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).