Introduction

Cells depend on sugars to generate energy and to build the molecules required for cellular form and function. Sugars can also act as signalling molecules with various roles in regulating growth and development, physiological processes, metabolic feedback, and modulating abiotic or biotic stress responses (Rolland et al., 2006). Plants generate their own sugars from photosynthesis. This dependence on light for energy supply creates specific challenges for plant cells, which must maintain these processes under both predictable and unpredictable fluctuations in the growth environment. This requires multiple sugar signalling pathways to coordinate dynamic supply and demand throughout the plant.

There are four well-recognised sugar signalling pathways in plants. HEXOKINASE 1 (HXK1) is responsible for the first enzymatic step in glycolysis but has glucose signalling functions independent of its enzymatic activity (Moore et al., 2003). G-protein signalling plays a role in extracellular glucose sensing and cell proliferation (Chen et al., 2003; Urano et al., 2012). TARGET OF RAPAMYCIN (TOR) kinase functions in numerous signalling pathways and is activated under carbon (C)-replete conditions (Xiong et al., 2013). By contrast, Snf1 RELATED KINASE 1 (SnRK1) is active under C starvation (Baena-González et al., 2007). SnRK1 activity is inhibited by the signalling sugar trehalose-6-phosphate (T6P) (Zhang et al., 2009), which is very tightly connected to sucrose levels (Figueroa & Lunn, 2016). SnRK1, and perhaps also TOR, can directly affect activity of transcription factors (Xiong et al., 2013; Mair et al., 2015). HXK1 can localize to the nucleus and associate with DNA-binding complexes (Cho et al., 2006).

The critical importance of sugar signalling in plant cells makes genetic analysis of these pathways challenging. Loss-of-function mutants in TOR or T6P SYNTHASE 1 (TPS1) are embryo lethal (Eastmond et al., 2002; Menand et al., 2002) and a double mutant in both catalytic subunits of SnRK1 is not viable (Ramon et al., 2019). Therefore, most studies on these pathways have used hypomorphic mutants or inducible transgenic lines (Baena-González et al., 2007; Gómez et al., 2010; Xiong et al., 2013; Belda-Palazón et al., 2020). By contrast, growth effects in mutants in HXK1 or REGULATOR OF G-PROTEIN SIGNALLING 1 (RGS1) are relatively minor, but both mutants are hyposensitive to growth inhibition by high exogenous sugar (Moore et al., 2003; Chen et al., 2006).

Although there is substantial overlap between the cellular processes controlled by these sugar signalling pathways, particularly growth and energy metabolism, there are distinct features of their signalling outputs. For example, genetic experiments indicate additive effects of hxk1-3 and rgs1-2 mutants (Huang et al., 2015), suggesting functionally distinct pathways. TOR regulates proteostasis, autophagy and cell cycle control by sugars (Burkart & Brandizzi, 2021), whereas SnRK1 controls responses to energy deprivation and regulation of iron homeostasis (Peixoto et al., 2021).

The circadian clock is a gene regulatory network that integrates external and intrinsic signals to coordinate biological rhythms according to daily and seasonal changes in the environment. Photoautotrophic metabolism requires feedback between C availability and the circadian oscillator to optimise plant growth and fitness. Sugars affect circadian rhythms in Arabidopsis in several ways. C status contributes to entrainment, the process of setting the phase of the circadian clock (Haydon et al., 2013), and measurement of photoperiod (Liu et al., 2021). Reduced photosynthesis lengthens the circadian period, which can be suppressed by supplying sugar (Haydon et al., 2013). Period adjustment by sugars requires T6P-SnRK1 signalling affecting transcription of PSEUDO RESPONSE REGULATOR 7 (PRR7) (Frank et al., 2018). Sugars can also affect the amplitude of specific oscillator components. One mechanism occurs by posttranscriptional control of GIGANTEA (GI) and requires F-box protein ZEITLUPE (ZTL) (Haydon et al., 2017).

Circadian rhythms rapidly dampen in seedlings released into continuous darkness without supplied sugar. Application of sucrose to dark-adapted seedlings can reinitiate circadian rhythms and the phase is set according to the time of sugar application (Dalchau et al., 2011). This transcriptional response to sugar does not require GI and the signalling processes are not known. This simple assay provides a sensitive technique to define sugar responses in the absence of light signals. We recently used transcriptome analysis of this response to reveal a role for superoxide, a reactive oxygen species (ROS), in promoting circadian gene expression and growth by sugar (Román et al., 2021). To further understand the signalling underlying this transcriptional response to sugar, we screened the Library of Pharmacologically Active Compounds (LOPAC; Sigma-Aldrich) for chemicals that modify the response of a circadian reporter to sucrose in dark-adapted seedlings. From a list of 75 confident hit compounds, we selected 15 compounds to further characterize their effects on sugar-dependent processes. We focus on two compounds that inhibit a sugar-activated ROS-Ca²⁺ signalling pathway that affects circadian rhythms, primary metabolism and plant growth. Our data provide a resource of pharmacological tools to manipulate sugar signalling in plants and has revealed opportunities to define new components of metabolic signalling.

中文翻译：

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从糖激活昼夜节律基因表达修饰剂的化学筛选中鉴定出一种活性氧类 Ca2+ 信号通路

介绍

细胞依靠糖来产生能量并构建细胞形态和功能所需的分子。糖类还可以作为信号分子，在调节生长发育、生理过程、代谢反馈以及调节非生物或生物应激反应方面发挥多种作用 (Rolland et al .,  2006 )。植物通过光合作用产生自己的糖分。这种对光能供应的依赖给植物细胞带来了特殊的挑战，植物细胞必须在生长环境的可预测和不可预测的波动下维持这些过程。这需要多个糖信号通路来协调整个工厂的动态供需。

植物中有四种公认的糖信号通路。己糖激酶 1 (HXK1) 负责糖酵解中的第一个酶促步骤，但具有独立于其酶促活性的葡萄糖信号传导功能（Moore等人，  2003 年）。G 蛋白信号在细胞外葡萄糖传感和细胞增殖中发挥作用（Chen等人，  2003 年；Urano等人，  2012 年）。雷帕霉素靶标 (TOR) 激酶在许多信号通路中发挥作用，并在碳 (C) 充足的条件下被激活 (Xiong等人，  2013 年)。相比之下，Snf1 相关激酶 1 (SnRK1) 在 C 饥饿下具有活性（Baena-González等人.,  2007 ). SnRK1 活性被信号糖海藻糖-6-磷酸 (T6P) 抑制（Zhang等人，  2009 年），它与蔗糖水平密切相关（Figueroa & Lunn，  2016 年）。SnRK1，或许还有 TOR，可以直接影响转录因子的活性（Xiong等人，  2013 年；Mair等人，  2015 年）。HXK1 可以定位于细胞核并与 DNA 结合复合物结合 (Cho et al .,  2006 )。

糖信号在植物细胞中的至关重要性使得对这些途径的遗传分析具有挑战性。TOR或T6P SYNTHASE 1 ( TPS1 ) 中的功能丧失突变体是胚胎致死的（Eastmond等人，  2002 年；Menand等人，  2002 年）并且 SnRK1 的两个催化亚基中的双突变体是不可行的（Ramon等人.,  2019 年）。因此，大多数关于这些途径的研究都使用了亚型突变体或可诱导的转基因品系（Baena-González等人，  2007 年；Gómez等人，  2010 年；Xiong等人.,  2013 ; Belda-Palazón等人，  2020 年）。相比之下，HXK1或G 蛋白信号调节器 1 ( RGS1 ) 突变体的生长效应相对较小，但两种突变体都对高外源糖的生长抑制不敏感（Moore等人，  2003 年；Chen等人，  2006 年） ).

尽管由这些糖信号传导途径控制的细胞过程之间存在大量重叠，特别是生长和能量代谢，但它们的信号输出具有明显的特征。例如，基因实验表明hxk1-3和rgs1-2突变体的累加效应 (Huang et al .,  2015 )，表明功能不同的途径。TOR 通过糖调节蛋白质稳态、自噬和细胞周期控制（Burkart & Brandizzi，  2021 年），而 SnRK1 控制对能量剥夺的反应和铁稳态的调节（Peixoto等人，  2021 年）。

生物钟是一种基因调控网络，它整合外部和内部信号，根据环境的日常和季节变化协调生物节律。光合自养代谢需要 C 有效性和昼夜节律振荡器之间的反馈，以优化植物生长和适应性。糖以多种方式影响拟南芥的昼夜节律。C 状态有助于夹带、设置生物钟相位的过程（Haydon等人，  2013 年）和光周期测量（Liu等人，  2021 年）。光合作用减少会延长昼夜节律，这可以通过提供糖来抑制（Haydon等人，  2013 年）). 糖的周期调整需要 T6P-SnRK1 信号影响 PSEUDO RESPONSE REGULATOR 7 ( PRR7 ) 的转录（Frank等人，  2018 年）。糖还可以影响特定振荡器组件的振幅。一种机制通过 GIGANTEA (GI) 的转录后控制发生，并且需要 F-box 蛋白 ZEITLUPE (ZTL)（Haydon等人，  2017 年）。

在没有糖分供应的情况下，幼苗被释放到持续的黑暗中，昼夜节律迅速减弱。向适应黑暗的幼苗施用蔗糖可以重新启动昼夜节律，并且根据糖施用的时间设置阶段（Dalchau等人，  2011）。这种对糖的转录反应不需要 GI，并且信号传导过程未知。这种简单的检测提供了一种灵敏的技术，可以在没有光信号的情况下定义糖的反应。我们最近使用这种反应的转录组分析来揭示超氧化物（一种活性氧 (ROS)）在促进昼夜节律基因表达和糖生长方面的作用（Román等人，  2021 年）). 为了进一步了解这种对糖的转录反应背后的信号，我们在药理活性化合物库（LOPAC；Sigma-Aldrich）中筛选了可以改变昼夜节律报告基因对暗适应幼苗中蔗糖反应的化学物质。从 75 种可靠的命中化合物列表中，我们选择了 15 种化合物来进一步表征它们对糖依赖过程的影响。我们专注于抑制糖激活的 ROS-Ca ²⁺信号通路的两种化合物，该信号通路影响昼夜节律、初级代谢和植物生长。我们的数据提供了一种药理学工具资源来操纵植物中的糖信号，并揭示了定义代谢信号新成分的机会。