The Plant Cell, Vol. 9, 1781-1790, October 1997 O 1997 American Society of Plant Physiologists zyxw Arabidopsis Mutants Resistant to the Auxin Effects of Indole-3-Acetonitrile Are Defective in the Nitrilase Encoded by the zyxw NITI Gene Jennifer Normanly,a Paula Grisafi,b Gerald R. Fink,blc and Bonnie Barteld,i aDepartment of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts O1 003 bWhitehead lnstitute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142 cBiology Department, Massachusetts lnstitute of Technology, Cambridge, Massachusetts, 02142 Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1 892 Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demon- strated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fel1 into a single complementation group that mapped to chromosome zyxwv 3, within 0.5 centimorgans of a cluster of three nitrilase-encodinggenes, NIT7, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NlT7 gene, and the expression pattern of NlT7 is consistent with the IAN insensitivity observed in the nit7 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit7 mutant, confirming that Arabi- dopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo. INTRODUCTION Severa1 pathways have been proposed to account for indole- 3-acetic acid (IAA) biosynthesis; however, none of these has been shown definitively to be responsible for the synthesis of the hormone in vivo (Normanly et al., 1995; Bartel, 1997). Plant mutants disrupted in IAA biosynthesis should help to illumi- nate these pathways, because auxotrophic mutants have been invaluable in elucidating a wide variety of biosynthetic pathways, from amino acid biosynthesis in microbes (Jones and Fink, 1982) to gibberellin biosynthesis in higher plants (Reid, 1993; Reid and Howell, 1995). Despite considerable effort, researchers have not identified IAA biosynthetic mu- tants (Blonstein et al., 1988; Oetiker et al., 1990; Fracheboud and King, 1991), perhaps because IAA is essential for growth and because plants appear to have multiple pathways for IAA biosynthesis (Michalczuk et al., 1992). Indole-3-acetonitrile (IAN) has been proposed as an IAA precursor in certain higher plants (reviewed in Schneider and Wightman, 1974; Sembdner et al., 1981; Nonhebel et al., 1993). IAN was first purified from cabbage (Henbest et al., 1953) and is present in Arabidopsis at levels comparable to that of total IAA (Normanly et al., 1993; llic et al., 1996). Consistent with the possibility that IAN serves as a precur- To whom correspondence should be addressed. E-mail bartel@bioc. rice.edu; fax 71 3-285-51 54. sor to IAA, Arabidopsis mutants blocked in either of the last two steps of tryptophan biosynthesis accumulate both total IAA and IAN (Normanly et al., 1993). Extracts from a variety of plant families, including the Cruciferae, Gramineae, and Musaceae, hydrolyze IAN to zyxwv IAA (Thimann and Mahadevan, 1964). Genes encoding nitrilase enzymes have been cloned from Arabidopsis (Bartling et al., 1992, 1994; Bartel and Fink, 1994; Hillebrand et al., 1996; Zhou et al., 1996a, 1996b), and similar genes are present in tobacco (Tsunoda and Yamaguchi, 1995), Chinese cabbage (Bischoff et al., 1995), and rice (http://www.staff .or.jp/) IAA may also be derived from IAN in several plant-associ- ated microbes. These organisms convert IAN to IAA by us- ing either a nitrilase or the sequential action of a nitrile hydratase and an amidase. In zyxw Alcaligenes faecalis JM3, an IAN-specific nitrilase has been purified and the correspond- ing gene cloned (Kobayashi et al., 1993). This enzyme is -30% identical to the Arabidopsis nitrilase enzymes (Bartel and Fink, 1994). In contrast, the plant pathogen Agrobacte- rium and several species of the plant symbiont Rhizobium have been shown to convert IAN to IAA through the nitrile hydratase pathway (Kobayashi et al., 1995). We are exploring the role of nitrilase genes in IAA biosyn- thesis in the model crucifer Arabidopsis. Each enzyme en- coded by the four Arabidopsis nitrilase genes (NIT7, NIT2, NlT3, and NlT4) can hydrolyze IAN to IAA when expressed in