College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China.
College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China; National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China.
Biochem Biophys Res Commun. 2020 May 7;525(3):537-542. doi: 10.1016/j.bbrc.2020.02.078. Epub 2020 Feb 26.
Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play important roles in brassinosteroid (BR)-induced antioxidant defense and enhancing the tolerance of plants to drought stress. The autophosphorylation of CCaMK is a key step for the activation of CCaMK, thus promoting substrate phosphorylation. However, how CCaMK autophosphorylation function in BR-induced antioxidant defense is not known yet. Here, seven potential autophosphorylation sites of ZmCCaMK were identified using mass spectroscopy (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) analysis. The transient gene expression analysis in maize protoplasts showed that Thr420 and Ser454 of ZmCCaMK were important for BR-induced antioxidant defense. Furthermore, Thr420 and Ser454 of ZmCCaMK were crucial for improving drought tolerance and alleviating drought induced oxidative damage of plants via overexpressing various mutant versions of ZmCCaMK in tobacco (Nicotiana tabacum). Mutations of Thr420 and Ser454 in ZmCCaMK substantially blocked the autophosphorylation and substrate phosphorylation of ZmCCaMK in vitro. Taken together, our results demonstrate that Thr420 and Ser454 of ZmCCaMK are crucial for BR-induced antioxidant defense and drought tolerance through modulating the autophosphorylation and substrate phosphorylation activities of ZmCCaMK.
钙/钙调素依赖性蛋白激酶（CCaMK）已被证明在油菜素内酯（BR）诱导的抗氧化防御和增强植物对干旱胁迫的耐受性方面发挥重要作用。CCaMK 的自磷酸化是 CCaMK 激活的关键步骤，从而促进底物磷酸化。然而，CCaMK 自磷酸化如何在 BR 诱导的抗氧化防御中发挥作用尚不清楚。在这里，使用质谱（液相色谱-串联质谱 [LC-MS/MS]）分析鉴定了 ZmCCaMK 的七个潜在的自磷酸化位点。在玉米原生质体中的瞬时基因表达分析表明，ZmCCaMK 的 Thr420 和 Ser454 对于 BR 诱导的抗氧化防御很重要。此外，通过在烟草（Nicotiana tabacum）中过表达各种突变体版本的 ZmCCaMK，Thr420 和 Ser454 对于提高耐旱性和缓解干旱诱导的植物氧化损伤至关重要。ZmCCaMK 的 Thr420 和 Ser454 突变在体外显著阻断了 ZmCCaMK 的自磷酸化和底物磷酸化。总之，我们的结果表明，ZmCCaMK 的 Thr420 和 Ser454 通过调节 ZmCCaMK 的自磷酸化和底物磷酸化活性，对于 BR 诱导的抗氧化防御和耐旱性至关重要。
