Biochemical, cellular and developmental characterization of a temperature-sensitive mutant of Nicotiana tabacum and its second site revertant.

We have analyzed a temperature-sensitive mutant, ts4, of Nicotiana tabacum, together with its newly isolated second revertant, Rt1, both in cell culture and as a regenerated plant. Cells of the temperature-sensitive mutant will grow only below 30 degrees C, whereas the revertant can grow at temperatures above that but not at those as high as will wild-type cells. Enzyme assays of phenylalanine ammonia lyase and nitrate reductase showed that ts4 had substantial increases and decreases, respectively. The revertant resembled ts4 for these activities. The decrease in nitrate reductase is associated with resistance of ts4 to a pulse of chlorate that kills wild-type. Amino acid and polyamine analyses showed that ornithine levels rise in ts4 during 24 hr at the high temperature, while spermine and spermidine levels fall. The ts4 mutant has low levels of ornithine decarboxylase activity and s-adnosyl-methionine decarboxylase activity, whereas the revertant has restored levels of only ornithine decarboxylase. This is the only enzyme activity in which Rt1 resembles wild-type more than it resembles ts4, suggesting that it is the site of metabolic alteration of the mutation. In regenerated plants, ts4 has a low chlorophyll content, while the revertant has a fully restored chlorophyll content. Flowers on the revertant plant are both male and female sterile, with the male sterility associated with anthers converted into petals. The correlations among these ts4 and Rt1 alterations demonstrate novel regulatory interactions between nitrate reductase and the two decarboxylase enzymes, and also suggest that we have partially unravelled the molecular basis for a particular developmental endpoint.