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.