2. Main physiological and biochemical roles of Mg ²⁺ and symptoms of deficiency

The physiological importance of Mg²⁺ has been most extensively characterized in the context of photosynthesis. As the central atom of chlorophyll, Mg²⁺ directly participates in light capture. It also stabilizes pigment-protein complexes in Photosystems I and II and contributes to the maintenance of thylakoid membrane architecture. Because thylakoid membranes carry a negative surface charge, Mg²⁺ plays a crucial role in counteracting these charges, promoting the appression of adjacent membranes and ultimately driving grana stacking. When Mg²⁺ becomes limiting, electrostatic repulsion increases and grana becomes unstacked or disorganized. As a result, chlorophyll content is reduced and interveinal chlorosis develops, predominantly in older mature leaves, accompanied by a decrease in photosynthetic rate (Cakmak & Yazici, Reference Cakmak and Yazici2010; Levitt, Reference Levitt1954; Marschner & Marschner, Reference Marschner and Marschner2012; Pandey, Reference Pandey, Hasanuzzaman, Fujita, Oku, Nahar and Hawrylak-Nowak2018). Notably, however, chlorosis is a late symptom of Mg²⁺ shortage (Hermans et al., Reference Hermans, Johnson, Strasser and Verbruggen2004; Ogura et al., Reference Ogura, Kobayashi, Hermans, Ichihashi, Shibata, Shirasu, Aoki, Sugita, Ogawa, Suzuki, Iwata, Nakanishi and Tanoi2020)

Beyond photosynthesis, Mg²⁺ is essential for ribosome biogenesis and translation. It stabilizes ribosomal subunits and facilitates the binding of messenger RNA (mRNA) and transfer RNA (tRNA) (Klein et al., Reference Klein, Moore and Steitz2004; Kobayashi & Tanoi, Reference Kobayashi and Tanoi2015; Sperrazza & Spremulli, Reference Sperrazza and Spremulli1983). When Mg²⁺ is deficient, these processes are impaired, leading to reduced protein synthesis (de Melo et al., Reference de Melo, Gutsch, Caluwé, Leloup, Gonze, Hermans, Webb and Verbruggen2021; Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016). This effect is especially pronounced in chloroplasts, which house more than 25% of total cellular proteins (Marschner & Marschner, Reference Marschner and Marschner2012; Peng et al., Reference Peng, Qi, Lee, Yang, Guo, Jiang and Chen2015; Chen et al., Reference Chen, Peng, Li and Liao2018).

In addition to its function in translation, Mg²⁺ serves as an essential cofactor and allosteric modulator for more than 300 enzymes involved in respiration, glycolysis, nucleic acid metabolism, chlorophyll biosynthesis and photosynthetic carbon fixation (De Bang et al., Reference De Bang, Husted, Laursen, Persson and Schjoerring2021; Hermans et al., Reference Hermans, Conn, Chen, Xiao and Verbruggen2013). Key examples include ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate (PEP) carboxylase, underscoring the central role of Mg²⁺ in carbon assimilation (Andreo et al., Reference Andreo, Gonzalez and Iglesias1987; Lorimer et al., Reference Lorimer, Badger and Andrews1976; Walker & Weinstein, Reference Walker and Weinstein1994; Willows, Reference Willows2003). Mg²⁺ is also indispensable for energy metabolism as the binding partner of adenosine triphosphate (ATP). Between 50% and 90% of cytosolic Mg²⁺ is complexed with ATP, and Mg²⁺ scarcity impairs Mg-ATP formation, thereby compromising energy-dependent reactions across metabolic pathways (Maguire & Cowan, Reference Maguire and Cowan2002; Marschner & Marschner, Reference Marschner and Marschner2012; Shaul, Reference Shaul2002). Moreover, ATP synthesis itself is highly dependent on Mg²⁺, since ATP synthase requires Mg²⁺ as a cofactor to catalyse the phosphorylation of ADP (Gout et al., Reference Gout, Rébeillé, Douce and Bligny2014; Lin & Nobel, Reference Lin and Nobel1971).

A key outcome of Mg²⁺ deficiency is impaired phloem loading. Reduced activity of H⁺-ATPases in phloem companion cells compromises the proton motive force and restricts sucrose export from source to sink tissues in species relying on apoplastic loading of photoassimilates (Bush, Reference Bush1989; Cakmak & Yazici, Reference Cakmak and Yazici2010; Vaughn et al., Reference Vaughn, Harrington and Bush2002). Experimentally, Mg²⁺ deficiency has been shown to induce carbohydrate accumulation in source leaves, increase the shoot-to-root dry weight ratio (Cakmak et al., Reference Cakmak, Hengeler and Marschner1994; Cakmak & Kirkby, Reference Cakmak and Kirkby2008; Hermans et al., Reference Hermans, Johnson, Strasser and Verbruggen2004), repress photosynthetic gene expression and further reduce the activities of CO₂-assimilating and photosynthetic enzymes (Cakmak & Kirkby, Reference Cakmak and Kirkby2008; Chen et al., Reference Chen, Peng, Li and Liao2018).

At the structural level, prolonged Mg²⁺ deficiency leads to swelling and disorganization of chloroplasts, as well as damage to the photosynthetic electron transport chain (Hermans et al., Reference Hermans, Johnson, Strasser and Verbruggen2004; Laing et al., Reference Laing, Greer, Sun, Beets, Lowe and Payn2000). These alterations promote overproduction of reactive oxygen species (ROS) (Cakmak, Reference Cakmak1994; Guo et al., Reference Guo, Nazim, Liang and Yang2016), which in turn exacerbate disturbances in source-sink dynamics, growth and biomass allocation (Hermans et al., Reference Hermans, Johnson, Strasser and Verbruggen2004, Reference Hermans, Conn, Chen, Xiao and Verbruggen2013; Kobayashi & Tanoi, Reference Kobayashi and Tanoi2015; Peng et al., Reference Peng, Qi, Lee, Yang, Guo, Jiang and Chen2015; Koch et al., Reference Koch, Winkelmann, Hasler, Pawelzik and Naumann2020). High-light environments amplify these detrimental effects by accelerating photooxidative stress and further enhancing ROS production (Cakmak & Kirkby, Reference Cakmak and Kirkby2008; Kumar Tewari et al., Reference Kumar Tewari, Kumar and Nand Sharma2006; Marschner & Marschner, Reference Marschner and Marschner2012; Ye et al., Reference Ye, Gao, Xu, Li, Ren, Li, Cong, Lu, Cakmak and Lu2024).

The distribution of Mg²⁺ within leaves also reflects its nutritional status. Depending on Mg²⁺ availability, up to 35% of total leaf Mg²⁺ is found in chloroplasts, with up to 25% associated to chlorophyll; this percentage is increased by Mg²⁺ deficiency or low-light conditions (Broadley & White, Reference Broadley and White2010; Cakmak & Yazici, Reference Cakmak and Yazici2010; Dorenstouter et al., Reference Dorenstouter, Pieters and Findenegg1985; Scott & Robson, Reference Scott and Robson1990). The remainder is distributed among the vacuole (the main storage site), the cell wall and the water-soluble cytoplasmic pool (Leigh & Wyn Jones, Reference Leigh and Wyn Jones1986; Maguire & Cowan, Reference Maguire and Cowan2002; Marschner & Marschner, Reference Marschner and Marschner2012). The vacuole’s strong Mg²⁺ retention capacity limits both deficiency and toxicity (Shaul, Reference Shaul2002; Stelzer et al., Reference Stelzer, Lehmann, Kramer and Lüttge1990).

Finally, accumulating evidence indicates that Mg²⁺ supplementation can enhance plant resistance to both abiotic stresses, such as high light, cold, drought and heat, and biotic stresses, including fungal and bacterial infections (Shao et al., Reference Shao, Li, Gao, Sun, Hu, Ullah, Gao, Li, Liu, Jiang, Cao, Tian and Dai2021; Gupta et al., Reference Gupta, Leibman-Markus, Anand, Rav-David, Yermiyahu, Elad and Bar2022; Li et al., Reference Li, Muneer, Sun, Guo, Wang, Huang, Li and Zheng2023). For example, Mg²⁺ oxide (MgO) application in tomato induces immunity against Fusarium wilt by activating the jasmonic acid signalling pathway (Fujikawa et al., Reference Fujikawa, Takehara, Ota, Imada, Sasaki, Kajihara, Sakai, Jogaiah and Ito2021), and overall Mg²⁺ fertilization can improve crop yield (Wang, Hassan, et al., Reference Wang, Hassan, Nadeem, Wu, Zhang and Li2020). Consequently, Mg²⁺ deficiency not only disrupts photosynthesis and carbohydrate allocation but also alters broader nutrient homeostasis (see Section 3), plant stress responses and immunity, thereby reinforcing a feedback loop of physiological decline. Understanding these interdependencies is therefore critical for optimizing fertilization strategies, particularly in soils with variable pH, texture and ionic composition.

3. Magnesium availability in soils and interactions with other nutrients

In soils, adequate Mg²⁺ concentrations range between 0.12 and 8.5 mM to sustain optimal plant growth (Karley & White, Reference Karley and White2009; Marschner & Marschner, Reference Marschner and Marschner2012). Levels outside this range can cause deficiency or toxicity, both of which impair photosynthesis, carbohydrate partitioning and biomass accumulation (Farhat et al., Reference Farhat, Elkhouni, Zorrig, Smaoui, Abdelly and Rabhi2016; Guo et al., Reference Guo, Nazim, Liang and Yang2016). Therefore, understanding the geochemical, physiological and competitive factors regulating Mg²⁺ availability is critical to improve yield in agricultural systems. Importantly, soil and plant Mg²⁺ concentrations are shaped by numerous chemical, mineralogical and environmental factors (Senbayram et al., Reference Senbayram, Gransee, Wahle and Thiel2015).

A major determinant of soil Mg²⁺ concentration is the mineral composition and degree of weathering. Primary silicates like olivine and augite contain more Mg²⁺ than highly weathered minerals such as muscovite (Guo, Reference Guo, Hossain, Kamiya, Burritt, Tran and Fujiwara2017; Maguire & Cowan, Reference Maguire and Cowan2002; Mayland & Wilkinson, Reference Mayland and Wilkinson1989). In addition, due to its large hydrated ionic radius and low binding affinity for negatively charged soil colloids, Mg²⁺ is highly mobile and easily leached, particularly in sandy or acidic soils (Guo, Reference Guo, Hossain, Kamiya, Burritt, Tran and Fujiwara2017; Maguire & Cowan, Reference Maguire and Cowan2002). Acidic soils, which represent nearly 70% of global arable land, often have low cation exchange capacity, limiting Mg²⁺ retention. This problem is exacerbated under high rainfall or irrigation, further increasing the risk of Mg²⁺ deficiency (Aitken et al., Reference Aitken, Dickson, Hailes and Moody1999; Mengel et al., Reference Mengel, Kirkby, Kosegarten and Appel2001; Senbayram et al., Reference Senbayram, Gransee, Wahle and Thiel2015).

However, Mg²⁺ availability is not determined solely by its absolute concentration but also by its interactions with other ions in the rhizosphere and plant tissues. Such interactions can influence uptake, transport or utilization through precipitation, complexation or competition for binding and transport sites (Epstein & Bloom, Reference Epstein and Bloom1972; Fageria, Reference Fageria2001; Marschner & Marschner, Reference Marschner and Marschner2012). For instance, Mg²⁺ forms insoluble Mg²⁺ carbonate (MgCO₃) in alkaline or calcareous soils (Broadley & White, Reference Broadley and White2010) and often competes with cations of similar properties, including Ca²⁺, K⁺, Na⁺ and NH₄ ⁺, to maintain electrochemical and osmotic balance (Gransee & Führs, Reference Gransee and Führs2013; Lasa et al., Reference Lasa, Frechilla, Aleu, González-Moro, Lamsfus and Aparicio-Tejo2000; Peuke et al., Reference Peuke, Jeschke and Hartung2002). This mutual antagonism is especially relevant in the context of modern agriculture, where unbalanced use of nitrogen–phosphorus–potassium (NPK) fertilizers contributes to progressive Mg²⁺ depletion in soils (Cakmak & Yazici, Reference Cakmak and Yazici2010; Hermans et al., Reference Hermans, Conn, Chen, Xiao and Verbruggen2013; Kobayashi & Tanoi, Reference Kobayashi and Tanoi2015).

Mg²⁺ interactions with other ions can also be synergistic, enhancing physiological functions or antagonistic, where excess of one ion inhibits the uptake of others. The effects depend on soil properties, nutrient ratios, plant species, plant tissues, developmental stage and environmental conditions (Chaudhry et al., Reference Chaudhry, Nayab, Hussain, Ali and Pan2021; Hermans et al., Reference Hermans, Conn, Chen, Xiao and Verbruggen2013). At low to moderate concentrations, Mg²⁺ uptake shows synergistic or neutral interactions with Ca²⁺ and K⁺ but becomes antagonistic when Ca²⁺ or K⁺ are present in excess. Under Mg²⁺ limitation, antagonism is usually observed, although findings for K⁺ remain inconsistent (Heenan & Campbell, Reference Heenan and Campbell1981; Fageria, Reference Fageria1983; Lasa et al., Reference Lasa, Frechilla, Aleu, González-Moro, Lamsfus and Aparicio-Tejo2000; Hermans et al., Reference Hermans, Johnson, Strasser and Verbruggen2004; Ding et al., Reference Ding, Luo and Xu2006; Tang & Luan, Reference Tang and Luan2017; Rhodes et al., Reference Rhodes, Miles and Hughes2018; Xie et al., Reference Xie, Cakmak, Wang, Zhang and Guo2021; Garcia et al., Reference Garcia, Crusciol, Rosolem, Bossolani, Nascimento, McCray, Reis and Cakmak2022). Interactions with micronutrients such as Mn²⁺, Zn²⁺, Fe²⁺ and Cu²⁺ are context-dependent: high concentrations of these metals can inhibit Mg²⁺ uptake, whereas Mg²⁺ deficiency may induce their accumulation or have little effect (Heenan & Campbell, Reference Heenan and Campbell1981; Kumar et al., Reference Kumar, Bhatia and Shukla1981; Agarwala et al., Reference Agarwala, Chatterjee, Gupta and Nautiyal1988; Le Bot et al., Reference Le Bot, Goss, Carvalho, Van Beusichem and Kirkby1990; Lu et al., Reference Lu, Liang, Lakshmanan, Guan, Liu and Chen2021; Sadeghi et al., Reference Sadeghi, Rezeizad and Rahimi2021; Xu et al., Reference Xu, Liu, Gu, Zhan, Li, Zhou, Zhang and Zou2024). Mg²⁺ generally supports P acquisition, while deficiency reduces or has little impact on tissue P content (Fageria, Reference Fageria1983; Skinner & Matthews, Reference Skinner and Matthews1990; Ogura et al., Reference Ogura, Kobayashi, Hermans, Ichihashi, Shibata, Shirasu, Aoki, Sugita, Ogawa, Suzuki, Iwata, Nakanishi and Tanoi2020; Weih et al., Reference Weih, Liu, Colombi, Keller, Jäck, Vallenback and Westerbergh2021). The form of N also affects Mg²⁺ uptake: NH₄ ⁺ tends to inhibit it, whereas NO₃ ⁻ promotes acquisition. Conversely, Mg²⁺ fertilization can enhance NO₃ ⁻ uptake by upregulating nitrate transporter genes such as NRT2.1 and NRT2.2 (Mayland & Wilkinson, Reference Mayland and Wilkinson1989; Lasa et al., Reference Lasa, Frechilla, Aleu, González-Moro, Lamsfus and Aparicio-Tejo2000; Peng et al., Reference Peng, Qi, Nie, Xiao, Liao and Chen2020; Tian et al., Reference Tian, Liu, Xu, Xing, Liu, Lyu, Feng, Zhang, Qin, Jiang, Zhu, Jiang and Ge2023). Finally, Mg²⁺ often mitigates toxic ions such as Al³⁺, Cd²⁺, Na⁺ and Li⁺, exerting protective effects by competing for transporters, stabilizing membranes, promoting organic acid exudation and maintaining cytoplasmic pH or metal homeostasis. For example, the overexpression of Mg²⁺ transporters genes (AtMGT1 and OsMGT1) increases cytosolic Mg²⁺ and enhances tolerance to Al³⁺ stress (Deng et al., Reference Deng, Luo, Li, Zheng, Wei, Smith, Thammina, Lu, Li and Pei2006; de Wit et al., Reference de Wit, Eldhuset and Mulder2010; Chou et al., Reference Chou, Chao, Huang, Hong and Kao2011; Hermans et al., Reference Hermans, Chen, Coppens, Inzé and Verbruggen2011; Rengel et al., Reference Rengel, Bose, Chen and Tripathi2015; Chen et al., Reference Chen, Yamaji, Horie, Che, Li, An and Ma2017; Lyu et al., Reference Lyu, Liu, Xu, Liu, Qin, Zhang, Tian, Jiang, Jiang, Zhu and Ge2023; Garcia-Daga et al., Reference Garcia-Daga, Fischer and Gilliham2025).

Taken together, the multiple cellular roles of Mg²⁺ emphasize the need for strict control of its concentration and distribution within plant tissues and organelles. These functional requirements have driven the evolution of specialized transport systems and regulatory networks that ensure Mg²⁺ homeostasis.

4. Magnesium transport systems in plants

While Mg²⁺ concentration in plants is estimated at 25 mM, most of it is tightly bound to nucleotides, ribosomes, chlorophyll or stored in vacuoles, making only a small fraction (0.2–5 mM) available for metabolism. This free Mg²⁺ pool is dynamically regulated by Mg²⁺ transporters and the binding of Mg²⁺ to nucleotides involved in cellular reactions (Kleczkowski & Igamberdiev, Reference Kleczkowski and Igamberdiev2021, Reference Kleczkowski and Igamberdiev2023).

Mg²⁺ transport in plants involves coordinated uptake, long-distance translocation and intracellular partitioning, ensuring proper allocation across tissues and organelles. The core Mg²⁺ transport system in higher plants is formed by the Mitochondrial RNA Splicing 2 (MRS2)/Mg²⁺ transporter (MGT) family, the Mg²⁺/H+ exchanger (MHX) and members of the Magnesium release (MGR)/Cyclin M (CNNM)/Cation Outward Rectifier C (CorC) family, which are more recently identified key players of Mg²⁺ transport. Additional systems like cyclic nucleotide–gated ion channels (CNGCs) contribute in specific contexts.

4.3. Subcellular transport

Mg²⁺ is vital at the subcellular level, and its transport has been especially studied in chloroplasts where it is the central atom of chlorophyll and participates in photosynthetic enzyme function and thylakoid stacking. Internal Mg²⁺ concentration can be measured by using an Mg²⁺-sensitive fluorescent indicator, mag-fura-2. Upon illumination, release of Mg²⁺ from thylakoid membranes has been observed in intact chloroplasts, and Mg²⁺ concentration typically increases from 0.5 mM to 2.0 mM in the stroma (Ishijima et al., Reference Ishijima, Uchibori, Takagi, Maki and Ohnishi2003). Stromal ion homeostasis depends on the activity of both thylakoid and envelope ion channels. While the identity of the Mg²⁺ transport system across the thylakoid membrane is unknown, transport across chloroplast inner envelope is mediated by AtMRS2-11/AtMGT10 in Arabidopsis (with OsMGT3 as its orthologue in rice) and the MGR family members MGR8 and MGR9 (ACDP/CNNM/CorC-related) (Figure 1). These three transporters have different roles, as evidenced by distinct phenotypes of their respective mutants (Dukic et al., Reference Dukic, Van Maldegem, Shaikh, Fukuda, Töpel, Solymosi, Hellsten, Hansen, Husted, Higgins, Sano, Ishijima and Spetea2023). For instance, thylakoid stacking is disrupted in mgt10 mutants but remains largely unaffected in mgr8 or mgr9. Conversely, grana size is reduced in the mgr8 mgr9 double mutant (Dukic et al., Reference Dukic, Van Maldegem, Shaikh, Fukuda, Töpel, Solymosi, Hellsten, Hansen, Husted, Higgins, Sano, Ishijima and Spetea2023; Zhang et al., Reference Zhang, Zhang, Tang, Zheng, Zhao, Fu, Lan and Luan2022) but increased in mgt10 plastids (Dukic et al., Reference Dukic, Van Maldegem, Shaikh, Fukuda, Töpel, Solymosi, Hellsten, Hansen, Husted, Higgins, Sano, Ishijima and Spetea2023; Sun et al., Reference Sun, Yang, Li and Huang2017). In Arabidopsis, AtMRS2-11/MGT10 has been shown to mediate Mg²⁺ export from chloroplasts using dye-based Mg²⁺ imaging, whereas MGR8 and MGR9 are responsible for Mg²⁺ import into the stroma (Ishijima et al., Reference Ishijima, Shiomi and Sagami2021; Kunz et al., Reference Kunz, Armbruster, Mühlbauer, De Vries and Davis2024). In addition to these envelope transporters, the thylakoid cation/H⁺ antiporter KEA3 may also contribute to Mg²⁺ movements within the chloroplast. KEA3 exports protons from the lumen in exchange for another cation, most likely K⁺, yet transport of Mg²⁺ cannot be excluded (Uflewski et al., Reference Uflewski, Mielke, Correa Galvis, Von Bismarck, Chen, Tietz, Ruß, Luzarowski, Sokolowska, Skirycz, Eirich, Finkemeier, Schöttler and Armbruster2021).

Figure 1. Schematic overview of chloroplastic Mg²⁺ flux under illumination in Arabidopsis.

Upon illumination, photosynthetic electron transport drives proton pumping from the stroma into the thylakoid lumen, generating a steep trans-membrane proton gradient (lumen ~ pH 5; stroma ~ pH 8). This proton motive force powers ATP synthase (AS), which restores H⁺ to the stroma while producing ATP. Lightning symbol indicates light-driven reactions of the photosynthetic electron transport chain. The resulting stromal alkalinization triggers Mg²⁺ release from the thylakoid lumen into the stroma, thereby increasing stromal Mg²⁺ concentration. At the chloroplast inner envelope, Mg²⁺ import from the cytosol into the stroma is mediated by transporter proteins MGR8 and MGR9, supporting Mg²⁺ requirements for ATP stabilization, enzyme activation, chlorophyll-binding protein function and other chloroplastic-related functions. The AtMGT10/OsMGT3 channel is likely responsible for Mg²⁺ efflux or exchange across the inner envelope, maintaining charge balance during light-dependent H⁺ movements. After a sudden light decrease, KEA3-driven H⁺ export from the lumen to the stroma in exchange for another cation, most likely K⁺, is required for the prompt relaxation of non-photochemical quenching. Since the substrate of KEA3 has not been demonstrated in plants, transport of Mg²⁺ via the antiporter KEA3 cannot be excluded (Uflewski et al., Reference Uflewski, Mielke, Correa Galvis, Von Bismarck, Chen, Tietz, Ruß, Luzarowski, Sokolowska, Skirycz, Eirich, Finkemeier, Schöttler and Armbruster2021) (figure created with BioRender.com).

Vacuolar sequestration of Mg²⁺ is another important aspect of intracellular Mg²⁺ partitioning. The MHX transporter, a vacuolar metal/H⁺ exchanger, contributes mainly to detoxification and buffering of excess Mg²⁺, along with other divalent cations such as Zn²⁺ and Cd²⁺ (Shaul, Reference Shaul2002). Conn et al. (Reference Conn, Conn, Tyerman, Kaiser, Leigh and Gilliham2011) suggested that MGT2/MRS2-1 and MGT3/MRS2-5 might contribute to vacuolar accumulation of Mg²⁺ in the mesophyll cells, especially under serpentine conditions (high Mg²⁺/Ca²⁺ ratio in the soil) (Conn et al., Reference Conn, Conn, Tyerman, Kaiser, Leigh and Gilliham2011). However, subsequent work rather showed that MGT2 functions in Mg²⁺ efflux from the vacuole (as detailed below), whereas the tonoplast MGR1 mediates Mg²⁺ sequestration into the vacuole (Tang et al., Reference Tang, Yang, Yan, Mao, Yuan, Wang, Zhao and Luan2022).

The remobilization of Mg²⁺ from the vacuole to the cytoplasm is another key step in Mg²⁺ homeostasis. This process is mediated by MRS2-10/MGT1 and MRS2-1/MGT2 in Arabidopsis, two redundant vacuolar transporters (only the double mutant displays a phenotype) (Tang et al., Reference Tang, Yang, Yan, Mao, Yuan, Wang, Zhao and Luan2022). Note that AtMGT1 was initially mislocalized in the plasma membrane (Li et al., Reference Li, Tutone, Drummond, Gardner and Luan2001). Mg²⁺ efflux via MGT1 and MGT2 constitutes a rate-limiting step in Mg²⁺ remobilization from old leaves to young tissues such as seeds (Tang et al., Reference Tang, Yang, Yan, Mao, Yuan, Wang, Zhao and Luan2022).

5. Magnesium homeostasis signalling pathways

Magnesium homeostasis ensures that plants maintain an optimal internal Mg²⁺ concentration, supporting metabolic processes while preventing excessive accumulation that could become toxic. The main mechanisms contributing to Mg²⁺ homeostasis include regulated uptake by roots, controlled long-distance transport and tissue partitioning, dynamic storage and remobilization from intracellular compartments and signalling pathways that adjust transporter activity according to both internal and external Mg²⁺ status.

Regulation of Mg²⁺ transporters operates at multiple levels, including transcriptional control (Franken et al., Reference Franken, Huynen, Martínez-Cruz, Bindels and De Baaij2022). However, unlike other macro-nutrients, Mg²⁺ deficiency generally induces only limited transcriptional regulation of the Mg²⁺ transporter genes involved in root uptake (Gebert et al., Reference Gebert, Meschenmoser, Svidová, Weghuber, Schweyen, Eifler, Lenz, Weyand and Knoop2010; Hermans, Vuylsteke, Coppens, Craciun, et al., Reference Hermans, Vuylsteke, Coppens, Craciun, Inzé and Verbruggen2010; Ogura et al., Reference Ogura, Kobayashi, Hermans, Ichihashi, Shibata, Shirasu, Aoki, Sugita, Ogawa, Suzuki, Iwata, Nakanishi and Tanoi2020). While Ogura et al. (Reference Ogura, Kobayashi, Suzuki, Iwata, Nakanishi and Tanoi2018) showed that Mg²⁺ uptake system was up-regulated in roots within 1 h in response to low Mg²⁺, this induction was not seen in mgt6 or mgt7 mutants, and the expression of AtMRS2-4/AtMGT6 and AtMRS2-7/AtMGT7 was not responsive to this condition (Ogura et al., Reference Ogura, Kobayashi, Suzuki, Iwata, Nakanishi and Tanoi2018). In rice, OsMGT1 was only induced in shoots but not in roots under Mg²⁺ deficiency (Zhang, Peng, et al., Reference Zhang, Meng, Li, Zhou, Ma, Fu, Schwarzländer, Liu and Xiong2019). However, differences between species or experimental conditions may occur, as illustrated in tomato, where Mg²⁺ deficiency led to a clear transcriptional induction of several Mg²⁺ transporter genes (SlMGT1, SlMGT6, SlMGT7, and SlMGT10) in roots (Ishfaq et al., Reference Ishfaq, Zhong, Wang and Li2021). Overall, this suggests that Mg²⁺ uptake is primarily controlled by post-transcriptional or post-translational mechanisms or by changes in transporter activity and localization rather than gene induction.

A key regulation is by Mg²⁺ or Mg-ATP binding. CorA/MRS2/MGT channels constitute the major Mg²⁺ uptake system in plants and are believed to be regulated similarly as their prokaryotic ancestors. Electrophysiological, electronic paramagnetic resonance and molecular-dynamics studies of bacterial CorA show that cytoplasmic Mg²⁺ acts as a ligand gating the pentameric channel (Dalmas et al., Reference Dalmas, Sompornpisut, Bezanilla and Perozo2014). CorA crystal structures revealed that Mg²⁺ binds to both the central pore and the intracellular region rich in acidic residues. When intracellular Mg²⁺ is high (i.e., >5 mM), binding to sensor sites induces a closed conformation by increasing inter-subunit contacts. Conversely, when internal Mg²⁺ drops and unbinding occurs, the channel undergoes asymmetric domain rearrangement, opening to allow Mg²⁺ influx. In eukaryotic MRS2 (yeast or human), with resolved crystal structures, Mg²⁺-sensing sites have been confirmed (Khan et al., Reference Khan, Sponder, Sjöblom, Svidová, Schweyen, Carugo and Djinović-Carugo2013; Li et al., Reference Li, Liu, Wallerstein, Villones, Huang, Lindkvist-Petersson, Meloni, Lu, Steen Jensen, Liin and Gourdon2025). Plant homologs show structural similarity suggesting a comparable architecture, though direct functional gating studies (e.g., patch-clamp or conformational assays) are limited. Therefore, Mg²⁺ regulation is likely, but experimental confirmation in plants is less extensive than in bacteria or human mitochondria.

Members of the CNNM/CorC family (including plant MGR proteins) carry cytosolic CBS-pair domains that in other systems bind Mg-ATP and trigger conformational changes that regulate transporter activity. Structural and functional work on bacterial/archaeal CorB/CorC and on animal CNNMs supports Mg-ATP as a regulatory ligand. High-resolution structures (apo and Mg-ATP-bound) of archaeal CorB/CorC reveal an ATP-binding site in the cytosolic domain and conformational differences between ligand-free and Mg-ATP-bound states. Binding induces a conformational rearrangement of the CBS dimer, which is communicated to the transmembrane DUF21 domain and alters the transporter’s conductive or regulatory state (promoting or inhibiting Mg flux depending on the system). Functional assays indicate that Mg-ATP binding is important for Mg²⁺ transport/export activity in these proteins (Chen et al., Reference Chen, Kozlov, Moeller, Rohaim, Fakih, Roux, Burke and Gehring2021). For plant MGRs, homology, domain architecture and physiological data strongly suggest similar regulation, but direct biochemical demonstration of Mg-ATP binding controlling plant MGR activity is absent in the peer-reviewed literature so far. MGRs have been mainly studied in Arabidopsis (9 members) and in wheat (15 members). In wheat, all 15 MGR genes contain conserved ABA-responsive elements in the promoter region (Chen et al., Reference Chen, Zhang, Zhao, Liu, Zhao, Xu, Wang, Zong, Zhang, Ji, Ma, Zhao and Li2025) although direct regulation by ABA remains to be demonstrated. This observation is reminiscent of the clear overlap between Mg-deficiency-induced genes and ABA-responsive genes described by Hermans, Vuylsteke, Coppens, Cristescu, et al. (Reference Hermans, Vuylsteke, Coppens, Cristescu, Harren, Inzé and Verbruggen2010).

Additional regulation derives from proton-motive forces that drive antiporters such as MHX and from crosstalk with Ca²⁺ signalling pathways that modulate transporter expression and ion channel activity. In Arabidopsis, tonoplast-localized Ca²⁺ sensors, CBL2 and CBL3, contribute to Mg²⁺ homeostasis via a V-ATPase-independent mechanism (Tang et al., Reference Tang, Zhao, Garcia, Kleist, Yang, Zhang and Luan2015). CBL2 and CBL3 recruit a set of four functionally redundant CBL-interacting protein kinases (CIPK 3/9/23/26) to the tonoplast, establishing a CBL-CIPK signalling module that regulates vacuolar Mg²⁺ sequestration in response to elevated cytosolic Mg²⁺ (as seen in plants living on serpentine soils characterized by high Mg²⁺ content and low Ca²⁺/Mg²⁺ ratios). The targets at the tonoplast of CIPK3/9/23/26 remain unidentified. Since the mutants of MHX or MGT2/3 showed wild-type response to high Mg²⁺, those transporters are not involved in this CBL-CIPK regulation. Because tolerance to high Mg²⁺ was dependent on external Ca²⁺ (Tang et al., Reference Tang, Zhao, Garcia, Kleist, Yang, Zhang and Luan2015), Ródenas and Vert (Reference Ródenas and Vert2021) proposed that TPC1 (a slowly activated, non-selective, Ca²⁺-activated vacuolar channel) is the target of CBL2/3-CIPK3/9/23/26. In strong support, the activity of TPC1 is modulated by high Mg²⁺. In addition, the sucrose non-fermenting-1-related protein kinase 2 (SnRK2) has recently been implicated in Arabidopsis high-Mg²⁺ response (Mohamadi et al., Reference Mohamadi, Babaeian Jelodar, Bagheri, Nematzadeh and Hashemipetroudi2023).

Genetic evidence reinforces the Mg²⁺-Ca²⁺ relationship: loss-of-function cax1 mutants, lacking a major vacuolar H⁺/Ca²⁺ antiporter, exhibit enhanced tolerance to serpentine soils, likely because reduced vacuolar Ca²⁺ sequestration increases cytosolic Ca²⁺ available to counteract excess Mg²⁺ (Bradshaw, Reference Bradshaw2005). Conversely, low external Ca²⁺ supply can partially rescue Mg²⁺-deficiency phenotypes in mutants lacking Mg²⁺ transporters (Lenz et al., Reference Lenz, Dombinov, Dreistein, Reinhard, Gebert and Knoop2013).

Besides, Mg²⁺ transporter activity can also be modulated by the ionic context of the environment. Mg²⁺ flux inside chloroplasts illustrates how Mg²⁺ transport operates within a broader ionic network (Figure 1). In illuminated chloroplasts, proton pumping into the thylakoid lumen during photosynthetic electron transport acidifies the lumen and generates a proton gradient between the stroma and the lumen. To maintain electrochemical balance, Mg²⁺ and other cations (e.g., K⁺) move from the lumen to the stroma, causing a rapid and reversible increase in free stromal Mg²⁺. This process is pH-dependent, as light-induced stromal alkalinization is required for Mg²⁺ release (Ishijima et al., Reference Ishijima, Uchibori, Takagi, Maki and Ohnishi2003). Because several chloroplast enzymes, including Rubisco, depend on Mg²⁺ for activation, this light-driven redistribution likely helps coordinate photosynthetic metabolism with light availability (Dukic et al., Reference Dukic, Van Maldegem, Shaikh, Fukuda, Töpel, Solymosi, Hellsten, Hansen, Husted, Higgins, Sano, Ishijima and Spetea2023). In addition, Mg²⁺ flux affects the ΔpH across the thylakoid membrane and contributes to the regulation of non-photochemical quenching (NPQ). Mg²⁺ influx into the stroma may lower stromal pH through reversible cation/H⁺ exchange across the chloroplast envelope (Dukic et al., Reference Dukic, Van Maldegem, Shaikh, Fukuda, Töpel, Solymosi, Hellsten, Hansen, Husted, Higgins, Sano, Ishijima and Spetea2023). KEA3-driven H⁺ export from the thylakoid is also critical for rapid NPQ relaxation after a sudden decrease in light, and a potential Mg²⁺ transport function of KEA3 would further link this process to stromal Mg²⁺ dynamics (Uflewski et al., Reference Uflewski, Mielke, Correa Galvis, Von Bismarck, Chen, Tietz, Ruß, Luzarowski, Sokolowska, Skirycz, Eirich, Finkemeier, Schöttler and Armbruster2021).

A main strategy to study regulation of mineral homeostasis is to expose plants to nutrient deficiency. Understanding how plants sense and signal Mg²⁺ deficiency remains challenging because stress signalling networks are inherently complex and often overlap with other physiological responses (Wilkins et al., Reference Wilkins, Matthus, Swarbreck and Davies2016). Transcriptomic analyses under Mg²⁺ starvation have identified numerous differentially expressed genes (DEGs), providing a global view of adaptive responses (Hermans, Vuylsteke, Coppens, Craciun, et al., Reference Hermans, Vuylsteke, Coppens, Craciun, Inzé and Verbruggen2010; Liang et al., Reference Liang, Lin, Wu, Ye, Qu, Xie, Lin, Gao, Wang, Ke, Li, Guo, Lu, Tang, Chen and Li2024; Ogura et al., Reference Ogura, Kobayashi, Hermans, Ichihashi, Shibata, Shirasu, Aoki, Sugita, Ogawa, Suzuki, Iwata, Nakanishi and Tanoi2020; Yang et al., Reference Yang, Zhou, Wang, Wu, Ye, Guo and Chen2019). Although these DEGs do not directly reveal the molecular identity of Mg²⁺ sensors, they highlight early-responding candidates potentially involved in deficiency signalling. Mg²⁺ deficiency responses are spatially and temporally asynchronous, typically appearing first in roots before shoots (Hermans, Vuylsteke, Coppens, Cristescu, et al., Reference Hermans, Vuylsteke, Coppens, Cristescu, Harren, Inzé and Verbruggen2010). Notably, Ca²⁺ has emerged as a likely secondary messenger in Mg²⁺ deficiency signalling. Several Ca²⁺ transporter genes are upregulated during Mg²⁺ starvation, and cytosolic Ca²⁺ levels increase, consistent with the known Mg²⁺-Ca²⁺ antagonism (Hermans, Vuylsteke, Coppens, Craciun, et al., Reference Hermans, Vuylsteke, Coppens, Craciun, Inzé and Verbruggen2010; Hermans, Vuylsteke, Coppens, Cristescu, et al., Reference Hermans, Vuylsteke, Coppens, Cristescu, Harren, Inzé and Verbruggen2010). Supporting this model, Wiesenberger et al. (Reference Wiesenberger, Steinleitner, Malli, Graier, Vormann, Schweyen and Stadler2007) showed in yeast that Mg²⁺ deficiency rapidly elevates cytosolic free Ca²⁺, triggering the activation of Ca²⁺-binding proteins (CaBPs), suggesting a central role for Ca²⁺ in mediating Mg²⁺-deficiency responses. Similar Ca²⁺-dependent mechanisms appear to operate under Mg²⁺ excess with the CBL-CIPK module (see above, Tang et al., Reference Tang, Zhao, Garcia, Kleist, Yang, Zhang and Luan2015; Tang & Luan, Reference Tang and Luan2017).

Beyond indirect signalling effects, Mg²⁺ directly modulates the activity of several ion channels, highlighting its dual role as nutrient and signalling regulator. At the tonoplast, cytosolic Mg²⁺ activates slow vacuolar (SV) channels while inhibiting fast vacuolar (FV) channels, reducing K⁺ leakage and supporting ionic stability (Lemtiri-Chlieh et al., Reference Lemtiri-Chlieh, Arold and Gehring2020). Mg²⁺ also inhibits outward non-selective cation channels (NSCCs), such as MgC in guard and subsidiary cells of broad bean and maize, and participates in NH₄ ⁺/NH₃ transport across the peribacteroid membrane in N₂-fixing plants, often coordinated with Ca²⁺- signalling. At the plasma membrane, Mg²⁺ further regulates Ca²⁺ influx through hyperpolarization-activated Ca²⁺ channels (HACCs), believed to correspond to cyclic nucleotide-gated channels (CNGCs). Physiological concentrations of cytosolic Mg²⁺, mainly in the form of Mg–ATP, strongly inhibit HACC activity in guard cells at highly negative voltages (≤ −200 mV), via interaction with a conserved diacidic Mg²⁺-binding motif (Lemtiri-Chlieh et al., Reference Lemtiri-Chlieh, Arold and Gehring2020). Thus, Mg²⁺ not only competes with Ca²⁺ for permeation but also seems to fine-tune Ca²⁺-dependent signalling.

Taken together, these findings reveal a tight interplay between Mg²⁺ and Ca²⁺ homeostasis, potentially involving shared transporters, regulatory sites and signalling components, although the underlying molecular mechanisms remain incompletely understood. Beyond its effects on Mg²⁺-related pathways, Ca²⁺ appears to act as a general mediator of nutrient signalling, influencing plant responses to K⁺, NO₃ ⁻, B³⁺ and possibly PO₄ ³⁻ (Behera et al., Reference Behera, Long, Schmitz-Thom, Wang, Zhang, Li, Steinhorst, Manishankar, Ren, Offenborn, Wu, Kudla and Wang2017; Matthus et al., Reference Matthus, Wilkins, Swarbreck, Doddrell, Doccula, Costa and Davies2019; Quiles-Pando et al., Reference Quiles-Pando, Navarro-Gochicoa, Herrera-Rodríguez, Camacho-Cristóbal, González-Fontes and Rexach2019; Wilkins et al., Reference Wilkins, Matthus, Swarbreck and Davies2016; Xu et al., Reference Xu, Li, Chen, Wang, Liu, He and Wu2006).

Parallels can also be drawn with animal systems, where Mg²⁺ functions as a second messenger regulating diverse cellular processes. For example, Li et al. (Reference Li, Chaigne-Delalande, Kanellopoulou, Davis, Matthews, Douek, Cohen, Uzel, Su and Lenardo2011) showed that defective Mg²⁺ flux underlies human T-cell immunodeficiency, while Stangherlin and O’Neill (Reference Stangherlin and O’Neill2018) demonstrated that Mg²⁺ dynamics modulate signal transduction. Collectively, these studies support the emerging view that Mg²⁺, beyond its metabolic and structural roles, contributes to the fine-tuning of cellular signalling pathways in plants.

6. Interactions of magnesium with the circadian clock

Recent findings have revealed a dynamic role for Mg²⁺ as a temporal regulator in plants. Mg²⁺ appears to modulate the circadian clock, and reciprocally, the clock influences Mg²⁺ homeostasis.

6.2. Circadian regulation of ion homeostasis

Historically, circadian rhythms were hypothesized to arise from feedback loops involving ion gradients and membrane transporters (Nitabach et al., Reference Nitabach, Holmes, Blau and Young2005; Njus et al., Reference Njus, Sulzman and Hastings1974). While the gene-centric model now dominates in circadian research, ion fluxes remain a critical interacting layer, that both modulate and is modulated by transcriptional and non-transcriptional feedback loops (Henslee et al., Reference Henslee, Crosby, Kitcatt, Parry, Bernardini, Abdallat, Braun, Fatoyinbo, Harrison, Edgar, Hoettges, Reddy, Jabr, von Schantz, O’Neill and Labeed2017; Mihut et al., Reference Mihut, O’Neill, Partch and Crosby2025). Mineral nutrients are now attracting renewed interest for their ability to influence circadian timing and improve crop productivity through chronoculture (Hastings et al., Reference Hastings, Maywood and O’Neill2008; Ogasawara et al., Reference Ogasawara, Hotta and Dodd2025; Steed et al., Reference Steed, Ramirez, Hannah and Webb2021).

Nutrient availability can affect clock dynamics in diverse ways (Table 1). Nitrogen deficiency shortens the circadian period in the dinoflagellate Gonyaulax polyedra (Sweeney & Folli, Reference Sweeney and Folli1984), and NO₃ ⁻ pulses entrain circadian gene expression in Arabidopsis, where clock components regulate many nitrogen-responsive genes (Covington et al., Reference Covington, Maloof, Straume, Kay and Harmer2008; Gutiérrez et al., Reference Gutiérrez, Stokes, Thum, Xu, Obertello, Katari, Tanurdzic, Dean, Nero, McClung and Coruzzi2008; Haydon et al., Reference Haydon, Bell and Webb2011; Porco et al., Reference Porco, Yu, Liang, Snoeck, Hermans and Kay2024). Transcripts associated with transport and homeostasis of K⁺, PO₄ ³⁻ and SO₄ ²⁻ are also circadian regulated (Lebaudy et al., Reference Lebaudy, Vavasseur, Hosy, Dreyer, Leonhardt, Thibaud, Véry, Simonneau and Sentenac2008; Haydon et al., Reference Haydon, Bell and Webb2011; Wang et al., Reference Wang, Shi, Ng, Battle, Zhang and Lu2011; Cao et al., Reference Cao, Wang, Wirtz, Hell, Oliver and Xiang2013; Uemoto et al., Reference Uemoto, Mori, Yamauchi, Kubota, Takahashi, Egashira, Kunimoto, Araki, Takemiya, Ito and Endo2023). In Arabidopsis, cytosolic and chloroplastic Ca²⁺ display circadian oscillations, peaking between midday and dusk in mesophyll cells (Johnson et al., Reference Johnson, Knight, Kondo, Masson, Sedbrook, Haley and Trewavas1995; Love et al., Reference Love, Dodd and Webb2004; Martí Ruiz et al., Reference Martí Ruiz, Jung and Webb2020; Sai & Johnson, Reference Sai and Johnson2002). Although some Ca²⁺ channels and transporters exhibit rhythmic transcription, these oscillations are thought to be primarily driven by post-transcriptional regulation (Dodd et al., Reference Dodd, Gardner, Hotta, Hubbard, Dalchau, Love, Assie, Robertson, Jakobsen, Gonçalves, Sanders and Webb2007; Haydon et al., Reference Haydon, Bell and Webb2011, Reference Haydon, Román and Arshad2015).

Table 1 Interactions between nutrients and the plant circadian clock. Information is for Arabidopsis unless specified.

Among micronutrients, Cu²⁺ and Fe²⁺ transport and homeostasis are under circadian regulation (Duc et al., Reference Duc, Cellier, Lobréaux, Briat and Gaymard2009; Hong et al., Reference Hong, Kim, Guerinot and McClung2013; Perea-García et al., Reference Perea-García, Andrés-Colás and Peñarrubia2010). Transcripts for Cu²⁺ transporters show circadian rhythms and their promoters contain conserved circadian elements (Covington et al., Reference Covington, Maloof, Straume, Kay and Harmer2008; Dodd et al., Reference Dodd, Gardner, Hotta, Hubbard, Dalchau, Love, Assie, Robertson, Jakobsen, Gonçalves, Sanders and Webb2007; Perea-García et al., Reference Perea-García, Andrés-Colás and Peñarrubia2010). Excess Cu²⁺ reduces clock amplitude and may lengthen circadian period, likely through GI-dependent pathways (Andrés-ColÁs et al., Reference Andrés-ColÁs, Perea-García, Puig and Peñarrubia2010; Haydon et al., Reference Haydon, Bell and Webb2011).

Similarly, genes involved in Fe²⁺ transport and storage, including ferritin-encoding genes are regulated by both Fe²⁺ availability and circadian components such as PRR7 and TIME FOR COFFEE (TIC) (Ding et al., Reference Ding, Millar, Davis and Davis2007; Duc et al., Reference Duc, Cellier, Lobréaux, Briat and Gaymard2009; Liu et al., Reference Liu, Carlsson, Takeuchi, Newton and Farré2013). Fe²⁺ deficiency lengthens the circadian period by ~1–2 hours in a light-dependent manner, requiring evening components such as GI and ZTL, while morning components CCA1 and LHY directly regulate Fe²⁺ uptake genes (Chen et al., Reference Chen, Wang, Shin, Wu, Shanmugam, Tsednee, Lo, Chen, Wu and Yeh2013; Hong et al., Reference Hong, Kim, Guerinot and McClung2013; Salomé et al., Reference Salomé, Oliva, Weigel and Krämer2013; Xu et al., Reference Xu, Jiang, Wang and Lin2019).

6.2.1. Magnesium oscillations and clock function

Magnesium is currently the nutrient with the most well-characterized connection to the circadian clock (Siqueira et al., Reference Siqueira, Zsögön, Fernie, Nunes-Nesi and Araújo2023). Oscillations in intracellular Mg²⁺ levels have been observed across multiple organisms, including the green unicellular alga Ostreococcus tauri, the fungus Neurospora crassa and human U2OS cells (Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016), as well as at the subcellular level in rice chloroplasts (Li et al., Reference Li, Yokosho, Liu, Cao, Yamaji, Zhu, Liao, Ma and Chen2020; Chen et al., Reference Chen, Tian, Li, Bai, Zhang, Li, Cao and Chen2022). These oscillations influence global translational activity by modulating ATP stability and ribosomal function (de Barros Dantas et al., Reference de Barros Dantas, Eldridge, Dorling, Dekeya, Lynch and Dodd2023; Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016). Mg²⁺ rhythms peak around dusk in Ostreococcus but at dawn in rice chloroplasts, suggesting that Mg²⁺ regulation differs among species and subcellular compartments (Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016; Li et al., Reference Li, Yokosho, Liu, Cao, Yamaji, Zhu, Liao, Ma and Chen2020).

In cyanobacteria, Mg²⁺ directly modulates the KaiC-based oscillator, and artificial Mg²⁺ cycles can drive rhythmic gene expression even in the absence of core clock genes KaiA and KaiB, highlighting the importance of non-transcriptional circadian regulation (Rust et al., Reference Rust, Golden and O’Shea2011; Jeong et al., Reference Jeong, Dias, Diekman, Brochon, Kim, Kaur, Kim, Jang and Kim2019; Li et al., Reference Li, Buckley and Haydon2022). Similarly, in Ostreococcus, intracellular Mg²⁺ rhythms persist without transcriptional activity in constant darkness, supporting post-translational control of Mg²⁺ transport and Mg²⁺ oscillations (Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016). These observations emphasize the importance of non-gene-centric mechanisms in circadian regulation.

In mammals, the Mg²⁺ transporter TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL SUBFAMILY M MEMBER 7 (TRPM7) exhibits rhythmic expression that affects intracellular Mg²⁺ levels, linking Mg²⁺ homeostasis to the mammalian circadian clock (Uetani et al., Reference Uetani, Hardy, Gravel, Kiessling, Pietrobon, Wong, Chénard, Cermakian, St-Pierre and Tremblay2017; Zhang et al., Reference Zhang, Lahens, Yue, Arnold, Pakstis, Schwarz and Sehgal2021).

In Arabidopsis, Mg²⁺ deficiency consistently lengthens the circadian period and dampens oscillation amplitude, a response also conserved in Ostreococcus and human U2OS cells (de Melo et al., Reference de Melo, Gutsch, Caluwé, Leloup, Gonze, Hermans, Webb and Verbruggen2021; Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016; Hermans, Vuylsteke, Coppens, Craciun, et al., Reference Hermans, Vuylsteke, Coppens, Craciun, Inzé and Verbruggen2010). This effect is amplified under long-day photoperiods and in the presence of sucrose, suggesting that both light and metabolic status modulate the interaction between Mg²⁺ and the clock (de Melo et al., Reference de Melo, Gutsch, Caluwé, Leloup, Gonze, Hermans, Webb and Verbruggen2021; Hermans, Vuylsteke, Coppens, Cristescu, et al., Reference Hermans, Vuylsteke, Coppens, Cristescu, Harren, Inzé and Verbruggen2010). Mg²⁺ also interacts with light-perception pathways, notably phytochromes, feeding back to regulate the clock (Rivière et al., Reference Rivière, Xiao, Gutsch, Defrance, Webb and Verbruggen2021). However, unlike NO₃ ⁻, Mg²⁺ pulses fail to reset circadian phase, suggesting that Mg²⁺ does not act as an entrainment signal in Arabidopsis (de Melo et al., Reference de Melo, Gutsch, Caluwé, Leloup, Gonze, Hermans, Webb and Verbruggen2021).

In rice, chloroplast Mg²⁺ oscillations are directly circadian-regulated via OsMGT3 (AtMGT10), which exhibits rhythmic expression peaking around dawn (J. Li et al., Reference Li, Yokosho, Liu, Cao, Yamaji, Zhu, Liao, Ma and Chen2020), and is repressed by two PRR proteins (OsPRR59, OsPRR95) (Chen et al., Reference Chen, Tian, Li, Bai, Zhang, Li, Cao and Chen2022). OsMGT3 rhythmic expression generates diel fluctuations of Mg²⁺ in rice chloroplasts, influencing Rubisco activity and photosynthetic carbon fixation rates (C.-Q. Chen et al., Reference Chen, Tian, Li, Bai, Zhang, Li, Cao and Chen2022). Knockout mutants osmgt3 show reduced chloroplast Mg²⁺ rhythms and photosynthetic efficiency, while mesophyll-specific overexpression enhances growth and carbon assimilation (Li et al., Reference Li, Buckley and Haydon2022). This remains the only confirmed example of a clock-controlled Mg²⁺ transporter in plants.

6.3. Magnesium, metabolism and circadian integration

Magnesium's regulation of circadian rhythms is deeply intertwined with its role in cellular energy metabolism. As the physiological cofactor of ATP, Mg²⁺ is required for most energy-dependent reactions, including photosynthesis, ATP synthase activity and translation (Kleczkowski & Igamberdiev, Reference Kleczkowski and Igamberdiev2021; Ko et al., Reference Ko, Hong and Pedersen1999). In chloroplasts, ATP synthesis increases with external Mg²⁺ supply in a light-dependent manner (Lin & Nobel, Reference Lin and Nobel1971; Ishijima et al., Reference Ishijima, Uchibori, Takagi, Maki and Ohnishi2003; Chen et al., Reference Chen, Peng, Li and Liao2018), whereas Mg²⁺ deficiency causes ADP accumulation, impaired respiration and growth arrest (Gout et al., Reference Gout, Rébeillé, Douce and Bligny2014).

Through its tight coupling with adenylate metabolism, Mg²⁺ functions upstream of the Target of Rapamycin (TOR) pathway, a conserved kinase energy-sensor complex that modulates the circadian clock and integrates nutrient, hormone and environmental signals to promote growth and translation while repressing autophagy in both mammals and plants (Khapre et al., Reference Khapre, Patel, Kondratova, Chaudhary, Velingkaar, Antoch and Kondratov2014; Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016; Wu et al., Reference Wu, Shi, Li, Fu, Liu, Xiong and Sheen2019; Zhang, Meng, et al., Reference Zhang, Meng, Li, Zhou, Ma, Fu, Schwarzländer, Liu and Xiong2019; Liu et al., Reference Liu, Wang, Zhang, Fu, Zhou, Long, He, Yang, Li and Peng2021; Meng, Zhang, Li, et al., Reference Meng, Zhang, Li, Shen, Sheen and Xiong2022; Urrea-Castellanos et al., Reference Urrea-Castellanos, Calderan-Rodrigues, Artins, Musialak-Lange, Macharanda-Ganesh, Fernie, Wahl and Caldana2025). Mammalian TOR activity is highly Mg²⁺-dependent, since MgATP functions as its substrate and an additional Mg²⁺ ion binds the active site to enable target phosphorylation (Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016; Kleczkowski & Igamberdiev, Reference Kleczkowski and Igamberdiev2023). TOR form an antagonistic regulatory module with the Sucrose non-fermenting-Related protein Kinase 1 (SnRK1). TOR inhibits SnRK1 signalling under energy-replete conditions, whereas SnRK1 suppress TOR activity during energy-deplete conditions, shifting metabolism from growth towards conservation, autophagy and stress responses. Activated SnRK1 phosphorylates and activates the transcription factor basic leucine Zipper 63 (bZIP63), which in turn binds to the PRR7 promoter to increase its transcription. Elevated PRR7 expression delays circadian phase and is required for sugar-induced shortening of the circadian period (Figure 2) (Haydon et al., Reference Haydon, Mielczarek, Robertson, Hubbard and Webb2013; Frank et al., Reference Frank, Matiolli, Viana, Hearn, Kusakina, Belbin, Wells Newman, Yochikawa, Cano-Ramirez, Chembath, Cragg-Barber, Haydon, Hotta, Vincentz, Webb and Dodd2018).

Figure 2. Hypothetical working model linking magnesium homeostasis, metabolism, calcium signaling, stomatal regulation and circadian clock in plants.

Under optimal conditions of light, sugar and nutrient availability, TOR is activated. TOR promotes anabolic processes while repressing catabolic pathways, partially through inhibition of abscisic acid (ABA) signalling. TOR-mediated suppression of ABA signalling inhibits SnRK1 and SnRK2 kinases, which modulate the bZIP63-PRR7 regulatory module. This module feeds back to the circadian clock, resulting in phase advancement and period shortening.

TOR also represses SnRK2 and cADPR-dependent Ca²⁺ signalling pathways, promoting stomatal opening under favourable growth conditions. Oscillations in Ca²⁺ are sensed by Ca²⁺-binding proteins CML23 and CML24, which in turn feed back to the circadian clock.

In rice, the rhythmically expressed Mg²⁺ transporter gene OsMGT3 (AtMGT10 in Arabidopsis) is transcriptionally repressed by PRR proteins; disruption of this regulation diminishes Mg²⁺ oscillations, photosynthesis and overall growth.

Arrow codes: Black solid arrows, established regulation; brown solid arrows, established transcriptional regulation; black dashed arrows, proposed or indirect regulation requiring further validation; blue arrows, upregulation or downregulation under optimal growth conditions. Lightning bolt = optimal growth conditions, warning sign = stress conditions (e.g., energy or nutrient limitation) and NAM = nicotinamide (figure created with BioRender.com).

In human and algal cells, TOR inhibition lengthens the circadian period and abolishes the Mg²⁺-depletion lengthening effect, indicating that Mg²⁺ regulates the clock, at least in part, via TOR signalling (Feeney et al., Reference Feeney, Hansen, Putker, Olivares-Yañez, Day, Eades, Larrondo, Hoyle, O’Neill and van Ooijen2016; Rubin, Reference Rubin2007; van Ooijen & O’Neill, Reference van Ooijen and O’Neill2016). In Arabidopsis, TOR inhibition similarly lengthens the circadian period in a dose-dependent manner (Zhang, Meng, et al., Reference Zhang, Meng, Li, Zhou, Ma, Fu, Schwarzländer, Liu and Xiong2019; Wang, Qin, et al., Reference Wang, Qin, Li, Zhang and Wang2020; Urrea-Castellanos et al., Reference Urrea-Castellanos, Caldana and Henriques2022). Restoration of sugar under low-energy condition reactivates TOR and normalizes/shortens period length, whereas this recovery is lost when TOR is silenced. Conversely, nicotinamide, a precursor of NAD⁺, inhibits sugar-driven ATP production, suppresses TOR activity, and lengthens the circadian period. Together, these observations demonstrate that both sugar-induced period shortening and nicotinamide-induced period lengthening are TOR-dependent (Figure 2) (Dodd et al., Reference Dodd, Gardner, Hotta, Hubbard, Dalchau, Love, Assie, Robertson, Jakobsen, Gonçalves, Sanders and Webb2007; Haydon et al., Reference Haydon, Mielczarek, Robertson, Hubbard and Webb2013; Zhang, Meng, et al., Reference Zhang, Meng, Li, Zhou, Ma, Fu, Schwarzländer, Liu and Xiong2019; de Melo et al., Reference de Melo, Gutsch, Caluwé, Leloup, Gonze, Hermans, Webb and Verbruggen2021). Mg²⁺ deficiency also delays the phase of PRR7 expression, which may relate to the finding that prr7-11 mutation abolishes nicotinamide- and sugar-induced clock adjustments (de Melo et al., Reference de Melo, Gutsch, Caluwé, Leloup, Gonze, Hermans, Webb and Verbruggen2021; Farré & Weise, Reference Farré and Weise2012; Haydon et al., Reference Haydon, Mielczarek, Robertson, Hubbard and Webb2013; Mombaerts et al., Reference Mombaerts, Carignano, Robertson, Hearn, Junyang, Hayden, Rutterford, Hotta, Hubbard, Maria, Yuan, Hannah, Goncalves and Webb2019). The convergent period-lengthening effects of Mg²⁺ deficiency, sugar deprivation and nicotinamide, along with their shared TOR-dependent influence on circadian timing, indicate that Mg²⁺ most likely modulates the circadian clock, at least in part, upstream of TOR signaling through conserved regulatory pathways in plants (Figure 2).

Conversely, there is growing evidence that the circadian clock exerts feedback on TOR signalling. In bzip63 mutants, rhythmic expression of Ribosomal Protein S6 Kinase 1 (S6K1) – a key downstream effector of TOR that phosphorylates ribosomal protein S6 to stimulate translation and cell growth – is disrupted, indicating that TOR signalling is at least partly under circadian control (Frank et al., Reference Frank, Matiolli, Viana, Hearn, Kusakina, Belbin, Wells Newman, Yochikawa, Cano-Ramirez, Chembath, Cragg-Barber, Haydon, Hotta, Vincentz, Webb and Dodd2018; Urrea-Castellanos et al., Reference Urrea-Castellanos, Caldana and Henriques2022). Moreover, PRR proteins (specifically PRR5, PRR7 and PRR9) repress the transcription of TANDEM ZINC FINGER 1 (TZF1); this repression prevents TZF1-mediated destabilization of TOR mRNA, thereby maintaining TOR signalling (Li et al., Reference Li, Wang, Zhang, Tian, Chong, Jang and Wang2019; Wang, Qin, et al., Reference Wang, Qin, Li, Zhang and Wang2020).

Sucrose influences the plant circadian clock in a context-dependent manner. Under low-energy conditions, sucrose shortens the circadian period, but has little impact in energy-repleted condition. Likewise, TOR signalling is activated by sugar availability but is repressed during sugar starvation or sugar excess, reflecting the complex integration of cellular energy status, sugar specificity, and stress signals in plants. Together, these observations highlight the intricate interplay between ionic cues, metabolic cues and circadian regulation (Haydon et al., Reference Haydon, Mielczarek, Robertson, Hubbard and Webb2013; Han et al., Reference Han, Hua, Li, Qiao, Yao, Hao, Li, Fan, De Jaeger, Yang and Bai2022; Wang et al., Reference Wang, Steed and Webb2022; Pereyra et al., Reference Pereyra, Parola, Lando, Rodriguez and Martínez-Noël2023). Therefore, the stronger impact of Mg²⁺ deficiency under sucrose-supplemented and long-day conditions (de Melo et al., Reference de Melo, Gutsch, Caluwé, Leloup, Gonze, Hermans, Webb and Verbruggen2021) likely arises from interactions between nutrient limitation and cellular energy status. Importantly, Mg²⁺ also plays a critical role in sucrose transport, further linking Mg²⁺ availability to carbon metabolism.

Mg²⁺ status, metabolism and circadian regulation also influence stomatal movement. TOR negatively regulates abscisic acid (ABA) signalling by phosphorylating Pyrabactin Resistance 1/PYR1-Like/Regulatory Component of ABA Receptor (PYR/PYL/RCAR) receptors, favouring growth over stress responses. Upon stress, ABA accumulates and binds to PYR/PYL receptors, inhibiting type 2C protein phosphatases (PP2Cs), and activating SnRK1 and SnRK2 kinases, which phosphorylate downstream targets and inhibit TOR (Figure 2) (Baena-González & Hanson, Reference Baena-González and Hanson2017; Belda-Palazón et al., Reference Belda-Palazón, Adamo, Valerio, Ferreira, Confraria, Reis-Barata, Rodrigues, Meyer, Rodriguez and Baena-González2020, Reference Belda-Palazón, Costa, Beeckman, Rolland and Baena-González2022).

Mg²⁺ deficiency upregulates numerous ABA-responsive genes (Hermans et al., Reference Hermans, Johnson, Strasser and Verbruggen2004; Hermans, Vuylsteke, Coppens, Cristescu, et al., Reference Hermans, Vuylsteke, Coppens, Cristescu, Harren, Inzé and Verbruggen2010), whereas Mg²⁺ excess activates a subclass of SnRK2 kinases in an ABA-dependent manner which, together with Ca²⁺-binding proteins, contributes to the maintenance of Mg²⁺ homeostasis, likely via vacuolar transport (Mogami et al., Reference Mogami, Fujita, Yoshida, Tsukiori, Nakagami, Nomura, Fujiwara, Nishida, Yanagisawa, Ishida, Takahashi, Morimoto, Kidokoro, Mizoi, Shinozaki and Yamaguchi-Shinozaki2015).

Upon ABA perception, activated SnRK2s activate slow anion channel-associated 1 (SLAC1) and related ion channels, promoting anion efflux and membrane depolarization, a key step in stomatal closure (Wang et al., Reference Wang, Zhao, Li, Hsu, Liu, Fu, Hou, Du, Xie, Zhang, Gao, Cao, Huang, Zhu, Tang, Wang, Tao, Xiong and Zhu2018; Wei et al., Reference Wei, Peng and Zhou2025). ABA also triggers cyclic ADP-ribose (cADPR)-mediated Ca²⁺ release, generating transient cytosolic Ca²⁺ spikes that reinforce stomatal closure (Wu et al., Reference Wu, Kuzma, Maréchal, Graeff, Lee, Foster and Chua1997; Leckie et al., Reference Leckie, McAinsh, Allen, Sanders and Hetherington1998). Notably, stomatal movement is known to be modulated by sugar, nicotinamide and TOR activity. TOR inhibition impairs light-induced stomatal opening, while nicotinamide, an inhibitor of both cADPR synthesis and TOR activity, inhibits ABA-induced stomatal closure in a dose-dependent manner (Han et al., Reference Han, Hua, Li, Qiao, Yao, Hao, Li, Fan, De Jaeger, Yang and Bai2022; Kottapalli et al., Reference Kottapalli, David-Schwartz, Khamaisi, Brandsma, Lugassi, Egbaria, Kelly and Granot2018; Leckie et al., Reference Leckie, McAinsh, Allen, Sanders and Hetherington1998; Siegel et al., Reference Siegel, Xue, Murata, Yang, Nishimura, Wang and Schroeder2009). In addition, cytosolic Mg²⁺ levels, regulated in part by the tonoplast-localized Mg²⁺ transporter MGR1, are essential for stomatal opening, with vacuolar Mg²⁺ sequestration particularly important under high Mg²⁺ conditions (Inoue et al., Reference Inoue, Hayashi, Huang, Yokosho, Gotoh, Ikematsu, Okumura, Suzuki, Kamura, Kinoshita and Ma2022).

Cytosolic and stromal Ca²⁺ levels display circadian rhythmicity, peaking around dusk in mesophyll cells, and feed back to the clock via Calmodulin-Like proteins 23 and 24 (CML23 and CML24), which are upregulated by ABA and darkness (Johnson et al., Reference Johnson, Knight, Kondo, Masson, Sedbrook, Haley and Trewavas1995; Love et al., Reference Love, Dodd and Webb2004; Delk et al., Reference Delk, Johnson, Chowdhury and Braam2005; Ruiz et al., Reference Ruiz, Hubbard, Gardner, Jung, Aubry, Hotta, Mohd-Noh, Robertson, Hearn, Tsai, Dodd, Hannah, Carré, Davies, Braam and Webb2018; Frank et al., Reference Frank, Happeck, Meier, Hoang, Stribny, Hause, Ding, Morsomme, Baginsky and Peiter2019). These oscillations are thought to depend on cADPR-mediated Ca²⁺ release from internal stores, as in animals, although the plant cADPR-sensitive channel and cADPR cyclase remain unidentified (Dodd et al., Reference Dodd, Gardner, Hotta, Hubbard, Dalchau, Love, Assie, Robertson, Jakobsen, Gonçalves, Sanders and Webb2007; Ikeda et al., Reference Ikeda, Sugiyama, Wallace, Gompf, Yoshioka, Miyawaki and Allen2003). Strikingly, sucrose starvation and nicotinamide, which inhibit Mg²⁺-sensitive TOR signalling, not only lengthen the circadian period and alter stomatal movement but are also known to abolish Ca²⁺ oscillations (Figure 2) (Dodd et al., Reference Dodd, Gardner, Hotta, Hubbard, Dalchau, Love, Assie, Robertson, Jakobsen, Gonçalves, Sanders and Webb2007; Johnson et al., Reference Johnson, Knight, Kondo, Masson, Sedbrook, Haley and Trewavas1995; Martí Ruiz et al., Reference Martí Ruiz, Jung and Webb2020).

Together, these findings support a model in which Mg²⁺, Ca²⁺, NAD⁺, sugars and TOR form an integrated metabolic–ionic network that couples cellular energy status to circadian regulation (Figure 2). Mg²⁺ acts as a metabolic integrator, connecting adenylate pools to TOR signalling and circadian timing. Daily fluctuations in nutrients and metabolites, including Mg²⁺, Ca²⁺ and sugars, provide a direct mechanism by which changes in cellular energy are mirrored into circadian adjustments, feeding back to modulate clock function, stomatal dynamics, and stress responses. This framework highlights a complex non-transcriptional layer of circadian regulation, in which ionic and metabolic cues converge to coordinate temporal control of growth, metabolism, and environmental responses.

7. Conclusion and perspectives

Mg²⁺ plays a pivotal role in plant physiology, acting both as a structural element and as a versatile regulatory cofactor. It is essential for chlorophyll coordination, activation of ATP-dependent enzymes and maintenance of ion homeostasis and signalling processes. Beyond these classical roles, recent findings have revealed that Mg²⁺ also influences circadian regulation, positioning it as an integrator of temporal, metabolic and nutritional cues in plants.

Despite its broad functional spectrum, Mg²⁺ remains comparatively understudied relative to other macronutrients. Considerable progress has been made in identifying Mg²⁺ transporters, yet major questions remain regarding their regulation, subcellular dynamics and integration into whole-plant nutrient allocation strategies. The complex interactions of Mg²⁺ with other ions, such as K⁺, Ca²⁺, NH₄ ⁺ and transition metals, represent another underexplored frontier, both at physiological and molecular scales.

In agronomic contexts, Mg²⁺ deficiency remains a widespread and often overlooked issue, particularly in acidic or intensively cultivated soils. Improving crop resilience and nutrient use efficiency will require a deeper understanding of how Mg²⁺ uptake, redistribution and sensing are modulated under stress and throughout plant development. Such knowledge will be instrumental for optimizing fertilization practices and guiding breeding strategies towards genotypes with enhanced Mg efficiency.

The recently established link between Mg²⁺ and circadian regulation opens promising research directions. Mg²⁺ may contribute to adaptive rhythmicity, fine-tuning metabolic and physiological processes in response to nutrient availability and environmental fluctuations. Investigating how Mg²⁺ homeostasis interacts with diurnal cycles and metabolic rhythms could uncover new regulatory mechanisms relevant to growth, yield and stress adaptation.

Future research should focus on the post-transcriptional and post-translational regulation of Mg²⁺ transporters, including the identification of upstream regulators such as kinases, phosphatases, small RNAs and protein interactors. Understanding how these components integrate into broader nutrient signalling and environmental response networks, such as those governed by circadian cues, light quality, or abiotic stress, will be key to grasping the dynamic control of Mg²⁺ homeostasis. Finally, exploring natural variation in transporter regulation across species or genotypes adapted to contrasting soils may reveal evolutionary strategies for Mg²⁺ acquisition and utilization, providing valuable targets for sustainable crop improvement.