Introduction

Magnesium (Mg) is an essential macronutrient. As a cofactor for many enzymes, Mg is required for fundamental cellular processes including energy metabolism, photosynthesis, and the synthesis of nucleic acids and proteins (Guo, Nazim, Liang & Yang, 2016). Mg is also involved in stress resistance (Huber & Jones, 2013, Mengutay, Ceylan, Kutman & Cakmak, 2013) and well-balanced Mg fertilization enhances crop yield and quality (Wang et al. , 2019). The intracellular level of Mg2+ is tightly regulated, and its deficiency or excess affects plant growth and development. Although there is a relatively good understanding of the physiological mechanisms responding to magnesium deficiency, not much is known about cellular response to high levels of Mg2+. For example, serpentine soils expose plants to high amounts of Mg and low levels of calcium. Similarly, in semi-arid regions, water stress can lead to the accumulation of Mg2+. For non-adapted plants, high Mg2+ conditions inhibit growth. In Arabidopsis, high-Mg2+ treatment results in a reduction of primary root length, fresh weight, and epicotyl length (Niu et al. , 2018). Plants grown in high-Mg2+ soil may avoid Mg2+ toxicity by limiting internal Mg2+ accumulation and/or Mg2+excretion from leaves. Sequestration of excess Mg2+ in the vacuole under high-Mg2+ conditions seems to play a pivotal role in Mg2+ tolerance. Vacuoles in leaf mesophyll cells can hold up to 80 mM of Mg2+ (Hermans, Conn, Chen, Xiao & Verbruggen, 2013).
As with other essential nutrients, magnesium (in its ionic form) is absorbed by plants from the soil. Recently, the signaling mechanism behind the response to high-Mg2+ was described. Network of calcineurin B-like calcium sensor proteins (CBL) CBL2/3, CBL-interacting protein kinases (CIPK) CIPK3/9/23/26, and sucrose nonfermenting-1-related protein kinase2 (SnRK2) SRK2D/E/I participate in the regulation of unknown downstream target(s) to confer Mg2+ tolerance. Knockout mutants cbl2/3,cipk3/9/23/26 , and srk2dD/E/I showed hypersensitivity to high levels of Mg2+. srk2d/e/i showed reduced shoot growth, and cbl2/3 and cipk3/9/23/26 showed reduced shoot and root growth under high Mg2+ conditions compared to wild type (wt). Moreover, cbl2/3 andcipk3/9/23/26 showed significantly less vacuolar Mg2+ influx than wt plants, which resulted in a decrease in the cellular concentration of Mg2+. Additionally, SRK2D protein kinase, which is involved in abscisic acid (ABA)-mediated drought response, physically interacts with CIPK3, 9, 23, and 26 (Chen, Peng, Li & Liao, 2018b, Mogami et al. , 2015, Tanget al. , 2015).
In addition to this signaling network, there is another group of proteins that are involved in high-Mg2+ response. Increased ABA content and expression of ABA biosynthesis genes have been reported under high-Mg2+ conditions (Guo et al. , 2014, Visscher et al. , 2010). Moreover, the ABA-insensitive mutant abi1-1 is less sensitive to high-Mg2+treatment than wt (Guo et al. , 2014). These results suggest that ABA signaling is involved in the response to high-Mg2+conditions. Additionally, several other proteins were identified by the increased sensitivity to high-Mg2+ of the corresponding knockout mutants. Vacuolar-type H+-pyrophosphatase (AVP1) (Yang et al. , 2018), magnesium transporter 6 (MGT6) (Yan et al. , 2018) and mid1-complementing activity (MCA) (MCA1/2 (Yamanaka et al. , 2010) are required for high Mg2+ tolerance because their knockout mutants are hypersensitive to high-Mg2+. In contrast, knock out mutants of cation exchanger 1 (CAX1) (Bradshaw, 2005, Cheng, Pittman, Barkla, Shigaki & Hirschi, 2003) and nucleoredoxin 1 (NRX1) (Niu et al. , 2018) were more resistant to high Mg2+. Interestingly, the last four proteins, MCA1/2, CAX1, and NRX1, are involved in the regulation of cytosolic Ca2+ concentration, but the exact molecular mechanisms of their involvement in high Mg2+ response are not yet understood. However, CAX1 serves as a calcium-proton antiporter localized in the tonoplast and helps maintain cytoplasmic Ca2+ levels (Cheng et al. , 2003). The authors speculated that the cax1 might have higher calcium content, which may have a positive effect under high-Mg2+ conditions. Additionally, supplementation of high-Mg2+ growth media with calcium alleviates the growth defects typically observed under excess Mg2+ (Tang et al. , 2015, Yamanakaet al. , 2010). Similar to magnesium-calcium, an antagonistic relationship has also been described for magnesium – potassium (Senbayram, Gransee, Wahle & Thiel, 2015). Potassium (K+) is an essential macronutrient, and its homeostasis is involved in response to abiotic stress caused by salt (Maathuis & Amtmann, 1999, Sun, Kong, Li, Liu & Ding, 2015) or high iron (Zhang et al. , 2018). K+ uptake in Arabidopsis roots is largely controlled by two channels, HAK5 and Arabidopsis K+ transporter 1 (AKT1) (Santa-Maria, Oliferuk & Moriconi, 2018). HAK5 is activated when the external potassium concentration is below 20 μM (Pyo, Gierth, Schroeder & Cho, 2010). At K+ concentrations higher than 0.5 mM, AKT1 is crucial (Nieves-Cordones, Martinez, Benito & Rubio, 2016). CIPKs/CBLs are important regulators of K+ uptake. In yeast (Saccharomyces cerevisiae ), HAK5 has been shown to be activated by CIPK23-CBL1/8/9/10 complexes. HAK5 is activated after phosphorylation by CIPK23 (Ragel et al. , 2015). CBL1/9-CIPK23 also interacts with and activates AKT1 via phosphorylation (Li, Kim, Cheong, Pandey & Luan, 2006). Additionally, translocation of the Shaker-type K+ Arabidopsis thaliana channel AKT2 from the endoplasmic reticulum to the plasma membrane as well as its activity is modulated by the CBL4-CIPK6 complex (Held et al. , 2011).
Plant phospholipase D (PLD) cleaves common phospholipids, such as phosphatidylcholine, to release phosphatidic acid (PA) and free head groups. PA can act as a signaling molecule (Pokotylo, Kravets, Martinec & Ruelland, 2018). In Arabidopsis, there are 12 members of the PLD family, which are sorted by domain structure and biochemical properties. PLDα1, the most abundant PLD in Arabidopsis, reportedly plays a role in stress response, including plant-microbe interactions, wounding, freezing, dehydration, and salinity (Hong et al. , 2016, Ruellandet al. , 2015, Wang, Guo, Wang & Li, 2014). The protein levels of PLDα1 remain unchanged, but its activity and the amount of PA increase transiently after treatment with NaCl (Zhang et al. , 2012). Additionally, transcript levels of PLDδ increase with NaCl treatment (Katagiri, Takahashi & Shinozaki, 2001). Compared to wt, plants with genetically impaired PLDα1 have decreased seedling root growth in high-salt medium. A similar phenotype was observed inpldδ plants. However, observation of the double mutantpldα1, pldδ suggested that individual PLDs act in distinct pathways in the salt stress-response (Bargmann et al. , 2009). Moreover, RNAi suppression of both PLDγ1 and PLDγ2 confers aluminum resistance (Zhao et al. , 2011) while genetic manipulation of PLDε expression revealed its role in nitrogen signaling (Hong et al. , 2009). To summarise, PLDs are involved in a range of abiotic stress responses, including ion toxicity and nutrient sensing, though the exact molecular mechanisms are mostly unknown. However, PA, the product of PLD activity, seems to play a pivotal role.
In this study, we show that Arabidopsis pldα1 is hypersensitive to high levels of magnesium, and that enzymatically-active PLDα1 is necessary for Arabidopsis to respond to high-Mg 2+