Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca(2+) -affinity targeted aequorin reporter.
Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca(2+) -affinity targeted aequorin reporter.
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DOI：
10.1111/tpj.15610
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发表时间：
2022-03
期刊：
影响因子：
7.2
通讯作者：
Navazio, Lorella
中科院分区：
文献类型：
--
作者：
Cortese, Enrico;Moscatiello, Roberto;Pettiti, Francesca;Carraretto, Luca;Baldan, Barbara;Frigerio, Lorenzo;Vothknecht, Ute C.;Szabo, Ildiko;De Stefani, Diego;Brini, Marisa;Navazio, Lorella
关键词：
迄今为止，缺乏对植物内质网（ER）腔中游离Ca 2+浓度动态变化的精确测量，尽管越来越多的证据表明该细胞内隔室对植物细胞中Ca 2+稳态和信号传导的贡献。在本研究中，我们针对一个水母发光蛋白嵌合体与降低钙离子亲和力的ER膜和面对ER腔。为此目的，将低-Ca 2 +-亲和力水母发光蛋白变体（AEQmut）的cDNA与编码不可切割的N-末端ER信号肽（fl 2）的核苷酸序列融合。通过在转基因拟南芥（Arabidopsis thaliana）（拟南芥）幼苗中的免疫细胞化学分析证实了fl 2-AEQmut的正确靶向。应用在动物细胞中建立良好的实验方案-由光蛋白重建期间的ER Ca 2+消耗和随后的ER Ca 2+再填充组成-在植物中进行ER Ca 2+测量。记录了响应于不同环境应力的ER腔Ca 2+浓度（[Ca 2 +]ER）的快速和短暂增加，显示刺激特异性Ca 2+特征。ER和叶绿体Ca 2+动力学的比较分析表明，这些细胞器在塑造胞质Ca 2+信号在信号转导事件的复杂的相互作用。我们的数据突出了显着差异基础[Ca 2 +]ER和Ca 2+处理植物ER相比，动物对应。ER靶向水母发光蛋白嵌合体的建立扩展并补充了目前可用的细胞器靶向Ca 2+指标工具包，增加了一个报告基因，提高了我们对植物内膜系统中Ca 2+稳态的定量理解。为了更好地了解植物内质网（ER）中的Ca 2+稳态，需要准确测量该室内腔中的游离Ca 2+浓度。我们设计了一种特异性ER靶向的水母发光蛋白嵌合体，可以定量监测拟南芥幼苗中ER Ca 2+水平响应环境线索的动态变化，我们分析了ER和叶绿体在塑造胞质Ca 2+信号中的相互作用。
Precise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low‐Ca2+‐affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non‐cleavable N‐terminal ER signal peptide (fl2). The correct targeting of fl2‐AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well‐established in animal cells – consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling – was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+]ER) were recorded in response to different environmental stresses, displaying stimulus‐specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set‐up of an ER‐targeted aequorin chimera extends and complements the currently available toolkit of organelle‐targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system. A better understanding of Ca2+ homeostasis in the plant endoplasmic reticulum (ER) requires accurate measurements of the free Ca2+ concentration in the lumen of this compartment. We designed a specifically ER‐targeted aequorin chimera that allowed quantitative monitoring of dynamic changes in ER Ca2+ levels in Arabidopsis seedlings in response to environmental cues and we analysed the interplay of ER and chloroplasts in shaping cytosolic Ca2+ signals.
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DOI：
10.1073/pnas.1107912108
发表时间：
2011-08-23
影响因子：
11.1
作者：
Capoen, Ward;Sun, Jongho;Oldroyd, Giles E. D.
通讯作者：
Oldroyd, Giles E. D.
影响因子：
4
作者：
Chung WY;Jha A;Ahuja M;Muallem S
通讯作者：
Muallem S
影响因子：
7.2
作者：
Clough, SJ;Bent, AF
通讯作者：
Bent, AF
影响因子：
5.6
作者：
De Vriese K;Costa A;Beeckman T;Vanneste S
通讯作者：
Vanneste S
影响因子：
7.5
作者：
Block, Maryse A.;Jouhet, Juliette
通讯作者：
Jouhet, Juliette