Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea.
Gene Engineering Division, Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 55365, Republic of Korea.
Mol Plant. 2017 Sep 12;10(9):1190-1205. doi: 10.1016/j.molp.2017.08.003. Epub 2017 Aug 18.
The abscisic acid (ABA) signaling pathway is regulated by clade A type 2C protein phosphatases (PP2CAs) in plants. In the presence of ABA, PP2Cs release stress/ABA-activated protein kinases by binding to ABA-bound receptors (PYL/RCARs) for activation. Although the wedging tryptophan in PP2Cs is critical in the interaction with PYL/RCARs in Arabidopsis and rice, it remains elusive as to how other interface regions are involved in the interaction. Here, we report the identification of a conserved region on PP2Cs, termed the VxGΦL motif, which modulates the interaction with PYL/RCARs through its second and fourth residues. The effects of the second and fourth residues on the interaction of OsPP2C50 with several OsPYL/RCAR proteins were investigated by systematic mutagenesis. One OsPP2C50 mutant, VFGML ("FM") mutant, lowered the affinity to OsPYL/RCAR3 by ∼15-fold in comparison with the wild-type. Comparison of the crystal structures of wild-type OsPP2C50:ABA:OsPYL/RCAR3 with those composed of FM mutant revealed local conformational changes near the VxGΦL motif, further supported by hydrogen-deuterium exchange mass spectrometry. In rice protoplasts, ABA signaling was altered by mutations in the VxGΦL motif. Transgenic Arabidopsis plants overexpressing OsPP2C50 and OsPP2C50FM showed altered ABA sensitivity. Taken together, the VxGΦL motif of PP2Cs appears to modulate the affinity of PP2Cs with PYL/RCARs and thus likely to alter the ABA signaling, leading to the differential sensitivity to ABA in planta.
脱落酸（ABA）信号通路在植物中受到 clade A 型 2C 蛋白磷酸酶（PP2CAs）的调节。在 ABA 存在的情况下，PP2C 与结合 ABA 的受体（PYL/RCARs）结合，释放应激/ABA 激活的蛋白激酶，从而被激活。尽管在拟南芥和水稻中，PP2C 中的楔入色氨酸在与 PYL/RCARs 的相互作用中是关键的，但其他界面区域如何参与相互作用仍然难以捉摸。在这里，我们报告了在 PP2Cs 上鉴定到一个保守区域，称为 VxGΦL 基序，它通过第二个和第四个残基来调节与 PYL/RCARs 的相互作用。通过系统诱变研究了第二个和第四个残基对 OsPP2C50 与几种 OsPYL/RCAR 蛋白相互作用的影响。一个 OsPP2C50 突变体，VFGML（"FM"）突变体，与野生型相比，与 OsPYL/RCAR3 的亲和力降低了约 15 倍。比较野生型 OsPP2C50：ABA：OsPYL/RCAR3 的晶体结构与由 FM 突变体组成的晶体结构揭示了 VxGΦL 基序附近的局部构象变化，这进一步得到了氢氘交换质谱的支持。在水稻原生质体中，VxGΦL 基序的突变改变了 ABA 信号。过表达 OsPP2C50 和 OsPP2C50FM 的转基因拟南芥植物表现出改变的 ABA 敏感性。总之，PP2C 的 VxGΦL 基序似乎调节了 PP2C 与 PYL/RCARs 的亲和力，从而可能改变 ABA 信号，导致植物体内对 ABA 的敏感性不同。
