Wang Peng, Yang Qiaofeng, Sang Sihong, Chen Yao, Zhong Yujiao, Wei Zhaoyun
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China.
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Biochem Biophys Res Commun. 2017 Aug 19;490(2):441-446. doi: 10.1016/j.bbrc.2017.06.060. Epub 2017 Jun 14.
Arabidopsis inositol polyphosphate kinase 2β (AtIpk2β) has multiple functions in plant development and in responding to abiotic stress. Although some related clues suggested a potential role of AtIpk2β in ABA signaling, the defined evidence was still lack. Here we discovered that a key ABA signaling component calcium-dependent protein kinase 4 (CPK4) can interact with AtIpk2β under ABA treated conditions through affinity purification and mass spectrometry detection. The interaction between CPK4 and AtIpk2β were further confirmed by yeast two hybrid and bimolecular fluorescence complementation assays. Expression of AtIpk2β also can be rapidly induced by ABA. In addition, we found that CPK4 can phosphorylate AtIpk2β in vitro and identified five novel phosphorylation sites of AtIpk2β by CPK4 kinase, including Tyr46, Ser48, Ser51, Thr128, Ser147. Overexpression of AtIpk2β in Arabidopsis was more sensitive to ABA in seed germination, primary root inhibition, ABA-responsive gene expression than wild type plants, whereas knockout mutant atipk2β exhibited no significant difference. The AtIpk2β variants containing Tyr46, Thr128, Ser147 mutated to Ala cannot complement the yeast mutant ipk2 growth in high temperature, suggesting that those three amino acid residues are critical for AtIpk2β. These findings provide insight into the modulation of ABA signaling by AtIpk2β.
拟南芥肌醇多磷酸激酶2β（AtIpk2β）在植物发育和应对非生物胁迫方面具有多种功能。尽管一些相关线索表明AtIpk2β在脱落酸（ABA）信号传导中可能发挥作用，但仍缺乏明确的证据。在此，我们发现，在ABA处理条件下，ABA信号传导的关键组分钙依赖性蛋白激酶4（CPK4）可通过亲和纯化和质谱检测与AtIpk2β相互作用。通过酵母双杂交和双分子荧光互补试验进一步证实了CPK4与AtIpk2β之间的相互作用。ABA也能快速诱导AtIpk2β的表达。此外，我们发现CPK4可在体外磷酸化AtIpk2β，并鉴定出CPK4激酶作用于AtIpk2β的5个新的磷酸化位点，包括Tyr46、Ser48、Ser51、Thr128、Ser147。在拟南芥中过表达AtIpk2β，在种子萌发、主根生长抑制、ABA响应基因表达方面比野生型植物对ABA更敏感，而敲除突变体atipk2β则无显著差异。将含有Tyr46、Thr128、Ser147突变为丙氨酸的AtIpk2β变体导入酵母突变体ipk2中，不能使其在高温下生长得到互补，这表明这三个氨基酸残基对AtIpk2β至关重要。这些发现为AtIpk2β对ABA信号传导的调控机制提供了深入见解。
