Physiologia Plantarum 130: 290–300. 2007 Copyright ª Physiologia Plantarum 2007, ISSN 0031-9317 Effects of tetracycline on wild-type and inducible P35So IPT-5/TETR transgenic tobacco plants Lieve Quanten a , Laury Chaerle b , Jean-Paul Noben c , Harry Van Onckelen d , Els Prinsen e , Dominique Van Der Straeten b and Roland Valcke a, * a Laboratory of Molecular and Physical Plant Physiology, Department SBG, Centre for Environmental Sciences, Building D, Hasselt University, Agoralaan, B-3590 Diepenbeek, Belgium b Unit Plant Hormone Signalling and Bio-imaging (HSB), Department of Molecular Genetics, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium c Laboratory of Immunology – Biochemistry, Building A, Hasselt University, Biomedisch Onderzoeksinstituut, Agoralaan, B-3590 Diepenbeek, Belgium d Laboratory of Plant Biochemistry and Physiology, University of Antwerp, Middelheimcampus G.U513, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium e Laboratory of Plant Biochemistry and Physiology, University of Antwerp, Middelheimcampus G.U515, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium Correspondence *Corresponding author, e-mail: roland.valcke@uhasselt.be Received 12 December 2006; revised 12 January 2007 doi: 10.1111/j.1399-3054.2007.00888.x Introduction of the Agrobacterium ipt gene, coding for isopentenyl transferase, under control of a tetracycline (Tc)-inducible promoter results in a very specific system in which cytokinin levels can be changed. Because Tc belongs to the group of antibiotics that affect 70S ribosomes, it is important to study the effects of Tc on untransformed plants. Although 1 mg l 21 Tc was previously reported to have no physiological effects, this study revealed several changes in hydro- ponically grown wild-type Nicotiana tabacum L. cv. Wisconsin. Therefore, lower Tc concentrations (0.1 and 0.2 mg l 21 Tc) were used to induce ipt- transgenic (tr) plants. Upon induction, real-time PCR analysis showed that the ipt gene was expressed several times higher in roots of tr plants, but not in leaves. Consequently, cytokinin levels were also elevated to a large extent in roots. This resulted in a disturbance of the cytokinin to auxin ratio, leading to an obstructed root growth. In leaves, no significant increase in cytokinins was observed. However, phenotypic and physiological effects, which could be attributed to cytokinin, were apparent in leaves of ipt-induced trs: chlorophyll and carotenoid content were elevated and grana stacking increased. Our study demonstrates that caution has to be taken to determine the ‘safe’ concentration of inducers when using inducible gene-expression systems. Introduction One of the main goals of plant biologists is to gain insight into the relation between structure and function in higher plants. To investigate complex processes such as devel- opment, signal transduction and photosynthesis, model organisms (e.g. Arabidopsis thaliana, rice), a diversity of mutants, and transgenics (trs) with a higher or lower expression level of a gene of interest are widely used. Several systems have been designed to overexpress a transgene. As a first approach, constitutive promoter-driven Abbreviations – ACC, 1-aminocyclopropane-1-carboxylic acid; CK, cytokinin; Ct, cycle threshold; DZRMP, dihydrozeatin riboside- 5#-monophosphate; EM, electron microscopy; iP, N 6 -(D 2 -isopentenyl) adenine; iPRMP, N 6 -(D 2 -isopentenyl) adenosine-5# monophosphate; ipt, isopentenyl transferase; RT-PCR, real-time polymerase chain reaction; Tc, tetracycline; TetR, Tet repressor; tr, transgenic plant; wt, wild-type; Z, zeatin; Z9G, zeatin-9-glucoside; ZR, zeatin riboside; ZRMP, zeatin riboside-5#-monophosphate. 290 Physiol. Plant. 130, 2007