Biochemical aspects of xylem differentiation in plant tissue culture

Markland, William (1983). Biochemical aspects of xylem differentiation in plant tissue culture. PhD thesis The Open University.



Plant cytodifferentiation is discussed in general terms with special reference to the 'model system' of xylem differentiation. Previous findings relating to polysaccharide, nucleic acid and lignin synthesis are reviewed in the introduction, while detailed reviews relating to protein synthesis, protein covalent modification and plant hormone binding are located under the appropriate chapter heading.

A list of criteria necessary for the use of plant tissue cultures in detailed biochemical analysis of xylogenesis is discussed with reference to other reported xylogenic cultures, none of which fully meet these requirements. A previously utilised Jerusalem artichoke (Helianthus tuberosus) tuber explant system has been modified and now appears to meet the above criteria. This two step culture allows for the separation of infected explants prior to their further culture on a xylogenic medium or a control medium (on which the explants grow at the same rate as those on the xylogenic medium but without a significant formation of xylem elements). Using this culture regime it should be possible to separate the biochemical events associated with the initial excision wound response of the artichoke tissue, tissue growth and xylem differentiation.

An investigation into a particulate and cytosol 2,4-D specific binding protein showed fluctuations in tissue concentration throughout the culture periods and while these fluctuations did not correlate with the xylogenic process they did appear to coincide with a wound or subculture response of the cultured artichoke tissues.

Utilising the two step artichoke culture system, protein synthesis was analysed by one and two dimensional gel electrophoresis, following radiolabelled amino acid incorporation. Investigations of protein covalent modification (namely glycosylation and phosphorylation) using radiolabelled precursor incorporation, one dimensional gel electrophoresis and autoradiography/fluorography have also been undertaken. Several, fluctuations were noted in the syntheses, which may be of significance in the xylogenic process, the most marked alterations being in the phosphorylation of cytosol located polypeptides which coincided with the peak rate of xylem differentiation. To complement the phosphorylation studies, protein kinase activity was extracted from cultured artichoke tissue, and found to be modulated by the in vitro addition of kinetin and cAMP (which appears to act as a cytokinin) and by 2,4-D. This allows for the possibility of a direct hormonal control of xylem differentiation in plant tissues, acting, at least in part, through the modulation of protein kinase activity and the phosphorylation of possible key proteins associated with the cytodifferentiation process.

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