Institute for Plant Biology, Technische Universität Braunschweig, Mendelssohnsstr. 4, 38106, Braunschweig, Germany; Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt.
Institute for Plant Biology, Technische Universität Braunschweig, Mendelssohnsstr. 4, 38106, Braunschweig, Germany.
Phytochemistry. 2019 Dec;168:112110. doi: 10.1016/j.phytochem.2019.112110. Epub 2019 Sep 5.
Based on the occurrence of indole alkaloids in so-called "chloroform leaf surface extracts", it was previously deduced that these alkaloids are present in the cuticle at the leaf surface of Catharanthus roseus and Vinca minor. As no symplastic markers were found in these extracts this deduction seemed to be sound. However, since chloroform is known to destroy biomembranes very rapidly, these data have to be judged with scepticism. We reanalyzed the alleged apoplastic localization of indole alkaloids by employing slightly acidic aqueous surface extracts and comparing the corresponding alkaloid patterns with those of aqueous total leaf extracts. Whereas in the "chloroform leaf surface extracts" all alkaloids are present in the same manner as in the total leaf extracts, no alkaloids occur in the aqueous leaf surface extracts. These results clearly show that chloroform had rapidly destroyed cell integrity, and the related extracts also contain the alkaloids genuinely accumulated within the protoplasm. The related decompartmentation was verified by the massively enhanced concentration of amino acids in aqueous surface extracts of chloroform treated leaves. Furthermore, the chloroform-induced cell disintegration was vividly visualized by confocal laser scanning microscopical analyses, which clearly displayed a strong decrease in the chlorophyll fluorescence in chloroform treated leaves. These findings unequivocally display that the indole alkaloids are not located in the apoplastic space, but exclusively are present symplastically within the cells of V. minor and C. roseus leaves. Accordingly, we have to presume that also other leaf surface extracts employing organic solvents have to be re-investigated.
基于所谓的“氯仿叶面提取物”中吲哚生物碱的存在，先前推断这些生物碱存在于长春花和长春花的叶表面的角质层中。由于在这些提取物中未发现质外体标记物，因此这种推断似乎是合理的。然而，由于众所周知氯仿会非常迅速地破坏生物膜，因此必须持怀疑态度来判断这些数据。我们通过使用微酸性的水表面提取物重新分析了吲哚生物碱的假定质外体定位，并将相应的生物碱图谱与水总叶提取物的图谱进行了比较。虽然在“氯仿叶面提取物”中所有生物碱都以与总叶提取物相同的方式存在，但在水叶面提取物中不存在生物碱。这些结果清楚地表明，氯仿迅速破坏了细胞完整性，相关提取物也含有真正在细胞质内积累的生物碱。通过对氯仿处理叶片的水表面提取物中氨基酸浓度的大量增加进行验证，相关的隔室化也得到了证实。此外，通过共聚焦激光扫描显微镜分析生动地可视化了氯仿诱导的细胞崩解，该分析清楚地显示了氯仿处理叶片中叶绿素荧光的强烈下降。这些发现明确显示，吲哚生物碱不在质外体空间中，而是仅存在于长春花和长春花叶片细胞的质中。因此，我们必须假定其他使用有机溶剂的叶面提取物也需要重新研究。
