Resistance to Acetohydroxamate Acquired by Slow Adaptive Increases in Urease in Cultured Tobacco Cells

Abstract

Urease activity of tobacco XD cells (1U cells) had undergone a 4-fold increase (4U cells) during a year of growth on urea (Skokut and Filner, 1980). A clone of 4U cells gave rise to 12U cells during another year of growth on urea. The doubling time of 12U cells on urea is 2.2 days, compared to .apprx. 4 days for 1U cells, while 1U and 12U cells double in 2 days on NO3. Acetohydroxamic acid (AHA), a specific inhibitor/reversible inactivator of jack bean urease, affects tobacco cell urease similarly. Fifty percent inhibition of growth by AHA occurred at 20 .mu.M in 1U cells growing on urea and at 165 .mu.M in 12U cells growing on urea but at 600 .mu.M for either 1U or 12U cells growing on NO3. When 12U cells were grown on urea with 100 .mu.M AHA, extractable urease activity decreased 80% within 2.5 h and remained at this level for 2 wk; the doubling time increased to 3.7 days and intracellular urea rose 2-fold, compared to 12U cells grown on urea without AHA. Urease of 12U cells inactivated by AHA in vivo could be reactivated to its pre-AHA level by incubation at 30.degree. C after extraction and separation from free AHA. AHA inhibited incorporation of 15N from [15N]urea into Kjeldahl N in the cells, in spite of the increased intracellular urea. AHA evidently acts primarily by inhibiting urease action, rather than by inhibition of formation of urease protein or of uptake of urea. Because 12U cells are 8 times more tolerant of AHA than 1U cells, it is likely that growth on urea in the presence of AHA should select strongly for cells with high urease.