Geelen Danny, Leyman Barbara, Batoko Henri, Di Sansebastiano Gian-Pietro, Moore Ian, Blatt Michael R
Laboratory of Plant Physiology and Biophysics, Imperial College of Science, Technology, and Medicine at Wye, Kent TN25 5AH, United Kingdom.
Plant Cell. 2002 Feb;14(2):387-406. doi: 10.1105/tpc.010328.
Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion, and secretion. Previously, we isolated the syntaxin-related protein NtSyr1 (NtSyp121) from tobacco in a screen for abscisic acid-related signaling elements, demonstrating its role in determining the abscisic acid sensitivity of K(+) and Cl(-) channels in stomatal guard cells. NtSyr1 is localized to the plasma membrane and is expressed normally throughout the plant, especially in root tissues, suggesting that it might contribute to cellular homeostasis as well as to signaling. To explore its functions in vivo further, we examined stably transformed lines of tobacco that expressed various constructs of NtSyr1, including the full-length protein and a truncated fragment, Sp2, corresponding to the cytosolic domain shown previously to be active in suppressing ion channel response to abscisic acid. Constitutively overexpressing NtSyr1 yielded uniformly high levels of protein (>10 times the wild-type levels) and was associated with a significant enhancement of root growth in seedlings but not with any obvious phenotype in mature, well-watered plants. Similar transformations with constructs encoding the Sp2 fragment of NtSyr1 showed altered leaf morphology but gave only low levels of Sp2 fragment, suggesting a strong selective pressure against plants expressing this protein. High expression of the Sp2 fragment was achieved in stable transformants under the control of a dexamethasone-inducible promoter. Sp2 expression was correlated positively with altered cellular and tissue morphology in leaves and roots and with a cessation of growth in seedlings. Overexpression of the full-length NtSyr1 protein rescued the wild-type phenotype, even in plants expressing high levels of the Sp2 fragment, supporting the idea that the Sp2 fragment interfered specifically with NtSyr1 function by competing with NtSyr1 for its binding partners. To explore NtSyr1 function in secretion, we used a green fluorescent protein (GFP)-based section assay. When a secreted GFP marker was coexpressed with Sp2 in tobacco leaves, GFP fluorescence was retained in cytosolic reticulate and punctate structures. In contrast, in plants coexpressing secreted GFP and NtSyr1 or secreted GFP alone, no GFP fluorescence accumulated within the cells. A new yellow fluorescent protein-based secretion marker was used to show that the punctate structures labeled in the presence of Sp2 colocalized with a Golgi marker. These structures were not labeled in the presence of a dominant Rab1 mutant that inhibited transport from the endoplasmic reticulum to the Golgi. We propose that NtSyr1 functions as an element in SNARE-mediated vesicle trafficking to the plasma membrane and is required for cellular growth and homeostasis.
syntaxin蛋白和其他SNARE蛋白对于细胞内囊泡运输、融合及分泌至关重要。此前,我们在筛选脱落酸相关信号元件时,从烟草中分离出了 syntaxin相关蛋白NtSyr1(NtSyp121),证明其在决定气孔保卫细胞中K(+)和Cl(-)通道对脱落酸敏感性方面的作用。NtSyr1定位于质膜,在整个植物中正常表达,尤其在根组织中,这表明它可能有助于细胞内稳态以及信号传导。为了进一步探究其在体内的功能,我们检测了稳定转化的烟草品系,这些品系表达了NtSyr1的各种构建体,包括全长蛋白和一个截短片段Sp2,Sp2对应于之前显示在抑制离子通道对脱落酸反应中具有活性的胞质结构域。组成型过表达NtSyr1产生了均匀高水平的蛋白(>野生型水平的10倍),并且与幼苗根生长的显著增强相关,但在成熟且水分充足的植物中没有任何明显表型。用编码NtSyr1的Sp2片段的构建体进行类似转化显示叶片形态改变,但Sp2片段水平较低,这表明对表达该蛋白的植物存在强大的选择压力。在糖皮质激素诱导型启动子的控制下,在稳定转化体中实现了Sp2片段的高表达。Sp2表达与叶片和根中细胞及组织形态的改变以及幼苗生长停止呈正相关。全长NtSyr1蛋白的过表达挽救了野生型表型,即使在表达高水平Sp2片段的植物中也是如此,这支持了Sp2片段通过与NtSyr1竞争其结合伙伴而特异性干扰NtSyr1功能的观点。为了探究NtSyr用基于绿色荧光蛋白(GFP)的切片分析来研究NtSyr1在分泌中的功能。当在烟草叶片中与Sp2共表达分泌型GFP标记物时,GFP荧光保留在胞质网状和点状结构中。相反,在共表达分泌型GFP和NtSyr1或仅分泌型GFP的植物中,细胞内没有积累GFP荧光。使用一种新的基于黄色荧光蛋白的分泌标记物表明,在Sp2存在下标记的点状结构与高尔基体标记物共定位。在存在抑制从内质网到高尔基体运输的显性Rab1突变体的情况下,这些结构没有被标记。我们提出NtSyr1作为SNARE介导的囊泡运输到质膜的一个元件发挥作用,并且是细胞生长和内稳态所必需的。 1在分泌中的功能,我们使用基于绿色荧光蛋白(GFP)的切片分析。当在烟草叶片中与Sp2共表达分泌型GFP标记物时,GFP荧光保留在胞质网状和点状结构中。相反,在共表达分泌型GFP和NtSyr1或仅分泌型GFP的植物中,细胞内没有积累GFP荧光。使用一种新的基于黄色荧光蛋白的分泌标记物表明,在Sp2存在下标记的点状结构与高尔基体标记物共定位。在存在抑制从内质网到高尔基体运输的显性Rab1突变体的情况下,这些结构没有被标记。我们提出NtSyr1作为SNARE介导的囊泡运输到质膜的一个元件发挥作用,并且是细胞生长和内稳态所必需的。