Nutrient antagonism and ion homeostasis in plant
More than the Mg2+ concentration alone, the ratios of Mg2+ to Ca2+ and Mg2+ to K+ appear to contribute to the plda1 phenotype (Fig. 6). Interference in the uptake of Mg2+, Ca2+, and K+by plants (sometimes called “nutrient antagonism”) has been widely reported (Diem & Godbold, 1993, Fageria, 2001, Pathak & Kalra, 1971). However, the molecular mechanism of nutrient antagonism is not yet fully understood.
High levels of external Ca2+ result in reduced uptake of Mg2+, and vice versa (Fageria, 2001, Mogamiet al. , 2015, Tang et al. , 2015, Yan et al. , 2018). In agreement with these reports, we found that Arabidopsis seedlings accumulate less Ca2+ upon treatment with high-Mg2+. We also observed that addition of Ca2+ alleviates the reduction in growth typically seen under high-Mg2+ conditions (Fig. 6). Moreover, altered sensitivity to high-Mg2+ of plants with genetically-impaired Ca2+ homeostasis proteins MCA1/2, CAX1, and NRX1 has been demonstrated (Bradshaw, 2005, Niu et al. , 2018, Yamanaka et al. , 2010). However, Ca2+content in pldα1 does not appear to differ from wt under high-Mg2+ (Fig. 5); thus, pldα1Mg2+ hypersensitivity is most likely not caused by altered Ca2+ homeostasis.
Likewise, high levels of external K+ result in reduced uptake of Mg2+ (Ding, Luo & Xu, 2006, Fageria, 2001), and an effect of high-Mg2+ on K+uptake has also been reported in Arabidopsis (Mogami et al. , 2015), though a more in-depth study of this phenomenon is needed. Authors observed lower K+-content in the aerial parts of plants growth under high external concentrations of Mg2+. It is possible that K+ and Mg2+ compete for the use of Mg2+transporters, as it has been reported that the monocot K+ transporters Os HKT2;4 and Ta HKT2;1 can transport Mg2+ (Horie et al. , 2011). Shabala and Hariadi (2005) suggest that at least two mechanisms are involved in Mg2+-uptake through the plasma membrane, one of which allows for uptake of K+ and Ca2+. Later, Guo et al. (2010) observed in Arabidopsis that suppression of the cyclic nucleotide-gated channel (CNGC10) led to decreased influx of K+, Ca2+, and Mg2+, implicating involvement of CNGC10 in Ca2+ and Mg2+ transport, and by extension, K+ transport.
We found that wt seedlings treated with high-Mg2+ had lower concentrations of K+ (Fig. 5), and that K+ was even lower in pldα1 . Additionally, we impaired transcription of HAK5 and CIPK9 (genes involved in K+ homeostasis) in pldα1 treated with high-Mg2+ (Fig. 7). In low-K+conditions, CIPK9 regulates K+ homeostasis (Liu, Ren, Chen, Wang & Wu, 2013, Pandey et al. , 2007), while HAK5 is largely responsible for its uptake (Rubio, Aleman, Nieves-Cordones & Martinez, 2010). We hypothesize that high external Mg2+ concentrations lead to a decrease in intracellular K+ concentrations; thus, activating a not yet fully understood compensation mechanism regulated by PLDα1, HAK5, and potentially CIPK9. The significance of the K+ compensation mechanism is seen in Arabidopsis mutants for two proteins involved in K+ uptake, HAK5 and AKT1, which display increased sensitivity to high-Mg2+ (Fig. 8). Importance of AKT1 and HAK5 for K+ uptake in high-Mg2+ conditions was also shown by Caballero et al. (2012), where a significant decrease in K+ uptake in mature akt1, hak5 Arabidopsis plants was found. However, altered K+-accumulation in the pldα1 vacuole cannot be excluded as well.

PLDα1 and PA are involved in stress responses

We found that PLDα1 activity (prepared from Arabidopsis roots) was rapidly and transiently increased in response to high-Mg2+ (Fig. 3). Phospholipase Dα1 belongs to the C2 subfamily of plant PLDs, and is activated by millimolar concentrations of Ca2+. PLDα1 prefers phosphatidylcholine to phosphatidylethanolamine as a substrate (Kolesnikov et al. , 2012, Wang et al. , 2014). Protein phosphorylation may also regulate PLDα1 activity, as it is predicted to have phosphorylation sites (Takáč et al. , 2016) and phosphorylated PLDα1 has been detected in response to drought stress (Umezawa et al. , 2013). Phospholipase Dα1 localizes predominantly to the cytosol; however, when stressed (such as through wounding or dehydration), it translocates to membranes (Chen et al. , 2018a, Wang et al. , 2000).
PLDα1 releases PA, which serves as an important secondary messenger and as a precursor in lipid biosynthesis. Elevated levels of PA have been described in response to many abiotic stresses, including salinity, drought, cold, injury, and heat, as well as biotic stresses (Hou, Ufer & Bartels, 2016, Testerink & Munnik, 2005, Vergnolle et al. , 2005, Wang et al. , 2014, Zhao, 2015). The molecular mechanism of PA as a signaling molecule appears fairly diverse, as a wide range of PA-binding proteins have been identified, including lipid transporters, protein kinases, and enzymes such as NADPH oxidase respiratory burst oxidase homologs D and F (RbohD/F) (Hong et al. , 2016, Pokotyloet al. , 2018, Yao & Xue, 2018).

Possible mechanisms of PLDa1 activity in magnesium and potassium homeostasis

We found that high-Mg2+ hypersensitivity of Arabidopsis pldα1 and plants producing inactive PLDα1 protein was the same (Fig. 4), demonstrating that PLDα1 activity is key in regulating the response to increased Mg2+concentrations. Although it cannot be ruled out that choline also plays a role, we assume that PA functions as a key molecule. Several molecular mechanisms for PA regulation have been hypothesized. ABA is known to be involved in response to high-Mg2+ conditions in Arabidopsis (Guo et al. , 2014), and PA is known to participate in ABA signaling in various ways. The PA produced through PLDα1 activity interacts with protein phosphatase 2C (PP2C), heterotrimetric GTP-binding protein, and RbohD/F (Mishra, Zhang, Deng, Zhao & Wang, 2006, Zhang, Qin, Zhao & Wang, 2004, Zhang et al. , 2009), all of which mediate ABA signaling. PA produced by PLDα1 also interacts directly with regulator of G-protein signaling 1 (RGS1), modulates the level of active Gα, and consequently, ABA signaling (Choudhury & Pandey, 2017, Zhao & Wang, 2004). Phytosphingosine-1-phosphate (phyto-S1P) has been identified as a lipid messenger, generated by sphingosine kinases (SPHKs), that mediates ABA response. PA binds to SPHK1 and SPHK2, stimulating their activity; thus, regulating ABA response (Guo, Mishra, Taylor & Wang, 2011). Arabidopsis PA has also been shown to interact directly with class 1 protein kinases SnRK2.4, and SnRK2.10 (McLoughlin et al. , 2012). Proteins from the same family (but in class 3), SnRK2d (SnRK2.2), SnRK2e (SnRK2.6), and SnRK2i (SnRK2.3) are known to be part of the high-Mg2+response. However, further research is needed to confirm the participation the proteins discussed here in the Arabidopsis PA-high Mg2+ response.
There are also several ways in which PA affects K+homeostasis. PA has been shown to bind to potassium channel β subunit 1 (KAB1) (McLoughlin et al. , 2013), which, in Arabidopsis, physically associates with the inward-rectifying potassium channel 1 (KAT1) (Tang, Vasconcelos & Berkowitz, 1996). KAT1 appears to be crucial for turgor-pressure changes in guard cells (Pilot et al. , 2001). Whether KAT1 is functional in the roots has yet to be investigated. Recently, PA‐mediated inhibition of Shaker K+ channel AKT2 in Arabidopsis and rice was reported (Shen et al. , 2020).
The mode of action of PA in the regulation of the rat voltage-gated potassium channel Kv1 has been studied in detail, with experiments revealing two effects of PA on Kv1 gating. The first method is generic, where the negative charge in PA shifts the membrane voltage. The second method is more specific to phosphatidic acid, where the negatively-charged end of the molecule interacts with the part portion of the channel that senses voltage changes in order to keep the pore closed. Whether a similar mechanism is used in the regulation of plant K+ channels remains to be investigated (Hite, Butterwick & MacKinnon, 2014).
In conclusion, we found that Arabidopsis PLDα1 is involved in response to high-Mg2+ conditions. We also demonstrate that PLDα1 activity is an essential part of this response. Moreover, high external concentrations of Mg2+ were found to disrupt K+ homeostasis, and PLDα1 is involved in the response to this disruption (Fig. 9).