The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures
The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures
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DOI：
10.1007/s00425-004-1430-3
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发表时间：
2005-05-01
期刊：
影响因子：
4.3
通讯作者：
Fitzpatrick, T
中科院分区：
文献类型：
--
作者：
Dean, JV;Mohammed, LA;Fitzpatrick, T
烟草（Nicotiana tabacum L. cv）中水杨酸（SA）的代谢。将[7-C-14] SA加入细胞培养24 h，通过高效液相色谱分析鉴定代谢物。SA的3个主要代谢产物是SA 2-O-β- d -葡萄糖（SAG）、2-O-β- d -葡萄糖甲基水杨酸（MeSAG）和水杨酸甲酯。对代谢物胞内定位的研究表明，与烟草原生质体相关的SAG全部定位于液泡内。然而，大多数MeSAG位于液泡外。烟草细胞含有一种SA诱导的SA葡萄糖基转移酶（SAGT），形成SAG。SAGT酶与液泡无关，似乎是一种细胞质酶。通过测定烟草细胞培养物中[C-14] SAG在细胞质囊泡中的吸收，表征了SAG的空泡运输。添加MgATP后，对SAG的吸收增加了8倍。巴菲霉素A(1)（液泡H+- atp酶的特异性抑制剂）和转质H+-电化学梯度的耗散抑制了atp依赖性SAG的摄取。钒酸盐不是SAG摄取的抑制剂。几种β-葡萄糖偶联物是SAG摄取的强抑制剂，而谷胱甘肽和葡萄糖醛酸偶联物只有轻微的抑制作用。SAG吸收表现为Michaelis - Menten型饱和动力学，K-m和V-max分别为11 μ M和205 pmol min(-1) mg（-1）。根据运输特性，烟草细胞对SAG的空泡摄取似乎是通过H+-反端口型机制发生的。
The metabolism of salicylic acid ( SA) in tobacco ( Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7-C-14] SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O-β-D-glucose (SAG), methylsalicylate 2-O-β-D-glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [C-14] SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A(1) ( a specific inhibitor of the vacuolar H+-ATPase) and dissipation of the transtonoplast H+-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several β-glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis - Menten type saturation kinetics with a K-m and V-max value of 11 μ M and 205 pmol min(-1) mg(-1), respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H+-antiport-type mechanism.