Domingo C, Roberts K, Stacey N J, Connerton I, Ruíz-Teran F, McCann M C
Department of Cell Biology, John Innes Centre, Colney, Norwich, UK.
Plant J. 1998 Jan;13(1):17-28. doi: 10.1046/j.1365-313x.1998.00002.x.
The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to reproducibly trans-differentiate into tracheary elements (TE) after 96 h, while in the presence of auxin alone the cells simply elongate. In a search for genes involved in modifications to cell-wall architecture before any overt signs of cell differentiation, a differential hybridization of a 72-h cDNA library with probes from mRNA at time-points of 24 h and 72 h was done revealing a number of transcripts up-regulated between these times. One of these cDNAs shows homology to pectate lyase, a pectin-degrading enzyme. The complete cDNA sequence (ZePel) corresponds to a translated protein of 44 kDa with an N-terminal signal peptide of about 2 kDa, and one potential N-glycosylation site. Northern analysis confirms that the strong expression of this gene during TE induction occurs at a very early stage of the process and is due solely to the presence of auxin in the induction medium. In situ hybridization studies in young Zinnia stems show that ZePel expression is associated with vascular bundles and shoot primordia. Recombinant protein made in Escherichia coli possesses calcium-dependent pectate lyase activity. Pectate lyase activity is detected in elongating and differentiating in vitro cell populations. The role of this enzyme in remodelling the cell wall during cell elongation and differentiation is discussed.
百日草叶肉细胞系统由培养中的分离叶肉细胞组成，在生长素和细胞分裂素的作用下，这些细胞可在96小时后可重复地转分化为管状分子（TE），而仅在生长素存在的情况下，细胞只会伸长。为了寻找在细胞分化出现明显迹象之前参与细胞壁结构修饰的基因，我们用24小时和72小时时间点的mRNA探针与一个72小时的cDNA文库进行了差异杂交，结果显示在这些时间之间有许多转录本上调。其中一个cDNA与果胶裂解酶（一种果胶降解酶）具有同源性。完整的cDNA序列（ZePel）对应一个44 kDa的翻译蛋白，其N端有一个约2 kDa的信号肽和一个潜在的N-糖基化位点。Northern分析证实，该基因在TE诱导过程中的强烈表达发生在该过程的非常早期阶段，并且仅归因于诱导培养基中生长素的存在。对幼嫩百日草茎进行的原位杂交研究表明，ZePel的表达与维管束和芽原基有关。在大肠杆菌中产生的重组蛋白具有钙依赖性果胶裂解酶活性。在体外伸长和分化的细胞群体中检测到了果胶裂解酶活性。本文讨论了这种酶在细胞伸长和分化过程中重塑细胞壁的作用。
