of 14-day-old seedlings of Col-0, *atipk2*β-1, atipk2α-1 and *atipk2βatipk2α*^kd lines grown on solidified half-strength MS media supplemented with 25 µM dexamethasone. Equal amount of plant tissues was used for the InsP extraction.

In agreement with previous observations [36,50,62,63], PAGE analyses of seed extracts confirmed that indeed seed InsP₆ levels are compromised in the atipk2β-1 lines compared to wild-type plants (S4A Fig). Notably, our analysis further unveiled that AtIPK2α-defective seeds are also compromised in InsP₆ production (S4A Fig). Furthermore, PAGE analysis suggests that the seed phytate levels of the atipk2βatipk2α^kd lines are comparable to the single atipk2 knockout plants. Taken together, these findings further highlight the distinct contribution of AtIPK2 isoforms in InsP metabolism between seed and seedlings.

Heat stress specifically targets cellular levels of 4/6-InsP₇

Given that the transcripts of AtIPK2 isoforms could be altered during heat shock [72] (S3 Table), we sought to validate the transcriptomics data using qPCR analyses. We found that the transcript levels of both AtIPK2α and AtIPK2β are upregulated in response to heat stress (S4B Fig). Next, we asked whether heat stress influences inositol phosphate profile of the atipk2βatipk2α^kd lines compared to Col-0 plants. As depicted in Fig 3A, heat stress did not alter the InsP_3-4-5-6 levels of the wild-type and the knockdown plants. Under control condition (22°C), the atipk2βatipk2α^kd lines showed 26–37% reduction in 4/6-InsP₇ level compared to the Col-0 plants (Fig 3B and 3C). Strikingly, the atipk2βatipk2α^kd lines suffered severely in 4/6-InsP₇ production with a ~ 56% reduction compared to the Col-0 plants when exposed to heat stress (37°C) (Fig 3A-3D). Notably, we did not observe significant changes in 1/3-InsP₇ levels in the AtIPK2-deficient plants during heat stress (Fig 3A), suggesting that AtIPK2 proteins do not contribute to 1/3-InsP₇ production in planta. Intriguingly, only one of the atipk2βatipk2α^kd lines exhibited significant downregulation of 5-InsP₇ levels (Fig 3A). Similar to 5 h heat treatment, the atipk2βatipk2α^kd lines displayed a robust reduction in 4/6-InsP₇ levels compared to the wild-type Col-0 plants when exposed to heat stress for 3 h (S4C Fig). Heat treatment did not affect other InsP₇ isomers in the atipk2βatipk2α^kd lines compared to Col-0 plants (S4C Fig).

A. AtIPK2-deficient plants exhibit compromised 4/6-InsP₇ production. CE-MS analyses of inositol polyphosphate levels of 2-week-old Col-0 and *atipk2βatipk2α*^kd seedlings. Seedlings were subjected to heat shock at 37⁰C for 5 h and are harvested. Inositol phosphates were extracted by TiO2 pull down and were subjected to CE-MS analyses. The InsP₅, InsP₆ and InsP₇ species were assigned by mass spectrometry and identical migration time compared with their relative standards. One InsP₄ and two InsP₃ isomers were detected. Data are means ± SEM (n = 3, biological replicates). Statistical significance is determined in two-way ANOVA followed by Tukey’s test (*P < 0.05, ****P < 0.0001). B. Quantification of 4/6-InsP₇ in % of the data presented in (A). 4/6-InsP₇ level of wild-type plants grown under control condition was set to 100%. Data are means ± SE (n = 3, biological replicates). Statistical significance is determined in two-way ANOVA followed by Tukey’s test (****P < 0.0001). Data presented in Fig 2D served as the control group of Fig 3A and 3B. C. AtIPK2α and AtIPK2β regulate 4/6-InsP₇ production *in planta*. CE-MS analysis (extracted ion electropherograms) of InsP₇ in Col-0 and *atipk2βatipk2α*^kd lines (blue trace) with spiked [¹³C₆] (black plot) and [¹⁸O₂] (purple plot) labelled InsP₇. 4/6-InsP₇ was assigned by mass spectrometry and identical migration time compared with its heavy isotopic standards ([¹⁸O₂] 4-InsP₇). D. CE-MS analysis (extracted ion electropherograms) of InsP₇ in Col-0 and *atipk2βatipk2α*^kd line after heat shock of 5 h at 37⁰C (pink trace) with spiked [¹³C₆] (black plot) and [¹⁸O₂] (purple plot) labelled InsP₇. 4/6-InsP₇ was assigned by mass spectrometry and identical migration time compared with its heavy isotopic standards ([¹⁸O₂] 4-InsP₇). Note that Fig 3C and 3D are the representative CE-MS spectra of the experimental data presented in Fig 3A and 3B.

AtIPK2α and AtIPK2β cooperate to control heat stress acclimation in Arabidopsis

To elucidate the possible role of AtIPK2α and AtIPK2β in heat stress acclimation, we exposed Col-0, both the single knockout lines and the independent atipk2βatipk2α^kd lines to high ambient temperature and monitored the adaptive responses commonly referred as thermomorphogenesis [73–75]. Specifically, 7-day-old seedlings of the above-mentioned genotypes were grown either at 22°C or 28°C (high ambient temperature) up to five days and hypocotyl length was measured after 5 days. Expectedly, Col-0 showed heat-induced hypocotyl elongation (Fig 4A and 4B). Similar to Col-0, the atipk2α-1 and atipk2β-1 knockout plants also displayed heat-induced hypocotyl elongation. However, the atipk2βatipk2α^kd lines exhibited compromised hypocotyl elongation when grown under higher ambient temperature (Fig 4A and 4B), indicating that AtIPK2 contributes to shoot adaptive response to heat stress. To further interrogate the role of AtIPK2 isoforms in plant basal thermotolerance, defined as the ability of plant to survive under high temperature (37°C), we performed basal thermotolerance assays using Col-0, atipk2β-1, atipk2α-1 and the three independent knockdown lines (Fig 4C-4E). Seedlings were initially grown at 22⁰C for 7 days in plant chamber. After this period, they were subjected to heat stress at 37⁰C for 3 days, followed by a recovery phase at 22°C for 4 days (Fig 4D). Plant survival was then assessed, defined by their ability to maintain, and generate fresh green leaves [76]. Notably, under control condition (22⁰C), the genotypes did not exhibit obvious difference in survival or germination (S5A Fig). However, the atipk2βatipk2α^kd lines were severely affected by heat stress showing poor survival rate as compared to Col-0 and the single mutant lines (Fig 4C and 4E). These data highlight the role of AtIPK2 isoforms in maintaining a plant’s basal thermotolerance. Importantly, altered hypocotyl elongation during heat stress and compromised basal thermotolerance of the atipk2βatipk2α^kd plants were largely rescued by the expression of AtIPK2β under the control of a constitutive 35S promoter (Figs 4F-4J, S5B and S5C). Consistent with the role of AtIPK2 in heat stress acclimation, the independent Arabidopsis transgenic lines expressing AtIPK2α in translational fusion with a GFP tag under the control of a constitutive 35S promoter, exhibited increased hypocotyl length compared to their isogenic wild-type plants when exposed to heat stress (Figs 4K, 4L and S5D). In conclusion, our data suggest that AtIPK2α and AtIPK2β function redundantly to regulate heat stress acclimation in Arabidopsis.

Fig 4 — A. AtIPK2α and AtIPK2β regulate hypocotyl elongation during heat stress. Representative photograph of high temperature-induced hypocotyl elongation phenotype of Col-0, *atipk2*β-1, atipk2α-1 and *atipk2βatipk2α*^kd lines. 7-day-old seedlings were exposed to 28°C for 5 days. Control plates were maintained at 22⁰C. Images were captured after 5 days of heat stress. Scale bar = 1 cm. B. Quantification of high temperature-induced hypocotyl elongation of the designated genotypes grown at 22⁰C and 28⁰C. Hypocotyl elongation was evaluated by using ImageJ. Data are means ± SEM (n ≥ 13, biological replicates). Statistical significance is determined by two-way analysis of variance (ANOVA) followed by Tukey’s test (**** P < 0.0001). Numbers on the bar represent the fold change in hypocotyl length upon heat stress compared to control condition in the respective genotypes. Dashed line represents the comparable hypocotyl length of designated genotype in control condition. C. Activity of Arabidopsis IPK2 isoforms is critical for survival during heat stress. Photograph showing basal thermotolerance phenotype of Col-0, *atipk2*β-1, atipk2α-1 and *atipk2β atipk2α*^kd lines after heat stress at 37⁰C. Surviving seedlings maintain green leaves and show emerged new leaves. D. Simplified experimental setup used for analysis of the survival phenotype. 7-day-old plants were exposed to 37⁰C for 3 days and were subjected to subsequent recovery at 22⁰C for 4 days. Control plates were maintained at 22⁰C. E. Survival rate analysis of the designated genotypes at 37⁰C. Values are means ±SEM, (n = 3, biological replicates) with each data point indicated and significant difference is determined by one-way analysis of variance (ANOVA) followed by Dunnett’s test (**** P < 0.0001). The experiment was repeated several times with independent generation. F. Expression of AtIPK2β rescues attenuated hypocotyl elongation of the *atipk2βatipk2α*^kd lines during heat stress. Representative photograph of high temperature-induced hypocotyl elongation phenotype of Col-0, *atipk2βatipk2α*^kd lines and transgenic lines expressing *AtIPK2β* in the *atipk2βatipk2α*^kd lines (Comple. lines 1/2/3). 7-day-old seedlings were exposed to 28°C for 5 days. Control plates were maintained at 22⁰C. Images were captured after 5 days of heat stress. Scale bar = 6.5 mm. G. Quantification of high temperature-induced hypocotyl elongation of the designated genotypes grown at 22⁰C and 28⁰C. Hypocotyl elongation was evaluated by using ImageJ. Data are means ± SEM (n ≥ 10, biological replicates). Statistical significance is determined in two-way analysis of variance (ANOVA) followed by Tukey’s test (**** P < 0.0001). Numbers on the bar represent the fold change in hypocotyl length upon heat stress compared to control condition in the respective genotypes. Dashed line represents the comparable hypocotyl length of designated genotype in control condition. H. Functional complementation of the AtIPK2-deficient plants compromised in heat stress acclimation by the expression of AtIPK2β. Photograph showing basal thermotolerance phenotype of Col-0, *atipk2βatipk2α*^kd lines and transgenic lines expressing *AtIPK2β* in the *atipk2βatipk2α*^kd lines after heat stress at 37⁰C. Surviving seedlings maintain green leaves and show emerged new leaves. I. Simplified experimental setup used for analysis of the survival phenotype. 7-day-old plants were exposed to 37⁰C for 3 days and were subjected to subsequent recovery at 22⁰C for 4 days. Control plates were maintained at 22⁰C. J. Survival rate analysis of the designated genotypes at 37⁰C. Values are means ±SEM, (n = 3, biological replicates) with each data point indicated and significant difference is determined by one-way analysis of variance (ANOVA) followed by Dunnett’s test (*P < 0.05, ****P < 0.0001). **K and L.** Overexpression of AtIPK2α leads to enhanced thermomorphogenetic response. Representative photograph of high temperature-induced hypocotyl elongation phenotype of Col-0 and three independent *pro35S::AtIPK2*α overexpression lines. 7-day-old seedlings were exposed to 28°C for 5 days. Control plates were maintained at 22⁰C. Images were captured after 5 days of heat stress. Scale bar = 1 cm (K). Quantification of high temperature-induced hypocotyl elongation of the designated genotypes grown at 22⁰C and 28⁰C. Hypocotyl elongation was evaluated by using ImageJ. Data are means ± SEM (n ≥ 19, biological replicates). Statistical significance is determined in two-way analysis of variance (ANOVA) followed by Tukey’s test (**** P < 0.0001) **(L)**.

AtIPK2 homologs are ubiquitous across plant kingdom and PP-InsP synthase activity of IPK2 is conserved in the liverwort M. polymorpha

Next, to explore the functional conservation of InsP kinase activity of AtIPK2 proteins in land plants, we constructed a phylogenetic tree including diverse taxa of plant kingdom such as green algae (Chlorophyta), liverworts and mosses (Bryophyta), lycopods (Pteridophyta), monocot and eudicot (Angiosperms) (S5 Table). The phylogenetic analysis allowed us to identify genes encoding IPK2-type proteins across the plant kingdom (S6A Fig). The analyses further suggest that these IPK2 homologs are derived from a single ancestral gene, with subsequent radiation in the individual lineages (S6A Fig). To understand the ancestral function of AtIPK2-type kinases in inositol phosphate homeostasis, we began to characterize the homolog of AtIPK2 in the liverwort M. polymorpha, a bryophyte whose genome sequence is available and is emerging as a model plant species to study land plant evolution (Fig 5A) [77,78]. The M. polymorpha genome encodes a single IPK2 homologue, named MpIPMK (as per nomenclature guidelines of M. polymorpha) [79] (S6A Fig).

Fig 5 — A. Schematic representation of 4/6-InsP₇ synthesis in *M. polymorpha*. B. MpIPMK is a functional yeast Ipk2 homolog. The *ipk2∆* yeast strain transformed with episomal pCA45 (*URA3*) plasmid carrying MpIPMK with N-terminal GST translation fusion were spotted in 8-fold serial dilution onto uracil-free plate and were incubated at 28ºC and 37⁰C for 3 days. *AtIPK2β* served as positive control [51,53] and empty vector served as negative control. DDY1810 yeast strain was used for the experiment. Note: DM denotes MpIPMK^D130AK132A catalytic dead variants. C. Complementation of the altered InsP profile of *ipk2∆* yeast mutant by ectopic expression of MpIPMK. HPLC profiles of extracts from [³H]-*myo*-inositol-labelled yeast transformants. Extracts were resolved by SAX-HPLC, and fractions collected each minute for subsequent determination of radioactivity as indicated. Experiments were repeated two times with similar results. D. MpIPMK phosphorylates InsP₆ *in vitro*. Recombinant His₈-MBP-MpIPMK and the catalytic dead proteins were incubated with 12.5 mM ATP, and 10 nmol InsP₆ at 37⁰C for 12 h in reaction buffer. The reaction product was separated by 33% PAGE and visualized with toluidine blue. Experiments were repeated independent times with similar results. E. Production of 4/6-InsP₇ by MpIPMK *in vitro*. Quantification of MpIPMK-derived 4/6-InsP₇ using CE-MS. Data represent means ± SEM (n = 2, replicates). Experiments were repeated two times with similar results.

The multiple sequence alignment of MpIPMK with AtIPK2α and yeast Ipk2 showed that MpIPMK possesses the conserved residues of the PXXXDXKXG catalytic motif suggesting that MpIPMK could be a functional homolog of AtIPK2 proteins (S6 B and S7A Figs). Comparison of a structural model of MpIPMK with AtIPK2α further indicates that MpIPMK could be a functional IPK2-type InsP kinase (S7 B-S7D Fig). We validated this hypothesis by taking advantage of the yeast (S. cerevisiae) ipk2∆ knockout strain and studied the consequences of heterologous expression of MpIPMK in these mutant strains. Ectopic expression of MpIPMK could restore the ipk2∆-associated growth defects [51,53] at high temperature (37⁰C) (Fig 5B). Rescue of the ipk2∆-associated growth defects could only be noticed by the ectopic expression of wild-type MpIPMK but not by the catalytic dead mutants MpIPMK^D130A, MpIPMK^K132A and MpIPMK^D130AK132A, indicating that MpIPMK possesses similar catalytic activity to yeast Ipk2 and AtIPK2α/β (Fig 5B). This conclusion was further substantiated by the HPLC analyses of the yeast ipk2∆ transformants expressing MpIPMK and MpIPMK^D130AK132A (Fig 5C). The altered InsP profile of the yeast ipk2∆ strain could be largely rescued by the ectopic expression of MpIPMK (Fig 5C). In contrast, the catalytically dead variant of MpIPMK, MpIPMK^D130AK132A failed to rescue the defective InsP profile (Fig 5C). These data allowed us to conclude that MpIPMK is a functional yeast Ipk2 homolog encoded by the M. polymorpha genome. To delineate whether MpIPMK possesses PP-InsP synthase activity similar to Arabidopsis IPK2 isoforms (Fig 5A), we incubated the translational fusion polypeptides of MpIPMK along with its catalytic dead variants (MpIPMK^D130A, MpIPMK ^K132A, MpIPMK ^D130AK132A) with InsP₆ and ATP and the reaction products were resolved by PAGE (Figs 5D and S7 E). The PAGE analyses revealed the presence of a clear kinase product when InsP₆ was incubated with the wild-type IPMK protein (Figs 5D and S7 F) demonstrating that MpIPMK phosphorylates InsP₆ in vitro. The mutant MpIPMKs failed to synthesize more polar species of InsP₆ (Fig 5D). To get further insights into MpIPMK catalytic activity, we expressed MpIPMK in different mutant yeast strains defective in PP-InsP metabolism. The expression of MpIPMK did not rescue the kcs1∆-associated growth defect (S8A Fig). Similarly, MpIPMK was not able to rescue the vip1∆- associated growth defects (S8B Fig). Collectively, these data indicate that MpIPMK is a functional yeast Ipk2 homolog that neither possess Kcs1-type nor Vip1-type catalytic activity. To further corroborate the catalytic activity of MpIPMK and to deduce the structural identity of the MpIPMK reaction product, we subjected the in vitro MpIPMK-derivatives for CE-MS analyses. Notably, the MpIPMK reaction product showed exact comigration with the [¹⁸O₂]4-InsP₇ standard, demonstrating that MpIPMK is the kinase responsible for 4/6-InsP₇ synthesis in vitro (Fig 5E). Similar to the AtIPK2 reaction products, a small amount of 1/3-InsP₇ could be detected using CE-MS analyses in the MpIPMK-derived reaction products (S8C Fig). Collectively, all these data unveil that MpIPMK is a functional IPK2-type InsP kinase that phosphorylates InsP₆ generating PP-InsP isomers in vitro.

MpIPMK contributes to 4/6-InsP₇ synthesis in planta and controls heat stress responses

To decipher the contribution of MpIPMK in InsP and PP-InsP homeostasis, independent M. polymorpha knockout lines of MpIPMK were generated using CRISPR-Cas9 technology [80,81] (Figs 6A and S9 A). These mutant plants did not show any obvious growth defects compared to the wild-type plants (S9B Fig). To assess the consequence of altered expression of MpIPMK in inositol phosphate metabolism, we monitored InsP profile of wild-type and Mpipmk knockout plants using SAX-HPLC. As depicted in Fig 6B, the MpIPMK-deficient plants displayed accumulation of unknown InsP₃ isomer compared to the wild-type plants. Similar to the IPK2-defective Arabidopsis seedlings, MpIPMK deficiency did not affect the cellular InsP₆ levels of M. polymorpha thallus (Fig 6B). Given that SAX-HPLC offers limited information about structural identity of InsP isomers present in plant extracts, we performed CE-MS analyses of wild-type and the independent Mpipmk knockout plant extracts. In congruence with the HPLC analysis, our CE-MS measurements revealed that the Mpipmk knockout plants showed increased levels of different InsP₃ species (S9C Fig). One of the Mpipmk knockout lines accumulated an InsP₄ isomer with unknown isomeric identity (S9C Fig). Notably, MpIPMK-defective plants were compromised significantly in their 4/6-InsP₇ level, suggesting that indeed MpIPMK contributes to 4/6-InsP₇ production in planta (Fig 6C). Given the role of AtIPK2α and AtIPK2β proteins in heat stress acclimation and that MpIPMK transcript level is altered after exposure to heat stress (Fig 6D), we asked whether MpIPMK is involved in regulating PP-InsP metabolism during heat stress. Unlike Arabidopsis extracts, we were able to detect InsP₇ in the M. polymorpha extracts using PAGE, and thus, we employed PAGE analyses to monitor changes in cellular InsP₇ level during heat stress (Fig 6E). We found that the cellular levels of InsP₇ were severely affected in the independent Mpipmk mutant plants, compared to the wild-type line during elevated heat stress (Fig 6E).

Fig 6 — A. Generation of Mpipmk knockout lines. Schematic representation of the two independent Mpipmk knockout lines obtained by CRISPR-Cas9 gene editing technology. Mpipmk1-2 has one insertion and one substitution of nucleotides preceding PAM sequence while Mpipmk1-7 has deletion of two nucleotides preceding PAM sequence. The mutations resulted in a premature stop codon. B. IPMK activity is not critical for maintaining InsP₆ level in *M. polymorpha* thallus. SAX-HPLC analysis of [³H]-*myo*-inositol-labelled wild-type and Mpipmk knockout plants. Neutralized extracts were resolved by SAX-HPLC and fractions collected each minute for subsequent determination of radioactivity as indicated. C. MpIPMK-deficient plants are compromised in 4/6-InsP₇ production. CE-MS analyses of 4/6-InsP₇ level in 14-day-old thalli of wild-type, Mpipmk1-2 and Mpipmk 1-7 knockout lines. 4/6-InsP₇ is presented in percentage to InsP₆. Data are means ± SEM (n = 3, biological replicates). Significant difference is determined by one-way ANOVA followed by Dunnett’s test. D. Quantitative RT-PCR (qRT-PCR) analysis of MpIPMK expression in wild-type thallus exposed to heat stress at 37⁰C for different time intervals. MpACT7 was used for normalization. Statistical significance is determined by one-way analysis of variance (ANOVA) followed by Dunnett’s test. E. MpIPMK activity is critical to maintain the major pool of InsP₇ production during heat stress. Inositol phosphates were extracted using TiO₂ beads from 14-day-old thalli of wild-type and Mpipmk knockout lines grown at 22⁰C and exposed to heat stress at 37⁰C for 3 h. InsPs were separated by 33% PAGE and visualized by toluidine blue stain. **F-G.** MpIPMK activity is critical for inducing heat stress response. Photograph showing representative thallus of the indicated genotypes grown at 22⁰C (top panel) and grown at 37⁰C for 8 h in growth chamber with subsequent recovery at 22⁰C (bottom panel) for 25 days (F). Quantification of thallus area as a measure of thermomorphogenic response (G). 5-day-old gemmalings of wild-type and Mpipmk knockout lines were subjected to heat stress in plant chamber maintained at 37⁰C for 8 h followed by subsequent recovery at 22⁰C for 13 days. Values are means ±SEM (n = 3, biological replicates) and significant difference is determined by one-way analysis of variance (ANOVA) followed by Tukey’s test (****P < 0.0001). Number on the bar depicts the fold changes in the thallus area upon heat stress compared to control condition.

To investigate the consequences of loss of MpIPMK activity in thermomorphogenesis, we monitored the response of wild-type and the two independent Mpipmk mutant plants after exposing them to the elevated temperature, 37⁰C for 8 h. In line with previous reports [82,83], heat treatment resulted in increased thallus area of the wild-type plants (Fig 6F and 6G). In contrast, the Mpipmk mutants displayed severe reduction in thallus area compared to the wild-type plants (Fig 6F and 6G). Taken together, these data show that MpIPMK critically contributes to plant resilience to heat stress.

M. polymorpha IPMK rescues the altered heat stress tolerance of Arabidopsis IPK2-defective plants

To investigate the functional conservation of IPK2 proteins between M. polymorpha and Arabidopsis, we generated the atipk2βatipk2α^kd transgenic lines expressing MpIPMK under the control of a constitutive 35S promoter (S10 A and S10B Fig) and performed shoot thermomorphogenesis assays. Remarkably, heterologous expression of MpIPMK rescued the attenuated heat-induced hypocotyl elongation of the atipk2βatipk2α^kd plants (Fig 7A and 7B). Similarly, the expression of MpIPMK largely rescued the compromised survival rate of atipk2β atipk2α^kd plants during heat stress (Fig 7C). Altogether, our findings highlight the functional conservation of IPK2 proteins between liverworts and angiosperms.

Fig 7 — A. Heterologous expression of MpIPMK rescues attenuated hypocotyl elongation of the *atipk2βatipk2α*^kd lines during heat stress. Representative photograph of high temperature-induced hypocotyl elongation phenotype of the indicated genotypes grown at 22⁰C and 28⁰C. 7-day-old seedlings were exposed to 28°C for 5 days. Control plates were maintained at 22⁰C. Images were captured after 5 days of heat stress. Scale bar = 6 mm. B. Quantification of elongated hypocotyls. Data are means ± SEM (n ≥ 11, biological replicates). Statistical significance determined by two-way analysis of variance (ANOVA) followed by Tukey’s test (****P < 0.0001). Numbers on the bar represents the fold change in hypocotyl length of the designated genotypes. Dashed line depicts the comparable hypocotyl length of the studied genotypes. C. Heterologous expression of MpIPMK enhances the basal thermotolerance of *atipk2βatipk2α*^kd lines during heat stress. Quantification of survival rate of the designated genotypes after heat stress at 37⁰C. Surviving seedlings maintain green leaves and show emerged new leaves. Values are means ±SEM (n = 3, biological replicates) with each data point indicated and significant difference is determined by one-way analysis of variance (ANOVA) followed by Dunnett’s test (***P < 0.001).

IPK2 promotes the transcriptional activity of HSF through both catalytic-dependent and catalytic-independent mechanisms

To gain possible mechanistic insights about IPK2-controlled heat stress acclimation, we first tested whether genes encoding members of HSP families, PIF, and genes involved in auxin signalling pathway that play role in heat stress acclimation [76,84–88], are differentially expressed in the Arabidopsis AtIPK2 knockdown lines. Our analyses suggest that the expression of different HSPs including HSP70, HSP22, HSP17.6, and HSP18.1 is largely compromised in the atipk2βatipk2α^kd lines when compared to Col-0 plants during heat stress (Fig 8A). Furthermore, the atipk2βatipk2α^kd lines showed deregulated expression of heat stress responsive genes involved in auxin signaling pathways, e.g., IAA19 and YUC8 (S10C Fig). Considering that the atipk2βatipk2α^kd lines suffered with the compromised expression of genes that are regulated by different heat shock transcription factors (HSF-TFs) [87,89,90] and that IPMK/IPK2 is already implicated in transcriptional regulation in various eukaryotes [21,91–94], we asked whether IPK2 controls HSF activity in planta. To this end, we performed transient transcription assay using dual-luciferase (LUC) reporter plasmid in N. benthamiana leaves. The plasmid encodes a Renilla luciferase gene driven by the constitutive 35S promoter, and a firefly LUC gene driven by the AtHSP18.1 promoter. Our analyses indicate that the presence of AtIPK2 significantly enhanced the transcriptional activity of AtHSFA1b (Fig 8B). In contrast, the catalytic dead variant of AtIPK2α did not augment the activity of AtHSFA1b (Fig 8B). Collectively, these results suggest that the catalytic activity of Arabidopsis IPK2α enhances the transcriptional activity of AtHSFA1b, critical for plant adaptation to heat stress.

Fig 8 — A. Quantitative RT-PCR (qRT-PCR) analysis of different *HSP*s in Col-0 and *atipk2βatipk2α*^kd lines after heat shock. 14-day-old seedlings were exposed to 37⁰C for 3 h and were harvested for qRT-PCR analysis. *PP2AA3* was used as a reference gene. Values are means ± SEM (n = 3, biological replicates). Statistical significance is determined by two-way ANOVA followed by Tukey’s test (*P < 0.05, ****P < 0.0001). B. AtIPK2α potentiates transcription activity of heat shock transcription factor, AtHSFA1b *in planta*. Schematic diagrams of luciferase reporter and effector constructs used in transient transactivation assays in *Nicotiana benthamiana* leaves (left panel). Statistical analysis of the expression of *pHSP18.1-LUC* in presence of AtHSFA1b and AtIPK2α (right panel). YFP served as control. Values are means ±SEM (n = 3, biological replicates). Different letters indicate significance in one-way analysis of variance (ANOVA) followed by Tukey’s test (a and b, P < 0.0001; b and c, P < 0.0001; a and c, P < 0.0001).

Given that the catalytic-independent activity of IPMK/IPK2 has already been implicated in transcriptional regulation across various eukaryotes [21,91–95], we investigated whether IPMK directly regulates HSF activity. To elucidate the role of IPMK-type proteins in transcriptional regulation, we first assessed whether these proteins physically associate with heat shock factors (HSFs) and subsequently influence their DNA-binding activity. Using yeast two hybrid (Y2H) assay and Bimolecular Fluorescence Complementation (BiFC) assay, we show that AtIPK2β physically interacts with AtHSAF1b (Fig 9A and 9B). Next, to investigate the consequence of this physical interaction, we performed an electrophoretic mobility shift assay (EMSA). The in vitro DNA binding study was performed using a short double nucleotide fragment containing the heat shock promoter element (HSE) of AtHSP18.1. In agreement with the previous report [96], we found that AtHSFA1b binds specifically to the HSE element (Fig 9C, 9D and 9E). Notably, when AtIPK2β was incubated with AtHSFA1b, a strong shift of the DNA occurred in a dose-dependent manner compared to incubation with AtHSFA1b alone, suggesting that AtIPK2β modulates the DNA-binding activity of HSFA1b (Fig 9F). Notably, the DNA-binding activity of HSFA1b was not influenced by the presence of MBP (Fig 9G). Importantly, AtIPK2β and MBP alone were unable to bind HSE element, suggesting that the enhancement in promoter element binding activity of HSFs in presence of AtIPK2β is not due to direct binding of AtIPK2β proteins with the promoter element (S11A and S11B Fig). Notably, incubation of AtHSFA1b with Ins(1,4,5)P₃, InsP₆, and 4-InsP₇ didn’t affect the DNA binding activity of AtHSFA1b (S11C Fig). Similar to Arabidopsis IPK2 proteins, we found that Marchantia IPMK physically interacts with MpHSFB1 (S12A and 12B Fig). Our EMSA analyses revealed that similar to AtHSFA1b, MpHSFB1 shows specific binding to HSE element (Fig 9H-9J). Intriguingly, MpIPMK also potentiates the DNA binding activity of MpHSFB1 in a dose-dependent manner (Fig 9K), whereas MBP fails to elicit a similar effect (Fig 9L). Moreover, MpIPMK alone did not show binding to the promoter element (S12C Fig). Additionally, the tested InsP species could not influence the DNA binding activity of MpHSFB1 in vitro (S12D Fig). Collectively, these findings highlight the crucial catalytic-independent function of IPK2 in controlling the DNA-binding activity of HSF-type transcription factors.

Fig 9 — A. AtIPK2β exhibits physical interaction with AtHSFA1b *in vivo.* AH109 yeast strain carrying the pGADT7-AtIPK2β and pGBKT7-AtHSFA1b plasmids were spotted on selective media lacking leucine (Leu, L), tryptophan (Trp, W), histidine (His, H), adenine (Ade, A) and indicated amount of 3-AT. B. BiFC experiment showing physical interaction of IPK2β and AtHSFA1b in *N. benthamiana* leaves. Empty vectors (pMDC_nVenus/ pMDC_CFP) and different combinations of empty vectors and cloned constructs of *AtHSFA1b* and *AtIPK2β* served as negative control. A-D denotes different *A. tumefaciens* transformants harbouring combination of vectors co-infiltrated in *N. benthamiana* leaves where A = cCFP empty + nVENUS empty, B = cCFP_AtIPK2β + nVENUS_empty, C = cCFP_empty + nVENUS_AtHSFA1b, D = cCFP_AtIPK2β + nVENUS_AtHSFA1b. C. An electrophoretic mobility assay (EMSA) showing AtHSFA1b binds to FAM-labelled *HSP18.1* promoter element having canonical HSE element (GAAnnTTC) in a dose-dependent manner. 250 nM of the FAM-labelled probe was incubated with recombinant MBP-AtHSFA1b (25, 50, 100 and 200 nM) for 15 mins on ice and resolved on 6% of native PAGE. D. EMSA showing AtHSFA1b doesn’t bind to probe lacking HSE element. 250 nM of the mutant probe (HSE∆) was incubated with recombinant MBP-AtHSFA1b (50, 100 and 200 nM) for 15 mins on ice and resolved on 6% of native PAGE. E. EMSA showing competition between the FAM-labelled and unlabelled probe for AtHSFA1b binding. 250 nM of both FAM-labelled and different concentration of unlabelled probe (50, 100, 250 nM, 500 nM), was incubated with 50 nM of AtHSFA1b for 15 mins on ice and resolved using 6% native PAGE. F. EMSA showing AtIPK2β enhances DNA binding activity of AtHSFA1b in a dose-dependent manner. Recombinant MBP-AtIPK2β (25, 50, 100, 200 and 500 nM) was incubated with 50 nM of recombinant MBP-AtHSFA1b for 30 mins followed by post incubated with 250 nM of FAM-labelled probe for 15 mins on ice. The complexes were resolved using 6% PAGE. G. EMSA showing negative control MBP cannot enhance DNA-binding activity of MpHSFB1 in a dose-dependent manner. Recombinant His8-MBP (25, 50, 100 and 250 nM) was incubated with recombinant MBP-AtHSFA1b (50 nM) for 30 mins on ice followed by post incubation with 250 nM of FAM-labelled probe for 15 mins on ice. The complexes were resolved using 6% native PAGE. H. An electrophoretic mobility assay (EMSA) showing MpHSFB1 binds to FAM-labelled *Heat shock element (HSE)* in a dose-dependent manner. 250 nM of the FAM-labelled probe was incubated with recombinant MBP-MpHSFB1 (25, 50, 100 and 250 nM) for 15 mins on ice and resolved on 6% of native PAGE. I. EMSA showing MpHSFB1 doesn’t bind to probe lacking HSE element. 250 nM of the mutant probe (HSE∆) was incubated with recombinant MBP-MpHSFB1 (50 and 100 nM). J. EMSA showing competition between the FAM-labelled and unlabelled probe for MpHSFB1 binding. 250 nM of both FAM-labelled and unlabelled probe (50, 100, 250, 500 nM) was incubated with 50 nM of MpHSFB1 for 15 mins on ice and resolved using 6% native PAGE. K. EMSA showing MpIPMK enhances DNA binding activity of MpHSFB1 in a dose-dependent manner. Recombinant MBP-MpIPMK (50, 100, 250 and 500 nM) was incubated with 50 nM of recombinant MBP-MpHSFB1 for 30 mins followed by post incubated with 250 nM of FAM-labelled probe for 15 mins on ice. The complexes were resolved using 6% PAGE. L. EMSA showing negative control MBP cannot enhance DNA-binding activity of MpHSFB1. Recombinant His8-MBP (50, 100, 250 and 500 nM) was incubated with recombinant MBP-MpHSFB1 (50 nM) for 30 mins on ice followed by post incubation with 250 nM of FAM-labelled probe for 15 mins on ice. The complexes were resolved using 6% native PAGE.

Discussion

PP-InsPs serve as cellular messengers that control a wide-range of physiological processes in eukaryotes. Using CE-MS and nuclear magnetic resonance (NMR) spectroscopy, several PP-InsP species have been identified in Arabidopsis extracts [30,33,34,56]. In this study, we report that 4/6-InsP₇ is the predominant PP-InsP species present in the studied land plant tissue extracts. In future studies, we will monitor the distribution of PP-InsP species in different plant tissues, different stages of plant development and do so across diverse plant species. Currently, we are unable to determine whether it is 4-InsP₇ or 6-InsP₇, i.e., which enantiomer is produced by IPK2 preferentially. Although these are interesting questions to be addressed, the major roadblocks to answer them are: i) conventional CE-MS cannot differentiate between enantiomers, ii) although NMR spectroscopy in presence of a chiral solvating agent could be employed to illustrate the enantiomeric identity of 4/6-InsP₇ as described previously to identify the product of a bacterial effector protein [97], 4/6-InsP₇ purified from Arabidopsis seedling extracts is not adequate for such NMR analysis. Structural elucidation of a plant-derived InsP₇ species using NMR was reported for atmrp5 mutant seeds [30], that are defective in vacuolar InsP₆ loading, consequently, cyto/-nucleoplasmic levels of InsP₆-derived PP-InsP species are augmented in the AtMRP5-deficient plants [36,98,99]. Further investigations are required to optimize purification of 4/6-InsP₇ from plant extracts for the structural determination of plant-derived PP-InsP species by NMR.

In search for putative kinases responsible for 4/6-InsP₇ production in plants, we found AtIPK2α as one of the primary candidates through a structure-based homology screen. Previous studies had reported that similar to yeast Ipk2, AtIPK2 proteins phosphorylate Ins(1,4,5)P₃ at 6-OH and 3-OH positions, respectively to generate Ins(1,3,4,5,6)P₅ in vitro [21,22,51,53]. However, contribution of AtIPK2 homologs in inositol phosphate homeostasis in planta remained largely obscure, mostly due to their redundancy. Additionally, the role of AtIPK2 as a PP-InsP synthase was not established previously. We show that AtIPK2α and AtIPK2β bind to InsP₆ with strong affinity, suggesting that InsP₆ could be a substrate of AtIPK2. Our kinetics analysis revealed that Km ATP for InsP₆ of AtIPK2 is somewhat comparable to that of mammalian IP6K and Arabidopsis ITPK1, reinstating that InsP₆ could be a possible physiological substrate of AtIPK2 enzymes. Consistent with the role of AtIPK2 as a PP-InsP synthase, we show that Arabidopsis IPK2 proteins can phosphorylate InsP₆ to generate 4/6-InsP₇ in vitro.

Do AtIPK2α and AtIPK2β play a role in InsP and PP-InsP metabolism in planta? To explore this, we analyzed the individual atipk2α-1 and atipk2β-1 knockout plants and observed a similar profile when compared to Col-0 seedlings. Notably, the atipk2α-1 plants exhibited elevated levels of an InsP₅ isomer while InsP₆ and PP-InsP isomers remained unchanged. To mitigate possible functional redundancy between AtIPK2α and AtIPK2β, we generated Arabidopsis transgenic lines with reduced transcript of AtIPK2α in the atipk2β-1 knockout plant background. Our CE-MS analyses of knockdown lines revealed that certain InsP₄ and InsP₅ isomers are deregulated in the atipk2βatipk2α^kd plants, yet InsP₆ levels remained unaffected in the AtIPK2-deficient plants. Collectively, these data suggest that while AtIPK2 regulates lower inositol phosphate metabolism, and that deregulation of InsP_4-5 metabolism does not affect the global pool of InsP₆ in seedlings. This differs from yeast and mammalian systems where IPK2/IPMK activity is critical for maintaining cellular levels of InsP₆ [21,22,49]. The consequence of altered InsP₄ and InsP₅ isomers in the atipk2βatipk2α^kd seedlings is yet to be understood. In agreement with previous reports [36,50,62,63], we found that seed phytate level is controlled by both AtIPK2α and AtIPK2β highlighting specialized functions of the AtIPK2 members in different plant parts. CE-MS analyses revealed a significant decrease in 4/6-InsP₇ in the Arabidopsis IPK2-deficient seedlings, while the 5-InsP₇ and 1/3-InsP₇ isomers remained unaffected. This suggests that IPK2 specifically regulates the cellular level of 4/6-InsP₇. It remains to be determined whether Arabidopsis IPK2 can generate a PP-InsP isomer using monophosphate-containing InsPs other than InsP₆ as substrates. Future work awaits to clarify whether IPK2 can produce 4/6-InsP₇ in an InsP₆-independent manner using InsP_3/4/5 as substrates in planta. Since the atipk2βatipk2α double knockout plants are embryonic lethal and that the atipk2βatipk2α^kd plants may retain residual AtIPK2 activity, it is yet to be explored whether the Arabidopsis genome encodes protein(s) other than AtIPK2 homologs that contribute to the remaining pool of 4/6-InsP₇ present in the knockdown lines. In agreement with the role of AtIPK2 in heat stress acclimation, the atipk2βatipk2α^kd plants displayed compromised shoot thermomorphogenesis. Furthermore, basal thermotolerance of the knockdown lines is severely affected. Notably, the altered heat stress acclimation of the IPK2-deficient plants could be reversed by ectopic expression of AtIPK2β under the control of a constitutive promoter.

Our parallel investigation using M. polymorpha allowed us to conclude that IPK2-dependent 4/6-InsP₇ production is an ancestral function, and that MpIPMK contributes to 4/6-InsP₇ production in planta. Future work awaits to clarify whether the M. polymorpha genome encodes proteins other than MpIPMK to control 4/6-InsP₇ synthesis. As discussed above, M. polymorpha might possess both 4-InsP₇ and 6-InsP₇ in a different ratio compared to Arabidopsis yet, it is also conceivable and likely that IPMK/IPK2 catalyze the synthesis of only one of them with high enantioselectivity. These are unresolved questions that require further investigation. Despite the accumulation of certain InsP₃, InsP₄ and InsP₅ species in both Arabidopsis and M. polymorpha IPK2/IPMK-deficient lines, the unaltered InsP₆ level suggests that the archetypal activity of IPK2 in producing InsP₆ (also known as phytic acid) in yeast is not conserved in the vegetative tissues of land plants. Our data also indicates that plant IPK2 evolved a distinct PP-InsP synthase activity during their divergence from the fungal lineage.

The ectopic expression of MpIPMK fully rescued the altered thermomorphogenic responses of the atipk2βatipk2α^kd plants, suggesting that Arabidopsis and M. polymorpha share functional IPK2 homologs. Moreover, our study sheds light on mechanistic insights into the regulation of heat stress acclimation by IPK2. Specifically, AtIPK2α augments DNA-binding and transcription activity of AtHSFA1b. In contrast, the catalytic dead variant of AtIPK2α does not potentiate the transcription activity of AtHSFA1b in planta, highlighting role AtIPK2-derived InsP and PP-InsP species in heat stress acclimation. Future work awaits to clarify which of the AtIPK2-dependent inositol phosphates regulate the transcription activity of AtHSFA1b. Although the reduction of 4/6-InsP₇ during heat stress points towards a possible function of this PP-InsP isomer in heat stress acclimation, future studies need to address whether reduction of 4/6-InsP₇ is specific to certain duration of heat exposure and the type of heat exposure (i.e., 28⁰C vs. 37⁰C). Notably, our findings also suggest that IPK2 can augment DNA binding activity of HSFs in vitro, highlighting the importance of catalytic-independent activity of IPK2 in heat stress acclimation. Further investigation is required to dissect the contribution of catalytic vs. non-catalytic activity of IPK2 proteins during heat stress.

In conclusion, our study offers a mechanistic framework to understand the conserved role of IPK2-derived InsP and PP-InsP species in regulating various cellular processes, and how this contributes to land plant evolution.

Methods

Phylogenetic analysis

The phylogenetic tree was constructed as described previously [35]. BLAST search analyses (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) were performed using the full-length sequence of AtIPK2α retrieved from A. thaliana genome database (https://www.arabidopsis.org/). The retrieved sequences were filtered to include only those sequences with a percent identity of more than 30%, query cover of more than 35%, E-value of less than 10^-5 and a bit score of more than 80. Sequences from every species were screened for the presence of gene isoforms. The GUIDANCE2 server (http://guidance.tau.ac.il/) was used to align the sequences using the version of MAFFT available on the server. The GUIDANCE2 algorithm was used to estimate the reliability of alignment columns. Unreliable columns having a confidence score lesser than 0.93 were removed from the multiple sequence alignment (GUIDANCE Server - a web server for assessing alignment confidence score (tau.ac.il). Phylogenetic analysis was conducted in MEGA11 [100]. The best maximum likelihood model for the given alignment was estimated using the default settings and this model was used to estimate a maximum likelihood tree. Alignment of complete protein sequences were done using ClustalW and phylogenetic tree was constructed with the Maximum Likelihood method, using the Dayhoff’s Model and a bootstrap test of 1000 replicates. Values less than 50% are not displayed on the tree and branch lengths are given in terms of the expected number of amino acid substitutions per site.

Plant materials and growth conditions

Seeds of Arabidopsis thaliana T-DNA insertional mutant of ipk2β-1 (SALK_104995) was genotyped for homozygous T-DNA insertions using primer S1 Table. Surface sterilization of seeds was performed by incubating the seeds in solution containing 0.05% SDS in 70% ethanol for 15 min. Sterilized seeds were sown onto the solidified half-strength Murashige and Skoog (MS) media containing 0.8% agar (w/v). After stratification for 3 days at 4 °C, plates were transferred in a Percival plant chamber under conditions of 16 h light and 8 h dark at 22 °C with light intensity 100 μmol/m²/s. The germinated seedlings were transferred onto soil (perlite and soilrite in the ratio of 1:2) and maintained in growth room. The growth room condition was maintained at 22°C with 70% RH and long-day (LD) conditions (16 h:8 h; light: dark cycle) with light intensity 100 μmol/m²/s.

Marchantia polymorpha accession Takaragaike-1 (Tak-1, male accession) and Takaragaike-2 (Tak-2, female accession) were used as wild-type plants. M. polymorpha lines were propagated asexually by gemma cultured on half-strength Gamborg’s B5 medium with 1% phytagel under long-day (LD) conditions (16 h:8 h; light: dark cycle) with light intensity 50–60 µmol/m²/s in a Percival growth chamber at 22⁰C.

Mature sporophylls of Nephrolepis sp. and Dryopteris sp. were collected from IISc campus.

Molecular cloning

Cloning of AtIPK2 and MpIPMK in pET28b-His₈-MBP bacterial and yeast expression vector.

Full-length coding sequence of AtIPK2α, AtIPK2β and MpIPMK were amplified using cDNA prepared from total RNA extracts of Col-0 and Tak-1 plants, respectively with primers listed in S1 Table. The amplified products were cloned at the EcoRV site in pBLUESCRIPT via blunt end cloning followed by directional subcloning into pET28b between the BamHI and Not1 sites. PCR mutagenesis was used to generate a point mutation in the conserved PXXXDXKXG motif of AtIPK2α, AtIPK2β and MpIPMK. Primer used to generate site directed mutagens are enlisted in S1 Table. For cloning MpIPMK wild-type and catalytic dead variants in yeast expression vector pCA45, the amplified product of MpIPMK coding DNA sequence (CDS) from Tak-1 plants was subcloned with pBLUESCRIPT followed by directional cloning in pCA45 vector pre-digested with the BamH1 and Not1.

Protein expression and purification

To purify recombinant AtIPK2α and AtIPK2β proteins, E. coli BL21 (RIL) strains were transformed with the pET28b-His₈-MBP-AtIPK2α and pET28b-His₈-MBP-AtIPK2β vectors. A single colony of transformants carrying the respective constructs was used to inoculate in terrific broth (TB medium). After induction with 0.5 mM IPTG, the culture was allowed to incubate further for 3 days at 12⁰C. Cells were harvested at 6000 rpm for 10 min at 4⁰C and pellet was washed with lysis buffer (300 mM NaCl, 25 mM Na₂HPO₄, pH 7.5). The pellets were resuspended in lysis buffer containing 5 mM β-ME and 1 mM PMSF, followed by cell lysis using sonication at 10 pulse of 30 sec ‘ON’ and 10 sec ‘OFF’. After sonication, the lysates were centrifuged at high speed 18000 rpm for 45 min at 4⁰C. Meanwhile Ni-NTA resin (Qiagen) was prepared by washing with ultra-pure water and the equilibrated with lysis buffer twice. After centrifugation, the cleared protein supernatant was incubated with prewashed Ni-NTA beads for 6 h on rotor at 4⁰C. Next, the beads were washed thrice with wash buffer (lysis buffer, 5 mM β-ME, 10 mM imidazole) at 4000 rpm for 5 min at 4⁰C. For elution, the beads were incubated with 250 µL elution buffer (lysis buffer containing 5 mM β-ME, 250 mM imidazole) at 4⁰C for 5 min on a rotor and centrifuged at 4000 rpm for 5 mins. Three such elutions were collected. Aliquots of elution and different concentration of BSA standards were heated at 95⁰C after adding 1X SDS loading dye and loaded on 12% SDS-PAGE. Protein was visualized by coomassie staining and quantification of the band intensity was done using ImageJ. The mutant variants of AtIPK2α and AtIPK2β purification were performed as mentioned above. Recombinant MpIPMK protein and its catalytic dead variants were purified using Phosphate Buffer Saline (PBS) buffer [137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ (pH 7.4)].

Isothermal titration calorimetry (ITC)

For ITC, tag-less AtIPK2 proteins were generated by subjecting the purified His₈-MBP-TEV-AtIPK2α/β recombinant proteins to TEV protease dialysed in 50 mM Tris Cl pH 8, 0.5 mM EDTA followed by binding with Ni-NTA and amylose resins. All ITC experiments were performed at 25 °C using a MicroCal PEAQ-ITC system (Malvern Panalytical) equipped with a 280 μL sample cell and a 40 μL injection syringe. All proteins were dialyzed against buffer (50 mM Tris Cl pH 8.0 and 0.5 mM EDTA); Ins(1,4,5)P₃ and InsP₆ ligands were diluted in same buffer prior to all measurements. A typical titration consisted of 19 injections, the protein concentrations in the syringe and in the cell are provided in the respective figure legend. Data were analyzed using the MicroCal PEAQ-ITC analysis software (v1.21). InsP₃ and InsP₆ were obtained from SiChem GmbH.

In vitro kinase assay

The InsP₆ kinase assays were performed by incubating 0.16 µg µL^-1 of recombinant AtIPK2α and AtIPK2β and their catalytic dead variants in a 30 µL of reaction volume containing kinase buffer [5 mM MgCl₂, 20 mM HEPES (pH 7.5), 1 mM DTT], 12.5 mM ATP, and 10 nmol InsP₆ at 37⁰C for 12 h. MBP protein was used as negative control. The in vitro kinase assay of MpIPMK and its catalytic dead variants was performed using the above-mentioned reaction condition. The reaction products were resolved using PAGE and visualized by toluidine blue staining [64].

For Km and Vmax calculations of ATP, 0.06 µg µL^-1 of AtIPK2β and AtIPK2α was incubated with InsP₆ and assayed with 2–12 mM ATP for 10 h. For time course experiment AtIPK2β was incubated with 12.5 mM ATP, and 10 nmol InsP₆ at 37⁰C for different time intervals. The reaction products were analysed using CE-MS.

Enrichment of inositol phosphate using titanium dioxide bead

Inositol phosphate pull down was performed as described previously [71]. All steps were carried on ice. TiO₂ beads were weighed to 10–12 mg for each sample and washed twice in water and once in 1 M perchloric acid (PA). Liquid N₂-frozen plant material (approx. ~ 100 mg) was homogenized using a pestle and immediately resuspended in 800 µL ice-cold PA. Samples were kept on ice for 5 min with short intermediate vortexing and then centrifuged for 5 min at 20000 g at 4⁰C. The supernatants were transferred into fresh 1.5 mL tubes and centrifuged again for 5 min at 20000 g. The supernatants were resuspended in the prewashed TiO₂ beads and incubated at 4⁰C for 30 min. After incubation, the beads were pelleted by centrifuging at 8000 g for 1 min and washed twice in PA. The supernatants were discarded. To elute inositol polyphosphates, beads were resuspended in 200 µL of 10% ammonium hydroxide and then incubated for 5 min at room temperature. After centrifuging at 2600 g, the supernatants were transferred into fresh tubes. The elution process was repeated, and the second supernatants were pooled as well. Eluted samples were vacuum evaporated at room temperature. InsPs were resuspended in 40 µL ultra-pure water for the following CE-MS analysis.

CE-MS analysis

The analysis was performed on a CE-QQQ system (Agilent 7100 CE-with Agilent 6495C Triple Quadrupole and Agilent Jet Stream electrospray ionization source, adopting an Agilent CE-ESI-MS interface). An isocratic Agilent 1200 LC pump was used to deliver the sheath liquid (50% isopropanol in water) with a final splitting flow speed of 10 µL/min via a splitter. All separation was performed via a bare fused silica capillary with a length of 100 cm (50 µm internal diameter and 365 µm outer diameter). 40 mM ammonium acetate titrated with ammonium hydroxide to pH 9.0 was used as background electrolyte (BGE). Between runs of each sample, the capillary was flushed with BGE for 400s. Samples were injected by applying 100 mbar pressure for 15s (30 nL). The MS source parameters were as follows: gas temperature was 150˚C, gas flow was 11 L/min, nebulizer pressure was 8 psi, sheath gas temperature was 175˚C and with a flow at 8 L/min, the capillary voltage was -2000V, the nozzle voltage was 2000V. Negative high-pressure RF and negative low-pressure RF were 70 V and 40 V, respectively. Multiple reaction monitoring (MRM) transitions were setting as shown in S6 Table.

Internal standard (IS) stock solution of 8 µM [¹³C₆] 2-OH InsP₅, 40 µM [¹³C₆] InsP₆, 2 µM [¹³C₆] 1-InsP₇, 2 µM [¹³C₆] 5-InsP₇, 1 µM [¹⁸O₂] 4-InsP₇ (only for assignment of 4/6-InsP₇) and 2 µM [¹³C₆] 1,5-InsP₈ were spiked to samples for the assignment of isomers and quantification of InsPs and PP-InsPs. 5 µL of the IS stock solution was mixed into 5 µL samples. Quantification of InsP₈, 5-InsP₇, 1-InsP₇, InsP₆, and InsP₅ was performed with known amounts of corresponding heavy isotopic references spiked into the samples. Quantification of 4/6-InsP₇ was performed with [¹³C₆] 5-InsP₇ and Quantification of InsP₃ and InsP₄ of which no isotopic standards are available was performed with spiked [¹³C₆] InsP₆. After spiking, 4 μM [¹³C₆] 2-OH InsP₅, 20 μM [¹³C₆] InsP₆, 1 μM [¹³C₆] 5-InsP₇, 1 μM [¹³C₆] 1-InsP₇, and 1 µM [¹³C₆] 1,5-InsP₈ were the final concentration inside samples. All [¹³C₆] inositol references [66] were kindly provided by Dorothea Fiedler.

Yeast strains and transformation

The yeast strains were grown on YPD agar plates at 28⁰C. Transformed yeast strains were grown in complete minimal medium containing the appropriate amino acids, 2% glucose, and lacking uracil to maintain selection for URA3 plasmids at 28⁰C. Yeast transformations were performed by the lithium acetate method [101]. Briefly, single colony of streaked yeast strain was inoculated in 5 mL of YPD liquid medium and incubated at 28⁰C in shaker incubator for overnight. Fresh 4 mL YPD liquid media was inoculated with overnight grown culture to reach OD_600nm~ 0.6. And culture was allowed to grow for 4 h in shaker incubator. Once the culture reached OD ~ 1, the culture was harvested at 2600 rpm for 1 min at room temperature. The pellets were washed twice with 500 µL of TE/LiAc buffer at 2600 rpm for 2 min. After final wash, the cells were resuspended with 200 µL of TE/LiAc buffer and kept on ice. These yeast competent cells were used for transformation. A total of 3.5 µL of salmon sperm DNA (approx. 8 mg/mL) was heated at 95⁰C for 5 min and kept on ice for 2 min. 1 µL of plasmid (200 – 500 ng plasmid) was added to the salmon sperm DNA followed by adding 16.5 µL of yeast competent cells. The cells with plasmid were resuspended with PEG/LiAc buffer and incubated for 40 min at room temperature on rotor. After incubation, the cells were subjected to heat shock at 42⁰C for 20 min. 70 µL of the cell suspension was used for plating on selection plate. The plates were incubated at 28⁰C. Complementation assay was performed by dropping 8-fold serially diluted resuspension of transformed colony of yeast on selection and screening plate. S4 Table contains the list of yeast strains used in this study.

RNA extraction, cDNA synthesis and gene expression analyses

RNA extraction and cDNA synthesis was done as described previously [45]. Briefly, 100 mg of plant tissue was used for RNA extraction with TRIzol (Sigma Aldrich) reagent, followed by DNase treatment with DNase I (NEB, M0303). A total of 2–3 µg of RNA was used for cDNA synthesis using PhiScript cDNA Synthesis Kit (dx/dt). The qPCR was performed using the DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo-scientific) with CFX96 Touch Real-Time PCR Detection System (Bio-Rad Hercules, CA, USA) according to the manufacturer’s protocol (Bio-Rad). Relative expression was calculated according to relative quantitation method (ΔΔCT). PP2AA3 and MpACT7 were used as reference genes for the qPCR analysis in Arabidopsis and M. polymorpha, respectively. The primers used for qPCR analyses are detailed in S1 Table.

Extraction and HPLC analysis of inositol phosphates

Inositol polyphosphates were extracted from yeast and analyzed as described [35,102,103]. Yeast transformants were grown to midlog phase in minimal media, labelled in 2 mL of minimal media supplemented with 6 μCi/mL [³H]-myo-inositol (18 Ci mmol−¹; PerkinElmer). The cells were harvested, washed twice with ultra-pure water were extracted in 1 M perchloric acid extracted. For labelling of M. polymorpha lines, 2-week-old thalli were labelled in liquid half-strength Gamborg’s B5 media supplemented with 50 μCi/mL [³H]-myo-inositol. After 5 days of labeling, thalli were washed two times with ultrapure water before flash frozen into liquid N2. Extracted inositol phosphates from yeast and plants were resolved by strong anion exchange high performance liquid chromatography (SAX-HPLC) using a Partisphere SAX 4.6 x 125 mm column (Whatman) at a flow rate of 0.5 mL/min with a shallow gradient formed by buffers A (1 mM EDTA) and B [1 mM EDTA and 1.3 M (NH₄)₂HPO₄, pH 3.8, with H₃PO₄] [102].

Cloning and plasmid construction for plant transformation

MpIPMK guideRNA (gRNA) designing and cloning. CRISPR/Cas9-based genome editing of MpIPMK was performed as described previously [80,81]. Selection of the gRNA target site was done using CRISPRdirect web tool [104]. The gRNA protospacers were generated by annealing complementary oligonucleotides and inserted into the pMpGE_En03 vector [81] previously digested with BsaI. MpIPMK-gRNA was incorporated into the binary vector pMpGE010 [81] using Gateway LR Clonase II Enzyme mix (Invitrogen, 11791100). In total, eight gRNAs were designed at different part of the gene that were used for generating CRISPR lines.

Thallus transformation of M. polymorpha

Transformation of M. polymorpha was performed as described previously [80]. In short, 14-day-old gemmalings were sliced to eliminate the apical notches and kept for 3 days for regeneration on half strength Gamborg’s B5 medium supplemented with 1% sucrose. Regenerating thalli fragments were then co-cultured with Agrobacterium tumefaciens GV3101 cells carrying the corresponding vectors in half strength Gamborg’s B5 medium supplemented with 2% sucrose under white light and gentle shaking of 120 rpm at 22⁰C. After 3 days of co-culture, the plant fragments were washed three times with sterile water and placed on half strength Gamborg’s B5 medium supplemented with 100 µg/mL cefotaxime and 10 µg/mL hygromycin B.

Construction of RNAi plasmid and generation of knockdown lines of in A. thaliana

Full-length coding region of AtIPK2α (~900 bp) was cloned into pOpOff2 (Hyg) [69] by Gateway recombination (LR ClonaseII, Invitrogen). The resulting vector pOpOFF2(Hyg)-AtIPK2α was used to stably transform the atipk2β-1 knockout plants using Agrobacterium tumefaciens GV3101-mediated transformation by floral dipping method. Positive transformants were selected on solidified half-strength MS media containing 30 mg/mL hygromycin. Homozygous lines were identified from selected lines at the T3 generation. For induction of RNAi, plants were treated with 25 µM dexamethasone (Dex) as indicated for each experiment.

Thermotolerance assay

A. thaliana thermotolerance assays were performed as described previously [76,105–107]. For basal thermotolerance assay, 7-day-old seedlings of all the respective genotypes (Col-0, atipk2α, atipk2β and atipk2βatipk2α^kd), grown on solidified half-strength MS media for 7 days, were exposed to 37⁰C for 2½ days and were kept at 22⁰C for recovery of another 4 days. Photographs were recorded and survival rates were counted after 4 days of recovery. For hypocotyl elongation assay, the stratified seedlings were allowed to grow at 22⁰C on solidified half-strength MS media for 5 days and then transferred to 28⁰C or maintained at 22⁰C for another 4 days. Photographs were taken after 5 days of transfer and the hypocotyl length was measured using Image software. For basal thermotolerance assay, 7-day-old seedlings of the respective genotypes (Col-0, atipk2α, atipk2β and atipk2βatipk2α^kd), grown on solidified half-strength MS media for 7 days, were exposed to 37⁰C for 3 days and were kept at 22⁰C for recovery of another 4 days. Photographs were recorded and survival rates were counted after 4 days of recovery.

For thermomorphogesis study in M. polymorpha, gemmae of wild-type and Mpipmk knockout lines were transferred on half strength Gamborg’s B5 media with 1% agar and grown for 5 days at 22⁰C with white light of (16 h:8 h; light: dark cycle) for 5 days. On the 5^th day, the plates having wild-type and Mpipmk knockout lines were exposed to 37⁰C in a plant chamber for 8 h. The heat-exposed plants were kept back at 22⁰C. Images were taken after 13 days of recovery. Thallus area was calculated using ImageJ software. Data were analysed using GraphPad Prism 6.

GUS staining

The 14-day-old seedlings of Col-0 and atipk2βatipk2α^kd lines were fixed for 30 min in ice-cold 90% (v/v) acetone and rinsed with staining buffer (0.5 M sodium phosphate buffer pH 7.2, 10% Triton X, 10 100 mM potassium ferrocyanide, 100 mM potassium ferricyanide) without X-Gluc (500 µg ml−¹ 5-bromo-4-chloro-3-indolyl-β-D-glucuronide) followed by incubation in staining buffer supplemented with 2 mM X-Gluc at 37 °C in the dark for overnight. After a series of wash with 20%, 35% and 50% ethanol, the cleared seedlings were mounted on slide and imaged using a ZEISS Stemi 508 light microscope.

Dual Luciferase reporter assay

A Dual Luciferase Reporter System was used to study the transient transactivation activity and was performed as described previously [76]. The promoter of AtHSP18.1 was cloned upstream of LUC in the pGreenII 0800-LUC to generate proAtHSP18.1:LUC reporter. The CDS of AtHSFA1b and AtIPK2α were cloned in PEG101 vector for effector constructs. Subsequently the CDS of wild-type and catalytic dead variant of AtIPK2α were cloned in translational fusion with N-terminal GFP in pGWB652 vector. Agrobacterium (GV3101) strain transformants harboring the above constructs in desired combination were infiltrated in Nicotiana benthamiana leaves as described previously [34]. Discs of leaves were collected after 72 h of infiltration. Luciferase assay was performed by Dual-Luciferase Reporter Assay System (Promega; E1910) following manufacturer’s instruction. Bioluminescence was measured using GloMax Explorer multimode microplate reader (Promega). The LUC activity was normalized to REN.

Bimolecular fluorescence complementation assay (BiFC)

The pENTR constructs carrying CDS of AtIPK2β, AtHSFA1b, MpIPMK and MpHSFB were cloned in pMDC_nVENUS and pMDC_cCFP via Gateway LR recombination. Agrobacterium (GV3101) strain was transformed with the above constructs and different constructs were infiltrated in N. bethamiana leaves as described previously [34]. The lower epidermal layer was collected and mounted on slide and visualized under a confocal microscope.

Electrophoretic mobility shift assay (EMSA)

Recombinant proteins of AtHSFA1b and MpHSF in translational fusion with the N-terminal His₈-MBP were expressed in E. coli BL21 (RIL) and subsequently lysed using PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ pH 7.4) followed by Ni-NTA affinity-based purification. The heat shock element (HSE) (20 nt) was fluorescein-labelled. The FAM-labelled probes (250 nM per reaction) were incubated with purified MBP-AtHSFA1b or MBP-MpHSF with and without AtIPK2β or MpIPMK for 30 min followed by separation using 6% native PAGE. The bands were detected using Amersham ImageQuant 800 GxP biomolecular images at 460 nm.

Yeast two hybrid assay

The full length CDS of MpIPMK/AtIPK2β and MpHSF/AtHSFA1b were amplified from cDNA by PCR using phusion polymerase with attB containing primers and introduced into pDONR221 (Thermo Fisher Scientific) vector using Gateway BP Clonase II Enzyme mix (Thermo Fisher Scientific). Furthermore, the coding sequences of MpIPMK and MpHSF were transferred into pGBKT7 and pGADT7, respectively, via Gateway LR recombination. Yeast transformations were performed by the lithium acetate method [101]. AH109 yeast strain was transformed with above constructs and serially spotted onto a synthetic selection media.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 6 software. The details of statistical data have been provided in S7 Table.

Accession numbers

AtIPK2α (At5g07370), AtIPK2β (At5g61760), AtITPK1 (At5g16760), Actin2 (At3g18780), AtHSP22 (At4g10250), AtHSP70 (At3g12580), AtHSP90 (At4g24190), AtHSP17.6 (At5g12020), AtHSFA1b (At5g16820), AtHSP18.1 (At5g59720), PP2AA3, TUBULIN (At5g62690), MpIPMK (Mp1g22660.1) and MpACT7 (Mp6g11010).

Supporting information

S1 Fig. Profile of inositol phosphates in the M. polymorpha thalli (Tak-1 and Tak-2), sporophylls of pteridophyta (Nephrolepis sp. and Dryopteris sp.) and seedlings of a dicot (A. thaliana; Col-0).

Quantification of different inositol phosphates detected in the above-mentioned embryophytes through CE-MS. Purified InsP extracts of 14-day-old M. polymorpha thalli (Tak-1 and Tak-2), mature sporophylls of pteridophytes (Nephrolepis sp. and Dryopteris sp.) and 14-day-old A. thaliana (Col-0) seedlings were subjected to CE-MS. The InsP₅ species were assigned by mass spectrometry and identical migration time compared with relative standards. Data are means ± SEM (n ≥ 4 biological replicates).

(TIF)

pgen.1011838.s001.tif^{(10.3MB, tif)}

S2 Fig. AtIPK2α and AtIPK2β phosphorylates InsP₆ in vitro.

A. Structural models (overview) of AtIPK2α (Protein Data Bank entry 4FRF) (pink) and HsIP6K3 (golden). Models were obtained by the AlphaFold web portal (https://alphafold.ebi.ac.uk/) and built on the Pymol. Overlay of AtIPK2α (hot pink) and HsIP6K3 (golden) structures (RMSD value = 0.958). B and C. SDS-PAGE analyses of tag-free recombinant AtIPK2α and AtIPK2β proteins used for ITC experiments. Recombinant His₈-MBP-TEV-AtIPK2 proteins were subjected to TEV protease and the digested products were further purified using affinity-based chromatography. The tag-free proteins were loaded on gel. Resolved proteins were visualized by coomassie blue staining. D and E. Isothermal titration calorimetry (ITC) assays of AtIPK2α (10 µM; left panel) and AtIPK2β (10 µM; right panel) (in cell) with ITC buffer (in syringe), respectively. Raw heats per injection are shown in the top panel and the bottom panel represents the integrated heats of each injection. F and G. SDS-PAGE analysis of the recombinant AtIPK2α and its catalytic dead variants protein in translational fusion with N-terminal His₈-MBP-TEV tag. Resolved proteins were visualized by coomassie blue staining (F). SDS-PAGE analysis of AtIPK2β WT and the catalytic dead variants protein. Resolved proteins were visualized by coomassie blue staining (G). H. PAGE analysis of in vitro kinase assay reaction products of AtIPK2s. InsP₆ alone and MBP served as control. I. Table showing the kinetic parameters (K_m and V_max) of AtIPK2α and AtIPK2β for Ins(1,4,5)P₃ and InsP₆ at varying ATP concentration. K_m and V_max were obtained after fitting of the data against the Michaelis-Menten model. J. Quantification of the AtIPK2α reaction product using CE-MS analyses. A minor amount of 1/3-InsP₇ species could be detected in the reaction products. Data represent means ± SEM (n = 3). Letters depict the significance in one-way ANOVA followed by Dunnett’s test (a and b, P < 0.0001; a and c, P < 0.05). K. AtIPK2β phosphorylates InsP₆ to synthesize 4/6-InsP₇ as a major PP-InsP species in vitro. Quantification of AtIPK2β WT and catalytic dead variant-derived reaction product using CE-MS analyses. A minor amount of 5-InsP₇ and 1/3-InsP₇ species could be detected in the reaction products. Data represent means ± SEM (n = 4). Letters depict the significance in one-way ANOVA followed by Dunnett’s test (a and b, P < 0.0001). L. Time-dependent conversion of InsP₆ to 4/6-InsP₇, 5-InsP₇, and 1/3-InsP₇ by AtIPK2β. AtIPK2β was incubated with ATP and InsP₆ for different time points and the reaction products were resolved by CE-MS. Data represent means ± SEM (n = 2, replicates).

(TIF)

pgen.1011838.s002.tif^{(24.1MB, tif)}

S3 Fig. Generation of atipk2α knockdown lines in the atipk2β-1 knockout plants.

A. Schematic diagram of the pOpOff2 vector. RB, right border; T, terminator; hyg, hygromycin; LB, left border. B. Genotyping PCR of Col-0, atipk2β-1 and all the three atipk2α knockdown lines. A to E represents the primers set used for genotyping, details of the primers are mentioned S1 Table. ACTIN served as a reference gene. C. Representative images of the GUS signal in leaves of Col-0 and the three independent atipk2βatipk2α^kd lines used in this study after dexamethasone (DEX) treatment. D. Stability of AtIPK2α transcript is affected in the atipk2βatipk2α^kd lines. Expression analyses of AtIPK2α between Col-0 and independent atipk2βatipk2α^kd lines using RT-PCR. ACTIN served as reference gene.

(TIF)

pgen.1011838.s003.tif^{(10.9MB, tif)}

S4 Fig. Seeds of AtIPK2-deficient lines are defective in phytic acid metabolism and heat stress induces AtIPK2 expression.

A. PAGE analysis of seed extracts of Col-0, atipk2βatipk2α^kd, atipk2α-1 and atipk2β-1 lines. This result is in agreement with the previously published report [36,50,63] that AtIPK2 contributes to InsP homeostasis distinctively in different plant parts. B. Quantitative RT-PCR (qRT-PCR) analysis of AtIPK2α and AtIPK2β in Col-0 after heat shock. 14-day-old seedlings were exposed to 37⁰C for 3 h and were harvested for qRT-PCR analysis. TUBULIN was used as a reference gene. Values are means ± SEM (n = 3, biological replicates). C. CE-MS analyses of InsP extracts of Col-0 and atipk2βatipk2α^kd seedlings after heat shock of 3 h at 37⁰C. Graph represents the fold difference of InsP isomers of the designated genotypes upon heat stress. Values are ± SEM (n = 4, biological replicates). Statistical significance is determined in one-way ANOVA followed by Dunnett’s test.

(TIF)

pgen.1011838.s004.tif^{(8.1MB, tif)}

S5 Fig. Characterization of the atipk2βatipk2α^kd lines expressing AtIPK2β and characterization of AtIPK2α overexpression lines.

A. Photograph of the control plate maintained at 22⁰C throughout basal thermal tolerance assay. This is the control set for the experiment presented in the main Fig 4C. B. Genotyping PCR of atipk2βatipk2α^kd lines expressing AtIPK2β under the control of a constitutive 35S promoter. The primers used for genotyping are mentioned in S1 Table. TUBULIN served as a reference gene. C. Expression analyses of AtIPK2β using RT-PCR. The primers used for RT-PCR are mentioned in S1 Table. TUBULIN served as a reference gene. D. Genotyping PCR of AtIPK2α in pro35S::AtIPK2α overexpression lines. The primers used for genotyping PCR are mentioned in S1 Table. TUBULIN served as a reference gene.

(TIF)

pgen.1011838.s005.tif^{(7.5MB, tif)}

S6 Fig. AtIPK2α-type kinases are ubiquitous in green plants and share conserved catalytic motif.

A. The phylogenetic tree was estimated from an alignment of AtIPK2α amino acid sequences using maximum likelihood. Branch support was calculated from 1000 bootstrap replicates, and values below 50% are omitted. Branch lengths are given in terms of expected numbers of amino acid substitutions per site. B. Protein alignment of MpIPMK with AtIPK2α and ScIpk2. Red rectangle marks the conserved catalytic motif PXXXDXKXG of the InsP kinase.

(TIF)

pgen.1011838.s006.tif^{(15.1MB, tif)}

S7 Fig. Structural model and catalytic activity of MpIPMK.

A. Cartoon depicting the conserved PXXXDXKXG motif of M. polymorpha IPMK. The residues highlighted in red are the altered residues, forming catalytic dead variants of IPMK, i.e., MpIPMK^D130A, MpIPMK^K132A, MpIPMK^D130AK132A (referred as MpIPMK^DM). B. Structural model (Structural model (overview) of MpIPMK. Models were obtained by the AlphaFold web portal (https://alphafold.ebi.ac.uk/) and built on the Pymol. C. Structural overlay of AtIPK2α (hot pink), MpIPMK (blue) structures (RMSD value = 0.872). Note the similarity between the MpIPMK model and the AtIPK2α structure (Protein Data Bank entry 4FRF). D. Zoom-in-into view of the catalytic active site of MpIPMK. E. SDS-PAGE analysis of MpIPMK and its catalytic dead variants. Arrow head denotes MpIPMK and its catalytic dead variants. F. PAGE analysis of the in vitro kinase reaction products of MpIPMK. Recombinant His₈-MBP-MpIPMK was incubated with 12.5 mM ATP, and 10 nmol InsP₆ at 37⁰C for 12 h in reaction buffer. The reaction product was separated by 33% PAGE and visualized with toluidine blue. InsP₆ alone served as a control.

(TIF)

pgen.1011838.s007.tif^{(10.2MB, tif)}

S8 Fig. MpIPMK does not possess yeast Kcs1- or Vip1-like activity.

A. Complementation of the yeast kcs1∆-associated growth defects by the ectopic expression of MpIPMK. Wild-type and kcs1∆ yeast transformants (BY4741 background) carrying designated plasmids were spotted in 8-fold serial dilution onto YPD with and without NaCl incubated at 28ºC and 37⁰C. AtITPK1 served as positive control [30] and empty vector served as negative control. B. Complementation of vip1∆ -associated growth defects in yeast by ectopic expression of MpIPMK. The vip1∆ yeast strain transformed with the episomal pCA45 (URA3) plasmids carrying MpIPMK and kinase dead mutants were spotted in 8-fold serial dilutions onto uracil-free minimal medium in presence and absence of 6-azauracil. No rescue of phenotype was observed. AtVIH2 KD served as positive control [35] and empty vector served as negative control. C. Quantification of the reaction product of MpIPMK analyzed by CE-MS. Data represent means ± SEM (n = 2).

(TIF)

pgen.1011838.s008.tif^{(12.5MB, tif)}

S9 Fig. Mpipmk knockout plants do not exhibit any obvious growth defects and accumulate InsP₃, InsP₄ and InsP₅ isomers.

A. Chromatogram showing CRISPR/Cas9-edited nucleotide sequences of Mpipmk1.2 and Mpipmk1.7 compared with those of wild-type plants using chromatogram obtained from sequencing results. B. Photograph of 14-day-old thalli of wild-type, Mpipmk1.2 and Mpipmk1.7 plants. C. CE-MS analyses of different inositol phosphates isomers in wild-type and Mpipmk knockout plants. The InsP₅ and InsP₇ species were assigned by mass spectrometry and identical migration time compared with their relative standards. Two InsP₄ and two InsP₃ isomers were detected. Data are means ± SEM (n = 3, biological replicates). Inositol phosphates are represented as percentage to InsP₆. Significant difference is determined by one-way ANOVA followed by Dunnett’s test (*P < 0.05, **P = 0.002 ***P < 0.001).

(TIF)

pgen.1011838.s009.tif^{(14.5MB, tif)}

S10 Fig. Confirmation of the transgenic atipk2βatipk2α^kd lines expressing MpIPMK heterologously under the control of a 35S promoter and qPCR analyses of the heat responsive genes between Col-0 and the atipk2βatipk2α^kd plants.

A. Genotyping PCR of atipk2βatipk2α^kd lines expressing MpIPMK. The primers used for genotyping are mentioned in S1 Table. TUBULIN served as a reference gene. B. RT-PCR of atipk2βatipk2α^kd lines expressing MpIPMK. The primers used for RT-PCR are mentioned in S1 Table. TUBULIN served as a reference gene. C. Quantitative RT-PCR (qRT-PCR) analysis of different genes involved in thermomorphogenesis between Col-0 and the atipk2βatipk2α^kd lines after heat shock. 14-day-old seedlings were exposed to 37⁰C for 3 h and were harvested for qRT-PCR analysis. Transcript levels of the benchmark genes are presented relative PP2AA3 transcript. Values are means ± SEM (n ≥ 3, biological replicates). Statistical significance is determined by two-way ANOVA followed by Tukey’s test (*P < 0.05, **P < 0.001).

(TIF)

pgen.1011838.s010.tif^{(7.5MB, tif)}

S11 Fig. Ins(1,4,5)P_3, InsP₆ and 4/6-InsP₇ do not influence DNA-binding affinity of Arabidopsis heat shock transcription factor.

A and B. EMSA showing MBP, MBP-AtIPK2β do not bind directly to HSE element. 250 nM of the probe was used. MBP and MBP-AtIPK2β were used in the concentration ranging from 50- 200 nM. C. Ins(1,4,5)P₃, InsP₆ and 4/6-InsP₇ don’t influence DNA-binding activity of heat shock transcription factor. 100 nM of HSF was pre-incubated with InsP₆, InsP₃ and InsP₇ (10 nM, 50 nM, 100 nM and 10 µM) for 30 mins followed by incubation with FAM-labelled probe for 15 mins on ice. The complexes were resolved using 6% of native PAGE.

(TIF)

pgen.1011838.s011.tif^{(4.8MB, tif)}

S12 Fig. MpIPMK physically interacts with MpHSFB1 and InsPs do not directly influence the transcriptional activity of MpHSFB1.

A. MpIPMK shows physical interaction with MpHSFB1 in vivo. AH109 yeast strain carrying the pGADT7-MpHSF and pGBKT7-MpIPMK plasmids were spotted on selective media. B. Transiently expressed MpIPMK interacts with MpHSFB1 in the nucleus of N. benthamiana cells. Different combination of co-expressed nVENUS and cCFP constructs were infiltrated in N. benthamiana. YFP represents the images taken with YFP filter and merge represents the overlay of YFP and brightfiled. Scale bar = 50 µm. C and D. EMSA showing MBP-MpIPMK do not bind directly to HSE element and PP-InsPs do not affect MpHSFB1 binding to HSE element. 250 nM of the probe was used. MpIPMK was used in the concentration ranging from 50- 200 nM. Ins(1,4,5)P₃, InsP₆ and 4/6-InsP₇ don’t influence DNA-binding activity of heat shock transcription factor. 100 nM of HSFB1 was pre-incubated with InsP₆, InsP₃ and InsP₇ (10 nM, 50 nM, 100 nM and 10 µM) for 30 mins followed by incubation with FAM-labelled probe for 15 mins on ice. The complexes were resolved using 6% of native PAGE.

(TIF)

pgen.1011838.s012.tif^{(12MB, tif)}

S1 Table. List of primers used in the study.

(XLSX)

pgen.1011838.s013.xlsx^{(13KB, xlsx)}

S2 Table. List of hits from Phyre² analyses using Human IP6K3.

(XLSX)

pgen.1011838.s014.xlsx^{(16.7KB, xlsx)}

S3 Table. AtIPK2α and AtIPK2β expression analysis under heat stress.

(XLSX)

pgen.1011838.s015.xlsx^{(21KB, xlsx)}

S4 Table. Yeast Strains used in this study.

(XLSX)

pgen.1011838.s016.xlsx^{(9.2KB, xlsx)}

S5 Table. Sequences used in Phylogenetic analysis.

(XLSX)

pgen.1011838.s017.xlsx^{(12.5KB, xlsx)}

S6 Table. MS parameters for MRM transitions.

(XLSX)

pgen.1011838.s018.xlsx^{(10.1KB, xlsx)}

S7 Table. Details of statistical analysis.

(XLSX)

pgen.1011838.s019.xlsx^{(39.5KB, xlsx)}

S8 Table. Raw data file.

(XLSX)

pgen.1011838.s020.xlsx^{(49.3KB, xlsx)}

Acknowledgments

We acknowledge Cristina Azevedo for the pCA45 plasmid. We acknowledge Sandeep M. Eswarappa for their Luminometer facility. We are grateful to Manoj Majee for the pGreenII 0800-LUC and pEarlyGate101 vectors. We thank Saikat Bhattacharjee for providing us the Arabidopsis ipk2α-1 seeds. We thank Utpal Nath for the AH109 yeast strain. We thank all members of the Laha Lab for their critical feedback to this study.

Data Availability

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work is supported by the Department of Biotechnology (DBT) for grant no. BT/PR43116/BRB/10/2010/2021, and in part by the Anusandhan National Research Foundation (ANRF) SRG/2021/000951, the MoE-STARS/STARS-2/2023-0162, the Infosys Foundation, and the Indian Institute of Science start-up fund to DL. We are also thankful to the DST-FIST infrastructure fund. RY is supported by the IISc fellowship. PR is the recipient of the Prime Minister Research Fellowship (PMRF). NJP acknowledges Council of Scientific & Industrial Research (CSIR) for research fellowship. HJJ and GL acknowledge funding from the Volkswagen Foundation (VW Momentum Grant 98604) and DFG (JE 572/11-1). This study was supported by the German Research Foundation (DFG) under Germany’s excellence strategy (CIBSS-EXC-2189-Project ID 390939984) to HJJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Wilson MSC, Livermore TM, Saiardi A. Inositol pyrophosphates: between signalling and metabolism. Biochem J. 2013;452(3):369–79. doi: 10.1042/BJ20130118 [DOI] [PubMed] [Google Scholar]
2.Shears SB. Inositol pyrophosphates: why so many phosphates? Adv Biol Regul. 2015;57:203–16. doi: 10.1016/j.jbior.2014.09.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Thota SG, Bhandari R. The emerging roles of inositol pyrophosphates in eukaryotic cell physiology. J Biosci. 2015;40(3):593–605. doi: 10.1007/s12038-015-9549-x [DOI] [PubMed] [Google Scholar]
4.Monserrate JP, York JD. Inositol phosphate synthesis and the nuclear processes they affect. Curr Opin Cell Biol. 2010;22(3):365–73. doi: 10.1016/j.ceb.2010.03.006 [DOI] [PubMed] [Google Scholar]
5.Barker CJ, Illies C, Gaboardi GC, Berggren P-O. Inositol pyrophosphates: structure, enzymology and function. Cell Mol Life Sci. 2009;66(24):3851–71. doi: 10.1007/s00018-009-0115-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Nguyen Trung M, Furkert D, Fiedler D. Versatile signaling mechanisms of inositol pyrophosphates. Curr Opin Chem Biol. 2022;70:102177. doi: 10.1016/j.cbpa.2022.102177 [DOI] [PubMed] [Google Scholar]
7.Chabert V, Kim G-D, Qiu D, Liu G, Michaillat Mayer L, Jamsheer K M, et al. Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81. Elife. 2023;12:RP87956. doi: 10.7554/eLife.87956 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Laha D, Portela-Torres P, Desfougères Y, Saiardi A. Inositol phosphate kinases in the eukaryote landscape. Adv Biol Regul. 2021;79:100782. doi: 10.1016/j.jbior.2020.100782 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Wilson MS, Jessen HJ, Saiardi A. The inositol hexakisphosphate kinases IP6K1 and -2 regulate human cellular phosphate homeostasis, including XPR1-mediated phosphate export. J Biol Chem. 2019;294(30):11597–608. doi: 10.1074/jbc.RA119.007848 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Li X, Gu C, Hostachy S, Sahu S, Wittwer C, Jessen HJ, et al. Control of XPR1-dependent cellular phosphate efflux by InsP8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling. Proc Natl Acad Sci U S A. 2020;117(7):3568–74. doi: 10.1073/pnas.1908830117 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Lee Y-S, Mulugu S, York JD, O’Shea EK. Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science. 2007;316(5821):109–12. doi: 10.1126/science.1139080 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Szijgyarto Z, Garedew A, Azevedo C, Saiardi A. Influence of inositol pyrophosphates on cellular energy dynamics. Science. 2011;334(6057):802–5. doi: 10.1126/science.1211908 [DOI] [PubMed] [Google Scholar]
13.Qin N, Li L, Ji X, Pereira R, Chen Y, Yin S, et al. Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast. Cell. 2023;186(4):748-763.e15. doi: 10.1016/j.cell.2023.01.014 [DOI] [PubMed] [Google Scholar]
14.Gu C, Liu J, Liu X, Zhang H, Luo J, Wang H, et al. Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line. Proc Natl Acad Sci U S A. 2021;118(10):e2020187118. doi: 10.1073/pnas.2020187118 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Zhang X, Li N, Zhang J, Zhang Y, Yang X, Luo Y, et al. 5-IP7 is a GPCR messenger mediating neural control of synaptotagmin-dependent insulin exocytosis and glucose homeostasis. Nat Metab. 2021;3(10):1400–14. doi: 10.1038/s42255-021-00468-7 [DOI] [PubMed] [Google Scholar]
16.Saiardi A, Erdjument-Bromage H, Snowman AM, Tempst P, Snyder SH. Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases. Curr Biol. 1999;9(22):1323–6. doi: 10.1016/s0960-9822(00)80055-x [DOI] [PubMed] [Google Scholar]
17.Mulugu S, Bai W, Fridy PC, Bastidas RJ, Otto JC, Dollins DE, et al. A conserved family of enzymes that phosphorylate inositol hexakisphosphate. Science. 2007;316(5821):106–9. doi: 10.1126/science.1139099 [DOI] [PubMed] [Google Scholar]
18.Wang H, Falck JR, Hall TMT, Shears SB. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding. Nat Chem Biol. 2011;8(1):111–6. doi: 10.1038/nchembio.733 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Desfougères Y, Portela-Torres P, Qiu D, Livermore TM, Harmel RK, Borghi F, et al. The inositol pyrophosphate metabolism of Dictyostelium discoideum does not regulate inorganic polyphosphate (polyP) synthesis. Adv Biol Regul. 2022;83:100835. doi: 10.1016/j.jbior.2021.100835 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.York JD, Odom AR, Murphy R, Ives EB, Wente SR. A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science. 1999;285(5424):96–100. doi: 10.1126/science.285.5424.96 [DOI] [PubMed] [Google Scholar]
21.Odom AR, Stahlberg A, Wente SR, York JD. A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science. 2000;287(5460):2026–9. doi: 10.1126/science.287.5460.2026 [DOI] [PubMed] [Google Scholar]
22.Saiardi A, Caffrey JJ, Snyder SH, Shears SB. Inositol polyphosphate multikinase (ArgRIII) determines nuclear mRNA export in Saccharomyces cerevisiae. FEBS Lett. 2000;468(1):28–32. doi: 10.1016/s0014-5793(00)01194-7 [DOI] [PubMed] [Google Scholar]
23.Draskovic P, Saiardi A, Bhandari R, Burton A, Ilc G, Kovacevic M, et al. Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups. Chem Biol. 2008;15(3):274–86. doi: 10.1016/j.chembiol.2008.01.011 [DOI] [PubMed] [Google Scholar]
24.Dollins DE, Bai W, Fridy PC, Otto JC, Neubauer JL, Gattis SG, et al. Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling. P Natl Acad Sci USA. 2020;117(17):9356–64. doi: 10.1073/pnas.1908875117 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Lin H, Fridy PC, Ribeiro AA, Choi JH, Barma DK, Vogel G, et al. Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases. J Biol Chem. 2009;284(3):1863–72. doi: 10.1074/jbc.M805686200 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Lorenzo-Orts L, Couto D, Hothorn M. Identity and functions of inorganic and inositol polyphosphates in plants. New Phytol. 2020;225(2):637–52. doi: 10.1111/nph.16129 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Riemer E, Pullagurla NJ, Yadav R, Rana P, Jessen HJ, Kamleitner M, et al. Regulation of plant biotic interactions and abiotic stress responses by inositol polyphosphates. Front Plant Sci. 2022;13:944515. doi: 10.3389/fpls.2022.944515 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Cridland C, Gillaspy G. Inositol Pyrophosphate Pathways and Mechanisms: What Can We Learn from Plants? Molecules. 2020;25(12):2789. doi: 10.3390/molecules25122789 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Whitfield H, White G, Sprigg C, Riley AM, Potter BVL, Hemmings AM, et al. 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. 2020;477(14):2621–38. doi: 10.1042/BCJ20200423 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Laha D, Parvin N, Hofer A, Giehl RFH, Fernandez-Rebollo N, von Wirén N, et al. Arabidopsis ITPK1 and ITPK2 Have an Evolutionarily Conserved Phytic Acid Kinase Activity. ACS Chem Biol. 2019;14(10):2127–33. doi: 10.1021/acschembio.9b00423 [DOI] [PubMed] [Google Scholar]
31.Adepoju O, Williams SP, Craige B, Cridland CA, Sharpe AK, Brown AM, et al. Inositol Trisphosphate Kinase and Diphosphoinositol Pentakisphosphate Kinase Enzymes Constitute the Inositol Pyrophosphate Synthesis Pathway in Plants. bioRxiv. 2019;724914. doi: 10.1101/724914 [DOI] [Google Scholar]
32.Zong G, Shears SB, Wang H. Structural and catalytic analyses of the InsP6 kinase activities of higher plant ITPKs. FASEB J. 2022;36(7):e22380. doi: 10.1096/fj.202200393R [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Riemer E, Qiu D, Laha D, Harmel RK, Gaugler P, Gaugler V, et al. ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis. Mol Plant. 2021;14(11):1864–80. doi: 10.1016/j.molp.2021.07.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Laha NP, Giehl RFH, Riemer E, Qiu D, Pullagurla NJ, Schneider R, et al. INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-dependent inositol polyphosphates regulate auxin responses in Arabidopsis. Plant Physiol. 2022;190(4):2722–38. doi: 10.1093/plphys/kiac425 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Laha D, Johnen P, Azevedo C, Dynowski M, Weiß M, Capolicchio S, et al. VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis. Plant Cell. 2015;27(4):1082–97. doi: 10.1105/tpc.114.135160 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Desai M, Rangarajan P, Donahue JL, Williams SP, Land ES, Mandal MK, et al. Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants. Plant J. 2014;80(4):642–53. doi: 10.1111/tpj.12669 [DOI] [PubMed] [Google Scholar]
37.Couso I, Evans BS, Li J, Liu Y, Ma F, Diamond S, et al. Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas. Plant Cell. 2016;28(9):2026–42. doi: 10.1105/tpc.16.00351 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Zhu J, Lau K, Puschmann R, Harmel RK, Zhang Y, Pries V, et al. Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis. Elife. 2019;8:e43582. doi: 10.7554/eLife.43582 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Laha D, Parvin N, Dynowski M, Johnen P, Mao H, Bitters ST, et al. Inositol Polyphosphate Binding Specificity of the Jasmonate Receptor Complex. Plant Physiol. 2016;171(4):2364–70. doi: 10.1104/pp.16.00694 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Dong J, Ma G, Sui L, Wei M, Satheesh V, Zhang R, et al. Inositol Pyrophosphate InsP8 Acts as an Intracellular Phosphate Signal in Arabidopsis. Mol Plant. 2019;12(11):1463–73. doi: 10.1016/j.molp.2019.08.002 [DOI] [PubMed] [Google Scholar]
41.Land ES, Cridland CA, Craige B, Dye A, Hildreth SB, Helm RF, et al. A Role for Inositol Pyrophosphates in the Metabolic Adaptations to Low Phosphate in Arabidopsis. Metabolites. 2021;11(9):601. doi: 10.3390/metabo11090601 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Guan Z, Zhang Q, Zhang Z, Zuo J, Chen J, Liu R, et al. Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 - PHR2 complex. Nat Commun. 2022;13(1):1581. doi: 10.1038/s41467-022-29275-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Kuo H-F, Hsu Y-Y, Lin W-C, Chen K-Y, Munnik T, Brearley CA, et al. Arabidopsis inositol phosphate kinases IPK1 and ITPK1 constitute a metabolic pathway in maintaining phosphate homeostasis. Plant J. 2018;:10.1111/tpj.13974. doi: 10.1111/tpj.13974 [DOI] [PubMed] [Google Scholar]
44.Pullagurla NJ, Shome S, Liu G, Jessen HJ, Laha D. Orchestration of phosphate homeostasis by the ITPK1-type inositol phosphate kinase in the liverwort Marchantia polymorpha. Plant Physiol. 2025;197(2):kiae454. doi: 10.1093/plphys/kiae454 [DOI] [PubMed] [Google Scholar]
45.Pullagurla NJ, Shome S, Yadav R, Laha D. ITPK1 Regulates Jasmonate-Controlled Root Development in Arabidopsis thaliana. Biomolecules. 2023;13(9):1368. doi: 10.3390/biom13091368 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Gulabani H, Goswami K, Walia Y, Roy A, Noor JJ, Ingole KD, et al. Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalk between SA-dependent immunity and phosphate-starvation responses. Plant Cell Rep. 2022;41(2):347–63. doi: 10.1007/s00299-021-02812-3 [DOI] [PubMed] [Google Scholar]
47.Chalak K, Yadav R, Liu G, Rana P, Jessen HJ, Laha D. Functional Conservation of the DDP1-type Inositol Pyrophosphate Phosphohydrolases in Land Plant. Biochemistry. 2024;63(21):2723–8. doi: 10.1021/acs.biochem.4c00458 [DOI] [PubMed] [Google Scholar]
48.Laurent F, Bartsch SM, Shukla A, Rico-Resendiz F, Couto D, Fuchs C, et al. Inositol pyrophosphate catabolism by three families of phosphatases regulates plant growth and development. PLoS Genet. 2024;20(11):e1011468. doi: 10.1371/journal.pgen.1011468 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Dovey CM, Diep J, Clarke BP, Hale AT, McNamara DE, Guo H, et al. MLKL Requires the Inositol Phosphate Code to Execute Necroptosis. Mol Cell. 2018;70(5):936-948.e7. doi: 10.1016/j.molcel.2018.05.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Stevenson-Paulik J, Bastidas RJ, Chiou S-T, Frye RA, York JD. Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases. Proc Natl Acad Sci U S A. 2005;102(35):12612–7. doi: 10.1073/pnas.0504172102 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Stevenson-Paulik J, Odom AR, York JD. Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases. J Biol Chem. 2002;277(45):42711–8. doi: 10.1074/jbc.M209112200 [DOI] [PubMed] [Google Scholar]
52.Wang H, Shears SB. Structural features of human inositol phosphate multikinase rationalize its inositol phosphate kinase and phosphoinositide 3-kinase activities. J Biol Chem. 2017;292(44):18192–202. doi: 10.1074/jbc.M117.801845 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Xia H-J, Brearley C, Elge S, Kaplan B, Fromm H, Mueller-Roeber B. Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex. Plant Cell. 2003;15(2):449–63. doi: 10.1105/tpc.006676 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Chen Y, Han JM, Wang XY, Chen XY, Li YH, Yuan CY, et al. OsIPK2, a Rice Inositol Polyphosphate Kinase Gene, Is Involved in Phosphate Homeostasis and Root Development. Plant Cell Physiol. 2023;64(8):893–905. doi: 10.1093/pcp/pcad052 [DOI] [PubMed] [Google Scholar]
55.Bowman JL. The origin of a land flora. Nat Plants. 2022;8(12):1352–69. doi: 10.1038/s41477-022-01283-y [DOI] [PubMed] [Google Scholar]
56.Qiu D, Wilson MS, Eisenbeis VB, Harmel RK, Riemer E, Haas TM, et al. Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry. Nat Commun. 2020;11(1):6035. doi: 10.1038/s41467-020-19928-x [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Qiu D, Gu C, Liu G, Ritter K, Eisenbeis VB, Bittner T, et al. Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues. Chem Sci. 2022;14(3):658–67. doi: 10.1039/d2sc05147h [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Liu G, Riemer E, Schneider R, Cabuzu D, Bonny O, Wagner CA, et al. The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones. RSC Chem Biol. 2023;4(4):300–9. doi: 10.1039/d2cb00235c [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Fassetti F, Leone O, Palopoli L, Rombo SE, Saiardi A. IP6K gene identification in plant genomes by tag searching. BMC Proc. 2011;5 Suppl 2(Suppl 2):S1. doi: 10.1186/1753-6561-5-S2-S1 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10(6):845–58. doi: 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Zhan H, Zhong Y, Yang Z, Xia H. Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development, pollen tube guidance and embryogenesis. Plant J. 2015;82(5):758–71. doi: 10.1111/tpj.12846 [DOI] [PubMed] [Google Scholar]
62.Raboy V. The ABCs of low-phytate crops. Nat Biotechnol. 2007;25(8):874–5. doi: 10.1038/nbt0807-874 [DOI] [PubMed] [Google Scholar]
63.Kim S-I, Tai TH. Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach. Mol Genet Genomics. 2011;286(2):119–33. doi: 10.1007/s00438-011-0631-2 [DOI] [PubMed] [Google Scholar]
64.Pisani F, Livermore T, Rose G, Chubb JR, Gaspari M, Saiardi A. Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis. PLoS One. 2014;9(1):e85533. doi: 10.1371/journal.pone.0085533 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Voglmaier SM, Bembenek ME, Kaplin AI, Dormán G, Olszewski JD, Prestwich GD, et al. Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor. Proc Natl Acad Sci U S A. 1996;93(9):4305–10. doi: 10.1073/pnas.93.9.4305 [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Harmel RK, Puschmann R, Nguyen Trung M, Saiardi A, Schmieder P, Fiedler D. Harnessing 13C-labeled myo-inositol to interrogate inositol phosphate messengers by NMR. Chem Sci. 2019;10(20):5267–74. doi: 10.1039/c9sc00151d [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Haas TM, Mundinger S, Qiu D, Jork N, Ritter K, Dürr-Mayer T, et al. Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18 O-Phosphoramidites. Angew Chem Int Ed Engl. 2022;61(5):e202112457. doi: 10.1002/anie.202112457 [DOI] [PMC free article] [PubMed] [Google Scholar]
68.Ritter K, Jork N, Unmüßig A-S, Köhn M, Jessen HJ. Assigning the Absolute Configuration of Inositol Poly- and Pyrophosphates by NMR Using a Single Chiral Solvating Agent. Biomolecules. 2023;13(7):1150. doi: 10.3390/biom13071150 [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Parvin N, Carrie C, Pabst I, Läßer A, Laha D, Paul MV, et al. TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana. Mol Plant. 2017;10(4):575–89. doi: 10.1016/j.molp.2016.12.012 [DOI] [PubMed] [Google Scholar]
70.Wielopolska A, Townley H, Moore I, Waterhouse P, Helliwell C. A high-throughput inducible RNAi vector for plants. Plant Biotechnol J. 2005;3(6):583–90. doi: 10.1111/j.1467-7652.2005.00149.x [DOI] [PubMed] [Google Scholar]
71.Wilson MSC, Bulley SJ, Pisani F, Irvine RF, Saiardi A. A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine. Open Biol. 2015;5(3):150014. doi: 10.1098/rsob.150014 [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Kilian J, Whitehead D, Horak J, Wanke D, Weinl S, Batistic O, et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 2007;50(2):347–63. doi: 10.1111/j.1365-313X.2007.03052.x [DOI] [PubMed] [Google Scholar]
73.Delker C, Quint M, Wigge PA. Recent advances in understanding thermomorphogenesis signaling. Curr Opin Plant Biol. 2022;68:102231. doi: 10.1016/j.pbi.2022.102231 [DOI] [PubMed] [Google Scholar]
74.Casal JJ, Balasubramanian S. Thermomorphogenesis. Annu Rev Plant Biol. 2019;70:321–46. doi: 10.1146/annurev-arplant-050718-095919 [DOI] [PubMed] [Google Scholar]
75.Quint M, Delker C, Balasubramanian S, Balcerowicz M, Casal JJ, Castroverde CDM, et al. 25 Years of thermomorphogenesis research: milestones and perspectives. Trends Plant Sci. 2023;28(10):1098–100. doi: 10.1016/j.tplants.2023.07.001 [DOI] [PubMed] [Google Scholar]
76.Li J, Cao Y, Zhang J, Zhu C, Tang G, Yan J. The miR165/166-PHABULOSA module promotes thermotolerance by transcriptionally and posttranslationally regulating HSFA1. Plant Cell. 2023;35(8):2952–71. doi: 10.1093/plcell/koad121 [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Bowman JL, Kohchi T, Yamato KT, Jenkins J, Shu S, Ishizaki K, et al. Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome. Cell. 2017;171(2):287–304.e15. doi: 10.1016/j.cell.2017.09.030 [DOI] [PubMed] [Google Scholar]
78.Kohchi T, Yamato KT, Ishizaki K, Yamaoka S, Nishihama R. Development and Molecular Genetics of Marchantia polymorpha. Annu Rev Plant Biol. 2021;72:677–702. doi: 10.1146/annurev-arplant-082520-094256 [DOI] [PubMed] [Google Scholar]
79.Bowman JL, Araki T, Arteaga-Vazquez MA, Berger F, Dolan L, Haseloff J, et al. The Naming of Names: Guidelines for Gene Nomenclature in Marchantia. Plant Cell Physiol. 2016;57(2):257–61. doi: 10.1093/pcp/pcv193 [DOI] [PMC free article] [PubMed] [Google Scholar]
80.Sugano SS, Shirakawa M, Takagi J, Matsuda Y, Shimada T, Hara-Nishimura I, et al. CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L. Plant Cell Physiol. 2014;55(3):475–81. doi: 10.1093/pcp/pcu014 [DOI] [PubMed] [Google Scholar]
81.Sugano SS, Nishihama R, Shirakawa M, Takagi J, Matsuda Y, Ishida S, et al. Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha. PLoS One. 2018;13(10):e0205117. doi: 10.1371/journal.pone.0205117 [DOI] [PMC free article] [PubMed] [Google Scholar]
82.Ludwig W, Hayes S, Trenner J, Delker C, Quint M. On the evolution of plant thermomorphogenesis. J Exp Bot. 2021;:erab310. doi: 10.1093/jxb/erab310 [DOI] [PubMed] [Google Scholar]
83.Wu T-Y, Hoh KL, Boonyaves K, Krishnamoorthi S, Urano D. Diversification of heat shock transcription factors expanded thermal stress responses during early plant evolution. Plant Cell. 2022;34(10):3557–76. doi: 10.1093/plcell/koac204 [DOI] [PMC free article] [PubMed] [Google Scholar]
84.Hahn A, Bublak D, Schleiff E, Scharf K-D. Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato. Plant Cell. 2011;23(2):741–55. doi: 10.1105/tpc.110.076018 [DOI] [PMC free article] [PubMed] [Google Scholar]
85.Yang J, Qu X, Ji L, Li G, Wang C, Wang C, et al. PIF4 Promotes Expression of HSFA2 to Enhance Basal Thermotolerance in Arabidopsis. Int J Mol Sci. 2022;23(11):6017. doi: 10.3390/ijms23116017 [DOI] [PMC free article] [PubMed] [Google Scholar]
86.Yoshida T, Ohama N, Nakajima J, Kidokoro S, Mizoi J, Nakashima K, et al. Arabidopsis HsfA1 transcription factors function as the main positive regulators in heat shock-responsive gene expression. Mol Genet Genomics. 2011;286(5–6):321–32. doi: 10.1007/s00438-011-0647-7 [DOI] [PubMed] [Google Scholar]
87.Liu H-C, Liao H-T, Charng Y-Y. The role of class A1 heat shock factors (HSFA1s) in response to heat and other stresses in Arabidopsis. Plant Cell Environ. 2011;34(5):738–51. doi: 10.1111/j.1365-3040.2011.02278.x [DOI] [PubMed] [Google Scholar]
88.Sun J, Qi L, Li Y, Chu J, Li C. PIF4-mediated activation of YUCCA8 expression integrates temperature into the auxin pathway in regulating arabidopsis hypocotyl growth. PLoS Genet. 2012;8(3):e1002594. doi: 10.1371/journal.pgen.1002594 [DOI] [PMC free article] [PubMed] [Google Scholar]
89.Ohama N, Kusakabe K, Mizoi J, Zhao H, Kidokoro S, Koizumi S, et al. The Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression. Plant Cell. 2016;28(1):181–201. doi: 10.1105/tpc.15.00435 [DOI] [PMC free article] [PubMed] [Google Scholar]
90.Liu H, Charng Y. Common and distinct functions of Arabidopsis class A1 and A2 heat shock factors in diverse abiotic stress responses and development. Plant Physiol. 2013;163(1):276–90. doi: 10.1104/pp.113.221168 [DOI] [PMC free article] [PubMed] [Google Scholar]
91.Malabanan MM, Blind RD. Inositol polyphosphate multikinase (IPMK) in transcriptional regulation and nuclear inositide metabolism. Biochem Soc Trans. 2016;44(1):279–85. doi: 10.1042/BST20150225 [DOI] [PMC free article] [PubMed] [Google Scholar]
92.Sang S, Chen Y, Yang Q, Wang P. Arabidopsis inositol polyphosphate multikinase delays flowering time through mediating transcriptional activation of FLOWERING LOCUS C. J Exp Bot. 2017;68(21–22):5787–800. doi: 10.1093/jxb/erx397 [DOI] [PMC free article] [PubMed] [Google Scholar]
93.Beon J, Han S, Yang H, Park SE, Hyun K, Lee S-Y, et al. Inositol polyphosphate multikinase physically binds to the SWI/SNF complex and modulates BRG1 occupancy in mouse embryonic stem cells. Elife. 2022;11:e73523. doi: 10.7554/eLife.73523 [DOI] [PMC free article] [PubMed] [Google Scholar]
94.Sowd GA, Stivison EA, Chapagain P, Hale AT, Poland JC, Rameh LE, et al. IPMK regulates HDAC3 activity and histone H4 acetylation in human cells. bioRxiv. 2024;2024.04.29.591660. doi: 10.1101/2024.04.29.591660 [DOI] [PMC free article] [PubMed] [Google Scholar]
95.Ahn H, Yu J, Ryu K, Ryu J, Kim S, Park JY, et al. Single-molecule analysis reveals that IPMK enhances the DNA-binding activity of the transcription factor SRF. Nucleic Acids Res. 2025;53(1):gkae1281. doi: 10.1093/nar/gkae1281 [DOI] [PMC free article] [PubMed] [Google Scholar]
96.Luo J, Jiang J, Sun S, Wang X. Brassinosteroids promote thermotolerance through releasing BIN2-mediated phosphorylation and suppression of HsfA1 transcription factors in Arabidopsis. Plant Commun. 2022;3(6):100419. doi: 10.1016/j.xplc.2022.100419 [DOI] [PMC free article] [PubMed] [Google Scholar]
97.Blüher D, Laha D, Thieme S, Hofer A, Eschen-Lippold L, Masch A, et al. A 1-phytase type III effector interferes with plant hormone signaling. Nat Commun. 2017;8(1):2159. doi: 10.1038/s41467-017-02195-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
98.Raboy V, Gerbasi PF, Young KA, Stoneberg SD, Pickett SG, Bauman AT, et al. Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1. Plant Physiol. 2000;124(1):355–68. doi: 10.1104/pp.124.1.355 [DOI] [PMC free article] [PubMed] [Google Scholar]
99.Nagy R, Grob H, Weder B, Green P, Klein M, Frelet-Barrand A, et al. The Arabidopsis ATP-binding cassette protein AtMRP5/AtABCC5 is a high affinity inositol hexakisphosphate transporter involved in guard cell signaling and phytate storage. J Biol Chem. 2009;284(48):33614–22. doi: 10.1074/jbc.M109.030247 [DOI] [PMC free article] [PubMed] [Google Scholar]
100.Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38(7):3022–7. doi: 10.1093/molbev/msab120 [DOI] [PMC free article] [PubMed] [Google Scholar]
101.Gietz D, St Jean A, Woods RA, Schiestl RH. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992;20(6):1425. doi: 10.1093/nar/20.6.1425 [DOI] [PMC free article] [PubMed] [Google Scholar]
102.Azevedo C, Saiardi A. Extraction and analysis of soluble inositol polyphosphates from yeast. Nat Protoc. 2006;1(5):2416–22. doi: 10.1038/nprot.2006.337 [DOI] [PubMed] [Google Scholar]
103.Laha D, Kamleitner M, Johnen P, Schaaf G. Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC. Methods Mol Biol. 2021;2295:365–78. doi: 10.1007/978-1-0716-1362-7_20 [DOI] [PubMed] [Google Scholar]
104.Naito Y, Hino K, Bono H, Ui-Tei K. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. 2015;31(7):1120–3. doi: 10.1093/bioinformatics/btu743 [DOI] [PMC free article] [PubMed] [Google Scholar]
105.Guan Q, Lu X, Zeng H, Zhang Y, Zhu J. Heat stress induction of miR398 triggers a regulatory loop that is critical for thermotolerance in Arabidopsis. Plant J. 2013;74(5):840–51. doi: 10.1111/tpj.12169 [DOI] [PubMed] [Google Scholar]
106.Zhang LL, Shao YJ, Ding L, Wang MJ, Davis SJ, Liu JX. XBAT31 regulates thermoresponsive hypocotyl growth through mediating degradation of the thermosensor ELF3 in Arabidopsis. Sci Adv. 2021;7(19):eabf4427. doi: 10.1126/sciadv.abf4427 [DOI] [PMC free article] [PubMed] [Google Scholar]
107.Ibañez C, Delker C, Martinez C, Bürstenbinder K, Janitza P, Lippmann R, et al. Brassinosteroids Dominate Hormonal Regulation of Plant Thermomorphogenesis via BZR1. Curr Biol. 2018;28(2):303-310.e3. doi: 10.1016/j.cub.2017.11.077 [DOI] [PubMed] [Google Scholar]

PLoS Genet. doi: 10.1371/journal.pgen.1011838.r001

Decision Letter 0

Bao-Cai Tan

25 Feb 2025

PGENETICS-D-24-01385

Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants

PLOS Genetics

Dear Dr. Laha,

Thank you for submitting your manuscript to PLOS Genetics. After careful consideration, we feel that it has merit but does not fully meet PLOS Genetics's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 60 days (April 26, 2025). If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosgenetics@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgenetics/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Bao-Cai Tan

Academic Editor

PLOS Genetics

Bao-Cai Tan

Academic Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

Additional Editor Comments (if provided):

Dear Dr. Laha,

I apologize for the delay in reviewing this manuscript, as we have difficulty securing reviewers. As you may see from the review comments, both reviewers find the identification of IPK2s as inositol pyrophosphate synthesizing enzymes and their involvement in heat stress very interesting. I think that it is a new finding that advances the function of inositol pyrophosphate 4/6-InsP7 in plants. However, the reviewers also raised serious questions on data interpretation, experiment design (proper controls used), and the sufficiency of evidence for the conclusion. I agree with the reviewer that necessary controls should be used in the ITC experiments and the EMSA assays in order to draw a convincing conclusion. The IP3/4/5 kinase activities in this manuscript should be compared with these previously reported. In addition, how AtIPK2α enhances the DNA-binding and transcription activity of AtHSFA1b should be addressed. In light of these concerns, I recommend a major revision. I would encourage a resubmission after the questions and concerns are addressed.

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors conducted CE-MS analysis and identified that 4/6-InsP7 is the predominant PP-InsP species present in Arabidopsis and liverworts. They also identified the enzymes responsible for the biosynthesis of 4/6-InsP7 and further tested the evolutionary conservation of these enzymes in generating 4/6-InsP7 and regulating heat stress tolerance. The findings are quite interesting, and I have a few comments.

1. Line 118: The authors state, “To investigate whether 4/6-InsP7 is ubiquitous across land plants.” However, in the manuscript, they only examine the inositol phosphate profiles from liverworts and Arabidopsis, which seems insufficient to support the claim of “land plants.” It would be better to include several land plants of evolutionary significance or cite relevant literature to substantiate this claim.

2. If possible, it would be beneficial to generate an atipk2β(kd)atipk2α mutant to further support the conclusion that AtIPK2α and AtIPK2β act redundantly to regulate the synthesis of 4/6-InsP7 in Arabidopsis.

3. In Supplementary Data Set 3, which column represents the transcripts of IPK2 under heat stress? The authors need to label the treatments clearly.

4. Lines 342-355: The authors utilize the ipk2∆ knockout strain to test the functional conservation among yeast, M. polymorpha, and Arabidopsis. What is known about the role of IPK2 in yeast? The authors need to introduce the function of yeast IPK2 in the introduction.

5. I am curious whether the overexpression (OE) of IPK2 could significantly enhance the heat tolerance of Arabidopsis plants. The manuscript only discusses the phenotypes of complementary lines and does not mention the OE lines.

6. The authors performed EMSA assays and concluded that the AtIPK2α protein enhances the DNA-binding and transcription activity of AtHSFA1b. Is the IPK2 protein localized in the nucleus? Does AtIPK2α interact with AtHSFA1b at the protein level? BiFC, split-luciferase, or Co-IP experiments would be necessary to validate this interaction. The incubation of AtHSFA1b with Ins(1,4,5)P3, InsP6, and 4-InsP7 did not affect the DNA-binding activity of AtHSFA1b. Therefore, AtIPK2 appears to have biological functions in catalyzing the formation of 4/6-InsP7 and enhancing the DNA-binding and transcription activity of AtHSFA1b. Do these two functions occur in the same subcellular location, such as the nucleus? Is AtIPK2 localized in both the cytosol and the nucleus? The authors need to address these questions.

Reviewer #2: The manuscript by Yadav and colleagues describes the identification of IPK2s from the model plants Arabidopsis and Marchantia as inositol pyrophosphate synthesizing enzymes involved in heat stress adaptation. The manuscript reports a number of interesting findings including new mutant alleles for IPK2s and a strong genetic connection between IPK2 function and plant heat stress responses. However, it is unclear of indeed InsP6 is the best substrate for IPK2s and if the enzyme directly regulates cellular 4/6-InsP7 levels as well as the transcriptional activity of plant heat shock transcription factors. I summarize my comments and suggestions below:

1. MINOR Introduction, Line 77-78. There is, to the best of my knowledge, no strong evidence that plant PPIP5Ks are InsP6 kinases. A Mpvip1ge mutant in Marchantia polymorpha has wild-type like levels of 1-InsP7, increased levels of 5-InsP7 and decreased levels of 1,5-InsP8 (https://pubmed.ncbi.nlm.nih.gov/39531477/). This suggests that at least in planta, Vip1 is not an InsP6 kinase, but may use exclusively use 5-InsP7 as substrate to generate 1,5-InsP8.

2. MINOR Line 92-94. 4/6-InsP7 levels have been reported for Arabidopsis seedling and for Marchantia Tak-1 plants here as well https://pubmed.ncbi.nlm.nih.gov/39531477/.

3. MAJOR Line 126-127. 4/6-InsP7 levels have been previously determined in Arabidopsis (references cited in the introduction + https://pubmed.ncbi.nlm.nih.gov/39531477/, see above) seedlings and in Marchantia polymorpha Tak-1 and various mutants in the PP-InsP biosynthesis and catabolic pathway (https://pubmed.ncbi.nlm.nih.gov/39531477/). In this previous study, both in Arabidopsis and in Tak-1 4/6-InsP7 levels are not significantly higher compared to either 1-InsP7, 5-InsP7 or 1,5-InsP8 (compare Fig. 1F and 3G in https://pubmed.ncbi.nlm.nih.gov/39531477/, respectively). Given that the absolute abundance of a certain PP-InsP species is poorly correlated with its biological signaling capacity, I would consider removing the statement that 4,6-InsP7 is "the most abundant InsP7 isomer". It would also be helpful if the authors could plot the total concentrations of InsPs and PP-InsPs per g of tissue weight, rather than as a fraction of InsP6 levels.

5. MINOR A structural superposition comparing HsIP6K3 and AtIPK2 in Ca representation should be presented, with corresponding structural alignment statistics (root mean square deviation in Angstroems comparing X corresponding Calpha atoms matching between the two structures) in revised Figure S6C.

6. MAJOR: Lines 168-180 and Figure 1C. The ITC experiments should to be repeated with appropriate controls. It is not sufficient to report the raw heats from injections of substrate (in the syringe) into the enzyme (in the cell). Essential control experiments are 1) injection of substrate (in the syringe) into buffer (in the cell, 50 mM Tris Cl pH 7.0 and 0.5 mM EDTA) and 2) injection of buffer (in the syringe, 50 mM Tris Cl pH 7.0 and 0.5 mM EDTA) into enzyme (in the cell). The raw heats of these control experiments should be plotted alongside the actual experiment in the upper panels of Figure 1C. Titration of InsPs into free buffer is usually associated with large DPs, this needs to be checked and corrected for. Binding stoichiometries (N) should not be fixed during data fitting and should be reported alongside the derived dissociation constants. It is presently hard to understand how, for example, the raw heats in the top left experiment in panel 1C could be fitted at to the binding curve. The method section should include the injection volumes for each injection and the method of fitting. How was the pH of the ligand in ITC buffer dilutions adjusted? Why is the Kd derived by ITC (0.5 micromolar) drastically different from the Km estimate (~250 micromolar)?

7. MAJOR The newly reported kinase activity for IPK2a needs to be compared to the well established IP3/4/5 kinase activities previously reported for this enzyme. AtIPK2a has been previously characterized as an InsP3 kinase, which can also accept InsP4 and InsP5 as potential substrates (https://pubmed.ncbi.nlm.nih.gov/12226109/) Specifically, AtIPK2a has been characterized to phosphorylate 1,4,5-InsP3 to 1,4,5,6-InsP6, or 1,3,4,5-InsP4 to 1,3,4,5,6-InsP5 i.e. phosphorylating the 6-position of the inositol ring, with Km's in the low micromolar range, and with Vmax of ~ 100 nmol min-1 mg-1. In contrast, the Km for InsP6 is 250 micromolar, with a Vmax of only 2 nmol min-1 mg. This suggests that InsP6 is a very poor substrate for AtIPK2, when compared to InsP3 or InsP4 (https://pubmed.ncbi.nlm.nih.gov/12226109/). The authors should consider performing comparative enzyme kinetics with AtIPK2a and InsP3, InsP4 and InsP6 as substrate to define the best in vitro substrate for this enzyme. Based on the current data the statement line 216-219 is not supported by robust experimentation.

8. MAJOR: Figure 2C The atipk2b/atipk2a knock-down line contains higher levels of InsP3, InsP4 and InsP5 but only very small variations in 4/6-InsP7. How come the difference in for example InsP5 levels between Col-0 (~1.6 pmol mg-1 FW) and atipk2b/atipk2akd line2 (~3 pmol mg-1 FW) are labeled statistical insignificant, while in the case of 4/6-InsP7 much smaller differences (Col-0 ~0.6 pmol mg-1 FW vs. 0.4 pmol mg-1 FW) are labeled as highly significant? Why are the total levels of InsPs/PP-InsPs so different for the Col-0 control when comparing the experiments shown in Figure 2A and 2D)? The authors should provide further details on the statistical tests used and discuss the possibility that the altered InsP3/4/5 levels may indirectly affect 4/5-InsP7 concentrations rather than the IPK2 enzymes themselves (see above, point #7).

10. MAJOR 283-297 and Figure 3A. Consistent with Figure 2C, 4/6-InsP7 levels are reduced in Figure 3A when comparing the Col-0 wild type with the atipk2b/atipk2a knock-down lines. During heat stress, InsP4/6 levels are reduced in Col-0 and in the mutant lines, which to me would suggest that heat stress-induced changes of 4/6-InsP7 levels occur independent of atipk2a and atipk2b. Can the authors explain why (line 296-297) :"Collectively, our data suggests that AtIPK2α and AtIPK2β function together to control cellular levels of 4/6-InsP7 during heat stress in planta." ?

11. MAJOR: Figure 5D, as outlined above it would be worthwhile to compare the InsP6 kinase activity of MpIPMK with its ability to phosphorylate InP3 and InsP4.

12. MAJOR: Figure 6B. Would the accumulation of another InsP3 peak in the Mpipmk knock-out line not argue for this InsP3 being a preferred in vivo substrate for MpIPMK? In line with this, InsP3 levels are up in Supplementary Figure 8C, again indicating that the lower 4/6-InsP7 levels in this mutant could be caused by a missing InsP4 precursor required for 4/6-InsP7 biosynthesis.

13. MINOR: Figure 8A. Based on what criteria were the heat shock response target genes chosen for this analysis?

14. MINOR: Figure 8B. Is it known that HSFA1b is regulating expression of HSP70, HSP22, HSP101, HSP17.6, HSP17.4, HSP18.1 and HSFA2 or how was this particular transcription factor selected? Also, there are many possible molecular scenarios that could explain why expression of AtIPK2a may lead to enhanced transcriptional activity of AtHSFA1b. What is the rational to focus on the direct interaction of the enzyme with the transcription factor, a rather unusual mode of transcription factor regulation? Have other scenarios been tested experimentally?

15. MAJOR: Figure 9 all panels. All gels are not very well resolved (protein-DNA complexes too close to the wells). Consider using a lower percentage of acrylamide to better separate the different molecular species. The EMSA assays require additional controls: Currently, there is no competitor DNA in the EMSA reaction. With the current experimental settings, any positively-charged DBD will bind to the negatively charged probe. Consider including a control experiment with a competitor DNA (like salmon sperm DNA) to establish the binding specificity of the probe. Along these lines, a mutated probe that abolishes binding should be tested alongside. Report molar concentrations for all proteins and probes concentrations. The current figure legend provides an interpretation rather than a description of the experiment. Line 459 change 'a short nucleotide fragment' for 'a short double stranded oligonucletotide'. Figure 9B, the enhancement of DNA binding in Figure 9B could be due to an artifact. Use another enzyme to check that the observed effect is specific to IPK2a (or to a domain of IPK2). Also, which amount of TF is used in Figure 9B? The legend for Figure 9D is incomplete. Figure 9E, If the hypothesis is that the enzyme affects the TF binding by interacting with it, then there should be a band for the TF alone and an upper band corresponding to the TF + AtIPK2a complex binding to the probe. But there seems to be only one band? The best way to address this point would be to identity missense mutations in the enzyme that impair the interaction of this mutant version with the TF and the probe in EMSA assays. The enhancement of DNA-binding in Figure 9E is not convincing. If the AtIPK2a enzyme indeed promotes DNA binding, then the intensity of the free probe should at least decrease. To aid comparison of Figure 9D and Figure 9E, the reactions should be performed with the same amounts of the HSF alone + probe (lane2). The percentage of bound probe should be similar but it is clearly not the same. For consistency, the interaction between the enzyme and the TF should also be tested for the Arabidopsis proteins, not only for the Marchantia orthologs in the Y2H assays, and possibly a second method (co-IP, in vitro pull down) could be used to substantiate this interaction.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy , and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

Figure resubmission:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

PLoS Genet. 2025 Sep 11;21(9):e1011838. doi: 10.1371/journal.pgen.1011838.r002

Author response to Decision Letter 1

10 Jun 2025

Attachment

Submitted filename: Response to Reviewers.pdf

pgen.1011838.s021.pdf^{(380.4KB, pdf)}

PLoS Genet. doi: 10.1371/journal.pgen.1011838.r003

Decision Letter 1

Bao-Cai Tan

22 Jul 2025

PGENETICS-D-24-01385R1

Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants

PLOS Genetics

Dear Dr. Laha,

The revised manuscript has undergone substantial improvements. All concerns have been addressed, except for a few minor ones raised by Reviewer 1. I am pleased to accept this manuscript for publication pending a minor revision. Thanks for submitting your best work to PLOS Genetics.

Please submit your revised manuscript within 30 days. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosgenetics@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgenetics/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Bao-Cai Tan

Academic Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: All my questions have been addressed

Reviewer #2: The revised manuscript by Yadav et al. represents a substantial improvement and now more accurately reflects the experimental work presented. The EMSA assays have been refined, and the discussion surrounding the InsP₃ versus InsP₆ kinase activity has become more balanced.

Figure 1C: The ITC experiments are now supported by appropriate control experiments. However, the observed stoichiometries do not align with a simple 1:1 enzyme–substrate model, likely due to the small differential power signal and considerable data noise. While this does not undermine the experiment, it suggests that the derived dissociation constants may lack accuracy. I recommend leaving the experiments as they are.

Lines 198–200: The current reasoning is convoluted — implying that low activity toward a substrate confirms its correctness is not convincing. It would be more straightforward to state that AtIPK2 is a relatively inefficient InsP₆ kinase, and that InsP₃ appears to be the preferred in vitro substrate.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

Figure resubmission:

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

PLoS Genet. 2025 Sep 11;21(9):e1011838. doi: 10.1371/journal.pgen.1011838.r004

Author response to Decision Letter 2

29 Jul 2025

Attachment

Submitted filename: Response to Reviewer Comments_July 2025.pdf

pgen.1011838.s022.pdf^{(34.4KB, pdf)}

PLoS Genet. doi: 10.1371/journal.pgen.1011838.r005

Decision Letter 2

Bao-Cai Tan

13 Aug 2025

Dear Dr. Laha,

We are pleased to inform you that your manuscript entitled "Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Bao-Cai Tan

Academic Editor

PLOS Genetics

Bao-Cai Tan

Academic Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository . As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website .

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly:

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-24-01385R2

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org .

PLoS Genet. doi: 10.1371/journal.pgen.1011838.r006

Acceptance letter

Bao-Cai Tan

PGENETICS-D-24-01385R2

Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants

Dear Dr Laha,

We are pleased to inform you that your manuscript entitled "

Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Anita Estes

PLOS Genetics

On behalf of:

The PLOS Genetics Team

Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom

plosgenetics@plos.org | +44 (0) 1223-442823

plosgenetics.org | Twitter: @PLOSGenetics

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

(TIF)

pgen.1011838.s001.tif^{(10.3MB, tif)}

S2 Fig. AtIPK2α and AtIPK2β phosphorylates InsP₆ in vitro.

(TIF)

pgen.1011838.s002.tif^{(24.1MB, tif)}

S3 Fig. Generation of atipk2α knockdown lines in the atipk2β-1 knockout plants.

(TIF)

pgen.1011838.s003.tif^{(10.9MB, tif)}

S4 Fig. Seeds of AtIPK2-deficient lines are defective in phytic acid metabolism and heat stress induces AtIPK2 expression.

(TIF)

pgen.1011838.s004.tif^{(8.1MB, tif)}

S5 Fig. Characterization of the atipk2βatipk2α^kd lines expressing AtIPK2β and characterization of AtIPK2α overexpression lines.

(TIF)

pgen.1011838.s005.tif^{(7.5MB, tif)}

S6 Fig. AtIPK2α-type kinases are ubiquitous in green plants and share conserved catalytic motif.

(TIF)

pgen.1011838.s006.tif^{(15.1MB, tif)}

S7 Fig. Structural model and catalytic activity of MpIPMK.

(TIF)

pgen.1011838.s007.tif^{(10.2MB, tif)}

S8 Fig. MpIPMK does not possess yeast Kcs1- or Vip1-like activity.

(TIF)

pgen.1011838.s008.tif^{(12.5MB, tif)}

S9 Fig. Mpipmk knockout plants do not exhibit any obvious growth defects and accumulate InsP₃, InsP₄ and InsP₅ isomers.

(TIF)

pgen.1011838.s009.tif^{(14.5MB, tif)}

(TIF)

pgen.1011838.s010.tif^{(7.5MB, tif)}

S11 Fig. Ins(1,4,5)P_3, InsP₆ and 4/6-InsP₇ do not influence DNA-binding affinity of Arabidopsis heat shock transcription factor.

(TIF)

pgen.1011838.s011.tif^{(4.8MB, tif)}

S12 Fig. MpIPMK physically interacts with MpHSFB1 and InsPs do not directly influence the transcriptional activity of MpHSFB1.

(TIF)

pgen.1011838.s012.tif^{(12MB, tif)}

S1 Table. List of primers used in the study.

(XLSX)

pgen.1011838.s013.xlsx^{(13KB, xlsx)}

S2 Table. List of hits from Phyre² analyses using Human IP6K3.

(XLSX)

pgen.1011838.s014.xlsx^{(16.7KB, xlsx)}

S3 Table. AtIPK2α and AtIPK2β expression analysis under heat stress.

(XLSX)

pgen.1011838.s015.xlsx^{(21KB, xlsx)}

S4 Table. Yeast Strains used in this study.

(XLSX)

pgen.1011838.s016.xlsx^{(9.2KB, xlsx)}

S5 Table. Sequences used in Phylogenetic analysis.

(XLSX)

pgen.1011838.s017.xlsx^{(12.5KB, xlsx)}

S6 Table. MS parameters for MRM transitions.

(XLSX)

pgen.1011838.s018.xlsx^{(10.1KB, xlsx)}

S7 Table. Details of statistical analysis.

(XLSX)

pgen.1011838.s019.xlsx^{(39.5KB, xlsx)}

S8 Table. Raw data file.

(XLSX)

pgen.1011838.s020.xlsx^{(49.3KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.pdf

pgen.1011838.s021.pdf^{(380.4KB, pdf)}

Attachment

Submitted filename: Response to Reviewer Comments_July 2025.pdf

pgen.1011838.s022.pdf^{(34.4KB, pdf)}

Data Availability Statement

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

[pgen.1011838.ref001] 1.Wilson MSC, Livermore TM, Saiardi A. Inositol pyrophosphates: between signalling and metabolism. Biochem J. 2013;452(3):369–79. doi: 10.1042/BJ20130118 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref002] 2.Shears SB. Inositol pyrophosphates: why so many phosphates? Adv Biol Regul. 2015;57:203–16. doi: 10.1016/j.jbior.2014.09.015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref003] 3.Thota SG, Bhandari R. The emerging roles of inositol pyrophosphates in eukaryotic cell physiology. J Biosci. 2015;40(3):593–605. doi: 10.1007/s12038-015-9549-x [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref004] 4.Monserrate JP, York JD. Inositol phosphate synthesis and the nuclear processes they affect. Curr Opin Cell Biol. 2010;22(3):365–73. doi: 10.1016/j.ceb.2010.03.006 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref005] 5.Barker CJ, Illies C, Gaboardi GC, Berggren P-O. Inositol pyrophosphates: structure, enzymology and function. Cell Mol Life Sci. 2009;66(24):3851–71. doi: 10.1007/s00018-009-0115-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref006] 6.Nguyen Trung M, Furkert D, Fiedler D. Versatile signaling mechanisms of inositol pyrophosphates. Curr Opin Chem Biol. 2022;70:102177. doi: 10.1016/j.cbpa.2022.102177 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref007] 7.Chabert V, Kim G-D, Qiu D, Liu G, Michaillat Mayer L, Jamsheer K M, et al. Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81. Elife. 2023;12:RP87956. doi: 10.7554/eLife.87956 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref008] 8.Laha D, Portela-Torres P, Desfougères Y, Saiardi A. Inositol phosphate kinases in the eukaryote landscape. Adv Biol Regul. 2021;79:100782. doi: 10.1016/j.jbior.2020.100782 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref009] 9.Wilson MS, Jessen HJ, Saiardi A. The inositol hexakisphosphate kinases IP6K1 and -2 regulate human cellular phosphate homeostasis, including XPR1-mediated phosphate export. J Biol Chem. 2019;294(30):11597–608. doi: 10.1074/jbc.RA119.007848 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref010] 10.Li X, Gu C, Hostachy S, Sahu S, Wittwer C, Jessen HJ, et al. Control of XPR1-dependent cellular phosphate efflux by InsP8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling. Proc Natl Acad Sci U S A. 2020;117(7):3568–74. doi: 10.1073/pnas.1908830117 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref011] 11.Lee Y-S, Mulugu S, York JD, O’Shea EK. Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science. 2007;316(5821):109–12. doi: 10.1126/science.1139080 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref012] 12.Szijgyarto Z, Garedew A, Azevedo C, Saiardi A. Influence of inositol pyrophosphates on cellular energy dynamics. Science. 2011;334(6057):802–5. doi: 10.1126/science.1211908 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref013] 13.Qin N, Li L, Ji X, Pereira R, Chen Y, Yin S, et al. Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast. Cell. 2023;186(4):748-763.e15. doi: 10.1016/j.cell.2023.01.014 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref014] 14.Gu C, Liu J, Liu X, Zhang H, Luo J, Wang H, et al. Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line. Proc Natl Acad Sci U S A. 2021;118(10):e2020187118. doi: 10.1073/pnas.2020187118 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref015] 15.Zhang X, Li N, Zhang J, Zhang Y, Yang X, Luo Y, et al. 5-IP7 is a GPCR messenger mediating neural control of synaptotagmin-dependent insulin exocytosis and glucose homeostasis. Nat Metab. 2021;3(10):1400–14. doi: 10.1038/s42255-021-00468-7 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref016] 16.Saiardi A, Erdjument-Bromage H, Snowman AM, Tempst P, Snyder SH. Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases. Curr Biol. 1999;9(22):1323–6. doi: 10.1016/s0960-9822(00)80055-x [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref017] 17.Mulugu S, Bai W, Fridy PC, Bastidas RJ, Otto JC, Dollins DE, et al. A conserved family of enzymes that phosphorylate inositol hexakisphosphate. Science. 2007;316(5821):106–9. doi: 10.1126/science.1139099 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref018] 18.Wang H, Falck JR, Hall TMT, Shears SB. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding. Nat Chem Biol. 2011;8(1):111–6. doi: 10.1038/nchembio.733 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref019] 19.Desfougères Y, Portela-Torres P, Qiu D, Livermore TM, Harmel RK, Borghi F, et al. The inositol pyrophosphate metabolism of Dictyostelium discoideum does not regulate inorganic polyphosphate (polyP) synthesis. Adv Biol Regul. 2022;83:100835. doi: 10.1016/j.jbior.2021.100835 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref020] 20.York JD, Odom AR, Murphy R, Ives EB, Wente SR. A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science. 1999;285(5424):96–100. doi: 10.1126/science.285.5424.96 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref021] 21.Odom AR, Stahlberg A, Wente SR, York JD. A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science. 2000;287(5460):2026–9. doi: 10.1126/science.287.5460.2026 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref022] 22.Saiardi A, Caffrey JJ, Snyder SH, Shears SB. Inositol polyphosphate multikinase (ArgRIII) determines nuclear mRNA export in Saccharomyces cerevisiae. FEBS Lett. 2000;468(1):28–32. doi: 10.1016/s0014-5793(00)01194-7 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref023] 23.Draskovic P, Saiardi A, Bhandari R, Burton A, Ilc G, Kovacevic M, et al. Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups. Chem Biol. 2008;15(3):274–86. doi: 10.1016/j.chembiol.2008.01.011 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref024] 24.Dollins DE, Bai W, Fridy PC, Otto JC, Neubauer JL, Gattis SG, et al. Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling. P Natl Acad Sci USA. 2020;117(17):9356–64. doi: 10.1073/pnas.1908875117 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref025] 25.Lin H, Fridy PC, Ribeiro AA, Choi JH, Barma DK, Vogel G, et al. Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases. J Biol Chem. 2009;284(3):1863–72. doi: 10.1074/jbc.M805686200 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref026] 26.Lorenzo-Orts L, Couto D, Hothorn M. Identity and functions of inorganic and inositol polyphosphates in plants. New Phytol. 2020;225(2):637–52. doi: 10.1111/nph.16129 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref027] 27.Riemer E, Pullagurla NJ, Yadav R, Rana P, Jessen HJ, Kamleitner M, et al. Regulation of plant biotic interactions and abiotic stress responses by inositol polyphosphates. Front Plant Sci. 2022;13:944515. doi: 10.3389/fpls.2022.944515 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref028] 28.Cridland C, Gillaspy G. Inositol Pyrophosphate Pathways and Mechanisms: What Can We Learn from Plants? Molecules. 2020;25(12):2789. doi: 10.3390/molecules25122789 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref029] 29.Whitfield H, White G, Sprigg C, Riley AM, Potter BVL, Hemmings AM, et al. 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. 2020;477(14):2621–38. doi: 10.1042/BCJ20200423 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref030] 30.Laha D, Parvin N, Hofer A, Giehl RFH, Fernandez-Rebollo N, von Wirén N, et al. Arabidopsis ITPK1 and ITPK2 Have an Evolutionarily Conserved Phytic Acid Kinase Activity. ACS Chem Biol. 2019;14(10):2127–33. doi: 10.1021/acschembio.9b00423 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref031] 31.Adepoju O, Williams SP, Craige B, Cridland CA, Sharpe AK, Brown AM, et al. Inositol Trisphosphate Kinase and Diphosphoinositol Pentakisphosphate Kinase Enzymes Constitute the Inositol Pyrophosphate Synthesis Pathway in Plants. bioRxiv. 2019;724914. doi: 10.1101/724914 [DOI] [Google Scholar]

[pgen.1011838.ref032] 32.Zong G, Shears SB, Wang H. Structural and catalytic analyses of the InsP6 kinase activities of higher plant ITPKs. FASEB J. 2022;36(7):e22380. doi: 10.1096/fj.202200393R [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref033] 33.Riemer E, Qiu D, Laha D, Harmel RK, Gaugler P, Gaugler V, et al. ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis. Mol Plant. 2021;14(11):1864–80. doi: 10.1016/j.molp.2021.07.011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref034] 34.Laha NP, Giehl RFH, Riemer E, Qiu D, Pullagurla NJ, Schneider R, et al. INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-dependent inositol polyphosphates regulate auxin responses in Arabidopsis. Plant Physiol. 2022;190(4):2722–38. doi: 10.1093/plphys/kiac425 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref035] 35.Laha D, Johnen P, Azevedo C, Dynowski M, Weiß M, Capolicchio S, et al. VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis. Plant Cell. 2015;27(4):1082–97. doi: 10.1105/tpc.114.135160 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref036] 36.Desai M, Rangarajan P, Donahue JL, Williams SP, Land ES, Mandal MK, et al. Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants. Plant J. 2014;80(4):642–53. doi: 10.1111/tpj.12669 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref037] 37.Couso I, Evans BS, Li J, Liu Y, Ma F, Diamond S, et al. Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas. Plant Cell. 2016;28(9):2026–42. doi: 10.1105/tpc.16.00351 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref038] 38.Zhu J, Lau K, Puschmann R, Harmel RK, Zhang Y, Pries V, et al. Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis. Elife. 2019;8:e43582. doi: 10.7554/eLife.43582 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref039] 39.Laha D, Parvin N, Dynowski M, Johnen P, Mao H, Bitters ST, et al. Inositol Polyphosphate Binding Specificity of the Jasmonate Receptor Complex. Plant Physiol. 2016;171(4):2364–70. doi: 10.1104/pp.16.00694 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref040] 40.Dong J, Ma G, Sui L, Wei M, Satheesh V, Zhang R, et al. Inositol Pyrophosphate InsP8 Acts as an Intracellular Phosphate Signal in Arabidopsis. Mol Plant. 2019;12(11):1463–73. doi: 10.1016/j.molp.2019.08.002 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref041] 41.Land ES, Cridland CA, Craige B, Dye A, Hildreth SB, Helm RF, et al. A Role for Inositol Pyrophosphates in the Metabolic Adaptations to Low Phosphate in Arabidopsis. Metabolites. 2021;11(9):601. doi: 10.3390/metabo11090601 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref042] 42.Guan Z, Zhang Q, Zhang Z, Zuo J, Chen J, Liu R, et al. Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 - PHR2 complex. Nat Commun. 2022;13(1):1581. doi: 10.1038/s41467-022-29275-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref043] 43.Kuo H-F, Hsu Y-Y, Lin W-C, Chen K-Y, Munnik T, Brearley CA, et al. Arabidopsis inositol phosphate kinases IPK1 and ITPK1 constitute a metabolic pathway in maintaining phosphate homeostasis. Plant J. 2018;:10.1111/tpj.13974. doi: 10.1111/tpj.13974 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref044] 44.Pullagurla NJ, Shome S, Liu G, Jessen HJ, Laha D. Orchestration of phosphate homeostasis by the ITPK1-type inositol phosphate kinase in the liverwort Marchantia polymorpha. Plant Physiol. 2025;197(2):kiae454. doi: 10.1093/plphys/kiae454 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref045] 45.Pullagurla NJ, Shome S, Yadav R, Laha D. ITPK1 Regulates Jasmonate-Controlled Root Development in Arabidopsis thaliana. Biomolecules. 2023;13(9):1368. doi: 10.3390/biom13091368 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref046] 46.Gulabani H, Goswami K, Walia Y, Roy A, Noor JJ, Ingole KD, et al. Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalk between SA-dependent immunity and phosphate-starvation responses. Plant Cell Rep. 2022;41(2):347–63. doi: 10.1007/s00299-021-02812-3 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref047] 47.Chalak K, Yadav R, Liu G, Rana P, Jessen HJ, Laha D. Functional Conservation of the DDP1-type Inositol Pyrophosphate Phosphohydrolases in Land Plant. Biochemistry. 2024;63(21):2723–8. doi: 10.1021/acs.biochem.4c00458 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref048] 48.Laurent F, Bartsch SM, Shukla A, Rico-Resendiz F, Couto D, Fuchs C, et al. Inositol pyrophosphate catabolism by three families of phosphatases regulates plant growth and development. PLoS Genet. 2024;20(11):e1011468. doi: 10.1371/journal.pgen.1011468 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref049] 49.Dovey CM, Diep J, Clarke BP, Hale AT, McNamara DE, Guo H, et al. MLKL Requires the Inositol Phosphate Code to Execute Necroptosis. Mol Cell. 2018;70(5):936-948.e7. doi: 10.1016/j.molcel.2018.05.010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref050] 50.Stevenson-Paulik J, Bastidas RJ, Chiou S-T, Frye RA, York JD. Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases. Proc Natl Acad Sci U S A. 2005;102(35):12612–7. doi: 10.1073/pnas.0504172102 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref051] 51.Stevenson-Paulik J, Odom AR, York JD. Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases. J Biol Chem. 2002;277(45):42711–8. doi: 10.1074/jbc.M209112200 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref052] 52.Wang H, Shears SB. Structural features of human inositol phosphate multikinase rationalize its inositol phosphate kinase and phosphoinositide 3-kinase activities. J Biol Chem. 2017;292(44):18192–202. doi: 10.1074/jbc.M117.801845 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref053] 53.Xia H-J, Brearley C, Elge S, Kaplan B, Fromm H, Mueller-Roeber B. Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex. Plant Cell. 2003;15(2):449–63. doi: 10.1105/tpc.006676 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref054] 54.Chen Y, Han JM, Wang XY, Chen XY, Li YH, Yuan CY, et al. OsIPK2, a Rice Inositol Polyphosphate Kinase Gene, Is Involved in Phosphate Homeostasis and Root Development. Plant Cell Physiol. 2023;64(8):893–905. doi: 10.1093/pcp/pcad052 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref055] 55.Bowman JL. The origin of a land flora. Nat Plants. 2022;8(12):1352–69. doi: 10.1038/s41477-022-01283-y [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref056] 56.Qiu D, Wilson MS, Eisenbeis VB, Harmel RK, Riemer E, Haas TM, et al. Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry. Nat Commun. 2020;11(1):6035. doi: 10.1038/s41467-020-19928-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref057] 57.Qiu D, Gu C, Liu G, Ritter K, Eisenbeis VB, Bittner T, et al. Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues. Chem Sci. 2022;14(3):658–67. doi: 10.1039/d2sc05147h [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref058] 58.Liu G, Riemer E, Schneider R, Cabuzu D, Bonny O, Wagner CA, et al. The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones. RSC Chem Biol. 2023;4(4):300–9. doi: 10.1039/d2cb00235c [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref059] 59.Fassetti F, Leone O, Palopoli L, Rombo SE, Saiardi A. IP6K gene identification in plant genomes by tag searching. BMC Proc. 2011;5 Suppl 2(Suppl 2):S1. doi: 10.1186/1753-6561-5-S2-S1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref060] 60.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10(6):845–58. doi: 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref061] 61.Zhan H, Zhong Y, Yang Z, Xia H. Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development, pollen tube guidance and embryogenesis. Plant J. 2015;82(5):758–71. doi: 10.1111/tpj.12846 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref062] 62.Raboy V. The ABCs of low-phytate crops. Nat Biotechnol. 2007;25(8):874–5. doi: 10.1038/nbt0807-874 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref063] 63.Kim S-I, Tai TH. Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach. Mol Genet Genomics. 2011;286(2):119–33. doi: 10.1007/s00438-011-0631-2 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref064] 64.Pisani F, Livermore T, Rose G, Chubb JR, Gaspari M, Saiardi A. Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis. PLoS One. 2014;9(1):e85533. doi: 10.1371/journal.pone.0085533 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref065] 65.Voglmaier SM, Bembenek ME, Kaplin AI, Dormán G, Olszewski JD, Prestwich GD, et al. Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor. Proc Natl Acad Sci U S A. 1996;93(9):4305–10. doi: 10.1073/pnas.93.9.4305 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref066] 66.Harmel RK, Puschmann R, Nguyen Trung M, Saiardi A, Schmieder P, Fiedler D. Harnessing 13C-labeled myo-inositol to interrogate inositol phosphate messengers by NMR. Chem Sci. 2019;10(20):5267–74. doi: 10.1039/c9sc00151d [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref067] 67.Haas TM, Mundinger S, Qiu D, Jork N, Ritter K, Dürr-Mayer T, et al. Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18 O-Phosphoramidites. Angew Chem Int Ed Engl. 2022;61(5):e202112457. doi: 10.1002/anie.202112457 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref068] 68.Ritter K, Jork N, Unmüßig A-S, Köhn M, Jessen HJ. Assigning the Absolute Configuration of Inositol Poly- and Pyrophosphates by NMR Using a Single Chiral Solvating Agent. Biomolecules. 2023;13(7):1150. doi: 10.3390/biom13071150 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref069] 69.Parvin N, Carrie C, Pabst I, Läßer A, Laha D, Paul MV, et al. TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana. Mol Plant. 2017;10(4):575–89. doi: 10.1016/j.molp.2016.12.012 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref070] 70.Wielopolska A, Townley H, Moore I, Waterhouse P, Helliwell C. A high-throughput inducible RNAi vector for plants. Plant Biotechnol J. 2005;3(6):583–90. doi: 10.1111/j.1467-7652.2005.00149.x [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref071] 71.Wilson MSC, Bulley SJ, Pisani F, Irvine RF, Saiardi A. A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine. Open Biol. 2015;5(3):150014. doi: 10.1098/rsob.150014 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref072] 72.Kilian J, Whitehead D, Horak J, Wanke D, Weinl S, Batistic O, et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 2007;50(2):347–63. doi: 10.1111/j.1365-313X.2007.03052.x [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref073] 73.Delker C, Quint M, Wigge PA. Recent advances in understanding thermomorphogenesis signaling. Curr Opin Plant Biol. 2022;68:102231. doi: 10.1016/j.pbi.2022.102231 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref074] 74.Casal JJ, Balasubramanian S. Thermomorphogenesis. Annu Rev Plant Biol. 2019;70:321–46. doi: 10.1146/annurev-arplant-050718-095919 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref075] 75.Quint M, Delker C, Balasubramanian S, Balcerowicz M, Casal JJ, Castroverde CDM, et al. 25 Years of thermomorphogenesis research: milestones and perspectives. Trends Plant Sci. 2023;28(10):1098–100. doi: 10.1016/j.tplants.2023.07.001 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref076] 76.Li J, Cao Y, Zhang J, Zhu C, Tang G, Yan J. The miR165/166-PHABULOSA module promotes thermotolerance by transcriptionally and posttranslationally regulating HSFA1. Plant Cell. 2023;35(8):2952–71. doi: 10.1093/plcell/koad121 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref077] 77.Bowman JL, Kohchi T, Yamato KT, Jenkins J, Shu S, Ishizaki K, et al. Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome. Cell. 2017;171(2):287–304.e15. doi: 10.1016/j.cell.2017.09.030 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref078] 78.Kohchi T, Yamato KT, Ishizaki K, Yamaoka S, Nishihama R. Development and Molecular Genetics of Marchantia polymorpha. Annu Rev Plant Biol. 2021;72:677–702. doi: 10.1146/annurev-arplant-082520-094256 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref079] 79.Bowman JL, Araki T, Arteaga-Vazquez MA, Berger F, Dolan L, Haseloff J, et al. The Naming of Names: Guidelines for Gene Nomenclature in Marchantia. Plant Cell Physiol. 2016;57(2):257–61. doi: 10.1093/pcp/pcv193 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref080] 80.Sugano SS, Shirakawa M, Takagi J, Matsuda Y, Shimada T, Hara-Nishimura I, et al. CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L. Plant Cell Physiol. 2014;55(3):475–81. doi: 10.1093/pcp/pcu014 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref081] 81.Sugano SS, Nishihama R, Shirakawa M, Takagi J, Matsuda Y, Ishida S, et al. Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha. PLoS One. 2018;13(10):e0205117. doi: 10.1371/journal.pone.0205117 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref082] 82.Ludwig W, Hayes S, Trenner J, Delker C, Quint M. On the evolution of plant thermomorphogenesis. J Exp Bot. 2021;:erab310. doi: 10.1093/jxb/erab310 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref083] 83.Wu T-Y, Hoh KL, Boonyaves K, Krishnamoorthi S, Urano D. Diversification of heat shock transcription factors expanded thermal stress responses during early plant evolution. Plant Cell. 2022;34(10):3557–76. doi: 10.1093/plcell/koac204 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref084] 84.Hahn A, Bublak D, Schleiff E, Scharf K-D. Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato. Plant Cell. 2011;23(2):741–55. doi: 10.1105/tpc.110.076018 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref085] 85.Yang J, Qu X, Ji L, Li G, Wang C, Wang C, et al. PIF4 Promotes Expression of HSFA2 to Enhance Basal Thermotolerance in Arabidopsis. Int J Mol Sci. 2022;23(11):6017. doi: 10.3390/ijms23116017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref086] 86.Yoshida T, Ohama N, Nakajima J, Kidokoro S, Mizoi J, Nakashima K, et al. Arabidopsis HsfA1 transcription factors function as the main positive regulators in heat shock-responsive gene expression. Mol Genet Genomics. 2011;286(5–6):321–32. doi: 10.1007/s00438-011-0647-7 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref087] 87.Liu H-C, Liao H-T, Charng Y-Y. The role of class A1 heat shock factors (HSFA1s) in response to heat and other stresses in Arabidopsis. Plant Cell Environ. 2011;34(5):738–51. doi: 10.1111/j.1365-3040.2011.02278.x [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref088] 88.Sun J, Qi L, Li Y, Chu J, Li C. PIF4-mediated activation of YUCCA8 expression integrates temperature into the auxin pathway in regulating arabidopsis hypocotyl growth. PLoS Genet. 2012;8(3):e1002594. doi: 10.1371/journal.pgen.1002594 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref089] 89.Ohama N, Kusakabe K, Mizoi J, Zhao H, Kidokoro S, Koizumi S, et al. The Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression. Plant Cell. 2016;28(1):181–201. doi: 10.1105/tpc.15.00435 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref090] 90.Liu H, Charng Y. Common and distinct functions of Arabidopsis class A1 and A2 heat shock factors in diverse abiotic stress responses and development. Plant Physiol. 2013;163(1):276–90. doi: 10.1104/pp.113.221168 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref091] 91.Malabanan MM, Blind RD. Inositol polyphosphate multikinase (IPMK) in transcriptional regulation and nuclear inositide metabolism. Biochem Soc Trans. 2016;44(1):279–85. doi: 10.1042/BST20150225 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref092] 92.Sang S, Chen Y, Yang Q, Wang P. Arabidopsis inositol polyphosphate multikinase delays flowering time through mediating transcriptional activation of FLOWERING LOCUS C. J Exp Bot. 2017;68(21–22):5787–800. doi: 10.1093/jxb/erx397 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref093] 93.Beon J, Han S, Yang H, Park SE, Hyun K, Lee S-Y, et al. Inositol polyphosphate multikinase physically binds to the SWI/SNF complex and modulates BRG1 occupancy in mouse embryonic stem cells. Elife. 2022;11:e73523. doi: 10.7554/eLife.73523 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref094] 94.Sowd GA, Stivison EA, Chapagain P, Hale AT, Poland JC, Rameh LE, et al. IPMK regulates HDAC3 activity and histone H4 acetylation in human cells. bioRxiv. 2024;2024.04.29.591660. doi: 10.1101/2024.04.29.591660 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref095] 95.Ahn H, Yu J, Ryu K, Ryu J, Kim S, Park JY, et al. Single-molecule analysis reveals that IPMK enhances the DNA-binding activity of the transcription factor SRF. Nucleic Acids Res. 2025;53(1):gkae1281. doi: 10.1093/nar/gkae1281 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref096] 96.Luo J, Jiang J, Sun S, Wang X. Brassinosteroids promote thermotolerance through releasing BIN2-mediated phosphorylation and suppression of HsfA1 transcription factors in Arabidopsis. Plant Commun. 2022;3(6):100419. doi: 10.1016/j.xplc.2022.100419 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref097] 97.Blüher D, Laha D, Thieme S, Hofer A, Eschen-Lippold L, Masch A, et al. A 1-phytase type III effector interferes with plant hormone signaling. Nat Commun. 2017;8(1):2159. doi: 10.1038/s41467-017-02195-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref098] 98.Raboy V, Gerbasi PF, Young KA, Stoneberg SD, Pickett SG, Bauman AT, et al. Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1. Plant Physiol. 2000;124(1):355–68. doi: 10.1104/pp.124.1.355 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref099] 99.Nagy R, Grob H, Weder B, Green P, Klein M, Frelet-Barrand A, et al. The Arabidopsis ATP-binding cassette protein AtMRP5/AtABCC5 is a high affinity inositol hexakisphosphate transporter involved in guard cell signaling and phytate storage. J Biol Chem. 2009;284(48):33614–22. doi: 10.1074/jbc.M109.030247 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref100] 100.Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38(7):3022–7. doi: 10.1093/molbev/msab120 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref101] 101.Gietz D, St Jean A, Woods RA, Schiestl RH. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992;20(6):1425. doi: 10.1093/nar/20.6.1425 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref102] 102.Azevedo C, Saiardi A. Extraction and analysis of soluble inositol polyphosphates from yeast. Nat Protoc. 2006;1(5):2416–22. doi: 10.1038/nprot.2006.337 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref103] 103.Laha D, Kamleitner M, Johnen P, Schaaf G. Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC. Methods Mol Biol. 2021;2295:365–78. doi: 10.1007/978-1-0716-1362-7_20 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref104] 104.Naito Y, Hino K, Bono H, Ui-Tei K. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. 2015;31(7):1120–3. doi: 10.1093/bioinformatics/btu743 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref105] 105.Guan Q, Lu X, Zeng H, Zhang Y, Zhu J. Heat stress induction of miR398 triggers a regulatory loop that is critical for thermotolerance in Arabidopsis. Plant J. 2013;74(5):840–51. doi: 10.1111/tpj.12169 [DOI] [PubMed] [Google Scholar]

[pgen.1011838.ref106] 106.Zhang LL, Shao YJ, Ding L, Wang MJ, Davis SJ, Liu JX. XBAT31 regulates thermoresponsive hypocotyl growth through mediating degradation of the thermosensor ELF3 in Arabidopsis. Sci Adv. 2021;7(19):eabf4427. doi: 10.1126/sciadv.abf4427 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1011838.ref107] 107.Ibañez C, Delker C, Martinez C, Bürstenbinder K, Janitza P, Lippmann R, et al. Brassinosteroids Dominate Hormonal Regulation of Plant Thermomorphogenesis via BZR1. Curr Biol. 2018;28(2):303-310.e3. doi: 10.1016/j.cub.2017.11.077 [DOI] [PubMed] [Google Scholar]

PERMALINK

Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants

Ranjana Yadav

Guizhen Liu

Priyanshi Rana

Naga Jyothi Pullagurla

Danye Qiu

Henning J Jessen

Debabrata Laha

Roles

Abstract

Author summary

Introduction

Results

4/6-InsP7 is ubiquitous in the studied land plants

Fig 1. 4/6-InsP7 represents the major form of InsP7 isomer in the studied land plant tissues and is synthesized by the Arabidopsis IPK2 proteins in vitro.

Arabidopsis IPK2 proteins phosphorylate InsP6 to generate 4/6-InsP7 as the major PP-InsP species in vitro

AtIPK2α and AtIPK2β act redundantly to control the synthesis of 4/6-InsP7 in planta

Fig 2. Arabidopsis AtIPK2 α and AtIPK2 β regulate cellular level of 4/6-InsP7. A.

Heat stress specifically targets cellular levels of 4/6-InsP7

Fig 3. Heat stress specifically targets 4/6-InsP7.

AtIPK2α and AtIPK2β cooperate to control heat stress acclimation in Arabidopsis

Fig 4. AtIPK2α and AtIPK2β act redundantly to control heat stress acclimation in Arabidopsis.

AtIPK2 homologs are ubiquitous across plant kingdom and PP-InsP synthase activity of IPK2 is conserved in the liverwort M. polymorpha

Fig 5. M. polymorpha genome encodes a functional yeast Ipk2 homolog, MpIPMK that phosphorylates InsP6 to generate PP-InsP isomers in vitro.

MpIPMK contributes to 4/6-InsP7 synthesis in planta and controls heat stress responses

Fig 6. MpIPMK activity is essential for maintaining the major pool of InsP7 during heat stress and it regulates heat stress acclimation in M. polymorpha.

M. polymorpha IPMK rescues the altered heat stress tolerance of Arabidopsis IPK2-defective plants

Fig 7. Arabidopsis and M. polymorpha share a functional IPK2 homolog.

IPK2 promotes the transcriptional activity of HSF through both catalytic-dependent and catalytic-independent mechanisms

Fig 8. AtIPK2α modulates the transcriptional activity of HSFs.

Fig 9. IPK2-type proteins interact physically with HSFs and enhance the DNA-binding activity of HSF in vitro.

Discussion

Methods

Phylogenetic analysis

Plant materials and growth conditions

Molecular cloning

Protein expression and purification

Isothermal titration calorimetry (ITC)

In vitro kinase assay

Enrichment of inositol phosphate using titanium dioxide bead

CE-MS analysis

Yeast strains and transformation

RNA extraction, cDNA synthesis and gene expression analyses

Extraction and HPLC analysis of inositol phosphates

Cloning and plasmid construction for plant transformation

Thallus transformation of M. polymorpha

Construction of RNAi plasmid and generation of knockdown lines of in A. thaliana

Thermotolerance assay

GUS staining

Dual Luciferase reporter assay

Bimolecular fluorescence complementation assay (BiFC)

Electrophoretic mobility shift assay (EMSA)

Yeast two hybrid assay

Statistical analysis

Accession numbers

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Bao-Cai Tan

Roles

Author response to Decision Letter 1

Decision Letter 1

Bao-Cai Tan

Roles

Author response to Decision Letter 2

Decision Letter 2

Bao-Cai Tan

Roles

Acceptance letter

Bao-Cai Tan

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP₇ in land plants

4/6-InsP₇ is ubiquitous in the studied land plants

Fig 1. 4/6-InsP₇ represents the major form of InsP₇ isomer in the studied land plant tissues and is synthesized by the Arabidopsis IPK2 proteins in vitro.

Arabidopsis IPK2 proteins phosphorylate InsP₆ to generate 4/6-InsP₇ as the major PP-InsP species in vitro

AtIPK2α and AtIPK2β act redundantly to control the synthesis of 4/6-InsP₇ in planta

Fig 2. Arabidopsis AtIPK2 α and AtIPK2 β regulate cellular level of 4/6-InsP_7. A.

Heat stress specifically targets cellular levels of 4/6-InsP₇

Fig 3. Heat stress specifically targets 4/6-InsP₇.

Fig 5. M. polymorpha genome encodes a functional yeast Ipk2 homolog, MpIPMK that phosphorylates InsP₆ to generate PP-InsP isomers in vitro.

MpIPMK contributes to 4/6-InsP₇ synthesis in planta and controls heat stress responses

Fig 6. MpIPMK activity is essential for maintaining the major pool of InsP₇ during heat stress and it regulates heat stress acclimation in M. polymorpha.