Proteomic analysis reveals a novel set of cell wall proteins in a transformed tobacco cell culture that synthesises secondary walls as determined by biochemical and morphological parameters
Proteomic analysis reveals a novel set of cell wall proteins in a transformed tobacco cell culture that synthesises secondary walls as determined by biochemical and morphological parameters
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DOI：
10.1007/s004250000407
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发表时间：
2001-02-01
期刊：
影响因子：
4.3
通讯作者：
Bolwell, GP
中科院分区：
文献类型：
--
作者：
Blee, KA;Wheatley, ER;Bolwell, GP
以烟草（Nicotiana tabacum L.）CV. Petit Havana）细胞系，该细胞系来源于用来自农杆菌的Tcyt基因转化的栽培品种，其导致高内源水平的细胞分裂素。该细胞系显示出增加的细胞聚集、伸长的细胞和5倍的壁厚增加。如果允许继续生长，它可以形成一个单一的质量，而不会将细胞脱落到培养基中。当在早期生长阶段进行分析时，发现这些培养物比采用外源细胞分裂素的其他系统产生更高水平的血管结节形成。这种分化相对于蔗糖和生长素信号进行了优化，以诱导最大限度地生产具有加厚壁和纤维细胞和管胞的形态特征的细胞，以及保持分生组织的细胞。为了建立该系统的有效性，研究次生壁的形成，壁和相关的生物合成的变化进行了分析，在这些细胞中的化学分析的墙壁，木聚糖和monolithyl合成的酶的活性的变化，和表达的mRNA编码的酶的木质素生物合成。将转化细胞的细胞壁组成与从典型的未转化烟草细胞系测定的初生壁组成进行比较。在转化培养物中，壁材料的回收率高50%。在该材料中，在果胶级分中发现了主要差异，其中在尺寸分布方面存在明显差异，并且转化系的甲基化水平较低，这可能与增加的淀粉酶有关。木聚糖的量增加，尽管由于细胞类型的混合，木葡聚糖与木聚糖含量的比率没有实质性差异。纤维素和酚类成分也有所增加。参与木聚糖的合成作为次生壁的标记的酶的活性增加发生在管胞分化的时间周围，并与肉桂醇脱氢酶活性的宽峰相吻合。一般苯丙素途径，苯丙氨酸解氨酶，肉桂酸4-羟化酶，儿茶酚O-甲基转移酶的mRNA编码的表达是相对组成的文化，而阿魏酸5-羟化酶，肉桂酰CoA-还原酶，肉桂醇脱氢酶和木质素过氧化物酶的转录诱导。转化细胞的壁也显示出相当大的差异，在子集中的可提取的蛋白质从发现在烟草的初生壁时，这些进行蛋白质组学分析。这些蛋白质中的许多似乎是新的，并不存在于初生壁。然而，可以鉴定出M-r-32，000几丁质酶、M-r-34，000过氧化物酶、M-r-65，000多酚氧化酶/漆酶和可能的M-r-68，000木聚糖酶以及结构蛋白。
A cell suspension culture of a tobacco (Nicotiana tabacum L. cv. Petit Havana) cell line derived from a cultivar transformed with the Tcyt gene from Agrobacterium, which leads to high endogenous levels of cytokinin, has been established. This cell line shows increased cell aggregation, elongated cells and a 5-fold increase in wall thickness. If allowed to carry on growing it can form a single mass without shedding cells into the medium. When analysed at an earlier growth stage, these cultures were found to produce improved levels of vascular nodule formation than in other systems that employ exogenous cytokinin. This differentiation was optimised with respect to sucrose and auxin signals in order to induce maximum production of cells with thickened walls and a morphology characteristic of fibre cells and tracheids, in addition to cells that remain meristematic. In order to establish the validity of this system for studying secondary wall formation, the walls and associated biosynthetic changes were analysed in these cells by chemical analysis of the walls, changes in activities of enzymes of xylan and monolignol synthesis, and expression of mRNAs coding for enzymes of lignin biosynthesis. The wall composition of the transformed cells was compared with that determined for primary walls from a typical untransformed tobacco cell line. Recovery of wall material was 50% greater in the transformed culture. In this material a major difference was found in the pectin fraction where there was a distinct difference in size distribution together with a lower level of methylation for the transformed line, which may be related to increased adhesiveness. There were increased amounts of xylan, although the ratio of xyloglucan to xylan content was not substantially different due to the mixture of cell types. There was also an increase in cellulose and phenolic components. Increased activity of enzymes involved in the synthesis of xylan as a marker for the secondary wall occurred around the time of tracheid differentiation and coincided with a broad peak of cinnamyl alcohol dehydrogenase activity. The expression of mRNAs coding for enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, catechol O-methyl transferase was relatively constitutive in the cultures while transcripts of ferulate 5-hydroxylase, cinnamoyl CoA-reductase, cinnamyl alcohol dehydrogenase and lignin peroxidase were induced. The walls of the transformed cells also showed considerable differences in the subset of extractable proteins from that found in primary walls of tobacco when these were subjected to proteomic analysis. Many of these proteins appear to be novel and not present in primary walls. However an M-r-32,000 chitinase, an M-r-34,000 peroxidase, an M-r-65,000 polyphenoloxidase/laccase and possibly an M-r-68,000 xylanase could be identified as well as structural proteins.