Legume Genomics and Genetics 2025, Vol.16, No.5, 234-244 http://cropscipublisher.com/index.php/lgg 236 Figure 1 The Ca2+-dependent activation of CPK28 is required for freezing tolerance (Adopted from Ding et al., 2022) Image caption: (A) Microscale thermophoresis (MST) assay showing the Ca2+-binding affinity of CPK28 and CPK28EFm. Quantification is shown in the left; recombinant proteins were stained by CBB (right). Data are means ± SD of three technical replicates. Similar results were obtained from three independent experiments. (B to D) In-gel kinase assays showing CPK28 activation in response to cold. The 14-day-old wild-type plants (B and C) or Super:CPK28-Myc plants (D) were treated at 4°C for the time indicated without EGTA or for 10 s after pretreatment with 25 mM EGTA for 4 hours, and total proteins (B) or immunoprecipitated CPK28-Myc with anti-Myc agarose beads (D) were subjected to in-gel kinase assay. GST-BIK1K105E was used as substrate. cpk28-1 mutant treated at 4 °C for 10 s was used as controls (B). Autoradiograph (AR) and CBB staining (B) or CPK28-Myc (D) are shown in the top and bottom, respectively. Relative kinase activity is shown in (C), with the intensity at 0 s set to 1.0. Data are means ±SEM of three independent experiments (**P < 0.01, Student’s t test). (E to H) Freezing phenotypes (E and G) and survival rates (F and H) of wild-type, cpk28-1, cpk28-1 CPK28pro:CPK28EFm (EFm #1 and #4), and cpk28-1 CPK28pro:CPK28D188A (D188A #8 and #16) seedlings. In (F) and (H), data are means ±SEM of three independent experiments, each with three technical replicates (*P < 0.05 and **P < 0.01, Student’s t test) (Adopted from Ding et al., 2022)
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