Studies on the Metabolism of Plant Cells in Tissue Culture
1. A tobacco cell-suspension tissue culture system derived from pith cells of Nicotiana tabacum L. var Wisconsin 38 was established. This system was used to study methods of purifying nucleic acids, phosphate ester and nucleic acid metabolism in cells subjected to nutritional shifts, and problems relating to the control of cell division in plant cells.
2. Cell cultures consisting of single cells, groups of cells and small aggregates were maintained in liquid medium on rotary shakers. Cultures exhibited an exponential growth period during which the cell generation time was 2, 3 days.
3. Cells removed from culture medium and resuspended in dilute inorganic salt solutions (step-down nutritional shift) exhibited marked changes in their phosphate and sulphate accumulation rates and in phosphate ester metabolism, but not in the type and rate of respiration.
4. Such metabolic responses to culture shifts were not related to cell damage or shock effects, but appeared to be a feature of changes in the nutritional environment of cells. The response of cells to a step-down nutritional shift was maximal at exponential- and late-stationary phases of culture growth. Step-up nutritional shifts (resuspending cells in enriched culture medium) produced some effects similar to those found for step-down cells. Actinomycin D did not prevent the phosphate accumulation response of cells to culture shifts, and was shown to inhibit only 50 per cent of the synthesis of rapidly-labelled ribonucleic acid.
5. RNA prepared from tobacco cells or rat liver by conventional phenol extraction was contaminated with other materials which composed as much as sixty per cent of the weight of the total preparation. RNA prepared from (32P) tobacco cells contained minor radioactivity in RNA but major radioactivity in a wide range of contaminating phosphate esters of high specific radioactivity.
6. Purification of RNA by the Kirby two-phase partition procedure removed most major contaminants but failed to remove (32P) phosphate esters. The procedure also resulted in degraded RNA.
7. A new method was developed for the purification of RNA prepared by the phenol procedure. It involved recovery of RNA from the upper phase of the two-phase Kirby extraction mixture by precipitation as the cetyltrimethylammonium (CETA) salt instead of by dialysis. This procedure reduced manipulation times and much more effectively removed RNAase and (32P) phosphate esters from the RNA.
8. TMV-RNA prepared by the new procedure was fully infectious. RNA prepared and purified from a mixture of whole rat liver and TMV was almost equally as infectious as RNA prepared from TMV alone. Infectivity of purified RNA, prepared from a mixture of tobacco cells and TMV, was stable in solution at 30°C for four hours. TMV-RNA prepared in this way and stored at -12°C in vacuo over P2O5 retained infectivity for at least 6 months.
9. Extraction of (32P) exponential-phase cells by aqueous phenol plus detergent released only RNA with the ribosomal type of base composition. Similar extractions in the presence of high salt concentrations released this RNA together with DNA and rapidly-labelled RNA.
10. During short treatments with (32P) orthophosphate, exponential-phase cells synthesised RNA with a base composition intermediate between that of ribosomal RNA and DNA. Cells subjected to a step-down nutritional shift prior to treatment with (32P) orthophosphate, synthesised RNA with a base composition close to that of tobacco DNA.
11. Following treatment times with (32P orthophosphate, or (32P) plus (3H) uridine, and following a step-down or steady-state nutritional shift to non-radioactive medium, tobacco cells exhibited the following changes in phosphate ester and RNA metabolism:-
a. Changes in the levels of radioactivity present in the nucleoside triphosphate RNA precursors and other phosphate esters.
b. A reduction in the rate of increase of specific radioactivity of RNA of step-down cells, followed by a subsequent recovery.
c. Changes in the (32P) base composition of the RNA synthesised following the culture shift.
d. Changes in the radioactive sedimentation profiles of RNA.
12. Extracts prepared from exponential-phase tobacco cells contained cytokinin (cell division stimulant) activity. Fractionation of cells, and bioassay of levels of cytokinins present in the various fractions, demonstrated the presence of soluble compounds with cytokinin activity in cell debris and cytoplasmic fractions, but not in the nuclear fraction.
13. Unhydrolysed (polymeric) tobacco nucleic acids (RNA plus DNA), prepared from exponential-phase tobacco cells, showed no cytokinin activity. A ribonucleotide mixture prepared by alkaline hydrolysis of these tobacco-cell nucleic acid preparations, mixtures of purified ribonucleotides, or a mixture of tobacco deoxyribo- and ribonucleosides resulting from snake-venom digestion, contained no activity. Deoxyribonucleotides were inhibitory.
14. A ribonucleotide mixture prepared by alkaline hydrolysis of RNA isolated from rat liver and sheep liver, contained cytokinin activity. Deoxyribo- and ribonucleoside mixtures resulting from snake-venom digestion of sheep-liver RNA and DNA contained similar levels of cytokinin activity to that found in extracts prepared by alkaline hydrolysis.