Grosset J, Marty I, Chartier Y, Meyer Y
Laboratoire de Physiologie et Biologie Moléculaire Végétales, Unité de Recherche Associée 565 au C.N.R.S., Perpignan, France.
Plant Mol Biol. 1990 Sep;15(3):485-96. doi: 10.1007/BF00019165.
We have used 2-dimensional (2D) non-equilibrium pH gradient gel electrophoresis (NEPHGE) of in vitro synthesized proteins and northern hybridization with labelled cDNAs coding for three pathogenesis related (P.R.) proteins, to analyze the shift in mRNA content induced by the isolation and culture of tobacco mesophyll protoplasts. The in vitro protein pattern of mRNAs from freshly isolated protoplasts is characterized by the absence of most leaf spots and the appearance of 19 new spots. After 6 hours of culture, the mRNAs coding for the P.R. proteins become detectable and after 12 hours the protoplasts contain an mRNA population almost typical of callus cells. The different steps involved in the isolation and culture of protoplasts were analysed. Cutting off the leaf and sterilization do not change the mRNA set. In contrast, the mechanical injury applied to the leaf in order to facilitate the penetration of the enzymatic mixture induces a modification of the mRNA content identical to that resulting from protoplast isolation. Wounding is the essential event inducing dedifferentiation. Varying the culture medium and conditions leads to only limited modifications of the mRNA pattern. These results are discussed on the basis of present knowledge of the reaction of the plant to wounding and we suggest that wound healing callus and in vitro callus correspond to the same differentiation state.
我们利用体外合成蛋白质的二维（2D）非平衡pH梯度凝胶电泳（NEPHGE）以及与编码三种病程相关（P.R.）蛋白的标记cDNA进行Northern杂交，来分析烟草叶肉原生质体分离和培养所诱导的mRNA含量变化。新鲜分离的原生质体mRNA的体外蛋白质图谱特征是大多数叶斑消失，出现19个新斑点。培养6小时后，编码P.R.蛋白的mRNA变得可检测到，培养12小时后，原生质体含有几乎典型的愈伤组织细胞的mRNA群体。对原生质体分离和培养过程中的不同步骤进行了分析。切断叶片和灭菌不会改变mRNA组。相反，为便于酶混合物渗透而对叶片施加的机械损伤会诱导mRNA含量发生与原生质体分离相同的变化。创伤是诱导去分化的关键事件。改变培养基和培养条件只会导致mRNA图谱的有限变化。根据目前对植物创伤反应的了解对这些结果进行了讨论，我们认为创伤愈合愈伤组织和体外愈伤组织对应于相同的分化状态。
