Results
pldα1 is hypersensitive to high levels
of
Mg2+
We grew pldα1-1 (Bargmann et al. , 2009) seedlings in
varying concentrations of diverse nutrients including
Mg2+. pldα1-1 was hypersensitive to magnesium,
with reduced primary root length, fresh weight (Fig. 1a,b,c), and number
and length of lateral roots (Fig. S1.). A significant decrease inpldα1-1 primary root length was observed after application of 1
mM MgCl2; at this conditions, pldα1-1 primary
roots were found to be 6% shorter than wt (Fig. 1a,b). Concentrations
of 5 mM MgCl2 and higher had a severe effect on the
growth of wt plants; however, in all studied concentrations,pldα1-1 was more sensitive. The greatest difference in primary
root length between wt and pldα1-1 was observed in plants treated
with 15 mM MgCl2, where pldα1-1 roots were 40%
shorter (Fig. 1b). The greatest difference in fresh weight between wt
and pldα1-1 was observed with 10 mM MgCl2, wherepldα1-1 was half the weight of wt (Fig. 1c).
To determine whether the MgCl2 hypersensitivity observed
in seedlings persists in mature plants, wt and pldα1-1 were grown
hydroponically. MgSO4 (at 10 mM) was added to the
hydroponic solution and the plants were maintained for 10 days. Reduced
growth in pldα1-1 compared to wt plants was markedly visible
(Fig. 1d). However, because magnesium sulfate was used instead of
magnesium chloride, it was necessary to rule out the possible effects of
other ions. Plants were treated with 10 mM MgCl2,
MgSO4, or Mg(NO3)2, and
growth was assessed. Although there were visible variations in
the effect of individual anions, the significant difference between wt
and pldα1-1 was clearly detectable in all cases (Fig. S2).
Therefore, it is possible to rule out that the anion is responsible for
the observed pldα1-1 phenotype.
To ensure that the observed Mg2+ hypersensitivity was
due to an insertion in PLDα1 and no other genes, we used three
additional T-DNA insertion lines for PLDα1 , includingpldα1-2 (described by Bargmann et al. (2009)), pldα1-3(SALK), and pldα1-4 (GABI-KAT). We also made complementation
lines by transforming pldα1-1 plants with PLDα1 driven by
its native promoter. The levels of PLDα1 (PLDα1-Com) protein in seedling
extracts was verified using anti-PLDα1/2 antibody. PLDα1 was not
detected in any of the pldα1 lines (Fig. 2a). Levels of PLDα1 in
the complementation lines were lower than in wt; therefore, the two
PLDα1-Com lines with the highest PLDα1 protein levels were used for
subsequent analyses. pldα1-2, pldα1-3 , pldα1-4, and
complementation lines were phenotyped for Mg2+sensitivity. Primary root length was 23, 25, and 26% lower inpldα1-2 , pldα1-3, and pldα1-4 , respectively. Fresh
weight was 52, 53, and 54% lower in pldα1-2 , pldα1-3, andpldα1-4 , respectively, compared to wt when treated with high
Mg2+ (Fig. 2 c, d, e). Overall, all PLDα1 mutants were
similarly Mg2+-sentitive to pldα1-1 . In
contrast, primary root length and fresh weight in linespldα1-1 -Com1 and pldα1-1 -Com2 were similar to wt when
treated with high Mg2+ (Fig. S3).
Arabidopsis pldα1 plants are more sensitive to
high-Mg2+ conditions than wt; thus, PLDα1 appears to
be involved in response to high-Mg conditions. These results uncovered a
novel physiological role of PLDα1 in the context of
Mg2+-homeostasis.
PLDα1 activity increases after
treatment with
Mg2+
Next, we investigated whether high levels of Mg2+could trigger changes in PLDα1 activity. Arabidopsis has 12 genes
encoding PLDs, which differ biochemically and require different in
vitro conditions for activation (Hong et al. , 2016). PLDs cleave
ordinary phospholipids such as phosphatidylcholine, releasing PA and
free head group, e.g. choline. PA is also the product of diacylglycerol
kinase activity, as well as the substrate for PA phosphatase, among
other enzymes (Ruelland et al. , 2015). Hence, PA levels do not
necessarily correlate with PLD activity. A unique feature of PLDs is
their so-called transphosphatidylation activity, where, in the presence
of primary alcohols such as n -butanol, PLD transfers the
phosphatidyl group from its substrate to n -butanol, releasing
phosphatidylbutanol (PBut). PBut-formation therefore directly
corresponds to PLD activity (deVrije & Munnik, 1997).
PLDα1 is known to be both membrane-associated and cytosolic (Fan, Zheng,
Cui & Wang, 1999). Predominant cytosolic localization was reported by
Novák et al. (2018) in Arabidopsis expressing PLDα1-YFP,
therefore we determined PLDα activity in the soluble fraction. Plants
were treated with 10 or 40 mM MgSO4, after which root
samples were taken at 10, 30, and 180 min. The soluble fraction was
prepared, and the activity of PLDα was determined using fluorescently
labeled phosphatidylcholine as a substrate under conditions optimal for
PLDα (Hong, Zheng & Wang, 2008). Lipids, including PBut, were extracted
and separated using high-performance thin-layer chromatography (HP-TLC),
and the amount of fluorescently labeled PBut was quantified (Fig. 3).
PLDα activity was also measured in samples prepared from pldα1-1plants, where PLDα activity was found to be negligible (Fig. 3a). Hence,
we concluded that the quantity of released PBut corresponds to PLDα1
activity.
PLDα1 activity in the soluble fraction increased after
MgSO4 treatment (Fig. 3b). The increase was
concentration-dependent, as higher concentrations of
MgSO4 consistently led to an increase in PLDα1 activity
(Fig. 3b,c). Interestingly, the increase in PLDa1 activity was
transient, reaching 2.5-fold after 30-minutes of treatment with 10 mM
MgSO4 (Fig. 3d).
An increase in PLDα1 activity could be due to higher rates of
transcription of PLDα1 , activation of PLDα1, or a combination of
the two. Thus, we measured transcriptional levels of PLDα1 in
control and high-Mg2+ treated (10 mM
MgSO4, for 24h) plants using quantitative RT-PCR. We
found no increase in PLDα1 transcript levels following
Mg2+ treatment (Fig. 3e).
These results demonstrate that PLDα1 is activated by
Mg2+ shortly after treatment, though not at the
transcriptional level.
PLDα1 activity contributes to high-
Mg2+tolerance
To confirm that the activity of PLDα1 is essential for high-magnesium
tolerance in wt plants, we introduced an inactive mutant forPLDα1 into pldα1-1 plants
(p PLDα1::PLDα1-Mut/pldα1 ). Members of the PLD superfamily
retain the highly conserved HKD motif, which is encoded twice in
higher-plant PLDs (Wang et al. , 2014). Point mutations in HKD
motifs result in the complete loss of PLD activity in Brassica
oleracea (Lerchner, Mansfeld, Kuppe & Ulbrich-Hofmann, 2006), as well
as in humans and mice (Sung et al. , 1997).
Transgenic pldα1-1 plants expressingpPLDα1 ::PLDα1 K334R;K663R (lines Mut1 and
Mut2) at levels consistent with wt (Fig. 2a) showed similar sensitivity
to MgCl2 as pldα1-1 , for both primary root length
(Fig. 4a) and fresh weight (Fig. 4b).
These results demonstrate that Arabidopsis PLDα1 activity is critical in
mediation of the response to high-magnesium conditions.
pldα1 accumulates less
Mg2+ and K+ under
high-Mg2+conditions
To elucidate the possible mechanism responsible for the higher
susceptibility of pldα1 , we measured
Mg2+-content in wt and mutant plants under control and
high-Mg2+ conditions. After
high-Mg2+ (10 mM) treatment, seedling
Mg2+ levels were elevated by about five times in wt
and pldα1-1. Nevertheless, pldα1 showed significantly
lower Mg2+-content than wt (Fig. 5a).
There is known to be an antagonistic relationship between the uptake of
Mg2+ and Ca2+ (Guo, Babourina,
Christopher, Borsic & Rengel, 2010, Yamanaka et al. , 2010).
Moreover, increased levels of Mg2+ application in rice
results in lower uptake of calcium and potassium (Fageria, 2001), and
transcription of the potassium transporter HAK5 increases
following treatment with Mg2+ in Arabidopsis (Tang &
Luan, 2017, Visscher et al. , 2010). Therefore, we measured
Ca2+ and K+ content in the wt andpldα1-1 plants to investigate these relationships.
In agreement with Mg-Ca antagonism, seedling Ca2+content was lower when 10 mM MgCl2 was added to the agar
medium. However, we did not observe any difference between wt andpldα1-1 (Fig. 5b). Interestingly, K+ levels in
wt and pldα1-1 were lower in Mg2+-treated
plants, with pldα1-1 plants retaining even less
K+ than wt (Fig. 5c).
These results demonstrate that PLDα1 is involved in the regulation of
Mg2+- and K+- content in Arabidopsis
seedlings grown in high-Mg2+ conditions.
Addition of Ca2+ and
K+ alleviates Mg2+-hypersensitivity
in pldα1
plants
Aware that there is an antagonistic relationship between some of the
essential nutrients, we investigated whether excess
Ca2+ or K+ could affect pldα1hypersensitivity to Mg2+. Application of both
Ca2+ and K+ ameliorate growth inpldα1-1 in high-Mg2+ (Fig. 6a). Addition of
Ca2+ completely restored the growth of pldα1-1to wt levels, in both root length and fresh weight. Roots from bothpldα1-1 and wt were smaller (Fig. 6b), while fresh weight for
both was similar, compared to control conditions (Fig. 6c). Application
of K+ lowered the root-length difference between wt
and pldα1 . With Mg2+, root length ofpldα1-1 was 82.5% that of wt. However, with
K+, root length of pldα1-1 increased to 94.9%
that of wt. Fresh weight under high-Mg2+ inpldα1-1 was brought up to wt levels with K+(Fig. 6c), though root length and fresh weight in both pldα1-1and wt were lower compared to the control conditions (Fig. 6).
Addition of Ca2+ increased plant growth in both wt andpldα1-1 plants (Fig. 6). However, no difference in
Ca2+ content between wt and pldα1-1 was
detected (Fig 5b). These results, along with what is known about the
antagonistic relationship between Ca and Mg, suggest that
Ca2+ deficiency is not the underlying factor behind
growth defects in pldα1 grown under high-Mg2+,
but that high Mg2+ or low K+ content
is responsible. Under high-Mg2+ conditions,pldα1‑1 retained less Mg2+ than wt, though the
lower Mg2+-content was more toxic to pldα1-1than the higher-Mg2+ content in wt. This phenomenon
may be explained by impairment of Mg2+ sequestration
in pldα1-1 , which would result in higher cytosolic concentrations
of Mg2+. Additionally, lower K+content may contribute to impaired growth in pldα1-1 , or a
combination of the two mechanisms.
K+-related genes
CIPK9 and HAK5 are not transcriptionally upregulated in
pldα1
Ten members of the MGT family were
identified in the Arabidopsis genome, (Li, Tutone, Drummond, Gardner &
Luan, 2001). Therefore, we investigated transcriptional response of MGT
family genes, of which Arabidopsis has 10 (Li et al. , 2001), to
high-magnesium stress. Transcript levels were determined using
quantitative RT-PCR in roots and leaves of wt and pldα1-1 plants,
separately (Fig. 7a). Transcript levels of MGT1 in roots andMGT7 in leaves was slightly elevated in
Mg2+-treated plants, though there was no difference
between wt and pldα1-1 (Fig. 7a). Transcript levels for two genes
are not shown, as MGT5 was under the detection limit andMGT8 was found to be a pseudogene (Zhang et al. , 2019).
In Arabidopsis, CAX1 is known to be involved in
high-Mg2+ resistance (Bradshaw, 2005). Moreover,
transcription of CAX1 is downregulated under
high-Mg2+ conditions (Visscher et al. , 2010).
Hence, we determined transcriptional levels of CAX1 in control
and Mg2+-treated wt and pldα1-1 plants.CAX1 transcription was decreased in the roots and leaves of
Mg2+-treated plants; however, as in case of MGT genes,
there was no difference between wt and pldα1-1 (Fig. 7b).
Next, we looked at transcription of CIPK9 and HAK5 , both
of which are known to be involved in potassium homeostasis under
low-potassium conditions (Coskun, Britto & Kronzucker, 2014), and are
reportedly upregulated in high-magnesium conditions (Tang et al. ,
2015, Visscher et al. , 2010). Furthermore, Arabidopsis CIPK9 has
also been shown to participate in high-Mg2+ response
(Tang et al. , 2015). In agreement with Visscher et al. (2010), we
observed high upregulation of CIPK9 and HAK5 in wt roots
after Mg2+ treatment. However, the increase inCIPK9 and HAK5 transcript levels was almost completely
abolished in pldα1-1 roots (Fig. 7b). In
Mg2+-treated pldα1-1 leaves, CIPK9transcript levels were slightly increased (~doubled),
and HAK5 was not detected; however, there was no difference
between wt and pldα1-1 (Fig. 7b).
These results indicate that PLDα1 is essential in a signaling mechanism
which leads to an increased expression of HAK5 and CIPK9in roots upon high-Mg2+ treatment.
The hak5, akt1 double mutant is
hypersensitive to
high-magnesium
Based on our previous observations, we speculated that proper regulation
of potassium homeostasis is essential in high
Mg2+-conditions. We examined whether hak5plants are hypersensitive to high-magnesium, and found no difference
between hak5 and wt (Fig. 8). Next, we tested the sensitivity of
a hak5 , akt1 double mutant to high-Mg2+and found that it was significantly more sensitive than wt. Root length
in hak5 , akt1 plants was 9.5% (Fig. 8b), and fresh weight
14.5%, less than in wt (Fig. 8c). Under control conditions,hak5, akt1 growth did not differ from wt (Fig. 8).
However, there was still a significant difference between pldα1and hak5, akt1 sensitivity to high-Mg2+.pldα1-1 roots were 24%,
and fresh weight 47%, less than wt; thus, hak5, akt1 is less
sensitive to high-Mg2+ compared to plda1-1(Fig. 8b,c).
These results revealed that plants impaired in K+uptake are also compromised in their tolerance to high levels of
Mg2+. Therefore, appropriate regulation of potassium
homeostasis is key to that of magnesium.
We conclude that in Arabidopsis, K+-homeostasis is
involved in response to high-Mg2+, and that this
mechanism is at least partially mediated by PLDα1.