Kinase MxMPK4-1 and calmodulin-binding protein MxIQM3 enhance apple root acidification during Fe deficiency

Abstract

Iron (Fe) deficiency is a long-standing issue in plant mineral nutrition. Ca²⁺ signals and the mitogen-activated protein kinase (MAPK) cascade are frequently activated in parallel to perceive external cues, but their interplay under Fe deficiency stress remains largely unclear. Here, the kinase MxMPK4-1, which is induced during the response to Fe deficiency stress in apple rootstock Malus xiaojinensis, cooperates with IQ-motif containing protein3 (MxIQM3). MxIQM3 gene expression, protein abundance, and phosphorylation level increased under Fe deficiency stress. The overexpression of MxIQM3 in apple calli and rootstocks mitigated the Fe deficiency phenotype and improved stress tolerance, whereas RNA interference or silencing of MxIQM3 in apple calli and rootstocks, respectively, worsened the phenotype and reduced tolerance to Fe deficiency. MxMPK4-1 interacted with MxIQM3 and subsequently phosphorylated MxIQM3 at Ser393, and co-expression of MxMPK4-1 and MxIQM3 in apple calli and rootstocks enhanced Fe deficiency responses. Furthermore, MxIQM3 interacted with the central-loop region of the plasma membrane (PM) H⁺-ATPase MxHA2. Phospho-mimicking mutation of MxIQM3 at Ser393 inhibited binding to MxHA2, but phospho-abolishing mutation promoted interaction with both the central-loop and C terminus of MxHA2, demonstrating phosphorylation of MxIQM3 caused dissociation from MxHA2 and therefore increased H⁺ secretion. Moreover, Ca²⁺/MxCAM7 (Calmodulin7) regulated the MxMPK4-1-MxIQM3 module in response to Fe deficiency stress. Overall, our results demonstrate that MxMPK4-1-MxIQM3 forms a functional complex and positively regulates PM H⁺-ATPase activity in Fe deficiency responses, revealing a versatile mechanism of Ca²⁺/MxCAM7 signaling and MAPK cascade under Fe deficiency stress.

Introduction

Iron (Fe) is one of the trace elements that are indispensable for plant growth and development. Although the soil is rich in Fe, plants cannot efficiently absorb and utilize it in neutral or alkaline soil (Jeong and Guerinot, 2009; Gratz et al., 2019). The stress of Fe deficiency causes serious adverse effects on plants (Kobayashi and Nishizawa, 2014), including deleterious effects on the productivity and quality of fruits (Alvarez-Fernandez et al., 2003). Plants have evolved two systems to manage Fe deficiency stress: strategy I and strategy II (Marschner and Romheld, 1994). Strategy I primarily occurs in dicotyledons and non-gramineous monocotyledons. These plants involve three processes in Fe deficiency stress: H⁺ secretion to acidify the rhizosphere, the reduction of Fe³⁺ to Fe²⁺, and the transportation of Fe²⁺ into root cells (Kobayashi and Nishizawa, 2012). Among these processes, functional genes are involved, such as the gene AHA2/AHA7 (Arabidopsis thaliana PLASMA MEMBRANE PROTON ATPASE2/7), which encodes the plasma membrane (PM) H⁺-ATPase to promote H⁺ secretion and enhance the solubility of Fe (Walker and Connolly, 2008; Yang et al., 2019). The ferric reduction oxidase2 (FRO2) is a trivalent Fe oxidoreductase, and the increase of its activity under Fe deficiency stress will promote the reduction of Fe³⁺ to Fe²⁺ (Robinson et al., 1999). The Fe-regulator transporter1 (IRT1) that is regulated by Fe encodes a transporter of Fe²⁺ across membranes, which facilitates the absorption and utilization of Fe²⁺ into root cells (Eide et al., 1996). Under Fe deficiency stress, these processes are generally regulated by the FER-like iron deficiency-induced transcription factor (FIT) to defend against this stress (Schwarz and Bauer, 2020; Wu et al., 2022).

Free Ca²⁺ is a universal signal that participates in diverse physiological and biochemical processes in plants, and the increases in cytoplasmic calcium [Ca²⁺]_cyt happen rapidly as a consequence of the perception of external stimuli (Gilroy et al., 2016; Ju et al., 2022). Ca²⁺ sensors or Ca²⁺-binding proteins decode the Ca²⁺ signal to activate downstream factors to adaptive extracellular alteration. For example, Fe deficiency stress induces [Ca²⁺]_cyt to activate CBL-INTERACTING PROTEIN KINASE11 (CIPK11), which subsequently phosphorylates FIT to regulate the Fe deficiency responses (Gratz et al., 2019). Moreover, Ca²⁺/calmodulin (CAM) is also one of the decoding Ca²⁺ signaling pathways, but individual CAMs are naturally unequipped with biochemical or enzymatic activity (Reddy et al., 2002; Kushwaha et al., 2008). CAMs typically bind to a calmodulin-binding protein (CaMBP) to regulate the physiological functions of plant cells (Bouche et al., 2005). CaMBP proteins can bind to CAMs through Ca²⁺-dependent or independent binding domains, and the IQ motif was the first Ca²⁺-independent CAM-binding domain discovered (Knight and Kendrickjones, 1993; Bhler and Rhoads, 2002). Six members of the IQ-motif containing protein (IQM) family have been identified in Arabidopsis (Arabidopsis thaliana) (AtIQM1-AtIQM6). AtIQM1 plays a positive role in the biosynthesis of jasmonic acid and defense against Botrytis cinerea by increasing the activities of enzymes catalase (CAT2) and acyl-coenzyme A oxidase (ACX2 and ACX3), and the binding of CaM5 to IQM1 further helps to increase CAT2 activity (Lv et al., 2019). AtIQM3 could be involved in regulation of lateral root number and main root length of seedlings by inhibiting the biosynthesis of gibberellin (Xu et al., 2019). The CAM-IQM module was recently reported to involve auxin-induced calli formation and lateral root development (Zhang et al., 2022). To our knowledge, the functional studies of IQ-motif proteins have not been reported in apple (Malus domestica).

Mitogen-activated protein (MAP) kinase (MAPK) cascades are highly conserved signaling modules that are composed of MAP kinase kinase kinases (MAPKKKs/MEKKs), MAP kinase kinase (MAPKKs/MKKs) and MAP kinase (MAPKs/MPKs) (Widmann et al., 1999). The MAPK cascade pathway is broadly involved in various plant stress responses, in which extracellular stimuli activate MAPKKKs, thus, regulating plant physiological responses through the successive phosphorylation (Danquah et al., 2014). FvMAPK3 is activated by FvMKK4 and SNF1-RELATED PROTEIN KINASE2.6 (FvSnRK2.6) under low temperature stress and then phosphorylates the downstream FvMYB10 to inhibit its transcription activity. Moreover, the phosphorylation of CHALCONE SYNTHASE1 (FvCHS1) by FvMAPK3 promotes its degradation and inhibits anthocyanin accumulation in strawberry (Fragaria x ananassa) fruit (Mao et al., 2022). MPK6 activity is induced and activated in low phosphorus environments in A. thaliana, which affects the expression of auxin-induced genes and promotes the growth of primary roots. This induction is enhanced by Fe supplementation, indicating that MPK6 plays a role in coordinating the balance of phosphorus (Pi)/Fe to regulate root growth (Lopez-Bucio et al., 2019). In addition, MPK3/6 is an important factor in the ethylene signaling pathway induced by Fe deficiency stress. A. thaliana mpk3 and mpk6 mutants produce less ethylene under Fe deficiency and are more sensitive to Fe deficiency stress (Ye et al., 2015). We have reported that expression of the MxMPK3-2, MxMPK4-1 and MxMPK6-2 genes is induced by Fe deficiency stress in the Fe-efficient apple rootstock Malus xiaojinensis, and MxMPK6-2 responds to reactive oxygen species (ROS) signals under Fe deficiency stress and phosphorylates basic Helix Loop Helix104 (MxbHLH104) to improve the resistance to Fe deficiency stress (Li et al., 2021). Moreover, we have also reported that MxMPK4-1 phosphorylates NADPH oxidase respiratory burst oxidase homologs 1/2 (MxRBOHD1/2) to regulate the ROS signals (Zhai et al., 2022). Multiple MAPK members perform a diverse array of biological functions in plants to collectively adapt to abiotic stress. How the specificity of MAPK-mediated signaling occurs has become a topic of interest. Therefore, the functional characterization of newly identified MAPK substrates is central to our understanding of MAPK and its specificity of signaling in plants.

The MAPK cascade and calcium signaling pathways are frequently activated in parallel. In addition, the two different pathways may also converge on the same target/substrate. In the resistance of A. thaliana to disease, both MPK3/MPK6 and calcium-dependent protein kinases CALCIUM DEPENDENT PROTEIN KINASE5/6 (CPK5/6) phosphorylate WRKY33 at different residues, respectively (Mao et al., 2011; Zhou et al., 2020). Accumulating evidence has demonstrated that Ca²⁺/CAM impacts the MAPK pathway, and the MAPK scaffold protein kinase suppressor of Ras1 (KSR1) couples Ca²⁺/CAM signaling to the MAPK cascade (Parvathaneni et al., 2021). In addition, the rice (Oryza sativa) Ca²⁺/CaM-dependent protein kinase CCaMK OsDMI3 (Doesn’t Make Infections3) -mediated phosphorylation of OsMKK1 plays an important role in abscisic acid (ABA) signaling (Chen et al., 2021). Collectively, the MAPK cascade and Ca²⁺ signal transduction in plant physiological processes have been reported to be associated (Mehlmer et al., 2010; Wurzinger et al., 2011), but most studies focus on how the Ca²⁺ pathway activates the MAPK cascade. There is little research about how MAPK affects the Ca²⁺ signaling-mediated pathway, particularly on the CaMBP mediated Ca²⁺ signaling pathway. Moreover, the crosstalk of the Ca²⁺/CAM-mediated CaMBP pathway and the MAPK cascade in the regulation of Fe absorption has not yet been reported to our knowledge.

In this study, we identified a calmodulin-binding protein MxIQM3 from the Fe-efficient apple rootstock M. xiaojinensis through a transcriptome analysis, which was induced by Fe deficiency stress and found to interact with MxMPK4-1 as a critical regulator of the Fe deficiency response. MxMPK4-1 phosphorylated MxIQM3 at the Ser393 site, which altered the interaction of MxIQM3 with PM H⁺-ATPase MxHA2 to elicit PM H⁺-ATPase activity under Fe deficiency stress. Furthermore, we proved that participation of the MxMPK4-1-MxIQM3 module in Fe deficiency responses was promoted by Ca²⁺ signals, and we also explored the candidate Ca²⁺/CAM signaling pathway related to the MxIQM3 protein using a yeast two-hybrid system. Our results suggest that the MxMPK4-1-MxIQM3 module constitutes a regulatory mechanism in response to Fe deficiency stress.

Results

Molecular characterization and spatial expression of MxIQM3

We identified 13 IQM gene members in the apple genome GDDH13 V1.1 (Supplemental Table S1). MD14G1019000 was expressed the most highly in apple roots as shown in the ArrayExpress database (E-EGOD-42873) (Supplemental Figure S1). Synchronously, this gene was highly expressed and substantially upregulated under Fe deficiency stress in the roots of Fe-efficient apple rootstock M. xiaojinensis but not in the Fe-inefficient apple rootstock M. baccata (Supplemental Figure S2; Sun et al., 2020), indicating that MD14G1019000 could function in Fe deficiency responses. We then analyzed the homology with AtIQM1-6 in A. thaliana by MEGA7.0 software using the neighbor-joining (NJ) method, and found that MD14G1019000 was homologous with AtIQM3, designated as MdIQM3 (Supplemental Figure S3).

IQM3 gene expression were significantly induced in the roots of M. xiaojinensis and M. baccata under Fe deficiency stress but were stronger in M. xiaojinensis than in M. baccata through a reverse transcription quantitative PCR (RT-qPCR) analysis (Figure 1, A and B), which suggests that IQM3 could be involved in the Fe deficiency stress tolerance mechanism of M. xiaojinensis. Through subcellular localization, MxIQM3 was localized in both cell membrane and nucleus of Nicotiana benthamiana leaves (Figure 1C).

MxIQM3 serves as a positive regulator of apple rootstocks in response to Fe deficiency stress

To preliminarily and quickly verify the gene function of MxIQM3 in Fe deficiency responses, we obtained overexpressed MxIQM3 (OE-MxIQM3) and MxIQM3 RNA interference transgenic apple calli (RNAi-MxIQM3) (Supplemental Figure S4). When subjected to Fe deficiency stress, the OE-MxIQM3 calli grew better than the WT calli, while the RNAi-MxIQM3 calli exhibited less growth (Figure 2A), which was consistent with the fresh weight of apple calli (Figure 2B). In addition, after the stress of Fe deficiency, the ratio of reduction of the fresh weight of the WT, OE-MxIQM3, and RNAi-MxIQM3 calli were 44.83%, 20.88%, and 56.52%, respectively (Supplemental Table S2). These data indicated that the overexpression of MxIQM3 in the apple calli enhanced the ability to resist Fe deficiency stress. In addition, a bromocresol purple color test showed that the secretion of H⁺ was not affected by overexpressing or interfering with the MxIQM3 gene in apple calli under Fe normal conditions, but Fe deficiency stress triggered enhanced and weakened H⁺ secretion and FCR activity in the OE-MxIQM3 and RNAi-MxIQM3 calli, respectively (Figure 2C; Supplemental Figure S5). These results demonstrated that MxIQM3 served as a positive regulator in Fe absorption and functioned under Fe deficiency stress. As a consequence, the following experiments were only treated with Fe deficiency stress.

To further identify the positive role of MxIQM3 in plants, we transiently silenced MxIQM3 in the roots of M. xiaojinensis using tobacco rattle virus (TRV)-mediated silencing technology and found that the rhizosphere acidification capacity decreased significantly in TRV-MxIQM3 plants under Fe deficiency stress (Supplemental Figure S6). Furthermore, we overexpressed MxIQM3 in the roots of M. baccata using A. rhizogenes-mediated stable transformation (Supplemental Figure S7A). When the plants encountered Fe deficiency stress, the overexpressed MxIQM3 plants (OE-MxIQM3) appeared to have mild leaf chlorosis compared with the control plants OE-EV (Figure 2D; Supplemental Figure S7B). Correspondingly, the leaf SPAD value was significantly higher in OE-MxIQM3 than that in OE-EV (Figure 2E). In addition, the overexpression of MxIQM3 dramatically enhanced the responses to Fe deficiency, including the efflux of H⁺ (Figure 2F) and FCR activity (Figures 2, G and H; Supplemental Figure S7C). Perls staining conclusively showed that the content of iron in the roots and leaves of OE-MxIQM3 had increased under Fe deficiency stress compared with that in the OE-EV (Figure 2I), showing that MxIQM3 is a positive regulator of Fe deficiency responses in apple rootstocks.

The Fe deficiency responsive kinase MxMPK4-1 directly interacts with the MxIQM3 protein

The induced gene expression of MxIQM3 prompted us to measure its level of protein in the roots of M. xiaojinensis under Fe deficiency stress. Not surprisingly, the abundance of MxIQM3 protein gradually increased along with the prolongation of the treatment time of Fe deficiency (Figure 3A). Protein phosphorylation, one of the major post-translational modifications, plays a crucial role in cell signaling (Ajadi et al., 2020), and MxIQM3 protein phosphorylation level obviously increased under Fe deficiency stress (Figure 3A), signifying that phosphorylation modification of MxIQM3 may function in Fe deficiency stress. In consideration of the involvement of MAPK cascade in the Ca²⁺ signaling transduction pathway (Gratz et al., 2019), we then tried to determine whether the MxMPKs could regulate MxIQM3 at the post-translational level. We determined that the activity of MPK was enhanced in the roots of M. xiaojinensis under Fe deficiency stress (Figure 3A). Unexpectedly, the Y2H assay indicated that MxIQM3 interacted with MxMPK4-1 (Figure 3B), which had been previously reported to positively regulate Fe deficiency responses in M. xiaojinensis in our laboratory (Zhai et al., 2022). A pull-down assay also demonstrated their interaction in vitro (Figure 3C). Moreover, bimolecular fluorescence complementation (BiFC) and luciferase complementation assays (LCA) showed that MxMPK4-1 could interact with MxIQM3 in vivo (Figures 3, D and E).

MxMPK4-1 phosphorylates MxIQM3 at the Ser393 site

Basing on the interaction of MxMPK4-1 and MxIQM3 and the increased level of phosphorylation of both MxMPK4-1 and MxIQM3 under Fe deficiency stress, we hypothesized that MxMPK4-1 could act on MxIQM3 through the modification of phosphorylation. To confirm this hypothesis, CA-MxMPK4-1-His and MxIQM3-GST fusion proteins were induced in vitro, and a phosphorylation test using Phos-tag was performed and showed that CA-MxMPK4-1-His could phosphorylate MxIQM3-GST (Figure 4A). Moreover, the level of phosphorylation of MxIQM3 was enhanced when MxMPK4-1 was expressed in the OE-MxIQM3 transgenic apple calli (Figure 4B; Supplemental Figure S8A), indicating that MxMPK4-1 could phosphorylate MxIQM3 in vivo.

To further identify the exact phosphorylation site, we conducted an liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. One potential phospho-site at Ser393 was detected (Figure 4C; Supplemental Tables S3 and S4). To demonstrate that MxMPK4-1 phosphorylates MxIQM3 at this site, we abolished the phosphorylation of MxIQM3 at Ser393 by a Ser to Ala mutation and conducted a phosphorylation test using a phosphoserine/threonine antibody in vitro (Figure 4, D and E). The amino acid mutation of MxIQM3 (Ser to Ala; MxIQM3^S393A) visibly decreased its level of phosphorylation compared with that of MxIQM3, demonstrating that the Ser393 site of MxIQM3 was phosphorylated by MxMPK4-1 (Figure 4E). Moreover, we determined the subcellular localization of MxIQM3^S393A and MxIQM3^S393D (a Ser to Asp phospho-mimicking mutation) and found that both MxIQM3^S393A and MxIQM3^S393D localized to the cell membrane and nucleus of Nicotiana benthamiana leaves (Figure 4F), which indicated that the phosphorylation of MxIQM3 at the Ser393 site did not change the localization of MxIQM3. A Y2H assay showed that CA-MxMPK4-1 interacted with MxIQM3^S393A (Figure 4G), also illustrating that this phospho-site did not affect their interaction. To verify whether this mechanism of phosphorylation of the MxMPK4-1-MxIQM3 module exists in other species, we analyzed whether this phospho-site was conserved in other species, including apple (Malus domestica), Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), Chinese white pear (Pyrus bretschneideri), jujube (Ziziphus jujuba), peach (Prunus persica), sweet orange (Citrus sinensis), wild strawberry (Fragaria vesca), wheat (Triticum aestivum), and rice (Oryza sativa). As expected, this phospho-site was conserved among all these species (Figure 4H), indicating that this phosphorylation modification of IQM3 by MPK4 could be widespread in plants.

The MxMPK4-1-MxIQM3 module enhances the responses to Fe deficiency

Although MxMPK4-1 interacted with MxIQM3, whether this module functions during the responses to Fe deficiency merits further identification. Therefore, we used the co-expressed transgenic apple calli OE-MxMPK4-1 + OE-MxIQM3 to perform an Fe deficiency experiment (Supplemental Figure S8A). Compared with that in the OE-MxIQM3 transgenic apple calli, OE-MxMPK4-1 + OE-MxIQM3 obviously enhanced the activity of FCR (Figure 5A), although there were no significant differences at the level of quantitative detection in lines #2 and #3; however, there were significant differences in line #4 (Figure 5B). At the molecular level, the relative expression of genes induced by Fe deficiency, such as FIT, FRO2, IRT1, HA2 and NRAMP1 (NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN), was enhanced when MxMPK4-1 was overexpressed in OE-MxIQM3 calli (Figure 5, C–E; Supplemental Figure S8, B and C).

Moreover, we co-expressed MxMPK4-1 and MxIQM3 in the roots of apple rootstocks M. baccata by Agrobacterium-mediated transient transformation (Supplemental Figure S9, A and B). The activities of PM H⁺-ATPase and FCR in OE-MxIQM3 plants increased substantially compared with that in the control (OE-EV), while the co-expression of MxMPK4-1 and MxIQM3 (OE-MxMPK4-1 + OE-MxIQM3) resulted in higher PM H⁺-ATPase activity than that in the OE-MxIQM3 plants (Figure 5, F and G; Supplemental Figure S9, C–E). These results demonstrated that MxMPK4-1 modulated MxIQM3 to enhance the responses to Fe deficiency in plants.

MxMPK4-1 phosphorylates MxIQM3 at the Ser393 site to enhance H⁺ secretion through dissociating from MxHA2 under Fe deficiency stress

Based on the enhanced Fe deficiency responses in the co-expression of MxMPK4-1 and MxIQM3 under Fe deficiency stress, we tried to explore the role of the phosphorylation modification of MxIQM3 at Ser393 site by MxMPK4-1 in response to Fe deficiency stress. Previous studies reported that a calcium-binding protein SCaBP3 modulates PM H⁺-ATPase activity to enhance root acidification and promote alkali tolerance in Arabidopsis (Yang et al., 2019). Rhizosphere acidification is the first step of mechanism I plants to cope with iron deficiency stress. Therefore, we focused on whether this MxMPK4-1-MxIQM3 module function under Fe deficiency stress through enhancing the acidification of roots, then, we transiently overexpressed MxIQM3, MxIQM3^S393A, and MxIQM3^S393D in the roots of M. baccata to assay root acidification using bromocresol purple color and a pH fluorescent probe (Supplemental Figure S10A). We found that the individual overexpression of MxIQM3^S393D and MxIQM3 under Fe deficiency stress both increased the root acidification, and OE-MxIQM3^S393D displayed stronger than OE-MxIQM3, while there was no obvious alteration in the overexpression of MxIQM3^S393A when compared with the empty vector control (OE-EV) (Figure 6, A and B; Supplemental Figure S10B), indicating MxIQM3 and its phospho-mimicking form function positive roles in root acidification. Therefore, these results indicated that MxMPK4-1 phosphorylated MxIQM3 at Ser393 to enhance the acidification of root under Fe deficiency stress.

To validate how MxIQM3 regulated root acidification, we examined whether MxIQM3 interacted with MxHA2, one gene that encoded PM H⁺-ATPase in M. xiaojinensis (Santi and Schmidt, 2009; Wang et al., 2014), and Y2H assay displayed that MxIQM3 interacted with the central-loop region of MxHA2 (Figure 6, C and D). Nonetheless, we further performed Y2H assays between truncated MxHA2 and MxIQM3^S393A, and MxIQM3^S393D, respectively, to determine whether this interaction could be affected by the phosphorylation modification of MxIQM3 by MxMPK4-1. Surprisingly, the phospho-mimicking MxIQM3^S393D did not interact with any regions of MxHA2; in contrast, MxIQM3^S393A exhibited strong interactions with both the C terminus and central loop of MxHA2 (Figure 6E). Moreover, LCA assays further showed the strong interaction of MxIQM3^S393A with both the C terminus and central loop of MxHA2, but no interaction for MxIQM3^S393D was detectable in vivo (Figure 6F), indicating that MxIQM3 was dissociated from the central loop of MxHA2 after phosphorylation at the Ser393 site. Since the C-terminal R domain contains two critical autoinhibitory regions (Region I [RI] and Region II [RII]) (Yang et al., 2019), we further identified that MxIQM3^S393A interacted with both regions (Figure 6G). These results demonstrated that Fe deficiency stress induced MxMPK4-1 to phosphorylate MxIQM3 at Ser393 site, which resulted dissociation of MxIQM3 from MxHA2, thus promoting root acidification.

The Ca²⁺/MxCAM7 signaling pathway involved in the MxMPK4-1-MxIQM3 module mediated the Fe deficiency responses

The CAM-IQM complex has been reported to sense and decode Ca²⁺ signals and then transmit them to downstream effectors (Lv et al., 2019). Here, we found that the level of Ca²⁺ was obviously enhanced in the roots of M. xiaojinensis at 3 h after Fe deficiency stress (Figure 7A). These findings indicated that Fe deficiency stress was able to induce Ca²⁺ signals in M. xiaojinensis, but whether it had any crosstalk with the MxMPK4-1-MxIQM3 module merits further study.

The activity of MPK4, the abundance of IQM3 protein, and its phosphorylation level in apple calli increased substantially under Fe deficiency conditions (Figure 7B). The application of Ca²⁺ donor CaCl₂ under Fe sufficiency conditions also induced the increase in MPK4 activity and the levels of IQM3 protein and phosphorylation; in contrast, these were inhibited when the Ca²⁺ inhibitor LaCl₃ was applied under Fe deficiency stress (Figure 7B). These results demonstrated that the Ca²⁺ signals induced under Fe deficiency could regulate the MxMPK4-1-MxIQM3 module.

It has been reported that Ca²⁺/CAM signaling transduction plays an important role in multiple physiological processes (Lv et al., 2019). A total of 11 CAM candidate genes were found in apple from the database AppleMDO (http://bioinformatics.cau.edu.cn/AppleMDO/) according to the gene annotation (Supplemental Table S5; Da et al., 2019), and we found that the MxCAM7 and MxCAM8 genes were highly expressed in the roots of apple plants (Supplemental Figure S11). Therefore, we conducted a Y2H analysis between them with MxIQM3 and found that MxCAM7-1 (MD06G1234300), MxCAM7-2/3/4 (MD12G1111300/MD11G1183900/MD14G1092700), and MxCAM7-5 (MD14G1241000) from M. xiaojinensis could interact with MxIQM3 (Figure 7C), and five MxCAM7 candidate proteins had highly similar protein sequence (Supplemental Figure S12). However, MxCAM7-5 interaction capacity with MxIQM3 was weaker than other four MxCAM7s (Figure 7C). Consequently, we proposed that the Ca²⁺ signals that induced MxCAM7 could bind to the MxIQM3 protein to involve it in the responses to Fe deficiency. In addition, both MxIQM3^S393A and MxIQM3^S393D interacted with the five MxCAM7 proteins (Supplemental Figure S13), indicating that phosphorylation of MxIQM3 by MxMPK4-1 did not influence MxCAM7-MxIQM3 complex.

Discussion

Fe deficiency stress seriously affects plant growth and development, and how woody plants respond to Fe deficiency stress is a long-standing issue in plant mineral nutrition (Marschner and Romheld, 1994). Both the MAPK cascade and Ca²⁺ signaling pathways play essential roles in the Fe deficiency response in plants, while whether there is any crosstalk between the two pathways remains unclear. In our study, we identified that the CaMBP MxIQM3 that responds to Fe deficiency interacted with MxMPK4-1. To our knowledge, the phosphorylation modification for IQM at the post-translational level has not been reported, while here we demonstrated that MxIQM3 was phosphorylated by MxMPK4-1 through in vitro and in vivo assays. Moreover, the phosphorylation of MxIQM3 by MxMPK4-1 regulated its ability to bind to the PM H⁺-ATPase MxHA2 to modulate root acidification. In conclusion, the results of this study provide substantial insights into a mechanism used to regulate Fe deficiency stress that involves the MAPK cascade and Ca²⁺ signaling pathway in apple rootstock.

The involvement of MAPK cascade in Fe absorption has been reported in succession (Ye et al., 2015; Li et al., 2021). MxMPK4-1 was one of the three genes induced by Fe deficiency in the roots of M. xiaojinensis (Li et al., 2021). We have illustrated that MxMPK4-1 played a positive role in the Fe deficiency responses of M. xiaojinensis that were mediated by ROS (Zhai et al., 2022). The function of MxMPK4-1 in the response to Fe deficiency is consistent with that of MxMPK6-2, which enhances the resistance to Fe deficiency stress (Li et al., 2021). MPKs, as the terminal participants of MAPK cascade, primarily function by phosphorylating target proteins to alter their enzyme activity or protein conformation (Widmann et al., 1999). MxMPK6-2 has been demonstrated to phosphorylate MxbHLH104 to upregulate genes that are induced by Fe deficiency (Li et al., 2021). For the Fe deficiency responses mediated by MxMPK4-1, we also previously demonstrated that the ROS synthetases MxRBOHD1/2 are the substrates of MxMPK4-1 (Zhai et al., 2022). In addition to its interactions with MxRBOHD1/2, MxIQM3 also interacted with and was phosphorylated by MxMPK4-1 to enhance the Fe deficiency responses in this study. The different regulatory pathways used by MxMPK6-2 and MxMPK4-1 to adaptive Fe deficiency stress, respectively, suggest that the involvement of MAPK cascade in Fe deficiency stress is fairly sophisticated.

IQMs, as one type of CaMBP protein, have been reported to regulate some pathogen defense and root developmental processes (Lv et al., 2019; Xu et al., 2019). The different levels of gene expression of IQM in M. baccata and M. xiaojinensis suggest the essential role of IQM in the Fe deficiency tolerance mechanism of M. xiaojinensis and that IQM could also be dissimilarly regulated by transcription factors at the transcription level in the two materials. Moreover, the enhanced Fe deficiency responses in the apple calli and apple rootstocks that overexpressed MxIQM3 under Fe deficiency stress demonstrated its participation in the absorption of Fe under Fe deficiency stress. The important representative responsive genes during iron absorption processes have been verified. These enhanced indexes in overexpression of MxIQM3 further demonstrated that MxIQM3 may directly or indirectly regulate HA2, FRO2 or IRT1 in apple rootstocks. Furthermore, it has been reported that IQM proteins can directly bind to some functional proteins, like catalase 2 (CAT2) (Lv et al., 2019). Here, MxIQM3 binds to PM H⁺-ATPase MxHA2. The enhanced PM H⁺-ATPase activity in the overexpression of MxIQM3 in apple calli and the roots of apple seedlings under Fe deficiency stress demonstrated that it acted as a positive regulator of PM H⁺-ATPase activity, which is consistent with the previous results that IQM positively responds to environmental stimuli (Lv et al., 2019). On the other hand, rhizosphere acidification is the first step caused by Fe deficiency stress, and accumulating evidence has demonstrated that root acidification affects the expression of genes including FRO and IRT. The activity of FRO and IRT transporter were related to its pH value, and substantially increased in acidic environments (Slatni et al., 2011; Gao et al., 2022b). This means that the three steps of iron absorption are not independent. Therefore, through our results, it is likely that IQM3 may involve in Fe deficiency responsiveness by modulating MxHA2 to enhance root acidification, which then promotes FRO and IRT subsequently. This can explain the results that FRO2 and IRT1 were both enhanced by overexpressing MxIQM3. However, we cannot exclude that whether IQM3 directly interacted with FRO or IRT in the response of Fe deficiency stress, which needs further study.

The regulation of PM H⁺-ATPase activity to enhance root acidification plays an essential role in responding to environmental stress (Havshoi and Fuglsang, 2022). Typically, PM H⁺-ATPase remains less active to maintain the H⁺ pump balance under non-stressed conditions (Yang et al., 2019). In our study, H⁺ secretion was low under normal Fe conditions in the presence of overexpressed MxIQM3. Moreover, a Y2H assay demonstrated that MxIQM3 interacted with the central loop of MxHA2, indicating that it is likely that MxIQM3 could be involved in maintaining low PM H⁺-ATPase activity through its interactions with the central-loop region under normal Fe conditions. Nonetheless, unlike Fe normal conditions, Fe deficiency stress induced the phosphorylation of MxIQM3 at the Ser393 site by MxMPK4-1 to enhance H⁺ secretion. Compared with MxIQM3, the difference is that the Ser393 to Asp phospho-mimicking mutation of MxIQM3 failed to bind to MxHA2. This indicated that Fe deficiency stress induced the phosphorylation of MxIQM3 by MxMPK4-1 to dissociate from the central-loop region of MxHA2 to increase PM H⁺-ATPase activity, which is similar to that under saline-alkali stress conditions, and SCaBP3 releases the interaction with C terminus of AHA2 to enhance the activity of PM H⁺-ATPase (Yang et al., 2019). And it alternatively indicated that MxIQM3 overexpression is not sufficient to increase H⁺ secretion upon Fe sufficiency, representing a large pool of non-phosphorylated MxIQM3. In contrast, the Ser393 to Ala mutation that abolished the phosphorylation activity of MxIQM3 instead results in its interaction with both the central-loop and C-terminal regions of MxHA2, while it did not increase H⁺ secretion. That means the phospho-dead mutation of MxIQM3 at Ser393 site is likely to promote the interaction between the C terminus and central loop, contributing to PM H⁺-ATPase activity repression, further signifying the great importance of this phospho-site. MxIQM3^S393A interacted with both the RI and RII regions of MxHA2, but SCaBP3 only interacts with the RI domain of AHA2 in A. thaliana (Yang et al., 2019), which indicated that SCaBP3 and MxIQM3 could regulate the activity of PM H⁺-ATPase through different regulatory mechanisms. This laterally explained that overexpression of SCaBP3 inhibited the activity of PM H⁺-ATPase under saline-alkali stress, but overexpression of MxIQM3 increased its activity under Fe deficiency stress.

The MAPK cascade pathway generally crosstalks with other pathways to form a complex regulatory network (Jalmi and Sinha, 2015). The involvement of Ca²⁺ signals in Fe deficiency stress has been reported in the model plant A. thaliana (Gratz et al., 2019). Similarly, an increased concentration of Ca²⁺ in the roots of M. xiaojinensis was observed, illustrating its role as a second messenger during responses to Fe deficiency. The non-changeable pH color in the transgenic apple calli of OE-MxIQM3 and RNAi-MxIQM3 under Fe normal conditions indirectly reflected that Ca²⁺ may be the driver of MxIQM3 during the regulation of PM H⁺-ATPase activity. This was demonstrated by an increase in the abundance of MxIQM3 protein under CaCl₂ treatment and the decrease following the application of LaCl₃. Considering that the IQMs are Ca²⁺ independent proteins, CAMs as the bridge of Ca²⁺ signal and IQM are of substantial importance. Arabidopsis CAM5 combines with AtIQM1 to enhance the activity of catalase (Lv et al., 2019), which is consistent with our preliminary Y2H assay results that the interaction of MxCAM7 with MxIQM3 signifies a potential Ca²⁺-MxCAM7-MxIQM3 signaling transduction pathway in the regulation of Fe deficiency responses. Chemical treatment of the Ca²⁺ signal also increased the activity of MPK4, and the level of phosphorylation of MxIQM3 was consecutively enhanced, demonstrating that the Ca²⁺ signals may also regulate the MAPK cascade to activate the MxMPK4-1-MxIQM3 module under Fe deficiency stress, i.e. the Ca²⁺-MxMPK4-1-MxIQM3 pathway. This is consistent with the finding that copper induced an increase in the intracellular level of Ca²⁺ that led to activation of the MAPK signaling pathways in the marine alga Ulva compressa (Laporte et al., 2020). These findings provide evidence of the characteristics of the Ca²⁺ signal under Fe deficiency stress and that the Ca²⁺ signal also enhances the MxMPK4-1-MxIQM3 module.

Basing on the results in this study, we proposed a working model about MxMPK4-1-MxIQM3 module in Fe absorption (Figure 7D). Under Fe normal conditions, MxIQM3 interacts with the central loop of MxHA2 to maintain H⁺ homeostasis. Once the plants encounter Fe deficiency stress, MxCAM7 decodes Ca²⁺ signals to target MxIQM3, and synchronously Ca²⁺ signals activate the MxMPK4-1 cascade to phosphorylate MxIQM3 at the Ser393 site, which ultimately leads to the dissociation of MxIQM3 from MxHA2 to promote H⁺ secretion. This study provides an insightful mechanism about Fe absorption in plants.

Materials and methods

Plant materials and growth conditions

Tissue cultured apple rootstock plantlets (M. xiaojinensis and M. baccata) were grown in Murashige and Skoog (MS) media supplemented with 0.3 mg/l 6-benzylaminopurine (6-BA), 0.5 mg/l indole-3-butyric acid (IBA), and 7.5% (w/v) agar (pH 5.8). The 1-month-old propagated plantlets were rooted in 1/2 MS media with 0.5 mg/l IBA and 7.5% agar (pH 5.8). The rooted plantlets were then transferred to Hoagland nutrient solution for future use.

Wild type and transgenic apple calli were propagated and grown in MS media that contained 0.4 mg/l 6-BA, 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 7.5% agar (pH 5.8) with or without the corresponding antibiotics and incubated in the dark for 10 d. The calli tissues were then treated under Fe normal (+Fe) and Fe deficient (−Fe) conditions with or without 5 mM CaCl₂ and 1 mM lanthanum chloride (LaCl₃) (Ma et al., 2018).

Plasmid construction and genetic transformation

For hairy root induction, the coding sequence of MxIQM3 from M. xiaojinensis was cloned into the pRI101-GFP vector (35S promoter) using In-fusion technology. The resulting pRI101-35S::MxIQM3-GFP plasmid was introduced into Agrobacterium rhizogenes strain K599. The information of all the constructs prepared in this study has been listed in Supplemental Table S6. Tissue cultured M. baccata shoots were cut using surgical scissors and then immersed in the suspended solution of Agrobacterium. The infected shoots were transferred into MS media for 2 days and then transferred into 1/2 MS media with 250 mg/l cephalosporin.

For apple calli transformation, the MxIQM3 coding sequence and the ∼350 bp specific fragment of the MxIQM3 sense and anti-sense strands were amplified and inserted into the pRI101-MYC vector and pRI101-RNAi vector, respectively, with eukaryotic resistance to kanamycin. The coding sequence of MxMPK4-1 was cloned into both the pSuper1300-GFP vector (eukaryotic resistance to hygromycin) and the pRI101-GFP vector (eukaryotic resistance to kanamycin), and the ∼350 bp specific fragment of MxMPK4-1 sense and anti-sense strands were amplified into the pRI101-RNAi vector. The resulting plasmids were then introduced into Agrobacterium strain GV3101. Agrobacterium-mediated transformation was conducted as previously described (Xie et al., 2012).

For transient expression in apple rootstocks, the coding sequences of MxIQM3^S393A (Ser to Ala mutation at 393 site) and MxIQM3^S393D (Ser to Asp mutation at 393 site) were inserted into the pRI101-GFP vector, respectively. Agrobacterium strain GV3101 was used to transiently overexpress MxIQM3 and MxMPK4-1 in the roots of M. baccata.

Protein subcellular localization

Agrobacterium strain GV3101 that harbored target genes was injected into Nicotiana benthamiana leaves. After 48 h, the infected leaves were immersed in 50 μM FM4-64 (plasma membrane stain) for 10-15 min in the dark and then transferred into 10 μg/ml DAPI for 5–10 min in the dark to stain the cell nuclei before confocal microscopic examination (FluoView FV3000; Olympus, Tokyo, Japan).

Etiolation phenotypic observation and detection by the soil and plant analyzer development (SPAD)

For A. rhizogenes-mediated transformation in M. baccata, the etiolation phenotype was observed and photographed after 7 d of Fe deficiency treatment, while it was observed 20 d after Fe deficiency treatment for TRV-mediated gene silencing in M. xiaojinensis.

The leaf SPAD values were measured using a portable chlorophyll meter (SPAD-502; Minolta, Osaka, Japan). Three plants were selected for each treatment, and three new functional leaves were analyzed per plant.

Quantitative and qualitative detection of Fe (III) reductase activity

The activity of FCR of tissues was measured using bathophenanthroline disulfonate (BPDS) as described by Pii et al. (2016). Briefly, root and apple calli tissues were immersed into the reaction solution and were then determined by UV–VIS spectrophotometry (UV 1800; Shimadzu, Tokyo, Japan) at 535 nm. Three replicates per treatment were performed.

For visualization of FCR activity, apple calli tissues were immersed in a 1.5-ml centrifuge tubes that contained the reaction solution. They were photographed immediately after 1 h in the dark at room temperature. To observe the FCR activity in the roots, the cooled reagent solution with 5% agar was poured into a petri dish with the roots for 1 h in the dark at room temperature. The red color around the roots was then photographed.

Bromocresol purple color for PM H⁺-ATPase activity

To visualize the PM H⁺-ATPase activity in apple calli, the tissues were gently tiled on the chromogenic media that contained 100 ml of 1 mM CaSO₄, including 0.006 g bromocresol purple and 0.5 g agar (pH 6.6), for 14 h and observed. The method for roots was the same as above, and the tissues were photographed after 30 min of incubation.

Detection of root pH fluorescence

Apple roots (∼5 mm) were soaked with 5 μM of the pH fluorescence probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF AM) (Beyotime Biotech., Inc., Haimen, China) and incubated in the dark for 1 h. The stained roots were then thoroughly washed three times with deionized water. The pH fluorescence was observed by a confocal microscope (LSM 710; Zeiss, Oberkochen, Germany) with the excitation wavelength of 488 and 458 nm.

Measurement of the root H⁺ efflux

The H⁺ flux in apple roots was measured using non-invasive micro-test technology equipment (NMT Physiolyzer®, YoungerUSA LLC, Amherst, MA, USA; Xuyue [Beijing] Sci. &Tech. Co., Ltd., Beijing, China) according to the previous description (Zhai et al., 2022). The mature zones of apple seedling root tips under the microscope were the test site. The H⁺ flow rate sensor was placed ∼10 μm away from the detection site.

Perls staining

Perls staining was conducted according to the previous study (Gao et al., 2022a). Briefly, leaf and root fresh tissues were fixed in FAA stationary liquid. The fixed tissues were immersed in staining solution [an equal volume of 4% HCl (v/v) and 4% potassium ferrocyanide (w/v)].

Detection of root Ca²⁺ fluorescence

The concentration of Ca²⁺ in the roots was detected using the calcium fluorescence probe Fluo-3-AM (Solarbio, Beijing, China) as previously described (Wu et al., 2020). Briefly, apple root samples (∼5 mm) were immersed in the incubation solution that contained 20 μM Fluo-3-AM, 0.5 M mannitol, 4 mM MES (pH 5.7), and 20 mM KCl for 1 h in the dark at room temperature. The stained roots were thoroughly washed three times with deionized water before confocal microscopy (Olympus FluoView FV3000).

Yeast two-hybrid (Y2H) assay

For the Y2H assay, vectors that included pGADT7-MxIQM3, pGADT7-MxIQM3^S393A, pGADT7-MxHA2-N, pGADT7-MxHA2-ccentral loop, pGADT7-MxHA2-C, pGADT7-MxCAM7-1, pGADT7-MxCAM7-2/3/4, pGADT7-MxCAM7-5, pGADT7-MxCAM8, pGBKT7-CA-MxMPK4-1, pGBKT7-MxIQM3, pGBKT7-MxIQM3^S393A, and pGBKT7-MxIQM3^S393D were constructed. The resulting plasmids were grouped for their introduction into the Y2H yeast cells. The co-expressed yeast cells were inoculated on synthetic dropout (SD) media (−Leu, −Trp; −Leu, −Trp, −His, and −Ade) at 28°C for 4 d to test the interactions of proteins. Negative controls were conducted concurrently.

BiFC assay

The pSPYCE-MxIQM3 and pSPYNE-CA-MxMPK4-1 plasmids were individually introduced into Agrobacterium strain GV3101. The same volume of Agrobacterium that harbored different genes (OD₆₀₀ = 1.0–1.2) was mixed and then injected into 1-month-old N. benthamiana leaves. The N. benthamiana plants were placed in low light, and the leaves were observed by confocal microscopy after 48 h.

LCA assay

The coding sequences of CA-MxMPK4-1 and MxIQM3 were inserted into the pCambia1300-nLUC and pCambia1300-cLUC vectors, respectively. The subsequent procedures were similar to those of the BiFC assay. The difference was that a CDD imaging system (NightShade LB 985 In Vivo Plant Imaging System; Berthold Technologies USA, LLC, Oak Ridge, TN, USA) was used to observe the N. benthamiana leaves. A volume of 1 mM D-luciferin was sprayed onto the back of leaves and placed in the dark for 7 min before the tissues were observed.

Pull-down assay

The pGEX-4T-1-MxIQM3 vectors (GST tag) and the pet32a (+)-CA-MxMPK4-1 vectors (His tag) were introduced into E. coli strain BL21 and E. coli strain BL21 (DE3), respectively. Proteins were induced using 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). Then mixed proteins were incubated together with His beads at 4°C in a shaker. The subsequent purification steps on the CWBIO Ni-Agarose His tag Protein Purification Kit (CoWin Biosciences, Cambridge, MA, USA) were conducted according to the manufacturer's instructions. The pull-down and input proteins were detected by anti-GST (CoWin Biosciences, CW0084) and anti-His antibodies (CoWin Biosciences, CW0285), respectively, using a western blot analysis.

In vitro and in vivo phosphorylation assay

For the in vitro phosphorylation assay, the phosphorylation reaction was performed at a 2:1 ratio of kinases to substrates in the reaction solution that contained 20 mM Tris–HCl (pH 7.4), 30 mM MgCl₂, and 1 mM DTT with 50 mM ATP at 30°C for 30 min. For the in vivo phosphorylation assay, the total soluble proteins of tissues were extracted using a plant protein extraction kit (CW0885M; CoWin Biosciences). The MxIQM3 protein phosphorylation level was determined using the Phos-tag^TM Acrylamide AAL-107 Kit (FUJIFILM Wako Chemicals USA Corp, Richmond, VA, USA) according to the manufacturer'. The GST antibody (CWBIO) was used for the in vitro assay, while the specific antibody IQM3 was used for the in vivo assay.

The in vitro phosphorylation reaction was performed as above for LC–MS/MS. The proteins were separated on SDS–PAGE gels after the reaction. The gels of target proteins were obtained for LC–MS/MS analysis with a nanoLC-LTQ-Orbitrap XL (Thermo, San Jose, CA, USA).

Gene expression analysis

The total RNA of tissues were extracted using the CTAB method as previously described (Gasic et al., 2004). The cDNA was synthesized using a TRUEscript One Step qRT-PCR Kit (Aidlab Biotechnologies Co., Ltd; Beijing, China). RT-qPCR analysis was performed on a QuantStudio 6 Flex Real time PCR machine (Applied Biosystems, Waltham, MA, USA) using a 2× M5 HiPer Realtime PCR Super mix (MF013-01; Mei5bio, Beijing, China). The amplification conditions were 95°C for 30 s, 40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 30 s. The 2^−△△CT method was used to analyze the relative levels of expression of the genes. The primers used in gene expression are listed in Supplemental Table S7.

Statistical analysis

The data were analyzed for significance using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). An ordinary one-way analysis of variance (ANOVA) was used for multiple comparisons, while a t-test was used for two group data.

Accession numbers

Sequence data from this article can be found in the Genome Database for Rosaceae (GDR) data libraries under accession numbers (MxMPK4-1, MD01G1069100; MxIQM3, MD14G101900; MxFRO2, MD01G1068200; MxIRT1, MD05G1255500; MxFIT, MD03G1129100; MxNRAMP1, MD17G1222500; MxHA2, MD08G1130200; MxCAM7-1, MD06G1234300; MxCAM7-2, MD12G1111300; MxCAM7-3, MD11G1183900; MxCAM7-4, MD14G1092700; MxCAM7-5, MD14G1241000).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Analysis of gene expression level of MdIQMs in roots of apple plants.

Supplemental Figure S2. IQM gene expression analysis in the transcriptome data of apple rootstocks Malus xiaojinensis and M. baccata.

Supplemental Figure S3. Evolutionary tree analysis of IQM gene family between apple and Arabidopsis thaliana.

Supplemental Figure S4. Identification of OE-MxIQM3 and RNAi-MxIQM3 transgenic apple callus

Supplemental Figure S5. Visualization of FCR activity in OE-MxIQM3 and RNAi-MxIQM3 transgenic apple callus.

Supplemental Figure S6. Silencing MxIQM3 in Malus xiaojinensis reduced rhizosphere acidification capacity through VIGS-mediated silencing technology.

Supplemental Figure S7. Leaf phenotype of OE-MxIQM3 transgenic Malus baccata seedlings.

Supplemental Figure S8. Co-expression of MxMPK4-1 and MxIQM3 in apple callus enhanced Fe deficiency responses.

Supplemental Figure S9. Co-expression of MxMPK4-1 and MxIQM3 in Malus baccata seedlings enhanced Fe deficiency responses.

Supplemental Figure S10. Phosphorylation modification of MxIQM3 at Ser 393 site regulates its role in Fe deficiency responses.

Supplemental Figure S11. Analysis of gene expression level of MdCAMs in roots of apple plants.

Supplemental Figure S12. Protein sequence analysis of five candidate CAM7s from Malus xiaojinensis.

Supplemental Figure S13. Phosphorylation of MxIQM3 at the Ser393 site did not affect its interaction between five MxCaM7s.

Supplemental Table S1. Characterization of the candidate MdIQM genes.

Supplemental Table S2. The reduction ratio of wild-type and transgenic apple callus under Fe deficiency stress.

Supplemental Table S3. The phosphorylation sites of MxIQM3 protein were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Supplemental Table S4. The phosphopeptide information of MxIQM3.

Supplemental Table S5. Characterization of the candidate MdCAM genes.

Supplemental Table S6. Information of all constructs prepared in this study.

Supplemental Table S7. Sequences of primers used in this study.

Author responsibility

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://dbpia.nl.go.kr/plphys/pages/General-Instructions) are: Yi Wang ([email protected]) and Zhenhai Han ([email protected]).

Acknowledgments

We are thankful to Prof. Yujin Hao of Shandong Agricultural University and Shuhua Yang of China Agricultural University for providing vectors and technical support. We thank the 2115 Talent Development Program of China Agricultural University, and the Laboratory of Matter Spectroscopy of China Agricultural University for LC–MS/MS analysis.

Funding

This work was supported by the National Natural Science Foundation of China (31972385 and 32172537), the Chinese Universities Scientific Fund and Graduate Independent Innovation Research Fund of China Agricultural University (2022TC171), the earmarked fund for China Agriculture Research System (CARS-27), and the Key Laboratory of Beijing Municipality of Stress Physiology and Molecular Biology for Fruit Trees.

Author contributions

Z.H. and Y.W. (Yi Wang) designed this research; Q.S. and L.Z. performed the experiments and data analysis; D.Z., Y.W. (Yue Wu), and M.G. provided plant materials; T.W., X.Z., and X.X. provided suggestions on experiments; L.Z., Q.S., Y.W. (Yi Wang), and Z.H. revised the manuscript. All the authors read and approved this manuscript submission.

References

Author notes