Journal: The New Phytologist

Article Title: A tonoplast‐localized magnesium transporter is crucial for stomatal opening in Arabidopsis under high Mg 2+ conditions

doi: 10.1111/nph.18410

Figure Lengend Snippet: Magnesium (Mg 2+ ) transport activity of MGR1/CST2, subcellular localization and expression pattern of MGR1 / CST2 . (a) Magnesium concentration in transgenic yeast cells. Yeast mutant plants ( mam3 ) expressing MAM3 , MGR1/CST2 cDNA or empty vector were grown on SD medium containing 3 mM Mg 2+ for 14 h. The Mg concentration in yeast was determined by inductively coupled plasma mass spectrometry (ICP‐MS). Data represent the mean ± SD ( n = 5). Asterisks indicate statistically significant differences from the empty vector/wild‐type (WT), calculated using one‐way analysis of variance (ANOVA) with Dunnett's multiple comparisons test (**, P < 0.01; ***, P < 0.001). DW, dry weight. (b) Subcellular localization of MGR1/CST2‐GFP. MGR1/CST2‐GFP was expressed alone (left) or co‐expressed with tonoplast target signal ( TTS ) ‐mCherry (center) or mCherry‐AHA1 (right) in leaf epidermal cells of Nicotiana benthamiana . Fluorescent images were obtained using a confocal laser microscope. (c) Expression pattern of MGR1/CST2 in different organs of Arabidopsis (Col‐0). Seedlings (6 wk old) precultured in 1/10 strength Hoagland solution were exposed to a nutrient solution containing 0, 0.25, and 5 mM Mg 2+ for 3 d, and the roots and leaves were sampled for RNA expression. The expression of MGR1/CST2 was determined by quantitative real‐time PCR (qRT‐PCR). Expression relative to roots not exposed to Mg 2+ is shown. Data are means ± SD ( n = 4). Different letters indicate significant differences at P < 0.01 according to the Tukey–Kramer test. (d) Time‐dependent change of MGR1/CST2 protein in response to high Mg 2+ concentrations. Wild‐type plants were grown on ½ Murashige & Skoog (½MS) agar medium (containing 0.75 mM Mg 2+ ) for 2 wk, and then transferred onto ½MS agar medium supplemented with 20 mM Mg 2+ . Proteins were extracted from rosette leaves at the indicated time points and subjected to immunoblotting using MGR1/CST2 and V‐PPase antibodies. (e) Magnesium (Mg 2+ ) concentration‐dependent MGR1/CST2 protein expression. Plants were grown on ½MS agar medium containing the indicated Mg 2+ concentrations for 2 wk. Protein samples were prepared from rosette leaves and subjected to immunoblotting using MGR1/CST2 and V‐PPase antibodies.

Article Snippet: The expression of MGR1 / CST2 was determined by qRT‐PCR with KOD SYBR qPCR Mix (Toyobo) on a real‐time PCR machine (model no. CFX96; Bio‐Rad) using the primers MGR1/CST2_qRT_F and MGR1/CST2_qRT_R (Table ).

Techniques: Activity Assay, Expressing, Concentration Assay, Transgenic Assay, Mutagenesis, Plasmid Preparation, Mass Spectrometry, Microscopy, RNA Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot