List of primers used in the study.

Inositol pyrophosphates (PP-InsPs) are soluble cellular messengers that integrate environmental cues to induce adaptive responses in eukaryotes. In plants, the biological functions of various PP-InsP species are poorly understood, largely due to the absence of canonical enzymes found in other eukaryotes. The recent identification of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP7 in the eudicot Arabidopsis thaliana, further highlights the intricate PP-InsP signalling network employed by plants. Yet, the abundance of 4/6-InsP7 in land plants, the enzyme(s) responsible for its synthesis, and the physiological functions of this species are all currently unknown. In this study, we show that 4/6-InsP7 is ubiquitous in the studied land plants. Our findings demonstrate that the Arabidopsis inositol polyphosphate multikinase (IPMK) homologs, AtIPK2α and AtIPK2β phosphorylates InsP6 to generate 4/6-InsP7 as the predominant PP-InsP species in vitro. Consistent with this, AtIPK2α and AtIPK2β act redundantly to control 4/6-InsP7 production in planta. Notably, activity of these IPK2 proteins is critical for heat stress acclimation in Arabidopsis. Our parallel investigations using the liverwort Marchantia polymorpha suggest that the PP-InsP synthase activity of IPK2 and role of IPK2 in regulating the heat stress response are conserved in land plants. Furthermore, we show that the transcription activity of heat shock factor (HSF) is regulated by IPK2 proteins, providing a mechanistic framework of IPK2-controlled heat stress tolerance in land plants. Collectively, our study indicates that IPK2-type kinases have played a critical role in transducing environmental cues for biological processes during land plant evolution.

肌醇焦磷酸盐（Inositol pyrophosphates, PP-InsPs）是一类可溶性细胞信使分子，可整合环境信号以诱导真核生物产生适应性应答。在植物中，各类PP-InsP亚型的生物学功能仍未得到充分阐明，这在很大程度上是由于植物缺乏其他真核生物中存在的经典相关酶类。近期在双子叶植物拟南芥（Arabidopsis thaliana）中鉴定出一种新的PP-InsP异构体，其对映体构型尚未明确，即4/6-InsP7，这进一步凸显了植物所拥有的复杂PP-InsP信号网络。然而，目前人们对4/6-InsP7在陆生植物中的丰度、负责其合成的酶类，以及该亚型的生理功能均一无所知。本研究证实，4/6-InsP7广泛存在于本次研究涉及的陆生植物中。实验结果表明，拟南芥肌醇多磷酸激酶（inositol polyphosphate multikinase, IPMK）同源蛋白AtIPK2α与AtIPK2β可在体外将InsP6磷酸化，生成作为主要PP-InsP亚型的4/6-InsP7。与此一致的是，AtIPK2α与AtIPK2β在植物体内可通过功能冗余的方式调控4/6-InsP7的产生。值得注意的是，这类IPK2蛋白的活性对于拟南芥的热胁迫适应至关重要。我们利用地钱（Marchantia polymorpha）开展的平行研究表明，IPK2的PP-InsP合酶活性以及其在调控热胁迫应答中的作用，在陆生植物中具有保守性。此外，本研究发现热激因子（heat shock factor, HSF）的转录活性受IPK2蛋白调控，这为陆生植物中IPK2介导的热胁迫耐受机制提供了理论框架。综上，本研究表明，IPK2类激酶在陆生植物演化过程中，于转导环境信号以调控生物学进程方面发挥了关键作用。