Calcium-regulated protein kinases are key components of are key components of intracellular signaling in plants that mediate rapid stress-induced responses to changes in the environment. To identify in vivo phosphorylation substrates of CALCIUM-DEPENDENT PROTEIN KINASE1 (CPK1), we analyzed the conditional expression of constitutively active CPK1 in conjunction with in vivo phosphoproteomics. We identified Arabidopsis thaliana ORESARA1 (ORE1), the developmental master regulator of senescence, as a direct CPK1 phosphorylation substrate. CPK1 phosphorylates ORE1 at a hotspot within an intrinsically disordered region. This augments transcriptional activation by ORE1 of its downstream target gene BIFUNCTIONAL NUCLEASE1 (BFN1). Plants that overexpress ORE1, but not an ORE1 variant lacking the CPK1 phosphorylation hotspot, promote early senescence. Furthermore, ORE1 is required for enhanced cell death induced by CPK1 signaling. Our data validate the use of conditional expression of an active enzyme combined with phosphoproteomics to decipher specific kinase target proteins of low abundance, of transient phosphorylation, or in yet undescribed biological contexts. Here, we have identified that senescence is not just under molecular surveillance manifested by stringent gene regulatory control over ORE1. In addition, the decision to die is superimposed by an additional layer of control towards ORE1 via its post-translational modification linked to the calcium-regulatory network through CPK1.

钙调节的蛋白激酶是植物中细胞内信号转导的关键成分，其介导对环境变化的快速胁迫诱导反应。为了鉴定钙依赖性蛋白激酶1（CPK1）的体内磷酸化底物，我们结合体内磷酸化蛋白质组学分析了组成型活性CPK1的条件表达。我们确定拟南芥拟南芥ORESARA1（ORE1），发展的衰老的主调节器，直接CPK1磷酸化底物。CPK1在本质上无序的区域内的热点处使ORE1磷酸化。这通过ORE1增强了其下游靶基因BIFUNCTIONAL NUCLEASE1（BFN1）的转录激活。过量表达ORE1而不缺少缺少CPK1磷酸化热点的ORE1变体的植物会促进早期衰老。此外，ORE1是CPK1信号转导诱导的细胞死亡增强所必需的。我们的数据验证了结合磷酸蛋白质组学使用活性酶的条件表达来破译低丰度，瞬时磷酸化或尚未描述的生物学背景下的特定激酶靶蛋白。在这里，我们已经确定衰老不仅受到对ORE1的严格基因调控控制所表现出的分子监视。此外，死亡决定由通过ORE1的翻译后修饰（通过CPK1与钙调节网络链接）对ORE1附加的控制层叠加而成。我们的数据验证了结合磷酸蛋白质组学使用活性酶的条件表达来破译低丰度，瞬时磷酸化或尚未描述的生物学背景下的特定激酶靶蛋白。在这里，我们已经确定衰老不仅仅受到对ORE1的严格基因调控的分子监测。此外，死亡决定由通过ORE1的翻译后修饰（通过CPK1与钙调节网络连接）对ORE1附加的控制层叠加而成。我们的数据验证了使用活性酶与磷酸化蛋白质组学相结合的条件表达来破译低丰度，瞬时磷酸化或尚未描述的生物学背景下的特定激酶靶蛋白。在这里，我们已经确定衰老不仅受到对ORE1的严格基因调控控制所表现出的分子监视。此外，死亡决定由通过ORE1的翻译后修饰（通过CPK1与钙调节网络连接）对ORE1附加的控制层叠加而成。