State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, National Plant Gene Research Centre, China Agricultural University, Beijing 100193, China.
Plant Cell. 2013 Feb;25(2):649-61. doi: 10.1105/tpc.112.103184. Epub 2013 Feb 28.
Potassium (K(+)) influx into pollen tubes via K(+) transporters is essential for pollen tube growth; however, the mechanism by which K(+) transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca(2+)-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca(2+)-dependent regulation of the inward K(+) (K(+)in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K(+)in currents of pollen tube protoplasts were inhibited by elevated [Ca(2+)]cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca(2+)-dependent inhibition of K(+)in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K(+)in channel is the main contributor to pollen tube K(+)in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca(2+)-dependent inhibition of K(+)in channels and participate in the regulation of pollen tube growth in Arabidopsis.
钾（K(+））通过 K(+)转运体流入花粉管对于花粉管的生长至关重要；然而，K(+)转运体在花粉管中是如何被调控的机制尚不清楚。在这里，我们报告拟南芥钙依赖性蛋白激酶 11（CPK11）和 CPK24 参与了花粉管中 Ca(2+)-依赖性内向 K(+)（K(+)in）通道的调控。通过膜片钳分析，我们证实了花粉管原生质体的 K(+)in 电流可被升高的胞质 Ca(2+)浓度抑制。然而，CPK11 或 CPK24 的缺失完全破坏了 Ca(2+)-依赖性对 K(+)in 电流的抑制作用，并增强了花粉管的生长。此外，cpk11 cpk24 双突变体表现出与相应的单突变体相似的表型，这表明这两种 CDPKs 在同一信号通路中发挥作用。双分子荧光互补和免疫共沉淀实验表明 CPK11 可以在体内与 CPK24 相互作用。此外，CPK11 在体外磷酸化 CPK24 的 N 端，这表明这两种 CDPK 作为激酶级联的一部分协同作用。电生理学分析表明，Shaker 花粉 K(+)in 通道是花粉管 K(+)in 电流的主要贡献者，并且是 CPK11-CPK24 途径的下游靶标。我们得出结论，CPK11 和 CPK24 共同介导 K(+)in 通道的 Ca(2+)-依赖性抑制，并参与拟南芥花粉管生长的调控。
