Arabidopsis Cytochrome P450 cyp83B1 Mutations Activate the Tryptophan Biosynthetic Pathway

Corresponding author: Judith Bender, Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205., jbender{at}welchlink.welch.jhu.edu (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In plants, the tryptophan biosynthetic pathway provides a number of important secondary metabolites including the growth regulator indole-3-acetic acid (IAA) and indole glucosinolate defense compounds. Genes encoding tryptophan pathway enzymes are transcriptionally induced by a variety of stress signals, presumably to increase the production of both tryptophan and secondary metabolites during defense responses. To understand the mechanism of transcriptional induction, we isolated altered tryptophan regulation (atr) mutants in Arabidopsis thaliana with activated transcription of tryptophan genes. One atr complementation group consisted of mutations in the cytochrome P450 gene CYP83B1. Mutant plants had constitutively activated expression of the ATR1 Myb factor gene, which was identified as a positive regulator of tryptophan genes via the atr mutant screen. cyp83B1 mutants were previously characterized as having defects in IAA homeostasis due to perturbation of secondary tryptophan metabolism. Our findings indicate that the upregulation of tryptophan pathway genes might also contribute to the overaccumulation of IAA in mutant plants. Moreover, we show that cyp83B1 mutants have lesion-mimic phenotypes, suggesting that multiple stress pathways are activated by loss of CYP83B1 function.

IN plants the tryptophan biosynthetic pathway leads to the synthesis of the amino acid tryptophan and a number of important secondary metabolites including the growth regulator indole-3-acetic acid (IAA) and indole glucosinolate defense compounds (RADWANSKI and LAST 1995 ). These metabolites differ in abundance, spatial distribution, developmental control, and environmental responsiveness, implying that tryptophan primary and secondary metabolic pathways are highly regulated.

The first committed enzyme in tryptophan biosynthesis is anthranilate synthase (AS). This enzyme provides a key point of control for the flow of metabolites through the pathway. AS consists of an -subunit that contains the catalytic activity and a ß-subunit that provides a glutamine amidotransferase activity. AS is the target of feedback inhibition by free tryptophan, which binds to an allosteric site on the -subunit to downregulate the enzyme when tryptophan is abundant. In Arabidopsis, the -subunit of AS is encoded by two somewhat divergent genes, ASA1 and ASA2 (NIYOGI and FINK 1992 ). ASA1 can be transcriptionally induced by several stress signals including bacterial pathogen infection and amino acid starvation, whereas ASA2 is not inducible (NIYOGI and FINK 1992 ; ZHAO and LAST 1996 ; ZHAO et al. 1998 ). Arabidopsis also encodes three AS ß-subunit genes, ASB1, ASB2, and ASB3 (NIYOGI et al. 1993 ). ASB transcription is induced by the same signals that up-regulate ASA1 expression.

Arabidopsis mutants with altered tryptophan regulation (atr) have been used to elucidate how the tryptophan pathway is controlled. The atr mutants were isolated as plants that are resistant to tryptophan feedback inhibition induced either by high levels of exogenous tryptophan or by a toxic analogue of tryptophan (BENDER and FINK 1998 ). The atr1D mutation, which creates a dominant overexpression allele of a Myb transcription factor ATR1, was isolated from this screen. The atr1D mutation increases ASA1 expression specifically in the hypocotyl of seedlings. This mutation is also likely to perturb the expression of other tryptophan metabolism genes because overexpression of the ASA1 gene alone is not sufficient to confer the atr1D resistance phenotype (NIYOGI 1993 ). The ATR1 transcription unit consists of three short open reading frames (ORFs) upstream of the main ATR1-encoding ORF. The atr1D mutation creates a premature stop codon in the first upstream ORF and causes an increase in the steady-state message levels of the ATR1 transcript. The mutation is presumed to act primarily by improving the translation of the ATR1 ORF, which then has the secondary effect of stabilizing the ATR1 messenger RNA. The deregulated phenotypes of the atr1D mutation can be phenocopied by expressing the ATR1 ORF, in the absence of the short upstream ORFs, from a strong constitutive promoter on a transgene (J. BENDER, unpublished observations). Thus, high levels of the ATR1 Myb transcription factor can positively regulate tryptophan genes and confer tryptophan feedback resistance. However, the physiological function of ATR1 remains to be characterized.

Another class of mutations recovered from the atr screen was the atr4 complementation group. We report here that the atr4 locus is the CYP83B1 cytochrome P450 gene. The CYP83B1 enzyme has been shown to catalyze the N-hydroxylation of indole-3-acetaldoxime (IAOx), which is an intermediate step in the biosynthesis of indole glucosinolates (BAK et al. 2001 ; HANSEN et al. 2001 ). Loss-of-function mutations in CYP83B1 confer adventitious root development from hypocotyl tissue, a phenotype diagnostic of increased IAA responses, and biochemical analysis indicates that cyp83B1 mutant plants have elevated levels of IAA (DELARUE et al. 1998 ; BARLIER et al. 2000 ). Thus, cyp83B1 mutations have been postulated not only to block the synthesis of indole glucosinolate defense compounds, but also to redirect the flow of indolic compounds toward IAA synthesis (BAK et al. 2001 ).

We show that the cyp83B1 mutants have upregulation of the ATR1 Myb factor, which is the likely cause of their atr phenotypes. Double mutants combining cyp83B1 and the dominant atr1D allele yield plants with enhanced adventitious root production. This observation suggests that increased tryptophan gene expression, stimulated by increased levels of the ATR1 Myb factor, contributes to the high IAA phenotype. We also show that cyp83B1 mutants display a number of morphological and gene regulation phenotypes diagnostic of constitutively upregulated stress responses. Thus, beyond the immediate perturbation of indole glucosinolate biosynthesis, cyp83B1 mutations confer an array of pleiotropic effects.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant growth conditions:
For all assays conducted with seedlings, surface-sterilized seeds were plated on plant nutrient 0.5% sucrose (PNS) medium (HAUGHN and SOMERVILLE 1986 ) with 0.75% agar in 100 x 100 x 15-mm square petri plates. Plates were sealed with Parafilm and grown under glass plates in a Percival CU-32L tissue culture incubator at 20° under continuous illumination. Glass plates were used to screen out ultraviolet (UV) light, which can contribute to the breakdown of chemical supplements, without blocking visible light. Adult plants were grown in Scott's Metromix 360 soil-less medium at 22° under continuous illumination. For all experiments except spontaneous lesion formation (see below), light intensity was 100 µE m^-2 sec^-1.

Mutant isolation and positional cloning:
The atr4-2 allele was isolated from a previously described screen for mutants that are resistant to feedback inhibition by high levels of exogenous tryptophan (BENDER and FINK 1998 ). This screen exploits the Columbia trp1-100 mutant, which carries a missense mutation in the PAT1 gene encoding the second enzyme in the tryptophan pathway (ROSE et al. 1997 ). The trp1-100 strain is weakly blue fluorescent under UV light due to accumulation of anthranilate compounds. However, when trp1-100 is grown on medium containing 100 µM tryptophan, the fluorescence is suppressed, presumably due to feedback inhibition of AS activity and reduction in anthranilate production. The atr4-2 allele was thus isolated by screening for ethyl methane sulfonate (EMS)-induced mutants in the trp1-100 background whose fluorescence persists on high tryptophan medium. The atr4-2 allele was subsequently segregated away from the trp1-100 mutation by crosses with wild-type Columbia. The atr4-1 allele was isolated from a screen of EMS-mutagenized Columbia seedlings for mutants that are resistant to the toxic tryptophan analogue 5-methyl-tryptophan (5MT; BENDER and FINK 1998 ). In this assay, seeds are germinated on PNS medium supplemented with 15 µM 5MT and scored at 10–14 days for root length.

The two atr4 mutations were shown to be allelic by complementation crosses: the F₁ progeny of the atr4-1 x atr4-2 cross were resistant to 5MT and displayed atr4 morphological defects such as adventitious root formation. The atr4-2 allele was mapped to chromosome 4 with standard cleaved amplified polymorphic sequence (CAPS; KONIECZNY and AUSUBEL 1993 ) and simple sequence length polymorphism (BELL and ECKER 1994 ) methods by crossing with the polymorphic strain Landsberg erecta (Ler). To localize the mutation, a second mapping cross was performed with a derivative of the Ler strain that carries three visible marker mutations along chromosome 4: bp1-1, cer2-2, and ap2-1 (strain CS8 from the Arabidopsis Biological Resource Center). Segregation of the atr4-2 morphology vs. the visible marker mutations in the progeny of this cross placed atr4-2 between cer2-2 and ap2-1, on the lower arm of the chromosome. A total of 419 plants with recombination breakpoints between cer2-2 and ap2-1, and thus near the atr4-2 locus, were used for fine-structure mapping. This analysis showed complete linkage to the g8300 cosmid-mapping marker (http://www.arabidopsis.org/maps/CAPS_Chr4.html). A contig of wild-type Columbia genomic clones spanning this region was isolated by hybridization from a plaque library (BENDER and FINK 1998 ), and fragments containing predicted ORFs were subcloned into the plant transformation vector pBIN19 (BEVAN 1984 ) to see if they could complement the atr4-2 mutation when introduced by Agrobacterium tumefaciens-mediated in planta transformation (CLOUGH and BENT 1998 ). This analysis showed that the atr4-2 morphological defects and 5MT resistance phenotype were complemented by a 5-kb EcoRI fragment containing the CYP83B1 gene. The mutant was also complemented by a cDNA of CYP83B1 driven from the cauliflower mosaic virus 35S promoter of transformation vector pBICaMV (HULL et al. 2000 ). The cDNA was isolated by hybridization screening of a Ler seedling cDNA library (MINET et al. 1992 ). The CYP83B1 Columbia cDNA sequence has been reported as GenBank accession no. D78598 (MIZUTANI et al. 1998 ) and the Columbia genomic sequence lies on bacterial artificial chromosome F3L17 (GenBank accession no. AL080283).

The CYP83B1 coding region was amplified by PCR from genomic DNA of both atr4 alleles. Products from two independent PCR reactions were cloned and sequenced for each allele. Both mutant alleles create a restriction site change, which allows PCR-based detection.

The atr4-1 mutation destroys one of two MspI sites in the 384-bp region amplified with the primers ATR4-A 5'-ATCTACCAGCAGAAACGTCC-3' and ATR4-B 5'-GTGTCGCAAGTTTCAGACCG-3'; the atr4-2 mutation destroys one of six HaeIII sites in the 711-bp region amplified with the primers ATR4-A and ATR4-C 5'-AAGACCAACCTTTCTCCATC-3'.

RNA gel blot analysis:
Total RNA was prepared from whole seedlings grown aseptically on PNS medium under glass plates for 10 days postgermination before RNA extraction. RNA gel blot analysis was performed by formaldehyde gel electrophoresis, transfer to nylon membranes, and hybridization with radiolabeled probes as previously described (MELQUIST et al. 1999 ). Probes were cDNA fragments from the indicated genes. A ß-tubulin probe (TUB) was used as a control to correct for loading differences. The ASA1, CYP79B2, CYP83B1, PDF1.2, and TUB probes were full-length cDNAs. The CYP79B2 probe has 80% nucleic acid identity with the related gene CYP79B3, and therefore should not significantly cross-hybridize under high stringency conditions. The PR1 probe was an internal cDNA fragment (ROGERS and AUSUBEL 1997 ). The ATR1 probe was a cDNA fragment extending from the 5' end of the transcript to an internal EcoRI site. Band intensities were quantitated using Fuji Phosphoimager and MACBAS 2.2 software. All results were reproduced in two or three independent experiments.

Plant strains:
The axr1-3 mutant was obtained from the Arabidopsis Biological Resource Center (CS3075). The atr4-1 axr1-3 double mutant was constructed by screening for F₂ progeny of the cross that were resistant to 150 nM 2,4-D, a phenotype diagnostic of the homozygous axr1-3 mutation (ESTELLE and SOMERVILLE 1987 ). The axr1-3 homozygous individuals were then screened for the atr4-1 mutation using the PCR genotype assay described above. The atr4-1 NahG strain was made by screening progeny of the cross for the kanamycin resistance marker on the NahG transgene and the atr4-1 genotype. The NahG transgenic strain in the Ler background (BOWLING et al. 1994 ) was a gift of X. Dong, Duke University. To eliminate potential phenotypic contributions from the erecta mutation in this background, plants were also scored for a wild-type ERECTA phenotype. The atr4-1 atr1D double mutant was constructed by screening progeny of the cross with PCR genotype assays for both atr4-1 and atr1D (BENDER and FINK 1998 ). Because this double mutant is sterile, it was propagated by segregation from an atr1D/atr1D homozygous atr4-1/ATR4 heterozygous parent. The cpr5-2 mutant (BOWLING et al. 1997 ) was obtained from the Arabidopsis Biological Resource Center (CS3770) and the rty mutant was obtained as a gift from J. Celenza, Boston University.

Assays for cyp83B1 phenotypes:
Spontaneous lesion formation was scored by growing seedlings aseptically on PNS medium for 20 days postgermination under glass plates and low light (25 µE m^-2 sec^-1) conditions. For each strain tested, a population of 48 seedlings was inspected for the presence of leaf lesions; if none of the plants in the population displayed lesions, the strain was scored as negative, and if any of the plants in the population displayed lesions, the strain was scored as positive. Under these conditions, both cyp83B1 and cpr5 mutant populations consisted of 75% seedlings with no lesions and 25% seedlings with one or two lesions. Adventitious root formation was scored by growing seedlings on PNS medium under glass plates and observing the phenotype at 14 days postgermination. 5MT resistance was scored by growing seedlings on PNS medium supplemented with 15 µM 5MT under glass plates and measuring root growth at 10 days postgermination.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and characterization of cyp83B1 mutants:
The atr4-1 and atr4-2 mutations in the CYP83B1 gene were originally isolated from EMS-mutagenized seedlings of the Columbia strain by screening for resistance to feedback inhibition induced by high levels of exogenous tryptophan or a toxic tryptophan analogue 5MT (see MATERIALS AND METHODS). 5MT presumably acts by feedback inhibiting the AS catalytic -subunit without substituting for the nutritional role of true tryptophan. One mechanism for 5MT resistance is creation of feedback resistance mutations in the AS -subunit gene, ASA1 (NIYOGI 1993 ; KREPS et al. 1996 ; LI and LAST 1996 ). Another mechanism of resistance is transcriptional upregulation of ASA1 and other tryptophan metabolism genes. For example, the dominant atr1D mutant carries an overexpression mutation in the ATR1 Myb transcription factor gene that leads to activation of ASA1 expression specifically in hypocotyl tissue (BENDER and FINK 1998 ). Under 5MT resistance screening conditions, wild-type Arabidopsis seedlings fail to develop roots whereas atr mutants display partial or full root development (Fig 1A).

View larger version (39K): In this window In a new window Download PPT slide   Figure 1. Altered tryptophan regulation phenotypes of the cyp83B1 mutant. (A) 5MT resistance. (B) RNA gel blot assays.

Unlike the previously characterized atr1D mutant, the atr4 mutants displayed a number of morphological abnormalities including the development of adventitious roots from the hypocotyl, epinastic (curled under) leaves, elongated petioles, formation of spontaneous necrotic lesions, delayed time to flowering, dark green pigmentation, reduced size, and reduced viability relative to the parental strain (Fig 2 and Fig 4; data not shown). The atr4-1 allele had morphological phenotypes stronger than those of the atr4-2 allele and was therefore used for all subsequent analyses unless otherwise specified.

View larger version (32K): In this window In a new window Download PPT slide   Figure 2. Pathogen response phenotypes of the cyp83B1 mutant. (A) Spontaneous lesion formation. (B) RNA gel blot assays.

View larger version (48K): In this window In a new window Download PPT slide   Figure 3. ATR1 steady-state message levels can accumulate in response to various hormones and trigger 5MT resistance in wild-type seedlings. (A) RNA gel blot assays. Wild-type Columbia seedlings were grown aseptically on PNS medium under glass plates for 10 days postgermination and then transferred to liquid PNS medium for a 6-hr induction before RNA extraction. The following concentrations of hormones were used: 20 µM methyl jasmonate (MeJA), 20 µM abscisic acid (ABA), 500 µM salicylic acid (SA), 20 µM IAA, 20 µM 6-benzylaminopurine (BAP), 20 µM gibberellic acid A3 (GA), 20 µM 1-aminocyclopropane 1-carboxylic acid (ACC), and 1 µM brassinolide (BR). (B) An inducer of ATR1 can elicit 5MT resistance in wild-type seedlings. Wild-type Columbia seedlings are shown after being grown for 10 days postgermination under glass plates on PNS medium containing no supplement, 15 µM 5MT, 10 µM MeJA, or both.

View larger version (51K): In this window In a new window Download PPT slide   Figure 4. The atr1D mutation enhances cyp83B1 adventitious roots. (A) Adventitious root development. (B) RNA gel blot assays.

For comparison, we examined other mutants with atr4-related morphological phenotypes for whether they were also resistant to 5MT. The rty or sur1 mutant has a strong adventitious root phenotype and accumulation of increased free IAA (BOERJAN et al. 1995 ; KING et al. 1995 ). This mutant displayed weak resistance to 5MT (Fig 1A), although the effect was probably underestimated because rty/sur1 has stunted root development even in the absence of 5MT. The necrotic lesion phenotype (Fig 2A) suggested that atr4 might have constitutive upregulation of pathogen response genes. Indeed, RNA gel blot analysis revealed that both the PR1 and PDF1.2 pathogen-responsive genes (REYMOND and FARMER 1998 ) were upregulated in the mutant (Fig 2B). The cpr5 mutant (BOWLING et al. 1997 ) constitutively activates a number of pathogen defense pathways and forms spontaneous necrotic lesions in the absence of pathogen challenge (Fig 2). However, this mutant was not resistant to 5MT (Fig 1A). Conversely, rty/sur1 did not display any necrotic lesions despite weak 5MT resistance. Together these results suggest that the atr4 mutant has a unique profile of gene expression changes that cause 5MT resistance, adventitious roots, and lesion-mimic phenotypes.

Cloning of cyp83B1 mutants:
The atr4 mutant locus was cloned on the basis of its map position (see MATERIALS AND METHODS). Standard mapping analysis placed the mutant locus on the lower arm of chromosome 4. Fine-structure mapping narrowed down the mutant region to an interval that contained several genes including the cytochrome P450 gene CYP83B1. This gene was shown to be the site of the atr4 lesions by two methods. First, the mutant alleles were sequenced, and both were found to have missense mutations relative to the wild-type Col sequence. The stronger atr4-1 allele corresponded to R438W (CGG to TGG), which alters a highly conserved residue in the heme-binding domain. The weaker atr4-2 allele corresponded to A291V (GCC to GTC), which affects an unconserved position. Second, the atr4-2 mutant phenotypes were completely complemented either by a wild-type genomic clone carrying only the CYP83B1 gene or by a CYP83B1 cDNA driven by the strong constitutive cauliflower mosaic virus 35S promoter. Furthermore, two other groups have reported cyp83B1 mutations with similar morphological defects to the atr4 isolates. The rnt1-1 mutant was isolated by reverse genetic screening for T-DNA insertions into cytochrome P450 genes (WINKLER et al. 1998 ), and the sur2 mutant was isolated from a En-1 insertional mutant collection on the basis of its adventitious root phenotype and cloned via the En-1 tag (BARLIER et al. 2000 ). The phenotypes of the stronger atr4-1 missense mutant allele are similar to those described for both insertional mutant alleles, suggesting that atr4-1 is a strong loss-of-function or even a null allele.

ATR1 is upregulated in the cyp83B1 mutant:
To determine whether the 5MT resistance phenotype of cyp83B1 correlates with activation of ASA1 expression, the transcription of ASA1 was monitored by RNA gel blot analysis of seedling tissues. This analysis revealed activation of ASA1 steady-state transcript levels in whole seedlings for cyp83B1 mutant vs. the wild-type parent strain (Fig 1B). These results were similar to those previously determined for the atr1D Myb overexpression mutant (BENDER and FINK 1998 ), although the whole-plant activation of ASA1 expression was higher in cyp83B1 than in atr1D (Fig 1B). The increased level of ASA1 expression might reflect input from elevated pathogen response signaling, as observed in the cpr5 mutant. Taken together, these results indicated that the atr phenotypes of cyp83B1 might be due to the upregulated tryptophan biosynthetic pathway and suggested the involvement of ATR1.

To investigate this possibility, we monitored steady-state levels of ATR1 transcripts by RNA gel blot analysis. ATR1 steady-state transcript levels were elevated in the cyp83B1 mutant relative to wild-type CYP83B1 plants, although not to as high an extent as in atr1D (Fig 1B). The rty mutant, which displayed weak 5MT resistance, also displayed weak activation of ATR1 expression. In contrast, the cpr5 mutant, which displayed no 5MT resistance, displayed no activation of ATR1 expression. These results show a correlation between increased ATR1 steady-state transcript levels and 5MT resistance.

The highly related Arabidopsis cytochrome P450 enzymes CYP79B2 and CYP79B3 were recently shown to convert tryptophan to IAOx, a precursor for both IAA and indole glucosinolate synthesis (HULL et al. 2000 ). Furthermore, overexpression of the CYP79B2 gene from the cauliflower mosaic virus 35S promoter on a transgene rendered wild-type Columbia seedlings weakly resistant to 5MT. This resistance is thought to be due to CYP79B2-mediated conversion of 5MT to nontoxic secondary metabolites. CYP79B2 is thus a candidate target gene for upregulation leading to 5MT resistance in atr1D and cyp83B1 mutants. To test this possibility, we monitored CYP79B2 steady-state transcript levels in wild-type and mutant strains by RNA gel blot analysis (Fig 1B). This analysis showed that CYP79B2 transcripts were indeed elevated in atr1D and cyp83B1 relative to wild-type Columbia. Therefore, CYP79B2 could contribute to 5MT resistance in these mutants.

We also used RNA gel blot analysis to determine whether CYP83B1 steady-state message levels were altered in either atr1D or cyp83B1 mutants relative to a wild-type control. CYP83B1 expression was increased in both of these mutants as well as in the cpr5 mutant (Fig 1B). However, because the CYP83B1 gene does not encode functional protein in the cyp83B1 mutant background, it is unlikely that its expression levels contribute significantly to the 5MT resistance phenotype. Interestingly, we found no change in CYP83B1 expression levels in the rty mutant background. This result contrasts with a previous report that CYP83B1 is upregulated in the sur1-3 allele (BARLIER et al. 2000 ). This difference might reflect a difference in alleles, a difference in the stage at which RNA was harvested, or a difference in growth conditions between the two experiments.

ATR1 expression is inducible:
To elucidate signals that might trigger the accumulation of the ATR1 transcript, we carried out induction experiments on wild-type plants using a variety of exogenously applied plant signaling molecules (Fig 3A). This analysis showed that several treatments, including methyl jasmonate, brassinolide, abscisic acid, and a cytokinin (6-benzylaminopurine), upregulated ATR1. Other treatments such as ACC, IAA, and salicylic acid decreased ATR1 expression. Consistent with a direct relationship between upregulation of ATR1 and 5MT resistance, addition of methyl jasmonate to 5MT medium was able to phenocopy an atr resistance phenotype in wild-type seedlings (Fig 3B). Thus, the ATR1 gene is regulated by multiple signaling pathways, one or more of which might be perturbed in the cyp83B1 mutant to yield activation of ATR1 and 5MT resistance.

The atr1D mutant enhances the adventitious root formation of cyp83B1:
The atr1D mutation is thought to improve the translation of the ATR1 protein (BENDER and FINK 1998 ). This mutation might thus have a stronger effect on ATR1 expression than the cyp83B1 mutation alone, especially if the cyp83B1 mutant effect is primarily at the level of transcription initiation (see DISCUSSION). We constructed a double mutant from these two alleles to determine whether atr1D could enhance cyp83B1 phenotypes. We found that the double mutant displayed markedly enhanced adventitious root formation (Fig 4A). Like the strong adventitious root mutant rty/sur1 (BOERJAN et al. 1995 ; KING et al. 1995 ), the cyp83B1 atr1D double mutant was sterile. The ATR1 steady-state message levels were also increased in the double mutant to approximately the same extent as in the atr1D single mutant (Fig 4B). These results suggest that increased deregulation of the genes controlled by ATR1, such as ASA1 and CYP79B2 (Fig 1B), contributes to the adventitious root phenotype in the cyp83B1 mutant background. This phenotype is correlated with increased levels of free IAA (BOERJAN et al. 1995 ; DELARUE et al. 1998 ). Therefore, IAA accumulation in cyp83B1 might reflect both the repartitioning of metabolites between indole glucosinolate and IAA biosynthesis (BAK et al. 2001 ) and an increased level of precursor compounds stimulated by ATR1.

AXR1 is necessary for cyp83B1 5MT resistance:
To further dissect the relationship between morphological defects and 5MT resistance in the cyp83B1 mutant, we constructed a double mutant with a block in IAA signaling using the axr1 mutation. axr1 was isolated by its resistance to exogenous auxin and confers a bushy plant morphology (ESTELLE and SOMERVILLE 1987 ). The AXR1 gene encodes a subunit of an E1 enzyme that activates the small protein RUB1 prior to its conjugation to target substrate proteins (DEL POZO et al. 1998 ). The emerging view is that AXR1 might contribute to the RUB1 regulation of an SCF ubiquitin ligase complex that controls the protein turnover of repressors of IAA-responsive genes (LEYSER 1998 ). By this model, in axr1 mutant strains the SCF complex would be inactive, IAA repressor proteins would accumulate to high levels, and IAA-responsive genes would remain quiescent, accounting for the IAA-resistant phenotype.

Double mutant axr1 cyp83B1 plants lacked adventitious root formation (Fig 5) and displayed the axr1 bushy morphology (data not shown). The double mutants also lacked upregulation of ATR1 and 5MT resistance (Fig 5). These results suggest that AXR1 function is required for the signal that triggers ATR1 activation in the cyp83B1 mutant background. However, the formation of spontaneous necrotic lesions was not affected in axr1 cyp83B1 plants. Therefore, AXR1 function is not required for this aspect of the cyp83B1 phenotype.

View larger version (30K): In this window In a new window Download PPT slide   Figure 5. AXR1 is necessary for 5MT resistance of cyp83B1 seedlings. (A) 5MT resistance. (B) RNA gel blot assays. (C) Spontaneous lesion formation. (D) Adventitious root development.

NahG expression does not block cyp83B1 5MT resistance:
The increased expression of the PR1 gene in cyp83B1 mutants is diagnostic of increased bacterial pathogen response signaling. Components of this response are mediated by elevated levels of salicylic acid (REYMOND and FARMER 1998 ). In fact, PR1 activation can be phenocopied by application of exogenous salicylic acid to uninfected plants (PENNINCKX et al. 1996 ). Salicylic acid levels can be modulated in planta by expression of the bacterial NahG salicylate hydroxylase enzyme (GAFFNEY et al. 1993 ; BOWLING et al. 1994 ). This enzyme acts by depleting salicylic acid through conversion into an inactive catechol.

To test whether salicylic acid might be involved in the 5MT resistance of cyp83B1 plants, we constructed a NahG cyp83B1 strain. In this strain the upregulation of ATR1 and 5MT resistance were unimpaired (Fig 6). The strain also displayed unimpaired adventitious root formation. However, the NahG cyp83B1 strain was suppressed for the formation of spontaneous necrotic lesions and displayed downregulation of PR1 steady-state transcript levels. These results suggest that tryptophan pathway responses are distinct from pathogen responses in the cyp83B1 mutant.

View larger version (34K): In this window In a new window Download PPT slide   Figure 6. NahG expression does not block 5MT resistance of cyp83B1 seedlings. (A) 5MT resistance. (B) RNA gel blot assays. (C) Spontaneous lesion formation. (D) Adventitious root development.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The tryptophan pathway in plants provides tryptophan for protein synthesis, IAA for regulation of development, and indole glucosinolate defense compounds. Genes in the pathway are transcriptionally responsive to a variety of biotic and abiotic stresses that cause increased demand for these metabolites (RADWANSKI and LAST 1995 ; ZHAO and LAST 1996 ; ZHAO et al. 1998 ). The atr screen in Arabidopsis was designed to elucidate the signals that converge on tryptophan pathway genes to control their expression. The screen has identified mutants that are resistant to high levels of exogenous tryptophan and toxic tryptophan analogues due to deregulation of the pathway.

In this work we show that loss-of-function alleles in the cytochrome P450 gene CYP83B1 are atr mutants of the atr4 complementation group. Loss-of-function cyp83B1 mutants were previously reported as sur2 mutants isolated on the basis of the adventitious root phenotype (DELARUE et al. 1998 ). In addition, the rnt1-1 allele of cyp83B1 was identified by reverse genetic screening for T-DNA insertions in cytochrome P450 genes (WINKLER et al. 1998 ). Because the adventitious root phenotype suggests a perturbation in IAA levels, work on both the sur2 and rnt1-1 mutations has focused on IAA metabolism. For example, sur2 alleles of cyp83B1 have been reported to have elevated levels of free IAA (DELARUE et al. 1998 ; BARLIER et al. 2000 ). Furthermore, the CYP83B1 enzyme has been shown in vitro to catalyze the conversion of IAOx to 1-aci-nitro-2-indolyl-ethane, a precursor for indole glucosinolates (BAK et al. 2001 ; HANSEN et al. 2001 ). Putting these observations together, the emerging view is that cyp83B1 mutants accumulate IAA because the block in the indole glucosinolate pathway causes an accumulation of IAOx, which is redirected into the IAA synthesis branch of tryptophan secondary metabolism (BAK et al. 2001 ; Fig 7).

View larger version (18K): In this window In a new window Download PPT slide   Figure 7. The tryptophan pathway in Arabidopsis. Dashed lines with solid arrowheads indicate multistep pathways. Lines with open arrowheads indicate positive regulation and lines with perpendicular bars indicate negative regulation. Abbreviations for signaling molecules are as described in Fig 3.

Here we have focused on the atr phenotypes of cyp83B1 mutants. One of the genes that is upregulated in cyp83B1 is the ATR1 Myb transcription factor-encoding gene (Fig 1). ATR1 was originally identified as a putative positive regulator of tryptophan genes via an overexpression allele, atr1D (BENDER and FINK 1998 ). Thus, a simple explanation for the cyp83B1 atr phenotypes is that they are also caused by the overexpression of ATR1. Consistent with this model, when ATR1 upregulation is blocked in the axr1 cyp83B1 double mutant, 5MT resistance is also blocked (Fig 5). However, because many genes are deregulated in cyp83B1 (Fig 1 and Fig 2), it remains possible that the atr phenotypes are due to other factors in addition to ATR1. We are currently pursuing various strategies to isolate loss-of-function atr1 alleles. If such mutants are viable, we can use double mutant analysis with cyp83B1 to determine whether 5MT resistance and perhaps other cyp83B1 phenotypes are ATR1 dependent.

In the atr1D and cyp83B1 mutants, ASA1 and CYP79B2 steady-state transcript levels are upregulated (BENDER and FINK 1998 ; Fig 1). Increased expression of both these genes is consistent with the mutant tryptophan feedback resistance phenotypes. Increased expression of ASA1 would create additional targets for tryptophan feedback inhibition, thus diluting the effects of high tryptophan or 5MT. Increased expression of CYP79B2 would allow increased conversion of tryptophan or 5MT to noninhibitory secondary metabolites (HULL et al. 2000 ). Thus, even though overexpression of ASA1 alone is not sufficient to confer 5MT resistance (NIYOGI 1993 ), and overexpression of CYP79B2 alone confers only weak resistance (HULL et al. 2000 ), the combined deregulation of both genes could account for the stronger resistance displayed in strains that overexpress ATR1. ATR1 could also have additional target genes that contribute toward tryptophan feedback resistance.

The upregulation of ASA1 and CYP79B2 is also consistent with our observation that the atr1D cyp83B1 double mutant has exaggerated adventitious root formation (Fig 4). ASA1 is a key point of control for the flow of metabolites through the tryptophan pathway (Fig 7). CYP79B2 produces IAOx (HULL et al. 2000 ), which presumably lies at the branchpoint between IAA and indole glucosinolate biosynthesis (BAK et al. 2001 ). Therefore, stimulation of ASA1 and CYP79B2 transcript levels by atr1D combined with the cyp83B1 block in the indole glucosinolate branch of the pathway would be predicted to increase the flow of tryptophan metabolites toward IAA (Fig 7). Interestingly, the characteristic phenotype of sur2 alleles of cyp83B1 was previously shown to be suppressed when plants were grown in the presence of exogenous IAA (BARLIER et al. 2000 ). Taken together with our observation that exogenous IAA suppresses ATR1 expression (Fig 3), a possible explanation for this result is that downregulation of ATR1 blocks activation of genes like ASA1 and CYP79B2 that contribute to the adventitious root phenotype.

Expression of ATR1 can be induced by exogenous application of several signaling compounds including methyl jasmonate, brassinolide, abscisic acid, and cytokinin (Fig 3). Thus, one or more of these compounds is likely to be elevated in the cyp83B1 mutant, counterbalancing potential negative effects from IAA and salicylic acid (see below). Consistent with the possibility that the mutant might have elevated levels of methyl jasmonate, methyl jasmonate-responsive genes including PDF1.2 (Fig 2) and LOX2 (data not shown) are upregulated in the mutant. However, these methyl jasmonate-responsive genes are also upregulated in the cpr5 mutant (BOWLING et al. 1997 ), and yet this strain does not display activation of ATR1 or 5MT resistance (Fig 1). Perhaps in cpr5, signals such as methyl jasmonate that act positively on ATR1 expression are overwhelmed by other signals, such as salicylic acid (BOWLING et al. 1997 ; Fig 2 and Fig 3), that act negatively on ATR1 expression.

It remains unknown whether the increase in ATR1 steady-state message levels in cyp83B1 reflects activation of transcription initiation, increased transcript stability, or both. The ATR1 transcript includes three short upstream ORFs (BENDER and FINK 1998 ). The atr1D overexpression allele creates a premature termination codon in the first and longest of these ORFs. The simplest interpretation of this observation is that the first upstream ORF inhibits translation of the downstream ATR1 ORF and that the atr1D mutation disrupts this translational inhibition, leading to improved ATR1 message stability. Thus, the atr1D mutation is predicted to have a strong positive effect on the levels of ATR1 protein. In contrast, signaling molecules such as methyl jasmonate that increase ATR1 steady-state message levels (Fig 3) might act solely at the level of transcription initiation, without a mechanism to override the translational repression of the upstream ORFs. ATR1 transcriptional activation alone would thus be predicted to yield only a modest increase in ATR1 protein levels. This scenario would explain our observation that the atr1D cyp83B1 double mutants display an exaggerated adventitious root phenotype (Fig 4), whereas growing the cyp83B1 mutant on medium supplemented with 20 µM methyl jasmonate does not confer this phenotype (data not shown).

The axr1 mutation, which was previously isolated on the basis of its resistance to high levels of exogenous auxin, blocks upregulation of ATR1 and 5MT resistance in cyp83B1 plants (Fig 5). One interpretation of this result is that the activation of ATR1 is mediated by IAA signaling. However, two observations argue against this view. First, exogenous IAA downregulates ATR1 (Fig 3). Second, the rty/sur1 mutant has stronger IAA responses than does the cyp83B1/sur2 mutant (BOERJAN et al. 1995 ; DELARUE et al. 1998 ), and yet it has only modest upregulation of ATR1 and weak 5MT resistance (Fig 1). An alternative possibility is that axr1 affects more than just IAA sensing. Because the AXR1 protein is involved in RUB1 post-translational modifications (LEYSER 1998 ), it could have multiple target substrates, some of which control the signals that activate ATR1. Loss of AXR1 function does not block spontaneous lesion formation in the cyp83B1 mutant (Fig 5), suggesting that AXR1-mediated signaling is not required for this phenotype.

Because the cyp83B1 mutant forms spontaneous necrotic lesions (Fig 2), it can be considered a "lesion-mimic" mutant (ALVAREZ 2000 ). Lesion formation is usually induced by the hypersensitive response (HR) to pathogen infection. During this response, tissue around the site of infection undergoes rapid cell death to block the spread of the pathogen and releases a signal to prime systemic acquired resistance (SAR) against subsequent pathogen infection. The salicylic acid-responsive gene PR1 is upregulated locally around HR lesions and globally in SAR (CARR et al. 1987 ; REYMOND and FARMER 1998 ). Thus, a possible explanation for the lesion-mimic phenotypes and PR1 upregulation of cyp83B1 is that the mutant has elevated levels of salicylic acid. Consistent with this possibility, expression of the NahG salicylate hydroxylase enzyme blocks lesion formation and PR1 activation (Fig 6). In fact, PR1 expression is suppressed well below the normal basal level in the NahG cyp83B1 strain. This reduced expression might represent the effects of other signaling pathways that are perturbed in cyp83B1 and that act contrary to the stimulatory effects of salicylic acid on PR1 expression.

If salicylic acid is elevated in cyp83B1, it presumably does not block ATR1 activation (Fig 3) because of its levels, tissue distribution, or competing signals from other pathways (Fig 7). It is interesting to note that when salicylic acid is depleted by NahG in the cyp83B1 background, ATR1 expression is elevated to a greater degree than in cyp83B1 (Fig 6). This observation suggests that there actually is partial suppression of ATR1 due to elevated salicylic acid in the cyp83B1 mutant, but that this suppression is antagonized by other activating signals. Blocking lesion-mimic phenotypes with NahG does not affect either 5MT resistance or adventitious root formation (Fig 6). Thus, salicylic acid is unlikely to be involved in the activation of these phenotypes.

Although the cyp83B1 mutation affects a single biosynthetic enzyme, it has multiple pleiotropic effects. The perturbation of IAA levels and the formation of adventitious roots in mutant plants can be explained by a primary disruption in tryptophan secondary metabolism (Fig 7). However, other mutant phenotypes, including activation of ATR1 and 5MT resistance or activation of PR1 and lesion formation, are not consistent with elevated IAA as the cause. As discussed above, ATR1 expression is suppressed by exogenous IAA. Furthermore, exogenous IAA does not activate PR1 expression in wild-type plants (data not shown), and the high IAA mutant rty/sur1 does not display activation of PR1 or obvious lesion formation (Fig 2). Instead, the loss of CYP83B1 function might activate non-IAA signaling pathways as a secondary consequence of the mutation. For example, the reduction of indole glucosinolate defense compounds in the cyp83B1 mutant (BAK et al. 2001 ; Fig 7) might upregulate other defense pathways such as those involving methyl jasmonate (accounting for the activation of ATR1 and PDF1.2) and salicylic acid (accounting for the activation of PR1). A promising direction for unraveling these complex signaling interactions is the isolation of second-site suppressor mutations that block some or all of the cyp83B1 mutant phenotypes.

	ACKNOWLEDGMENTS

We thank the Arabidopsis Biological Resource Center, Xinnian Dong, John Celenza, and Krishna Niyogi for plant strains and cloned probe fragments. This work was supported by the National Science Foundation under grant IBN 9723172 to J.B.

Manuscript received August 9, 2001; Accepted for publication October 12, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ALVAREZ, M. E., 2000  Salicylic acid in the machinery of hypersensitive cell death and disease resistance. Plant Mol. Biol. 44:429-442[Medline].

BAK, S., F. E. TAX, K. A. FELDMANN, D. W. GALBRAITH, and R. FEYEREISEN, 2001  CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis. Plant Cell 13:101-111[Abstract/Free Full Text].

BARLIER, I., M. KOWALCZYK, A. MARCHANT, K. LJUNG, and R. BHALERAO et al., 2000  The SUR2 gene of Arabidopsis thaliana encodes the cytochrome P450 CYP83B1, a modulator of auxin homeostasis. Proc. Natl. Acad. Sci. USA 97:14819-14824[Abstract/Free Full Text].

BELL, C. J. and J. R. ECKER, 1994  Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 19:137-144[Medline].

BENDER, J. and G. R. FINK, 1998  A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis. Proc. Natl. Acad. Sci. USA 95:5655-5660[Abstract/Free Full Text].

BEVAN, M., 1984  Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12:8711-8721[Abstract/Free Full Text].

BOERJAN, W., M. T. CERVERA, M. DELARUE, T. BEECKMAN, and W. DEWITTE et al., 1995  Superroot, a recessive mutation in Arabidopsis, confers auxin overproduction. Plant Cell 7:1405-1419[Abstract].

BOWLING, S. A., A. GUO, H. CAO, A. S. GORDON, and D. F. KLESSIG et al., 1994  A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance. Plant Cell 6:1845-1857[Abstract/Free Full Text].

BOWLING, S. A., J. D. CLARKE, Y. LIU, D. F. KLESSIG, and X. DONG, 1997  The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 9:1573-1584[Abstract].

CARR, J. P., D. C. DIXON, B. J. NIKOLAU, K. V. VOELKERDING, and D. F. KLESSIG, 1987  Synthesis and localization of pathogenesis-related proteins in tobacco. Mol. Cell. Biol. 7:1580-1583[Abstract/Free Full Text].

CLOUGH, S. J. and A. F. BENT, 1998  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16:735-743[Medline].

DELARUE, M., E. PRINSEN, H. V. ONCKELEN, M. CABOCHE, and C. BELLINI, 1998  Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis. Plant J. 14:603-611[Medline].

DEL POZO, J. C., C. TIMPTE, S. TAN, J. CALLIS, and M. ESTELLE, 1998  The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. Science 280:1760-1763[Abstract/Free Full Text].

ESTELLE, M. A. and C. SOMERVILLE, 1987  Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet. 206:200-206.

GAFFNEY, T., L. FRIEDRICH, B. VERNOOIJ, D. NEGROTTO, and G. NYE et al., 1993  Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261:754-756.

HANSEN, C. H., L. DU, P. NAUR, C. E. OLSEN, and K. B. AXELSEN et al., 2001  CYP83B1 is the oxime-metabolizing enzyme in the glucosinolate pathway in Arabidopsis. J. Biol. Chem. 276:24790-24796[Abstract/Free Full Text].

HAUGHN, G. W. and C. SOMERVILLE, 1986  Sulfonylurea-resistant mutants in Arabidopsis thaliana. Mol. Gen. Genet. 204:430-434.

HULL, A. K., R. VIJ, and J. L. CELENZA, 2000  Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis. Proc. Natl. Acad. Sci. USA 97:2379-2384[Abstract/Free Full Text].

KING, J. J., D. P. STIMART, R. H. FISHER, and A. B. BLEECKER, 1995  A mutation altering auxin homeostasis and plant morphology in Arabidopsis. Plant Cell 7:2023-2037[Abstract].

KONIECZNY, A. and F. M. AUSUBEL, 1993  A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J. 4:403-410[Medline].

KREPS, J. A., T. PONAPPA, W. DONG, and C. D. TOWN, 1996  Molecular basis of alpha-methyltryptophan resistance in amt-1, a mutant of Arabidopsis thaliana with altered tryptophan metabolism. Plant Physiol. 110:1159-1165[Abstract].

LEYSER, O., 1998  Auxin signalling: protein stability as a versatile control target. Curr. Biol. 8:R305-R307[Medline].

LI, J. and R. L. LAST, 1996  The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan. Plant Physiol. 110:51-59[Abstract].

MELQUIST, S., B. LUFF, and J. BENDER, 1999  Arabidopsis PAI gene arrangements, cytosine methylation and expression. Genetics 153:401-413[Abstract/Free Full Text].

MINET, M., M. E. DUFOUR, and F. LACROUTE, 1992  Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J. 2:417-422[Medline].

MIZUTANI, M., E. WARD, and D. OHTA, 1998  Cytochrome P450 superfamily in Arabidopsis thaliana: isolation of cDNAs, differential expression, and RFLP mapping of multiple cytochromes P450. Plant Mol. Biol. 37:39-52[Medline].

NIYOGI, K. K., 1993 Molecular and genetic analysis of anthranilate synthase in Arabidopsis thaliana, p. 213. Ph.D. Thesis, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA.

NIYOGI, K. K. and G. R. FINK, 1992  Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway. Plant Cell 4:721-733[Abstract/Free Full Text].

NIYOGI, K. K., R. L. LAST, G. R. FINK, and B. KEITH, 1993  Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit. Plant Cell 5:1011-1027[Abstract/Free Full Text].

PENNINCKX, I. A., K. EGGERMONT, F. R. TERRAS, B. P. THOMMA, and G. W. DE SAMBLANX et al., 1996  Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8:2309-2323[Abstract].

RADWANSKI, E. R. and R. L. LAST, 1995  Tryptophan biosynthesis and metabolism: biochemical and molecular genetics. Plant Cell 7:921-934[Medline].

REYMOND, P. and E. E. FARMER, 1998  Jasmonate and salicylate as global signals for defense gene expression. Curr. Opin. Plant Biol. 1:404-411[Medline].

ROGERS, E. E. and F. M. AUSUBEL, 1997  Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression. Plant Cell 9:305-316[Abstract].

ROSE, A. B., J. LI, and R. L. LAST, 1997  An allelic series of blue fluorescent trp1 mutants of Arabidopsis thaliana. Genetics 145:197-205[Abstract].

WINKLER, R. G., M. R. FRANK, D. W. GALBRAITH, R. FEYEREISEN, and K. A. FELDMANN, 1998  Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Isolation of mutations in the cytochrome P450 gene superfamily. Plant Physiol. 118:743-750[Abstract/Free Full Text].

ZHAO, J. and R. L. LAST, 1996  Coordinate regulation of the tryptophan biosynthetic pathway and indolic phytoalexin accumulation in Arabidopsis. Plant Cell 8:2235-2244[Abstract].

ZHAO, J., C. C. WILLIAMS, and R. L. LAST, 1998  Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor. Plant Cell 10:359-370[Abstract/Free Full Text].

This article has been cited by other articles:

				J. Stout, E. Romero-Severson, M. O. Ruegger, and C. Chapple Semidominant Mutations in Reduced Epidermal Fluorescence 4 Reduce Phenylpropanoid Content in Arabidopsis Genetics, April 1, 2008; 178(4): 2237 - 2251. [Abstract] [Full Text] [PDF]

				J. I. Kim, A. Sharkhuu, J. B. Jin, P. Li, J. C. Jeong, D. Baek, S. Y. Lee, J. J. Blakeslee, A. S. Murphy, H. J. Bohnert, et al. yucca6, a Dominant Mutation in Arabidopsis, Affects Auxin Accumulation and Auxin-Related Phenotypes Plant Physiology, November 1, 2007; 145(3): 722 - 735. [Abstract] [Full Text] [PDF]

				B. Dombrecht, G. P. Xue, S. J. Sprague, J. A. Kirkegaard, J. J. Ross, J. B. Reid, G. P. Fitt, N. Sewelam, P. M. Schenk, J. M. Manners, et al. MYC2 Differentially Modulates Diverse Jasmonate-Dependent Functions in Arabidopsis PLANT CELL, July 1, 2007; 19(7): 2225 - 2245. [Abstract] [Full Text] [PDF]

				C. Sorin, L. Negroni, T. Balliau, H. Corti, M.-P. Jacquemot, M. Davanture, G. Sandberg, M. Zivy, and C. Bellini Proteomic Analysis of Different Mutant Genotypes of Arabidopsis Led to the Identification of 11 Proteins Correlating with Adventitious Root Development Plant Physiology, January 1, 2006; 140(1): 349 - 364. [Abstract] [Full Text] [PDF]

				P. J. Overvoorde, Y. Okushima, J. M. Alonso, A. Chan, C. Chang, J. R. Ecker, B. Hughes, A. Liu, C. Onodera, H. Quach, et al. Functional Genomic Analysis of the AUXIN/INDOLE-3-ACETIC ACID Gene Family Members in Arabidopsis thaliana PLANT CELL, December 1, 2005; 17(12): 3282 - 3300. [Abstract] [Full Text] [PDF]

				A. W. WOODWARD and B. BARTEL Auxin: Regulation, Action, and Interaction Ann. Bot., April 1, 2005; 95(5): 707 - 735. [Abstract] [Full Text] [PDF]

				K. Ljung, A. K. Hull, J. Celenza, M. Yamada, M. Estelle, J. Normanly, and G. Sandberg Sites and Regulation of Auxin Biosynthesis in Arabidopsis Roots PLANT CELL, April 1, 2005; 17(4): 1090 - 1104. [Abstract] [Full Text] [PDF]

				J. L. Celenza, J. A. Quiel, G. A. Smolen, H. Merrikh, A. R. Silvestro, J. Normanly, and J. Bender The Arabidopsis ATR1 Myb Transcription Factor Controls Indolic Glucosinolate Homeostasis Plant Physiology, January 1, 2005; 137(1): 253 - 262. [Abstract] [Full Text] [PDF]