Figure legends
Fig. 1 pldα1-1 is hypersensitive to high external levels
of Mg2+. Plants were grown on half-strength MS for 5
days, after which they were transferred to plates supplemented with 0
(Control), 1, 5, 10, 15, and 20 mM MgCl2 for 7 days. (a)
Growth of pldα1-1 and wt seedlings on agar plates with excess
Mg2+. (b) Root length of pldα1-1 and wt
seedlings 7 d after transfer. Values represent mean ± SD, n=24 plants.
(c) Fresh weight of pldα1-1 and wt 7 d after transfer. Values
represent means ± SD, n=4 (pools of 6 plants). (c) Wt and pldα1-1plants were grown hydroponically for 3 weeks in half-strength Hoagland´s
media, followed by 10 d with or without 10 mM MgSO4. The
experiment was repeated three times with similar results. Asterisks
indicate significant differences compared to wt (Student’s t-test,
*p<0.05, **p<0.01).
Fig. 2 Detection of PLDα1 by western blot in the various lines
used. Knockout lines plda1-2 , plda1-3, and plda1-4had the same phenotype as plda1-1 under
high-Mg2+. (a) Western blot detection of PLDα1 in
protein extracts from 10-day-old seedlings. Each lane was run with 9.5
µg of protein. (b) Loading control stained with Novex. (c) Plants grown
on half-strength MS for 5 d, followed by transfer to agar plates with or
without 10 mM MgCl2 for 7 d. (d) Root length inpldα1-2, pldα1-3, pldα1-4, and wt plants. Values represent mean ±
SD, n=24. (e) Fresh weight of pldα1-2, pldα1-3, pldα1-4, and wt.
Values represent means ± SD, n=4 (pools of 6 plants). Asterisks indicate
significant differences compared to wt (Student´s t-test,
*p<0.05, **p<0.01).
Fig. 3 High-Mg2+ treatment triggers a
transient increase in PLDα1 activity in a dose-dependent manner, but and
does not induce transcription of PLDα1 . 4-week-old hydroponically
grown plants were treated with MgSO4 and sampled at 10,
30, and 180 min. (a) Thin layer plate showing phosphatidyl butanol
(PBut) levels in plants treated with MgSO4.
‑n- But indicates the control sample, where +n -But was
omitted. (b) Thin layer plate showing accumulation of
fluorescently-labeled PBut after MgSO4 treatment over
time. (c) Quantification of PBut accumulation in response to
MgSO4 over time. (d) Relative increase in PLDα1 activity
with MgSO4 treatment over time. Values represent mean ±
SD, n=3. (e) Transcription analysis of PLDα1 in roots and leaves
of wt plants after treatment with 10 mM MgSO4 for 24 h.
Transcript levels were measured in roots (R) and leaves (L) by
quantitative RT-PCR. Transcription was normalized to the reference gene
SAND, and transcription of non-treated plants was set to one. Values
represent the mean ± SD, n=3; C, control; R, roots; L, leaves;n- But, n -butanol; PBut, phosphatidyl butanol.
Fig. 4 Growth of Arabidopsis seedlings expressing inactive
PLDα1 is the same that of pldα1 on agar plates with excess
Mg2+. Plants were grown on half-strength MS for 5 d
and transferred to agar plates with or without 10 mM
MgCl2 for 7 d. (a) Root length of plants after
Mg2+ treatment. Values represent mean ± SD, n=24. (b)
Fresh weight of plants after Mg2+ treatment. Values
represent mean ± SD, n=8 (pools of 3 plants). Asterisks indicate
significant differences compared to wt (Student´s t-test, **
p<0.01).
Fig. 5 Under high-Mg2+ conditions,
concentrations of Mg2+ and K+ are
lower in pldα1-1 compared to wt. Seven-day-old seedlings were
transferred on ½ MS agar plates with or without 10 mM
MgCl2 and grown for 10 days. Bars represent mean ± SD,
n=5 (Student´s t-test, ** p<0.01). Asterisks indicate
significant differences compared to wt.
Fig. 6 Addition of Ca2+ and
K+ alleviates pldα1-1Mg2+-hypersensitivity. Plants were grown on
half-strength MS for 5 d and transferred to agar plates supplemented
with 10 mM MgCl2, 10 mM MgCl2 + 10 mM
CaCl2, or 10 mM MgCl2 + 50 mM KCl for 7
d. (a) Growth of plants on agar plates with additional
Mg2+ and Ca2+, or
K+. (b) Root length of plants after transfer to
supplemented plates. Values represent mean ± SD, n=24. (c) Fresh weight
of plants after transfer to supplemented plates. Values represent means
± SD, n=4 (pools of 6 plants). Asterisks indicate significant
differences compared to wt (Student´s t-test, * p<0.05, **
p<0.01).
Fig. 7 Transcription of CIPK9 and HAK5 is reduced
in the roots of pldα1 under high-Mg2+conditions. Transcription levels of (a) MGT , (b) CAX1 ,CIPK9 , and HAK5 genes in roots (R) and leaves (L) after
high-Mg2+ treatment. 4-week-old hydroponically grown
plants were treated with 10 mM MgSO4 for 24 h.
Transcript levels were measured in roots by quantitative RT-PCR.
Transcription was normalized to the reference gene SAND, and the
transcription of non-treated plants was set to one. Values represent
means ± SD, n=3, (Student´s t-test, *p<0.05). Asterisks
indicate significant differences compared to wt. nd indicated not
detected.
Fig. 8 The double knockout line hak5, akt1 is
hypersensitive to high-Mg2+ conditions. Growth ofhak5 and hak5, akt1 on agar plates with added
Mg2+. Plants were grown on half-strength MS for 5 d
and transferred to plates with or without 10 mM MgCl2for 7 d. (a) Growth of plants. (b) Root length in plants after
treatment. Values represent mean ± SD, n=24. (c) Fresh weight of plants
after treatment. Values represent mean ± SD, n=8 (pools of 3 plants).
Asterisks indicate significant differences compared to wt (Student´s
t-test, * p<0.05, ** p<0.01).
Fig. 9 Proposed model for PA-mediated response to
high-Mg2+ in Arabidopsis. High concentrations of
extracellular Mg2+ results in excess intracellular
Mg2+ and reduced K+-uptake, leading
to a lower intracellular concentrations of K+.
Meanwhile, PLDα1 is activated and produces PA and polar head group such
as choline (Cho). Activation of PLDα1 leads to transcription of the
K+ channel HAK5 and protein kinaseCIPK9 , possibly activating K+-uptake. CIPK9 is
reported to be involved in Mg2+ sequestration via the
CBL2/3-CIPK3/9/23/26 network and an unknown tonoplast-localized
Mg2+ transporter. Additionally, CIPK9 is involved in
the regulation of K+ homeostasis. PLDα1 activity may
also interact with machinery regulating K+ vacuole
homeostasis. AKT1 – Arabidopsis K+ transporter 1, CBL
- calcineurin B-like calcium sensor protein, CIPK-CBL-interacting
protein kinase, Cho – choline, HAK5 – high-affinity
K+ transporter 5, PA – phosphatidic acid, PLDα1 –
phospholipase Dα1, arrows in black solid lines – this study, arrows in
black dotted lines – possible interaction based on this study, arrows
in gray broken lines – reported study, arrows in gray dotted lines –
possible interaction.