Rice B2 and B3 subgroup RAF kinases play central roles in both ABA and osmotic stress signaling

Protein kinases in the mitogen-activated protein kinase kinase kinase (MAPKKK) supper family are known to be central actors in orchestrating cell differentiation, growth, and responses to extracellular stimuli in eukaryotes. However, the rice (Oryza sativa L.) MAPKKKs, especially the RAF subgroup ones have rarely been studied yet. In this study, we generated high-order mutants of B1, B2, and B3 RAF subgroup MAPKKKs. The resulting raf-b2, raf-b3q, and raf-b2/3q mutants are hyposensitive to ABA but show dramatic sensitivity to various osmotic stresses. The raf-b1 mutant only shows reduced growth but responds to ABA and osmotic stress normally. The rice B2 and B3 RAFs could phosphorylate SAPKs and rescue the ABA-induced expression of RD29B-LUC in mesophyll protoplasts of Arabidopsis OK100-nonu mutant. In the rice raf-b2/3q, the ABA-triggered activation of SAPKs is largely impaired, and the expression of stress-response genes is decreased compared to that in the wild type. We also compared the ABA and osmotic stress phenotypes among B2/3 RAF mutants in Arabidopsis, P. patens, and rice. Our study reveals a crucial role of B2 and B3 subgroup MAPKKKs in ABA signaling and osmotic stresses and sheds light on the functional divergence of RAF-SAPK cascade in environmental stress responses in different plant species.

促分裂原活化蛋白激酶激酶激酶（MAPKKK）超家族中的蛋白激酶是真核生物中调控细胞分化、生长及对胞外刺激应答的核心因子。然而，水稻（Oryza sativa L.）中的MAPKKK，尤其是RAF亚家族成员，相关研究仍较为匮乏。本研究构建了RAF亚家族B1、B2及B3成员的高阶突变体。获得的raf-b2、raf-b3q及raf-b2/3q突变体对脱落酸（ABA）敏感性降低，但对多种渗透胁迫（osmotic stress）表现出显著敏感性；raf-b1突变体仅表现出生长受抑制，但对ABA及渗透胁迫的应答正常。水稻B2和B3 RAF成员可磷酸化SAPK，并能恢复拟南芥OK100-nonu突变体叶肉原生质体中ABA诱导的RD29B-LUC表达。在水稻raf-b2/3q突变体中，ABA诱导的SAPK激活显著受损，且胁迫应答基因的表达水平较野生型降低。此外，我们比较了拟南芥、小立碗藓（P. patens）及水稻中B2/3 RAF突变体在ABA和渗透胁迫下的表型。本研究揭示了B2和B3亚家族MAPKKK在ABA信号通路及渗透胁迫中的关键作用，并为不同植物物种中RAF-SAPK级联在环境胁迫应答中的功能分化提供了新的见解。