Humboldt-Universität zu Berlin, Institute of Biology, Department of Plant Physiology, Philippstr. 13 Building 12, 10115 Berlin, Germany.
J Exp Bot. 2022 Dec 8;73(22):7401-7416. doi: 10.1093/jxb/erac378.
Sucrose transporters of the SUT4 clade show dual targeting to both the plasma membrane as well as to the vacuole. Previous investigations revealed a role for the potato sucrose transporter StSUT4 in flowering, tuberization, shade avoidance response, and ethylene production. Down-regulation of StSUT4 expression leads to early flowering, tuberization under long days, far-red light insensitivity, and reduced diurnal ethylene production. Sucrose export from leaves was increased and a phase-shift of soluble sugar accumulation in source leaves was observed, arguing for StSUT4 to be involved in the entrainment of the circadian clock. Here, we show that StSUT4, whose transcripts are highly unstable and tightly controlled at the post-transcriptional level, connects components of the ethylene and calcium signalling pathway. Elucidation of the StSUT4 interactome using the split ubiquitin system helped to prove direct physical interaction between the sucrose transporter and the ethylene receptor ETR2, as well as with the calcium binding potato calmodulin-1 (PCM1) protein, and a calcium-load activated calcium channel. The impact of calcium ions on transport activity and dual targeting of the transporter was investigated in detail. For this purpose, a reliable esculin-based transport assay was established for SUT4-like transporters. Site-directed mutagenesis helped to identify a diacidic motif within the seventh transmembrane spanning domain that is essential for sucrose transport activity and targeting, but not required for calcium-dependent inhibition. A link between sucrose, calcium and ethylene signalling has been previously postulated with respect to pollen tube growth, shade avoidance response, or entrainment of the circadian clock. Here, we provide experimental evidence for the direct interconnection of these signalling pathways at the molecular level by direct physical interaction of the main players.
蔗糖转运蛋白 SUT4 家族的成员具有双重靶向性，既能靶向质膜，也能靶向液泡。先前的研究表明，马铃薯蔗糖转运蛋白 StSUT4 在开花、块茎形成、避荫反应和乙烯产生中起作用。StSUT4 表达的下调导致开花提前、长日照下的块茎形成、远红光不敏感和昼夜乙烯产生减少。叶片中的蔗糖输出增加，源叶中可溶性糖积累出现相位移动，这表明 StSUT4 参与了生物钟的驯化。在这里，我们表明，StSUT4 的转录本在转录后水平高度不稳定且受到严格控制，它连接了乙烯和钙信号通路的组成部分。使用分裂泛素系统阐明 StSUT4 的相互作用组有助于证明蔗糖转运蛋白与乙烯受体 ETR2 以及钙结合的马铃薯钙调素-1（PCM1）蛋白和钙负载激活的钙通道之间存在直接的物理相互作用。详细研究了钙离子对转运活性和转运蛋白双重靶向性的影响。为此，建立了一种可靠的基于 esculin 的 SUT4 样转运蛋白运输测定法。定点突变有助于确定第七跨膜结构域内的一个二酸性基序，该基序对蔗糖转运活性和靶向性至关重要，但不需要钙依赖性抑制。先前曾提出蔗糖、钙和乙烯信号之间存在联系，涉及花粉管生长、避荫反应或生物钟的驯化。在这里，我们通过主要参与者的直接物理相互作用，提供了这些信号通路在分子水平上直接相互连接的实验证据。
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