Study On Process Of MAPK Signal Transduction Of Taxus Cuspidata Cells In Immobilized Cultures Microenvironments

The influences of immobilization microenvironments on the growth and production of plant cells were investigated by immobilizing Taxus cuspidata cells on the non-toxic polyurethane foam. The changes in cellular morphology, protein expressions and phosphorylation level were monitored to study the signal transduction of mitogen-activated protein kinase (MAPK) in Taxus cuspidata cells induced by local microenvironments in immobilized cultures.The cylinder-shaped polyurethane foam (7.5 mm in radius and 15 mm in height) was viewed as consisting of two zones, 0-3 mm and 3~7.5 mm from the edge to the center, respectively. The immobilized cells growing on the foam surface were defined as the outer zone cells, while the cells growing in the zones of 0~3 mm and 3~7.5 mm from edge to center were called as the middle zone cells and the central zone cells, respectively. The viability, mitotic index (MI), cellular differentiation and Taxol production of the immobilized T. csupidata cells were investigated. The immobilized cells had higher viability than the freely suspended cells in the lag and stationary stages. After day 20, the MI of the immobilized cells began to decrease gradually, while the cellular differentiation and Taxol production of the immobilized cells gradually enhanced from the outer to the central zones in the immobilization support matrices. These indicate the differences of the physical-chemical microenvironments in the different zones of the immobilization support matrices.The impacts of immobilized cultures on the cell wall signal molecules (arabinogalactan-proteins, AGPs) were detected with western blot and immunofluorescence. It has been found that the AGPs expressions of T. csupidata cells in the middle and central zones of the immobilized support matrices were higher than those of the cells in the outer zone and of the freely suspended cells after day 20. The enhanced Taxol production and cellular differentiation were accompanied with the higher AGPs expressions in the immobilized T. csupidata cells.The signal mechanisms of T. cuspidata cells involved in the responses to the local microenvironment in the immobilization support matrices were investigated by detecting the phosphorylation level of ERK, JNK and p38 MAPK of the cells.Western blot analysis showed that the immobilized cultures significantly altered the activation of the extracellular signal-regulated kinase-like (ERK-like, approximately 46 kDa) MAPK in T. cuspidata cells during the whole culture period. After day 20, the ERK-like MAPK activation in T. cuspidata cells was down-regulated from the outer to the central zone in the immobilization support matrices, which associated with the formation of nutrient concentration gradient in different zones of the immobilization support matrices. The ERK-like MAPK activation in the immobilized cells was positively correlated to the cellular MI but negatively correlated to the degree of cellular differentiation, AGPs expressions and Taxol production of T. cuspidata cells. Moreover, the immobilized cultures up-regulated 52-kDa JNK-like MAPK activation in T. cuspidata cells but down-regulated 45-kDa JNK-like and 41-kDa p38-like MAPK activation in comparison with the freely suspended cultures. Further experiments showed that it was not only the 52-kDa JNK-like and 41-kDa p38-like MAPK activation but also the 45-kDa and 47-kDa JNK-like MAPK activation in T. cuspidata cells were involved in the immobilization of the cultures protecting cells from the hydrodynamic shear stresses.The differentially expressed proteins were investigated by 2-DE and MALDI-TOF-MS to study the influences of microenvironments on the growth and production of plant cells from the view point of protein levels. It has been found that some differentially expressed proteins were involved in the regulation of carbohydrate, nitrogen and sulfur metabolisms. Among the proteins identified, the immobilized cultures enhanced the expressions of UDPG pyrophosphorylase, which was involved directly or indirectly in the biosynthesis of cell wall polysaccharides. The abundance of S-adenosylmethionine synthetase (SAMS) was positively correlated to the division activity of T. cuspidata cells.