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+