Discussion

Magnesium is an essential and abundant macronutrient, though its importance has been overlooked. Magnesium is the eighth-most widespread element on Earth, and is well soluble and highly mobile in soil (Guo, 2017). Typically, Arabidopsis is cultivated in vitro on full-strength (containing 1.5 mM Mg2+) or half-strength (with 0.75 mM Mg2+) MS media. We used half-strength MS in our study, and root growth differences between wt and pldα1 were observed with 1 mM more Mg2+added to the media (Fig. 1).
High extracellular concentrations of Mg2+ lead to higher intracellular concentrations (Guo et al. , 2014, Mogamiet al. , 2015, Tang et al. , 2015, Yan et al. , 2018). The concentration of Mg2+ in the plant cell differs significantly between cellular compartments. Less than 1 mM Mg2+ is found in mitochondria (0.2-0.5 mM), the cytosol (0.2-0.4 mM), and apoplast (0.2-0.5 mM). The vacuole (5-80 mM) and chloroplasts (1-5 mM) contain the highest concentrations (Hermanset al. , 2013). Sequestration of excess Mg2+ to the vacuole, or possibly to the endoplasmic reticulum, is presumably a key mechanism for Mg2+ tolerance. The plant-specific Mg2+/H+ exchanger (MHX), MGT2, and MGT3 reportedly play a role in the sequestration of Mg2+ in vacuoles (Conn et al. , 2011, Shaulet al. , 1999), though knockout lines for these genes exhibit wt-like response to high-Mg2+ conditions. Hence, the participation of these proteins in Mg2+ tolerance is not clear. Detoxification of high-Mg2+ via Mg2+-vacuolar sequestration is regulated by calcium sensors CBL2/3 and their downstream component CIPK3/9/23/26 protein kinases (Tang et al. , 2015). The cbl2, cbl3 double mutant is hypersensitive to high-Mg2+. Interestingly, when grown under under high-Mg2+ conditions, cbl2, cbl3 was found to retain lower concentrations of Mg2+compared to wt, though the lower Mg2+ concentration appeared to be more toxic to cbl2, cbl3 , as it grew less than wt (Tang et al. , 2015). This observation may be explained by reduced vacuolar-sequestration. We also found lower levels of Mg2+ in pldα1 plants (Fig. 5). Thus, reduced vacuolar Mg2+-sequestration should be considered. Interplay between PLDα1 and CIPK9 likely contributes to the Mg2+ hypersensitivity observed in pldα1 , as supported by the observed decrease in transcript levels of CIPK9in pldα1 roots (Fig. 7).
mgt6 plants were also found to be hypersensitive to high-Mg2+, and contained less Mg2+in the shoot, compared to wt. Under high-Mg2+conditions, MGT6 likely mediates the transport of Mg2+into shoot tissues (Yan et al. , 2018). Additionally, the MGT7 mutant mrs2-7 is more sensitive to high-Mg2+than wt (Oda et al. , 2016). As MGT6 and MGT7 reportedly localize to the endoplasmic reticulum, it has been suggested that they also act as a bi-directional transporters, thus maintaining cytosolic concentrations of Mg2+ using the ER as a storage location (Oda et al. , 2016).