Date of Award
Doctor of Philosophy (PhD)
Ross H. Hall
N⁶-(Δ²-Isopentenyl)adenosine is a cytokinin, that is, it promotes cell division, growth and differentiation in certain plant tissues. Some aspects of the metabolism and biological effects of i⁶Ado were studied. These studies have contributed to scientific knowledge in several areas: the biological effects of i⁶Ado on animal and plant cells; the metabolism of i⁶Ado by animal cells; and the origin of free cytokinins.
A. The effects of i⁶Ado on a cell line derived from the circulating leucocytes of a patient with chronic myeloid leukaemia, Roswell Park Memorial Institute line 6410, were studied.
(i) The growth of 6410 cells was not stimulated by i⁶Ado, but concentrations of i⁶Ado of 3 μM inhibited the growth of this cell line. N⁶-(Δ²-Isopentenyl)adenosine did not affect the duration of the lag phase of growth of these cells. There was no detectable maturation of these cells as judged by an alteration in alkaline phosphatase activity under these conditions. An increase in lactic dehydrogenase activity in 6410 cells that have been treated lath i⁶Ado is a non-specific effect related to the death of the cells.
(ii) N⁶-(Δ²-Isopentenyl)adenosine is a component of 6410 cell tRNA in quantities approximately equal to those found in other organisms. Neither i⁶Ado nor the corresponding base, i⁶Ade, nor the corresponding 5' monophosphate, i⁶AMP, were found free in the cells or culture medium.
(iii) The tRNA of cells cultured in the presence of i⁶Ado contains a normal complement of Δ²-isopentenYl groups. This indicates that there is no feedback mechanism between free i⁶Ado and the synthesis of i⁶Ado in the tRNA of these cells.
(iv) Because 6410 cells contain i⁶Ado in their tRNA they are exposed to the free nucleoside that is toxic to them during tRNA turnover. However, 6410 cells contain an enzyme that converts i 6Ado to the corresponding base, that is much less toxic to these cells. This is the first time that this mechanism of detoxification has been demonstrated. The finding is of importance in the development of new cytotoxic drugs related to i⁶Ado, because detoxification reactions potentially limit therapeutic value of a drug.
B. The effects of i⁶Ado on a variant strain of tobacco pith tissue were studied. This is the first report of the effects of this naturally occurring cytokinin on tobacco pith callus at a cellular level.
(i) The variant strain did not differentiate to form mature shoots and leaves as does normal tissue when treated with high concentrations i⁶Ado.
(ii) Administration of i⁶Ado to the variant tissue was followed by cell division and a reduction in cell size within 48 hours. After this period the cell size remained constant but the tissue continued to grow.
(iii) There was an increase in the starch content of the tissue for 10 days following the administration of i⁶Ado. After this time the starch levels remained constant. This is different from the rapid fall in starch concentrations after 12 days noted in tobacco pith tissue that forms organs.
(iv) The growth of the variant tissue was stimulated by i⁶Ado. Unlike normal tobacco pith tissue i⁶Ado in high concentrations, 3 μM, 9 μM did not significantly inhibit its growth.
(v) An hypothesis is proposed relating the effects of i⁶Ado energy metabolism, and tissue growth and organogenesis.
C. The biosynthesis of i⁶Ade was studied using a plant pathogen Corynebacterium fascians. Cultures of this bacteria have been reported to contain up to 100 μg/litre of i⁶Ade.
(i) [8-¹⁴C] Adenine and [8-¹⁴C] adenosine but not [2-¹⁴C] mevalonic acid added to cultures of C. fascians were incorporated by the bacteria. Because [2-¹⁴C] mevalonic acid was not incorporated, it could not be used as a specific label for the Δ²-isopentenyl side-chain of i⁶Ado in the tRNA of these bacteria.
(ii) [8-¹⁴C] Adenine was incorporated into free i⁶Ade extracted from cultures of these bacteria. The extent of labelling of i⁶Ade that was extracted when the cultures were labelled with divided doses of [8-¹⁴C] adenine throughout logarithmic growth phase, was the same as the extent of labelling that occurred when the same quantity of radioactivity was administered in one dose in late logarithmic growth phase.
(iii) When cultures of C. fascians that have been labelled with [8-¹⁴C] adenine are extracted by a procedure in which the pH never falls below 7.0, less than one fifth of labelled ¹⁴C-i⁶Ade is extracted compared with the amount extracted from cultures subjected to heating and mild acidification before the extraction procedure.
(iv) The quantity of i⁶Ade present in the cultures was estimated to be approximately 5.0 μg/litre. Heating and acidification increased the quantity of i⁶Ade present to 36 to 40 μg/litre. This increase in i⁶Ade was shown to be due to release of i⁶Ade from C. fascians tRNA by heating and mild acidification.
(v) Bioassay of extracts of C. fascians cultures showed that i⁶Ado is only a minor component of the total cytokinin activity present. Other cytokinin activity corresponded chromatographically to i⁶Ado, zeatin and ribosyl zeatin.
(vi) On this evidence, I propose that all free cytokinins are not derived directly from the corresponding cytokinin hypermodified nucleosides in the tRNA, because C. fascians tRNA has been shown to contain only one cytokinin.
tRNA may act as a store of relatively inactive cytokinins that are converted to more active cytokinins under appropriate physiological circumstances.
This would enable organisms to exert control over cytokinin activity in its tissues even in the event of tride fluctuations in the turnover of tRNA.
Inevitably, work of this nature generates new questions. An important area for future study appears to be the further definition of the metabolism of i⁶Ado. It is probable that metabolites of i⁶Ado could cause many of the effects that have previously been due to the parent compound. In addition, a better understanding of the mechanisms by which i⁶Ado causes growth differentiation and cytotoxicity may assist in the development of new therapeutic agents.
Rathbone, Michel Piers, "N⁶-(Δ²-ISOPENTENYL)ADENOSINE" (1972). Open Access Dissertations and Theses. Paper 3668.