THE ROLB GENE OF AGROBACTERIUM-RHIZOGENES DOES NOT INCREASE THE AUXINSENSITIVITY OF TOBACCO PROTOPLASTS BY MODIFYING THE INTRACELLULAR AUXIN CONCENTRATION

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a R olB-catalyzed hydrolysis of auxin conjugates (J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128). We have investigated this hypo thesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplas ts isolated from plants transformed with the rolB gene under the contr ol of its own promoter (BBGUS 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 pl asma membrane when triggered with exogenous auxin. Because this phenot ypical trait was homogeneously displayed over the entire population, p rotoplasts were judged to be a more reliable test system than the tiss ue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[H-3]naphthaleneacetic a cid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplas ts. Naphthyl-beta-glucose ester, the major NAA metabolite in protoplas ts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Further more, NAA accumulation and metabolism in BBGUS 6 protoplasts were inde pendent of the rolS gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a co nsequence of modifying the intracellular auxin concentration but likel y results from changes in the auxin perception pathway.