241 Plant Growth Regulation 19: 241-248,1996. @ 1996 Kluwer Academic Publishers. Printed in the Netherlands. Deregulated branched-chain amino acid synthesisin a Nicotiana plumbaginifolia cell line resistant to valine Giuseppe Forlani,M. Cristina Suardi & Erik Nielsen Department of Genetics and Microbiology, University of Pavia, I-27100 Pavia, Italy Received 7 September 1995; accepted 6 February 1996 Key words: acetohydroxyacid synthase, branched-chain amino acids/cell growth inhibitors, enzyme level regulation, valine tolerance Abstract A Nicotiana plumbaginifolia cell line able to grow in the presence of high doses of valine was isolated following y-rays mutagenesis. The selected clone, named D5R5, showed a growth rate higher than that of wild-type. It was less sensitive also to an equimolar mixture of the three branched-chain amino acids, but did not display cross-resistance to isoleucine and leucine. The increased tolerance was due to neither a reduced valine uptake, nor a modification in the level or sensitivity to feed-back inhibition by valine of the first common enzyme (and the main regulative site) in isoleucine, leucine and valine synthesis, acetohydroxyacid synthase (AHAS). When wild-type cells were fed with valine or equimolar mixtures of the three aminoacids, a decrease in AHAS level was found. On the contrary, the level of extractable AHAS activity from D5R5 cells was significantly less affected by similar treatments, suggesting that some alteration in enzyme modulation mechanism(s) could account for valine resistance. Abbreviations: AHAS = acetohydroxyacid synthase; BCAA = branched-chain amino acid; FAD = flavin adenine dinucleotide; ILV = equimolar mixture of isoleucine, leucine and valine; TPP = thiamine pyrophosphate 1. Introduction Exogenously supplied amino acids, singly or in com- bination, were found in several instances to inhibit the growth of plant cells [2, 16, 251. This effect is possibly due to the synthetic inter-dependency among protein precursors, whose carbon skeletons are derived from branched pathways often sharing long common sequences [ 181. Such relationships provide the cell with mechanisms whereby a balanced synthesis of amino acids may be approached. However, as a conse- quence of the exogenous availability of some of them at high concentration, an unbalanced regulation can occur, thus leading to starvation for other amino acid(s) [13,25]. In both microorganisms and plants, isoleucine, leucine and valine are synthesized through paths which share three initial steps [28, 291. The metabolic flow through this common sequence is modulated at the level of the first enzyme, acetohydroxyacid synthase (AHAS; EC 4.1.3.18). In enterobacteria the presence of distinct AHAS isozymes subjected to differential feed- back inhibition and trascriptional control allows each branched-chain amino acid (BCAA) to regulate its own synthesis without interfering with that of the others [I]. In higher plants, in which the occurrence of multiple forms of theenzyme has not been demonstrated to date, the inhibitory effect of valine and leucine on AHAS activity leads on the contrary to isoleucine starvation [4]. In addition, high concentrations of isoleucine are susceptible to deprive cells of leucine, as it mimicks the latter in feed-back inhibition of the branch-point enzyme 2-isopropylmalate synthase [3]. Thus the addi- tion of each BCAA to the culture medium exerts severe inhibition on plant cell growth [4,9,16]. A remarkable inhibition was nonetheless found when cells were fed with an equimolar mixture of isoleucine, leucine and valine (ILV) [9]. This has to be