A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings:: Identification of AtRALF1 peptide
A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings:: Identification of AtRALF1 peptide
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DOI：
10.1021/bi8001488
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发表时间：
2008-06-17
期刊：
影响因子：
2.9
通讯作者：
Sussman, Michael R.
中科院分区：
文献类型：
--
作者：
Haruta, Miyoshi;Monshausen, Gabriele;Sussman, Michael R.
细胞质Ca2+浓度的短暂增加是植物响应发育和环境线索启动许多细胞信号通路的关键事件；然而，迄今为止，只有少数细胞外介质调节细胞质Ca2+单态是已知的。为了鉴定调节细胞质Ca2+信号的内源性细胞信号肽，我们利用表达aequorin的拟南芥幼苗进行了体内Ca2+变化的发光实验。这些幼苗受到来自植物提取物的馏分的挑战。经高效液相色谱分离后，观察到多个热稳定、蛋白酶敏感的升钙活性峰。串联质谱法鉴定了一系列色谱分离分离得到的主要活性分子为49个氨基酸的多肽AtRALF1（快速碱化因子蛋白家族）。在用纳摩尔浓度的天然或合成肽处理后的40秒内，细胞质Ca2+水平升高并达到最大值。先前使用Ca2+螯合剂或ip3依赖性信号抑制剂治疗部分抑制atralf1诱导的Ca2+浓度增加，表明可能参与Ca2+内流穿过质膜以及从细胞内储备释放Ca2+。利用表达FRET-based Ca2+传感器yellow cameleon (YC) 3.6的幼苗进行Ca2+成像显示，AtRALF1可以诱导根表面细胞Ca2+浓度升高，这与aequorin报道的添加AtRALF1对Ca2+水平的快速影响一致。我们的数据支持一个模型，在这个模型中，RALF肽通过细胞表面受体介导Ca2+依赖性信号事件，它可能在引发与应激反应或生长调节相关的事件中发挥作用。
Transient increases in the cytoplasmic Ca2+ concentration are key events that initiate many cellular signaling pathways in response to developmental and environmental cues in plants; however, only a few extracellular mediators regulating cytoplasmic Ca2+ singling are known to date. To identify endogenous cell signaling peptides regulating cytoplasmic Ca2+ signaling, Arabidopsis seedlings expressing aequorin were used for an in vivo luminescence assay for Ca2+ changes. These seedlings were challenged with fractions derived from plant extracts. Multiple heat-stable, protease-sensitive peaks of calcium elevating activity were observed after fractionation of these extracts by high-performance liquid chromatography. Tandem mass spectrometry identified the predominant active molecule isolated by a series of such chromatographic separations as a 49-amino acid polypeptide, AtRALF1 (the rapid alkalinization factor protein family). Within 40 s of treatment with nanomolar concentrations of the natural or synthetic version of the peptides, the cytoplasmic Ca2+ level increased and reached its maximum. Prior treatment with a Ca2+ chelator or inhibitor of IP3-dependent signaling partially suppressed the AtRALF1-induced Ca2+ concentration increase, indicating the likely involvement of Ca2+ influx across the plasma membrane as well as release of Ca2+ from intracellular reserves. Ca2+ imaging using seedlings expressing the FRET-based Ca2+ sensor yellow cameleon (YC) 3.6 showed that AtRALF1 could induce an elevation in Ca2+ concentration in the surface cells of the root consistent with the very rapid effects of addition of AtRALF1 on Ca2+ levels as reported by aequorin. Our data support a model in which the RALF peptide mediates Ca2+-dependent signaling events through a cell surface receptor, where it may play a role in eliciting events linked to stress responses or the modulation of growth.