arabidopsis mutants resistant to the auxin effects of ... · the plant cell, vol. 9, 1781-1790,...

The Plant Cell, Vol. 9, 1781-1790, October 1997 O 1997 American Society of Plant Physiologists

Arabidopsis Mutants Resistant to the Auxin Effects of Indole-3-Acetonitrile Are Defective in the Nitrilase Encoded by the 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 021 42 cBiology Department, Massachusetts lnstitute of Technology, Cambridge, Massachusetts, 021 42

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 demonstrated 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 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, 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 mutants (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 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-associated microbes. These organisms convert IAN to IAA by using either a nitrilase or the sequential action of a nitrile hydratase and an amidase. In Alcaligenes faecalis JM3, an IAN-specific nitrilase has been purified and the corresponding 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 biosynthesis in the model crucifer Arabidopsis. Each enzyme encoded by the four Arabidopsis nitrilase genes (NIT7, NIT2, NlT3, and NlT4) can hydrolyze IAN to IAA when expressed in

1782 The Plant Cell

Escherichia coli (Bartling et al., 1992, 1994; Bartel and Fink,1994). These genes are differentially expressed during de-velopment, and the NIT2 gene is specifically induced in re-sponse to bacterial pathogen infiltration (Bartel and Fink,1994). NIT1 is the most highly expressed nitrilase gene, asdetermined by RNA gel blot analysis, expression of pro-moter-reporter gene fusions in transgenic plants, and cDNAabundance (Bartel and Fink, 1994). Here, we describe the iso-lation and analysis of point mutations in the NIT1 gene anduse these nitl mutants, along with transgenic plants overex-pressing the various nitrilase cDNAs, to investigate the possi-bility that IAN is an intermediate in IAA biosynthesis.

RESULTS

IAN Has Auxinlike Effects on GerminatingArabidopsis Seedlings

Arabidopsis seedlings germinated on IAN displayed severaleffects suggestive of but distinct from those caused by ex-ogenous IAA. The most dramatic alteration was seen at thebase of the hypocotyl, where the connection between thestele and the cortical and epidermal cells disintegrated andadventitious lateral root primordia developed on the hypo-cotyl. In addition, IAN inhibited root elongation and causedepinastic cotyledons, as shown in Figure 1B. This inductionof lateral root initiation and suppression of root elongationwas also observed with IAA (Figure 1C), although the magni-tude of the effects was different. In particular, exogenousIAN was more effective in producing auxin effects in theshoot, whereas roots were more sensitive to exogenousIAA. These differences might be due to differences in thetransport or metabolism of the two molecules.

Consistent with the possibility that IAN is acting as anauxin in Arabidopsis, IAN effects were decreased in severalmutants isolated on the basis of their resistance to variousnatural and synthetic auxins. In particular, the auxl (Pickettet al., 1990), axrl (Lincoln et al., 1990), and axr2 (Wilson etal., 1990) mutants were resistant to IAN (Figure 1B and datanot shown). The axrl mutant was resistant to both the hypo-cotyl disintegration and the inhibition of root elongationcaused by IAN (Figure 1B). However, the auxl mutant wasresistant to the root elongation inhibition but remained sen-sitive to the lAN-induced hypocotyl disintegration (Figure1B). This IAN sensitivity in the auxl shoot is consistent withother phenotypes of the auxl mutant, which is disrupted inauxin perception only in the root (Pickett et al., 1990),whereas the axrl mutation has a pleiotropic phenotype withdefects in both root and shoot tissues (Lincoln et al., 1990).In contrast, the ilrl mutant, which is resistant to certain IAA-amino acid conjugates but sensitive to free IAA (Bartel andFink, 1995), was no different from the wild type in its sensi-tivity to IAN (data not shown). Each of the mutants showed

Col-0 nit1-3 aux1-7 axr1-3Figure 1. IAN Effects on the Wild Type and nitl and Auxin-ResistantMutants.

Eight-day-old seedlings grown under yellow-filtered light on variousmedia were removed from the agar and photographed under thesame magnification. The nit1-3 mutants shown are homozygous F3

plants from the second backcross to ecotype Columbia (Col-0).(A) Seedlings grown on PNS medium.(B) Seedlings grown on PNS supplemented with 30 |o.M IAN.(C) Seedlings grown on PNS supplemented with 1 H.M IAA.Bar in (C) = 5 mm for (A) to (C).

normal root development in the absence of added IAA orIAN (Figure 1A).

The auxin effects of IAN may result from nitrilase-medi-ated hydrolysis, and the location of these effects might re-flect the tissue-specific expression of the various isozymes.Arabidopsis contains four nitrilase genes (Bartling et al.,1992, 1994; Bartel and Fink, 1994) that are differentially ex-pressed during development and in response to environ-mental stimuli (Bartel and Fink, 1994). Transgenic plantscarrying a promoter-reporter gene fusion show that the NIT1gene is strongly expressed at the base of the hypocotyl(Bartel and Fink, 1994). This region is particularly sensitive tothe effects of exogenous IAN (Figure 1B). The coincidentsensitivity to IAN and the expression pattern of NIT1 sug-gest that sensitivity to IAN results from hydrolysis to IAA bythe NIT1 enzyme.

Arabidopsis Nitrilase Mutants 1783

Mutants lnsensitive to IAN Map to the NITl Locus

To isolate mutants with disrupted nitrilase function, we screened for mutants resistant to the auxin effects of IAN but that retain IAA sensitivity. We isolated three such mutants from the progeny of seeds that had been mutagenized with ethyl methanesulfonate (see Methods). These mutants were recessive to the wild type and fel1 into a single complementation group, which we subsequently determined was disrupted in the NITl locus (see below). When germinated on concentrations of IAN ranging from 10 to 80 )LM, each of the three nit7 mutant alleles showed increased root elongation and increased hypocotyl integrity compared with that of the wild type (Figure 1 B and data not shown). Quantification of this phenotype is shown in Figure 2. After 8 days on 30 )LM IAN (a concentration that inhibits wild-type root elongation by >80%), the nit7 mutant showed three- to fourfold increased root elongation compared with that of the wild type (Figures 1B and 2). In contrast, nit7 root elongation did not differ from that of the wild type when germinated on plates containing 0.6 to 3 pM IAA (Figures 1C and 2 and data not shown), showing that IAA sensitivity was not altered in the mutant.

1 O0

80

C .o 60 m o) C O a,

c

- c

o 40 2 8

20

COLO nitl-1

c] nitl-2 nitl-3 auxl-7 axrl-3 T T

a 30 pM IAN 1 pM IAA

Figure 2. Root Elongation of nit l , auxl, ax r l , and Wild-Type Seed- lings on IAN and IAA.

Eight-day-old seedlings grown as given in the legend to Figure 1 were removed from the agar, and the length of the longest root was recorded. At least nine seedlinas of each aenotvDe were measured - - ,, for each condition. Shown is the percentage of elongation for either IAN or IAA compared with that of the PNS control. Error bars indicate the standard deviation of the mean.

We mapped the nitl mutant (see Methods) to the region of chromosome 3 that contains a cluster of three nitrilase genes, NlT7, NITZ, and NIT3, within 12 kb of each other (Bartel and Fink, 1994). This mapping data, along with the colocal- ization of IAN effects and NlTl expression discussed previously, suggested that the IAN-resistant mutants might be disrupted in the NIT7 gene. To test this directly, we sequenced the NITl gene from each of the nitl mutant alleles and compared these sequences with that of the wild-type (ecotype Columbia [Col-O]) sequence (Zhou et al., 1996a). Each mutant allele had a single base substitution that changed an amino acid (nit7-7 and nitl-2) or prematurely terminated the NIT1 polypeptide (nit7-3). As shown in Figure 3A, all three of the nitl mutant alleles were located in the fourth exon. The changes in the nitl-7 and nitl-2 alleles both substitute charged residues (aspartate or arginine) for glycine residues that are invariant not only in all four Arabidopsis genes (Fig- ure 38) but also in a similar gene from Chinese cabbage (Bischoff et al., 1995), two related tobacco genes (Tsunoda and Yamaguchi, 1995), and similar genes from the microbes A. faecalis JM3 (Kobayashi et al., 1993), Rhodococcus rhodochrous J1 (Kobayashi et al., 1992b), and R. rhodochrous K22 (Kobayashi et al., 1992a).

Overexpression of NIT Genes in Arabidopsis

Each of the four Arabidopsis nitrilase enzymes is capable of hydrolyzing IAN to IAA when expressed in E. coli (Bartling et al., 1992, 1994; Bartel and Fink, 1994), suggesting a role for nitrilases in plant IAA biosynthesis. The isolation of IAN- resistant nitl mutants described here demonstrates that the NIT1 enzyme is capable of performing this hydrolysis in vivo as well. To test whether any of the other Arabidopsis nitrilase isozymes can hydrolyze IAN in vivo, we made transgenic plants expressing the NIT7, NITZ, NIT3, and NIT4 cDNAs from the highly expressed cauliflower mosaic virus 35s promoter. RNA gel blot analysis with gene-specific probes was used to select the lines showing the greatest increase in NIT message. The results are shown in Figure 4. These overexpressing lines were tested for their sensitivity to IAN and IAA. Figure 5 shows that overexpression of the NITZ cDNA from the 35s promoter dramatically increased sensitivity to IAN compared with the wild type, whereas all of the lines retained similar sensitivity to IAA. This hypersensitivity suggests that NIT2, like NIT1, is capable of hydrolyzing IAN in vivo.

We also determined whether the various 35s-NIT constructs could suppress the IAN-resistant phenotype of the nitl mutant. We crossed the transgenic lines expressing the NIT cDNAs from the 35s promoter (Figures 4 and 5) to the nitl-7 mutant, and we plated the F, or F4 plants homozygous for the nit7-7 mutation and the transgene (see Methods) on plates containing IAN. The homozygous nitl mutant overexpressing NIT2 from the 35s promoter was as sensitive to IAN (data not shown) as the 35s-NIT2 construct in a

1784 The Plant Cell

TAG

B

——VTPEAHEFTHV ——— IGKATQDSV———VTQETLDML-EVGEHNASLLK

NIT1 (X.t.NIT2 (>f.t.NIT (B.r.NIT3 Ol.t.NIT4 (xl.t.NIT4A (A/, tNIT4B (A/, t(R.r. Jl)(K. r. K22)Ol.f. JM3)

Figure 3. Mutations in the NIT1 Gene in Three Mutant Alleles.

(A) Genomic structure of the NIT1 locus. Introns are indicated with thin lines between black rectangles (exons). The position of the active sitecysteine is indicated with an asterisk.(B) Alignment of the region encoded by exon 4 in the NIT1 gene, with corresponding regions from other plant and microbial nitrilase genes show-ing the positions of the mutations found in each of the three nitl mutant alleles. The nit1-1 mutation changes a GGT (Gly-228) codon to GAT (D,Asp), creating an Mboll restriction site; the nit1-2 mutation changes a GCA (Gly-277) codon to AGA (R, Arg); and the nit1-3 mutation changes aTGG (Trp-255) codon to TAG (stop). Sequences were aligned with the Megalign program (DNAStar, Madison, Wl), using the Clustal method.Dashes indicate gaps introduced to maximize alignment, and residues identical to the NIT1 sequence are boxed. Sequences shown are NIT1,NIT2, NITS, and NIT4from A thaliana (Af.), a nitrilase from Brassica rapa (B.r.) (Bischoff et al., 1995), NIT4A (Tsunoda and Yamaguchi, 1995) andNIT4B from Nicotians tabacum (N.t), nitrilases from R. rhodochrous (R.r.) strains J1 (Kobayashi et al., 1992b) and K22 (Kobayashi et al., 1992a),and a nitrilase from A. faecalis (A.f.) JM3 (Kobayashi et al., 1993).

wild-type background (Figure 5 and data not shown), indi-cating that NIT2 overexpression is able to suppress the nitlmutation fully.

NIT1 NIT2 NIT3 NIT4

Figure 4. RNA Gel Blot Analysis of NIT Genes in Transgenic Plants.

Eight micrograms of total RNA prepared from 19-day-old seedlingswas separated on a 1% agarose gel, transferred to a nylon mem-brane, and hybridized with gene-specific probes NIT1 to NIT4 de-rived from untranslated regions of the NIT transcripts, as describedpreviously (Bartel and Fink, 1994). Equal loading was confirmed byobservation of ethidium bromide-stained rRNAs in each lane. No-0,Nossen; 35SNIT, 35S-A//7.

Steady State IAN and IAA Levels in nitl and35S-A//T2 Plants

Given the IAN resistance of the nitl mutants (Figure 2) andthe increased IAN sensitivity of the 35S-A//T2 line (Figure 5),we quantified free IAA, total IAA (free IAA plus amide- andester-linked conjugates of IAA), and IAN by isotope dilutiongas chromatography-selective ion monitoring-mass spec-trometry (GC-SIM-MS) analysis (Normanly et al., 1993; Ilic etal., 1996) in these lines. The nit1-3 allele was selected forthis analysis because it showed the most dramatic IAN re-sistance of the three alleles (Figure 2) and because the le-sion in this strain was a premature stop codon (Figure 3B).As shown in Table 1, steady state levels of free IAA, totalIAA, and IAN did not differ from that of the wild type in thenit1-3 mutant. Similarly, the steady state levels of thesecompounds in 7-, 8-, 13-, or 14-day-old transgenic seed-lings overexpressing the various NIT genes from the 35S


100-

80-

promoter did not differ dramatically from those in control lines (Table 1 and data not shown).

IAN Metabolism in nitf and 35S-NIT2 Plants

To explore further IAN metabolism in these lines, we wished to follow the fate of labeled IAN, determining its half-life and conversion to IAA. Severa1 features of the experimental de- sign are critical for valid measurements. Exogenous I3C1- IAN must be applied in amounts that label the IAN pool but only minimally alter the steady state leve1 of IAN. In addition, steady state IAN levels must remain constant over the time period in which the half-life measurements are made.

We found that a 1-hr pulse of 60 p M I3C1-IAN 'adminis- tered to 7.5-day-old wild-type seedlings resulted in 30 to 50% labeling of the IAN pool. To determine whether 60 pM IAN perturbed endogenous IAN metabolism, we conducted mock pulse-labeling experiments in which 7.5-day-old seedlings were manipulated in the same manner as in the pulse-labeling experiments, except that either no IAN was present (mock pulse) or 60 p M unlabeled IAN was added (cold pulse). In duplicate experiments, total IAN levels varied by <12% from 1 to 3 hr after the mock pulse. Adding 60 p M IAN during the cold pulse increased IAN levels twofold. This doubling is expected for 50% labeling; therefore, we con- cluded that exposing the seedlings to 60 p M IAN did not drastically alter endogenous IAN metabolism. (Similar at- tempts to establish labeling conditions in Arabidopsis for the measurement of IAA turnover that do not substantially per- turb IAA metabolism have been unsuccessful [J. Normanly, unpublished data].)

Lastly, improper storage of IAN can result in its nonenzymatic conversion to IAA. Because endogenous IAN levels are at least two orders of magnitude higher than free IAA levels are (Normanly et al., 1993; Table l), any i3C1-IAA con- tamination of the 13C1-IAN used would artificially elevate the observed incorporation of I3C1 label into IAA. We used HPLC to determine that our 13C1-IAN contained <0.07% 13C1-IAA. This would result in at most 42 nM 13C1-IAA being present during a pulse with 60 p M I3C1-IAN. In a separate experiment performed in quadruplicate, we determined that a 1-hr pulse with 42 nM I3C6-IAA resulted in 7% labeling of

W NO-O EA 35SNIT1 Q 35SNIT2 T 1, H 35SNIT3

.. 10 pM IAN 0.2 FM IAA

Figure 5. Root Elongation of NIT Overexpressing and Wild-Type Seedlings on IAN and IAA.

Root elongation of 8-day-old seedlings was quantified as given in the legend to Figure 2, except that 10 p,M IAN was used to allow de- tection of increased sensitivity.

the free IAA pool. Therefore, 7% was determined to be the maximum background in our labeling experiments described below.

We then directly tested the ability of the nitl mutant and the 35S-NIT2 line to metabolize IAN. Seedlings were pulse- labeled with I3C1-IAN, and the incorporation of label from i3Ci-IAN into IAA was determined by GC-SIM-MS. In addition, we determined the rate at which IAN was metabolized (IAN half-life) in these lines. Adding an interna1 standard (WG-IAA) upon extraction enabled the determination of free IAA levels.

As shown in Table 2, all of the tested lines hydrolyzed exogenous I3C1-IAN to I3C1-IAA, directly demonstrating that Arabidopsis can hydrolyze IAN in vivo. The half-life of IAN in the 35s-NIT2 line was twofold shorter than was the untransformed control, and the levels of free IAA (13Cl-labeled plus unlabeled) increased almost fourfold by 1 hr after the i3C1-IAN

Table 1. IAN and IAA Levels in 7-Day-Old Wild-Type, nitl-3, and Transgenic 35S-NITZ Seedlingsa

Plants IAN Free IAA Total IAA

Wild type (Gol-O) 637 ? 108 7 + 1 1542 ? 150 nitl-3 (Gol-O) 579 2 20 12 t 1 1360 t 10 Wild type (No-O) 467 ?10 5 0.3 1455 ? 23 35s-NIT2 (NO-O) 845 t 101 8 2 1 2651 ? 146

aValues are given in nanograms per gram fresh weight of tissue, presented as the mean ?SE from three independent samples, except for free IAA in 35s-NIT2 and total IAA in Gol-O, in which case the sample sizes were two and the values are presented as the mean plus or minus the average deviation from the mean.

1786 The Plant Cell

Table 2. IAN Half-Life and lncorporation of Label from l3C1-IAN into IAA in 7.5-Day-01d Wild-Type, nitl-3, and Transgenic 35s-NIT2 Seedlingsa

Free IAA (nglg FW)

Unlabeled 13C1 Labeled l3G1-IAN IAN Free Half-Life 1 3 c 1 - l ~ (%)”

Plants t l t2 (hr) t l t l t2 t l t2 ~ ~

Wild type (COLO) 28 2 2 9 2 2 1.5 2 0.5 49 ? 4 8 2 1 9 2 3 7 2 0.3 3 2 0.3 nitl-3 (Gol-O) 23 2 1 10 2 2 1.7 2 0.3 4 ? 1 91243 7 2 2 3 2 2 1 2 0.5 Wild type (No-O) 26 2 8 20 2 8 3.5 2 1 24 ? 4 6 2 1 10 2 3 2 ir 0.3 1 2 0.3 35s-NK’ (NO-O) 32 2 0.4 14 * 0.8 1.8 2 0.2 86 ? 4 4 2 1 6 2 2 2 6 2 2 13 2 1

aAnalysis was performed in triplicate for COLO and nitl-3 (data presented as the mean ?SE) and in duplicate for No-O and 35s-NIT2 (data presented as the mean plus or minus average deviation). For Col-O and nitl-3: t l , 1 hr; t2, 3 hr; for No-O and 35s-NITZ: t l , 1 hr; t2, 2.5 hr. bThe percentage of l3C,-IAN was determined for each sample by dividing the abundance of ions with an mlz of 229 (corrected for natural abundance of carbon-13 and nitrogen-15, as described in Methods) by the sum of abundances for ions with an mlz of 228 and an mlz of 229. =The percentage of free I3C1-IAA was determined for each sample by dividing the nanograms per gram fresh weight of tissue (nglg FW) value for 13C,-IAA at t l by the sum of nanoqram per qram values for 13C,-IAA and unlabeied IAA.

pulse (Table 2). This alteration in IAN metabolism is consistent with the observed increase in sensitivity to IAN in the 35s-NIT2 line (Figure 5).

In contrast, the half-life of IAN in the n i t l -3 mutant did not differ significantly from that of the wild type (Table 2). The levels of i3Ci-IAA formed in the nit l-3 mutant during the pulse- labeling experiments were also similar to those of the wild type, possibly because of compensatory activity of other nitrilases. However, in two of three nitl-3 samples, the levels of endogenous unlabeled IAA were radically altered (10- to 27-fold above normal) by the i3Ci-IAN pulse but only briefly, indicating that in this mutant, IAA metabolism is altered in response to exogenous IAN.

DISCUSSION

It is likely that severa1 pathways for IAA biosynthesis coexist within a given plant, as has been demonstrated for carrot (Michalczuk et al., 1992), maize (Wright et al., 1991; Koshiba et al., 1995), and Arabidopsis (Normanly et al., 1993; J. Normanly, unpublished data), all of which show both tryptophan-dependent and tryptophan-independent pathways of IAA biosynthesis. The situation might be further compli- cated by the existence of multiple genes encoding isozymes of IAA biosynthetic enzymes, as is the case for numerous biosynthetic pathways in Arabidopsis (Lam et al., 1995; Radwanski and Last, 1995). Although this potential redundancy at the level of both pathways and isozymes compli- cates biochemical analysis, it may provide plants with the precise control necessary for IAA homeostasis.

The nature of the molecular defect of the IAN-resistant nitl mutants described here implies that IAN exerts its auxinlike effects through its hydrolysis to IAA and that this hydrolysis can be performed in vivo by the product of the NITl gene. The increased sensitivity to IAN of plants overex-

pressing the NIT2 gene and the observation that NIT2 overexpression can suppress the nitl defect suggest that the NIT2 enzyme is also capable of hydrolyzing IAN to IAA in vivo. These results are consistent with the observation that ectopic expression of the Arabidopsis NIT2 gene in transgenic tobacco results in plants with increased sensitivity to exogenous IAN (Schmidt et al., 1996).

The observation that the 35s-NITl, 35s-NIT3, and 35s- NIT4 lines did not display increased sensitivity to IAN (Figure 5) or suppress the nitl mutant phenotype on IAN (data not shown) despite an increase in message accumulation (Figure 4) may indicate that these lines are not overexpressing pro- tein because of post-transcriptional regulation. It is also possible that the NIT3 and NIT4 enzymes, which can hydrolyze IAN to IAA in vitro (Bartel and Fink, 1994), do not do so in vivo.

The data from i3Ci-IAN labeling studies directly demon- strate that Arabidopsis seedlings can hydrolyze IAN to IAA in vivo. In all tested lines, i3C-labeled IAN was hydrolyzed to i3C-labeled IAA (Table 2). The half-life of IAN was two times shorter and the percentage of incorporation of I3C from IAN into IAA was approximately fourfold higher in the 35s-NIT2 line than in the untransformed control. Thus, when the NIT2 enzyme was overexpressed in vivo, it hydrolyzed l3C1-IAN to IAA. These results agree with the observation that ectopic expression of the Arabidopsis NIT2 gene confers on transgenic tobacco the ability to hydrolyze exogenous 13C-IAN to I3C-IAA (Schmidt et al., 1996).

Although nitrilase activity in extracts from the transgenic 35s-NIT2 line was elevated (Jutta Ludwig-Müller, personal communication) and the level of i3C-labeled free IAA recov- ered after a 13C-IAN pulse was increased in the 35s-NIT2 line compared with that of the wild type (Table 2), steady state levels of IAN and unlabeled free IAA were not dramatically altered in this line (Tables 1 and 2). A possible explana- tion, supported by the data from pulse-labeling studies, is that endogenous IAN may be limiting or not accessible to


the overproduced nitrilase. In the 35s-NIT2 line after a 1 -hr pulse with 13C-IAN and a 1-hr chase, the total cellular IAN pool was 32% labeled with carbon-13, whereas the IAA pool was 86% carbon-13 labeled. If endogenous IAN and I3C- IAN were both readily utilized by the overproduced nitrilase, then incorporation of label into IAA should be the same or less than incorporation of label into IAN. The most likely ex- planation for a precursor (IAN) being less enriched than the product (IAA) would entail separate pools of IAN, with the labeled pool being preferentially used as a precursor.

If compartmentation of IAN plays a role in regulation of IAA biosynthesis, then overproduction of NIT2 may not result in increased hydrolysis of endogenous, unlabeled IAN. Biochemical fractionation indicates that NlTl is a soluble enzyme and that NIT2 is membrane associated (Bartling et al., 1994); however, the subcellular localization of IAN is not known, and we have not localized the overexpressed NIT2 enzyme. IAA has been localized both to the cytosol and to chloroplasts (Sitbon et al., 1993), and the tryptophan biosynthetic enzymes (the source of IAA precursors) have been localized to the chloroplast (Zhao and Last, 1995). Determi- nation of the subcellular location of IAN would be helpful in assessing the degree to which IAN participates in IAA biosynthesis under normal conditions.

Steady state levels of IAA and IAN, the half-life of IAN, and incorporation of carbon-13 from IAN to IAA were not dramatically altered in the nit7-3 mutant (Tables 1 and 2). Given the redundancy of nitrilases in Arabidopsis, it is possible that IAA levels and IAN metabolism are maintained at wild- type levels by the remaining functional nitrilases and other IAA biosynthetic pathways. In addition, subtle differences in IAN or IAA metabolism between the wild-type and nitl mutant strains may be masked by the necessity of performing biochemical analysis on entire seedlings, whereas a mutant phenotype can reflect local or tissue-specific differences in metabolism. In two of three nitl-3 samples, the endogenous, unlabeled IAA levels were significantly higher (10- to 27-fold) than those of the wild type after a pulse with 13C- IAN, returning to normal levels within 2 hr (Table 2). These observations suggest that IAA metabolism in response to exogenous IAN is altered in the mutant, perhaps contribut- ing to its IAN resistance. IAA levels appeared normal in one of the nit7-3 samples, which we attribute to inadvertent vari- ations in environmental parameters.

Our results provide another example in which redundancy at the leve1 of functionally similar genes does not preclude the isolation of conventional mutants. This was seen previously in the case of tryptophan biosynthetic genes, in which recessive mutants in genes encoding biosynthetic enzymes were isolated by selecting for resistance to toxic intermedi- ates (Last and Fink, 1988; Last et al., 1991; Radwanski et al., 1996). As expected, when the targets were members of gene families, these mutations tended to occur in the genes encoding the most highly expressed isozymes (Last et al., 1991; Niyogi et al., 1993). It is likely that the strong expression of the N lT l gene at the base of the hypocotyl and the

relative absence of expression of the other NIT genes in this tissue (Bartel and Fink, 1994) account for our ability to select for mutations in this gene. However, the observations that the nitl mutants do not have dramatic morphological phenotypes in the absence of IAN and are not severely disrupted in IAN metabolism suggest that if nitrilases are involved in IAA biosynthesis in Arabidopsis, at least one of the three NIT genes that remain active in the nitl mutant is able to compensate for its function in supplying IAA to the plant. Alternatively, other pathways of IAA biosynthesis might be induced in the absence of a functional N lT l gene. Ongoing efforts to identify mutations in the other NIT genes should resolve these possibilities.

METHODS

Arabidopsis thaliana Strains and Growth Conditions

Arabidopsis ecotypes Columbia (Col-O), Landsberg erecta (Ler), and Nossen (No-O) were grown in soil (Metromix 200; Scotts-Sierra Horticultural Products, Marysville, OH) under continuous illumination at 22 to 25°C. Plants were grown asceptically on plant nutrient medium with 0.5% sucrose solidified with 0.6% agar (PNS; Haughn and Somerville, 1986) or PNS supplemented with 10 to 80 IJ.M indole- 3-acetonitrile (IAN; from 100 mM stock in ethanol), 0.5 to 3 pM indole-3-acetic acid (IAA; from 10 mM stock in ethanol), or 20 pg/mL kanamycin (from 25 mg/mL stock in water). Plates were wrapped in gas-permeable surgical tape (3M, St. Paul, MN) and grown under continuous illumination (25 to 45 p E m-2 sec-l) with yellow long-pass filters to reduce the breakdown of indolic compounds (Stasinopoulos and Hangarter, 1990). Plants used for analysis of IAA and IAN were COLO (wild type) or homozygous nitl-3 mutant plants that had been backcrossed to Col-O twice. Transgenic plants were the T4 or T5 generation. Plants were grown asceptically as above in the absence of IAN or IAA in 150 X 25 mm Petri plates (Falcon 1013; Becton Dickinson Labware, Lincoln Park, NJ) at a density of 1000 seeds per plate under continuous illumination (100 IJ.E m-2 sec-l). Seedlings were harvested at varying times after germination, weighed, frozen in liquid nitrogen, and stored at -80°C.

For IAN turnover studies, seeds were surface sterilized (Last and Fink, 1988), resuspended in molten PNS made with 0.55% agar (ul- trapure; U.S. Biochemical), and sown on sterile nylon mesh discs (catalog No. 3-500-49; Tetko, Rochester, NY) that had been placed atop the same medium in 150 X 25 mm Petri plates. Plates were wrapped as given above and incubated at room temperature under continuous illumination (100 pE m2 sec-l). After 7.5 days, the seedlings were transferred to Petri plates containing 25 mL of sterile liquid PN medium (PNS lacking ferric EDTA and sucrose) and 60 pM of '3C1-IAN (Ilic et al., 1996). Transfer was accomplished by gently lifi- ing the nylon mesh discs from the agar by using sterile forceps and taking care to remove any clumps of solidified media that clung to the roots. Seedlings were incubated under continuous illumination (25 to 45 p E m-2 sec-l) with yellow long-pass filters for 1 hr. The seedlings were then washed extensively with sterile water to remove excess label and transferred to fresh liquid PN medium lacking both l3C,-IAN and ferric EDTA and incubated for 2.5 to 3 hr. Samples were harvested at 1 and 2.5 or 3 hr after transfer to fresh medium by gently pulling the seedlings from the nylon mesh with forceps, blotting

1788 The Plant Cell

excess moisture with Kimwipes, weighing, and freezing in liquid nitrogen. Samples were stored at -80°C.

Mutant Screen

The nitl mutant alleles were isolated as follows. Col-O seeds (1 0,000) were mutagenized with 0.24% (v/v) ethyl methanesulfonate for 16 hr at room temperature. M1 seeds were washed extensively with water and sown in 16 pools of -625 seeds and allowed to self-fertilize. Of the resulting M, seeds, 40,000 were surface sterilized (Last and Fink, 1988) and spread on 150 x 25 mm Petri plates containing 1 O0 mL of PNS supplemented with 80 pM IAN at a density of -500 seeds per plate. After 2 weeks, putative mutants with increased root length and hypocotyl integrity were transferred to soil and allowed to set seeds. The resultant M, seeds were screened for resistance to 50 pM IAN or 3 pM IAA. Four mutants from three pools were identified that were resistant to IAN and sensitive to IAA. These four mutants failed to complement one another and represented three independent nitl alleles (nitl-2 to nitl-3) based on sequence analysis.

Generation of Transgenic Plants

Full-length cDNAs encoding each of the nitrilase cDNAs were cloned downstream of the cauliflower mosaic virus 35s promoter in the vector pBlCaMV (a vector derived from pB1121 [Jefferson et al., 19871 in which the gene encoding 0-glucuronidase was replaced by a multiple cloning site [J. Celenza, personal communication]). These constructs were introduced into Agrobacterium tumefaciens LBA4404 by electroporation. The resultant strains were used to construct transgenic plants (ecotype No-O) by using the method of root transformation, as previously described (Valvekens et al., 1988). Seeds from regenerated plants were plated on PNS supplemented with 20 kg/mL kanamycin, and homozygous lines were identified in sub- sequent generations. RNA was prepared from homozygous plants, and relative levels of nitrilase gene expression were compared using RNA gel blot analysis with gene-specific probes (Bartel and Fink, 1994). One line from each of the four overexpressing constructs showing the highest increase in mRNA abundance for the gene of in- terest was selected for additional experiments.

Genetic Analysis

nitl plants were backcrossed to the Col-O ecotype for phenotypic analysis and outcrossed to the Ler ecotype for genetic mapping. DNA was prepared (Celenza et al., 1995) from nitl F, plants and analyzed by polymerase chain reaction (PCR) with primers that amplify the NlTI-NIT2 intragenic region on chromosome 3 (Bartel and Fink, 1994). These primers detect a length polymorphism between the Col-O and Ler ecotypes. Two nitl mutant alleles were analyzed and showed 100% linkage (38 plants fornitl-7 and 66 plants fornitl-2) to the Col-O ecotype in this analysis, corresponding to a map distance of < O 5 centimorgans.

The nitl-7 mutant was crossed to transgenic plants carrying the NlTl, NIT2, NlT3, or NIT4 cDNAs driven by the cauliflower mosaic virus 35s promoter. F, plants carrying the transgene were selected on plates containing 20 pg/mL kanamycin, and the genotype of these plants at the NlTl locus was determined by cleavage of a NlTl PCR product with the enzyme Mboll. A new Mboll site was generated by the G --t A base change of the nitl-7 allele. To avoid DNA from the

transgene in this analysis, DNA encompassing exon 3, intron 3, and exon 4 was amplified using primers designed from intron 2 (Nl-14: 5'-CTAATTAATGAGTGTTCTTGCTCTAG-3') and intron 4 (Nl -20: 5'-GATGACTATCAAATGAGAGTGT-3'). PCR conditions were 30 cycles of 30 sec at 940C, 30 sec at 55"C, and 1 min at 72°C. Kanamycin- resistant plants homozygous for the nitl-7 inutation were allowed to self-fertilize and set seeds. F3 or F4 families that gave 100% kanamycin-resistant seedlings (indicating that the transgene locus was homozygous) were plated on 50 pM IAN to assay suppression of the IAN-resistant phenotype.

Sequencing of Mutant NlTl Alleles

For sequencing the nitl mutant alleles, the NITl gene was amplified by PCR (using the primers 5'-GTCTATATATTGCTTCTCTCAAGTCTC-3' and 5'-CTTGACTCCATAGATCTTACCAlTGAAG-3') from genomic DNA prepared from each mutant allele. PCR conditions were 40 cycles of 30 sec at 9YC, 30 sec at 56"C, and 3 min at 72°C. The resultant 2.1 -kb PCR products were either sequenced directly (Ausubel et al., 1993) or cloned using the pT7Blue T-vector kit (Novagen, Madison, WI). DNA sequencing was with custom oligonu- cleotide-primed reactions analyzed on an Applied Biosystems (Foster City, CA) automated DNA sequencer by D. Needleman (Uni- versity of Texas-Houston Medical School Molecular Genetics Core Facility) using dye terminators or by dideoxy sequencing using Se- quenase DNA polymerase, as described by the manufacturer (U.S. Biochemical). Changes found in the mutant alleles were verified by sequencing from an independent PCR reaction.

Analysis of IAA and IAN

Up to 2 g of frozen tissue was ground in a mortar that had been chilled with liquid nitrogen. Glass beads (1 50 to 212 pm; Sigma) were added to aid tissue disruption. The frozen powder was added to cold (-10 to -20°C) extraction buffer (35% 0.2 M imidazole, pH 7.0, 65% isopropanol) to which 50 to 400 ng of l3C,-IAA (Cambridge lsotope Laboratories, Andover, MA) and up to 400 ng of 13C1-IAN (Ilic et al., 1996) had been added as interna1 standards. Samples were equili- brated at -20°C for 1 hr and then boiled for 20 min under a stream of nitrogen to destroy myrosinase activity. Samples were centrifuged briefly in a tabletop centrifuge to pellet tissue debris. The supernatant was removed to a new tube, and the pellet was washed with extraction buffer and centrifuged again. This process was repeated three times, the supernatants were pooled, and isopropanol was removed by rotary evaporation. Approximately 100,000 cpm of 3H-IAA (25.4 Ci/mmol; Amersham, Arlington Heights, IL) was added as a ra- diotracer. IAN, total IAA, and free IAA were isolated as described previously (Normanly et al., 1993), except that larger capacity (1 g; J & W Scientific, Folsom, CA) amino columns were used. For analysis of samples from 13C1-IAN pulse label experiments, only IAN and free IAA were isolated, and no additional 13C,-IAN was added upon extraction.

Gas chromatography-selective ion monitoring-mass spectrome- tty (GC-SIM-MS) was conducted on a Hewlett Packard (Rockville, MD) mode15890 GC coupled to a Hewlett Packard mode15972 MS. For IAA analysis, either a 15-m DB1701 or 30-m DB5MS column (J & W Scientific) was used. The initial temperature was 14o"C, ramped at 5°C per min with a helium flow rate of (1 mUmin to a final temperature of 280°C. IAN analysis was performed on either a 30-m DB5MS or 15-m DB5 GC column (J & W Scientific). The initial temperature was 140"C, ramped at 20°C per min with a helium flow rate of <1


mLlmin to a final temperature of 280°C. Each sample was analyzed two or three times by GC-MS. For determination of total IAA levels, molecular ions with an mlz of 189, 190, and 195 were monitored. For quantification of IAN, the molecular ions with an mlz of 228 and 229 were monitored. Corrections for the natural abundance of carbon-13 and nitrogen-15 were calculated based on empirical values (Ilic et al., 1996), and the correction factor for nonenzymatic conversion of IAN to IAA was calculated as described previously (Ilic et al., 1996). For free IAA quantification, quinolinium and molecular ions with an mlz of 130, 136, 189, and 195 were monitored. For free IAA samples resulting from 13C1-IAN pulse labeling experiments, molecular ions with an m/z of 189, 190, and 195 were monitored; for IAN samples, the molecular ions monitored were the same as for IAN quantification.

IAN half-life values were calculated as described previously (Zilversmit et al., 1943), according to the following formula: f1,2 = ln2/k, where k , the first-order rate constant, is In CJC, x l/f. C, is the percentage of I3C1 enrichment of IAN at the first time point (1 hr), Ct is the I3C1 enrichment of IAN at the second time point (2.5 or 3 hr), and t is the time elapsed in hours between C, and C,. The percentage carbon- 13 enrichment for IAN was calculated by dividing the value formlz 228 by the sum of the values for mlz 228 and m/z 229 after correcting for the natural abundance of carbon-13 and nitrogen-15. The percentage of carbon-13 enrichment of IAA was determined by dividing the value for 13C1-IAA (nanograms per gram fresh weight) by the sum of the values for 13C1-IAA and unlabeled IAA (nanograms per gram fresh weight).

ACKNOWLEDGMENTS

We gratefully acknowledge Mark Estelle for aux7 and ar7 seeds, John Celenza for pBICaMV, the Department of Chemistry at Amherst College for use of the gas chromatograph-mass spectrometer, and Seiichi Matsuda for critical comments on the manuscript. This research was supported by grants from the Department of Energy (No. DE-FG02- 95ER20204 to J.N.), the National Science Foundation (to G.R.F.), and the Texas Advanced Technology Program (No. 003604-065 to €3.6.).

Received June 2,1997; accepted August 18,1997.

REFERENCES

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., eds (1993). Current Protocols in Molecular Biology. (New York: Greene Publishing Associates and Wiley-lnterscience).

Bartel, B. (1997). Auxin biosynthesis. Annu. Rev. Plant Physiol. Plant MOI. Biol. 48, 51-66.

Bartel, B., and Fink, G.R. (1994). Differential regulation of an auxin- producing nitrilase gene family in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 91,6649-6653.

Bartel, B., and Fink, G.R. (1995). ILRI, an amidohydrolase that releases active indole-3-acetic acid from conjugates. Science

Bartling, D., Seedorf, M., Mithofer, A., and Weiler, E.W. (1992). Cloning and expression of an Arabidopsis nitrilase which can con-

268,1745-1 748.

vert indole-3-acetonitrile to the plant hormone, indole-3-acetic , acid. Eur. J. Biochem. 205, 417-424.

Bartling, D., Seedorf, M., Schmidt, R.C., and Weiler, E.W. (1994). Molecular characterization of two cloned nitrilases from Arabidop- sis thaliana: Key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid. Proc. Natl. Acad. Sci. USA 91, 6021-6025.

Bischoff, M., Low, R., Grsic, S., Rausch, T., Hilgenberg, W., and Ludwig-Müller, J. (1 995). lnfection with the obligate biotroph Plasmodiopbora brassicae, the causal agent of the club root dis- ease, does not affect expression of NITll2-related nitrilases in roots of Chinese cabbage. J. Plant Physiol. 147, 341-345.

Blonstein, A.D., Vahala, T., Koornneef, M., and King, P.J. (1988). Plants regenerated from auxin-auxotrophic variants are inviable. MOI. Gen. Genet. 215, 58-64.

Celenza, J.L., Grisafi, P.L., and Fink, G.R. (1995). A pathway for lateral root formation in Arabidopsis thaliana. Genes Dev. 9,

Fracheboud, Y., and King, P.J. (1 991). An auxin-auxotrophic mutant of Nicotiana plumbaginifolia. MOI. Gen. Genet. 227, 397-400.

Haughn, G.W., and Somerville, C. (1 986). Sulfonylurea-resistant mutants of Arabidopsis thaliana. MOI. Gen. Genet. 204, 430-434.

Henbest, H.B., Jones, E.R.H., and Smith, G.F. (1953). lsolation of a new plant-growth hormone, 3-indolylacetonitrile. J. Chem. SOC.,

Hillebrand, H., Tiemann, B., Hell, R., Bartling, D., and Weiler, E.W. (1 996). Structure of the gene encoding nitrilase 1 from Arabi- dopsis thaliana. Gene 170, 197-200.

Ilic, N., Normanly, J., and Cohen, J.D. (1996). Quantification of free plus conjugated indoleacetic acid in Arabidopsis requires correction for the nonenzymatic conversion of indolic nitriles. Plant Physiol. 111, 781-788.

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: p-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907.

Jones, E.W., and Fink, G.R. (1982). Regulation of amino acid and nucleotide biosynthesis in yeast. In The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, J.N. Strathern, E.W. Jones, and J.R. Broach, eds (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 181-299.

Kobayashi, M., Yanaka, N., Nagasawa, T., and Yarnada, H. (1992a). Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rbodocbrous K22 and expression of its gene and identification of its active site residue. Biochemistry 31, 9000-9007.

Kobayashi, M., Komeda, H., Yanaka, N., Nagasawa, T., and Yarnada, H. (1992b). Nitrilase from Rbodococcus rbodocbrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue. J. Biol. Chem. 267, 20746-20751.

Kobayashi, M., Izui, H., Nagasawa, T., and Yamada, H. (1993). Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: Cloning the Alcaligenes gene and site-

/ directed mutagenesis of cysteine residues. Proc. Natl. Acad. Sci.

Kobayashi, M., Suzuki, T., Fujita, T., Masuda, M., and Shimizu, S. (1 995). Occurrence of enzymes involved in biosynthesis of indole- 3-acetic acid from indole-3-acetonitrile in plant-associated bacte- ria, Agrobacterium and Rbizobium. Proc. Natl. Acad. Sci. USA 92,

2 I 31 -2 1 42.

3796-3801.

USA 90,247-251.

71 4-71 8.

1790 The Plant Cell

Koshiba, T., Kamiya, Y., and lino, M. (1995). Biosynthesis of indole-3-acetic acid from L-tryptophan in coleoptile tips of maize (Zea mays L.). Plant Cell Physiol. 36, 1503-1510.

Lam, H.-M., Coschigano, K., Schultz, C., Melo-Oliveira, R., Tjaden, G., Oliveira, I., Ngai, N., Hseih, M.-H., and Coruzzi, G. (1995). Use of Arabidopsis mutants and genes to study amide amino acid biosynthesis. Plant Cell 7, 887-898.

Last, R.L., and Fink, G.R. (1 988). Tryptophan-requiring mutants of the plant Arabidopsis tbaliana. Science 240, 305-31 O.

Last, R.L., Bissinger, P.H., Mahoney, D.J., Radwanski, E.R., and Fink, G.R. (1991). Tryptophan mutants in Arabidopsis: The conse- quences of duplicated tryptophan synthase p genes. Plant Cell 3,

Lincoln, C., Britton, J.H., and Estelle, M. (1990). Growth and development of the ar7 mutants of Arabidopsis. Plant Cell 2,

Michalczuk, L., Ribnicky, D.M., Cooke, T.J., and Cohen, J.D. (1 992). Regulation of indole-3-acetic acid biosynthetic pathways in carrot cell cultures. Plant Physiol. 100, 1346-1353.

Niyogi, K.K., Last, R.L., Fink, G.R., and Keith, B. (1993). Suppressors of trpl fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase p subunit. Plant Cell5, 101 1-1027.

Nonhebel, H.M., Cooney, T.P., and Simpson, R. (1993). The route, control and compartmentation of auxin synthesis. Aust. J. Plant Physiol. 20, 527-539.

Normanly, J., Cohen, J.D., and Fink, G.R. (1993). Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid. Proc. Natl. Acad. Sci. USA 90,

Normanly, J., Slovin, J.P., and Cohen, J.D. (1995). Rethinking auxin biosynthesis and metabolism. Plant Physiol. 107, 323-329.

Oetiker, J., Gebhardt, C., and King, P.J. (1990). A temperature- sensitive auxin auxotroph not deficient in indole-3-acetic acid. Planta 180,220-228.

Pickett, F.B., Wilson, A.K., and Estelle, M. (1990). The auxl mutation of Arabidopsis confers both auxin and ethylene resistance. Plant Physiol. 94, 1462-1466.

Radwanski, E.R., and Last, R.L. (1 995). Tryptophan biosynthesis and metabolism: Biochemical and molecular genetics. Plant Cell

Radwanski, E.R., Barczak, A.J., and Last, R.L. (1 996). Character- ization of tryptophan synthase alpha subunit mutants of Arabi- dopsis tbaliana. MOI. Gen. Genet. 253, 353-361.

Reid, J.B. (1993). Plant hormone mutants. J. Plant Growth Regul.

345-358.

1071-1080.

10355-1 0359.

7,921-934.

12,207-226.

Reid, J.B., and Howell, S.H. (1995). Hormone mutants and plant development. In Plant Hormones, P.J. Davies, ed (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 448-485.

Schmidt, R.C., Müller, A., Hain, R., Bartling, D., and Weiler, E.W. (1 996). Transgenic tobacco plants expressing the Arabidopsis tbaliana nitrilase II enzyme. Plant J. 9, 683-691.

Schneider, E.A., and Wightman, F. (1974). Metabolism of auxin in higher plants. Annu. Rev. Plant Physiol. 25,487-513.

Sembdner, G., Gross, D., Liebisch, H.-W., and Schneider, G. (1981). Biosynthesis and metabolism of plant hormones. In Hor- mona1 Regulation and Development. I. Molecular Aspects of Plant Hormones, J. MacMillan, ed (Berlin: Springer-Verlag), pp. 281-444.

Sitbon, F., Edlund, A., Gardestrom, P., Olsson, O., and Sandberg, G. (1 993). Compartmentation of indole-3-acetic acid metabolism in protoplasts isolated from leaves of wild-type and IAA-overpro- ducing transgenic tobacco plants. Planta 191, 274-279.

Stasinopoulos, T.C., and Hangarter, R.P. (1 990). Preventing pho- tochemistry in culture media by long-pass light filters alters growth of cultured tissues. Plant Physiol. 93, 1365-1369.

Thimann, K.V., and Mahadevan, S. (1 964). Nitrilase. I. Occurrence, preparation, and general properties of the enzyme. Arch. Bio- chem. Biophys. 105, 133-141.

Tsunoda, H., and Yamaguchi, K. (1995). The cDNA sequence of an auxin-producing nitrilase homologue in tobacco (accession no. D63331). Plant Physiol. 109,339.

Valvekens, D., Van Montagu, M., and Van Lijsebettens, M. (1988). Agrobacterium tumefaciens-mediated transformation of Arabi- dopsis tbaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536-5540.

Wilson, A.K., Pickett, F.B., Turner, J.C., and Estelle, M. (1990). A dominant mutation in Arabidopsis confers resistance to auxin, ethylene, and abscisic acid. MOI. Gen. Genet. 222, 377-383.

Wright, A.D., Sampson, M.B., Neuffer, M.G., Michalczuk, L., Slovin, J.P., and Cohen, J.D. (1991). Indole-3-acetic acid biosynthesis in the mutant maize orange pericarp, a tryptophan auxotroph. Science 254, 998-1000.

Zhao, J., and Last, R.L. (1 995). lmmunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis tbaliana. J. Biol. Chem. 270,608143087.

Zhou, L., Bartel, B., and Thornburg, R. (1996a). Nucleotide sequence of the Arabidopsis tbaliana nitrilase 1 gene (accession no. U38845). Plant Physiol. 110, 337.

Zhou, L., Bartel, B., and Thornburg, R. (1996b). Nucleotide sequence of a pathogen-induced nitrilase gene from Arabidopsis thaliana: Nit2 (accession no. U47114). Plant Physiol. 110, 1048.

Zilversmit, D.B., Enterman, C., and Fisher, M.C. (1943). On the calculation of turnover rate from experiments involving the use of labeling agents. J. Gen. Physiol. 26, 325-331.

DOI 10.1105/tpc.9.10.1781 1997;9;1781-1790Plant Cell

J Normanly, P Grisafi, G R Fink and B Bartelnitrilase encoded by the NIT1 gene.

Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the

This information is current as of July 8, 2018

Permissions 98X

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw15322

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

arabidopsis mutants resistant to the auxin effects of ... · the plant cell, vol. 9, 1781-1790,...

Documents