Modifying the lntracellular Auxin Concentration'

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates (J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128). We have investigated this hypothesis in detail using tobacco (Nicofiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBCUS 6 clone) or the cauliflower mosaic virus 35s promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBCUS 6, and wild-type protoplasts. Naphthyl-B-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBCUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBCUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, i t was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.