Ca2+ acts as a universal second messenger in eukaryotes. In animals, a wide variety of environmental and developmental stimuli trigger Ca2+ dynamics in organelles, such as the cytoplasm, nucleus, and endoplasmic reticulum (ER). However, ER Ca2+ ([Ca2+]er) homeostasis and its contributions in cytosolic and/or nucleosolic Ca2+ dynamics in plants remain elusive. GCaMPs are comprised of a circularly permutated form of enhanced green fluorescent protein fused to calmodulin and myosin light-chain kinase M13 and used for monitoring Ca2+ dynamics in mammalian cells. Here, we targeted a high-affinity variant of GCaMP with nuclear export signal in the cytoplasm (NES-GCaMP6m), with a nuclear-localised signal in the nucleus (NLS-GCaMP6m), and a low-affinity variant of GCaMP, also known as calcium-measuring organelle-entrapped protein indicators (CEPIA), with a signal peptide sequence of the ER-localised protein Calreticulin 1a in the ER lumen (CRT1a-R-CEPIA1er) for intraorganellar Ca2+ imaging in Arabidopsis. We found that cytosolic Ca2+ ([Ca2+]cyt) increases induced by 250 mM sorbitol as an osmotic stress stimulus, 50 μM abscisic acid (ABA), or 1 mM carbachol (CCh) were mainly due to extracellular Ca2+ influx, whereas nucleosolic Ca2+ ([Ca2+]nuc) increases triggered by osmotic stress, ABA, or CCh were contributed by [Ca2+]er release. In addition, [Ca2+]er dynamics presented specific patterns in response to different stimuli such as osmotic stress, ABA, or CCh, indicating that Ca2+ signalling occurs in the ER in plants. These results provide valuable insights into subcellular Ca2+ dynamics in response to different stresses in Arabidopsis root cells and prove that GCaMP imaging is a useful tool for furthering our understanding of plant organelle functions.

中文翻译：

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使用 GCaMP 变体 GCaMP6m 和 R-CEPIA1er 在拟南芥幼苗根中进行细胞内钙成像

Ca2+ 在真核生物中充当通用第二信使。在动物中，各种各样的环境和发育刺激会触发细胞器中的 Ca2+ 动力学，例如细胞质、细胞核和内质网 (ER)。然而，ER Ca2+ ([Ca2+]er) 稳态及其对植物细胞质和/或核质 Ca2+ 动力学的贡献仍然难以捉摸。GCaMP 由循环排列形式的增强型绿色荧光蛋白组成，该蛋白与钙调蛋白和肌球蛋白轻链激酶 M13 融合，用于监测哺乳动物细胞中的 Ca2+ 动力学。在这里，我们针对 GCaMP 的高亲和力变体，在细胞质中具有核输出信号 (NES-GCaMP6m)，在细胞核中具有核定位信号 (NLS-GCaMP6m)，以及 GCaMP 的低亲和力变体，也称为作为钙测量细胞器包裹的蛋白质指示剂 (CEPIA)，具有内质网腔内内质网定位蛋白钙网蛋白 1a 的信号肽序列 (CRT1a-R-CEPIA1er)，用于拟南芥中的细胞器内 Ca2+ 成像。我们发现由 250 mM 山梨糖醇作为渗透应激刺激物、50 μM 脱落酸 (ABA) 或 1 mM 卡巴胆碱 (CCh) 诱导的细胞质 Ca2+ ([Ca2+]cyt) 增加主要是由于细胞外 Ca2+ 流入，而核质 Ca2+（ [Ca2+]nuc) 由渗透压力、ABA 或 CCh 触发的增加是由 [Ca2+]er 释放引起的。此外，[Ca2+]er 动力学呈现出响应不同刺激的特定模式，如渗透胁迫、ABA 或 CCh，表明 Ca2+ 信号发生在植物的 ER 中。