O high external Mg2 concentrations or low external Ca2 concentrations, we generated transgenic Arabidopsis plants overexpressing CIPK26 beneath the D-Threonine Biological Activity control from the CaMV 35S promoter. We evaluated the development with the transgenic plants under higher external Mg2 concentrations or low external Ca2 concentrations. The expression of CIPK26 in two independent overexpressors was confirmed to become larger thanFigure 4. (Continued.) instances; a representative outcome is shown. Bars indicate SD (n = six). Asterisks indicate statistically considerable difference compared using the wild sort. , P , 0.01, oneway ANOVA followed by a post hoc Dunnett’s multiple comparison test. C, Representative pictures of rosette leaves of the wild sort, the cipk26/3/9 triple mutant, plus the cipk26/3/9/23 quadruple mutant. D, Growth phenotypes of plants grown on GM agar plates for two weeks then in soil for a different 14 d. Bars = 1 cm. E, Inflorescence height of each plant grown as described in D. Bars indicate SD (n = 6). Experiment was performed two occasions; a representative result is shown. Asterisks indicate statistically substantial difference compared using the wild sort as described in B. F, Representative photos of shoot apexes of your wild kind, the cipk26/3/9 triple mutant, and also the cipk26/3/9/23 quadruple mutant. G, Representative pictures of plants of the wild variety, the cipk26/3/9 triple mutant, and also the cipk26/3/9/23 quadruple mutant grown inside a hydroponic culture system for 24 d. Left, Plants grown hydroponically in modified lowcalcium answer (“Materials and Methods”) supplemented with indicated concentrations of CaCl2. Appropriate, Plants grown hydroponically in lowmagnesium resolution (“Materials and Methods”) supplemented with indicated concentrations of MgCl2. Bars = 1 cm. H, Fresh weight of aerial components of each plant grown as described in G. Columns marked with different lowercase letters represent significantly different means (P , 0.01) as outlined by twoway ANOVA followed by a post hoc TukeyKramer many comparison test. Experiment was performed two instances; a representative result is shown. Bars indicate SD (n = six). WT, Wild variety.Plant Physiol. Vol. 167, 2015Mogami et al.that in wildtype or vectorcontrol plants by quantitative RTPCR (Supplemental Fig. S10A). Then, we tested the plants’ susceptibility to high external Mg2 concentrations on agar plates. The CIPK26overexpressing plants were significantly extra tolerant than vectorcontrol plants to a high external Mg2 concentration (25 mM MgCl2) on agar plates (Supplemental Fig. S10, B and C). These CIPK26overexpressing plants also grew better below a low external Ca2 concentration (0.1 mM CaCl2) than did vectorcontrol plants in the hydroponic culture system (Supplemental Fig. S10, D and E). Taken together, these outcomes support the view that CIPK26 plays an important role in plant development under both higher external Mg2 and low external Ca2 circumstances in a dosedependent manner.Hypersusceptibility of srk2d/e/i Triple and srk2d/e/i/ cipk26/3/9/23 Septuple Mutants to a Higher External Mg2 ConcentrationConsidering that CIPK26 was identified as a novel interactor of subclass III SnRK2s (Fig. two, A ), we considered no matter if subclass III SnRK2s are also required for plant development beneath high external Mg2 concentrations. We investigated whether or not SRK2D can still physically interact with CIPK26 under a high external Mg2 concentration by coIP. We observed that the 43mycCIPK26 proteins were coimmunoprecipitated together with the SRK2DsGFP proteins in extracts from p.
